.

Advances in

MICROBIAL PHYSIOLOGY

This Page Intentionally Left Blank

Advances in

MICROBIAL PHYSIOLOGY

Edited by A. H. ROSE

School of Biological Sciences Bath University

England

and

D. W . TEMPEST Microbiological Research Esi~blishnient

Porton, Salisbury England

VOLUME 8

ACADEMIC PRESS, INC. iHart ourt Br,i( c lovanovic h. Publishrrrl

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PKINTF.I) INTHE UNlTEDSTATESOFAMIiRICA

8 5 86 87 88 9 8 7 6 5 4 3 2

Contributors to Volume 8

J. R. BENEMANN, Department of Ch.emistry, University of California, San

H. GOLDFINE, Department of Microl~iology, School of Medicine, University of

J. C.-C. HUANQ, Department of Bacteriology, University of California, Davis,

M. P. STARR, Department of RnrlPdoqy, Ilniversity of Cdifornia, Davis,

R. E. STRANUE. Microbiological Ke.wnrc,L E.otnbliahment, Porton, N r . Salisbury,

T. W . SUTHERLAND, Departnieiit o j Q ~ n ~ m l Microbiology, University of

R. C . VALENTINE, Department of Biochemiatrp, Univerdy of California,

Diego, Ln Jolla, California, 92037, IJ .8 .A .

Pennsylvania, Philadelphia, Penasylvania 19104, U.S.A.

California 95616, U.S.A.

California 95616, 1J.S.A.

Wilhhire, England.

Edinburgh, Edinburgh, Ncotland.

Berkeley, California 94720, U .S .A .

V


Contents

Contributors to Volume 8 . V

Comparative Aspects of Bacterial Lipids. HOWARD GOLDFINE

I. ~ ~ t r o ~ ~ c ~ , i o ~ . 11. Lipids of Bactesia C o ~ ~ ~ s e d With Thorte of Higher ~ a n ~ s r n s .

A. Apofar Componenta: F&ty Acids, Alk-I-Enyl Ethers and

B. ~ x t r a c ~ b l e Polar ids of Bacteria. . C. Non-Extractable Lipide in Bacteria , . D. Neutral Lipids of Bacteria . E, ~ o l ~ ~ - ~ y d r o x y b u t y r j c Acid . . A. Biosynthesie of the Apolar Components B. Biosynthesis of the Complex Lipids . A. Lipids of Gram-Negative Bacteria . B. Complex Lipids of Gram-Positive 3 ~ c ~ r i ~ C. Concluaions .

References.

Alkyl Ethers .

111. Bi~ayn~hee~s of BactpiaI Lipids . IV. Bacterial Lipid Compositions .

. t’. A ~ k i ~ o w ~ e ~ ~ ~ ~ n t ~ -

1 2

2 6

16 17 18 19 19 28 36 35 42 50 50 51

The Pathways of Nitrogen Fixation,

1. fntroduction . A. Nitrogen-Fixing Micro- Organisms B. B ioch~~ie t ry of Nitrogen Fixation

11. Electron Donors . A. fyruvate - 3% Fomate ,

C. ~ i ~ o t i n a m ~ d e ~ ~ ~ c l e o t ~ d ~ ~ , 23. Hydrogen . E. Photosynthetic Electron Donors

111. Electron Chmiers. A. Fencadaxins . B. Flavodaxins . C. Coupling Factors of Azotoltuclsr

A. Isolation and Prapertirw . B. ~ e c ~ ~ a n i ~ r n of Action

IV. Nitrogenam

vii

JOHN R. BENEMANN and R. C, VALENTINE

. 59 . 60 * M * 70 . 70 * 72 t 73 . 76 . 76 I 78 . (10 * 83 . 86 * 88 * Ht( * !t t

viii CONTENTS

V. Conclusions and Future Outlook . . 94 VI. Acknowledgements . . 97

References . . 98

Rapid Detection and Assessment of Sparse Microbial Pop u I at i o n s. R. E. STRANGE

1. Introduction . . 105 11. Principles of Itapid Microbisl Assessment Method8 . . 107

A. Physianl Properties of Microbes . . 108 B. Chemical Composition of Microbes . . 109 C. Microbial Growth and Metabolism . . 111 D. Bacterial Enzymes. . 113 E. Immunological Propertice of Microbes . . 114

Ill. Rapid Broad Spectrum Methods . . 118 A. Luminol Chemiluminescence . 118 B. Determination of ATP . . 120 C. Staining Methods . , 121

E. Methods Depending on Growth and Metabolism . . . 125 D. Physical Methods . . 124

I V . Rapid Specific Identification Methods . . 126 A. Immunofluorescence-Membrane Filtration Techniques . 126 B. Radioactive Antibody Technique . . 127 C. Analysis of Bactcrinl Growth Products . , 130 D. Gas Chromatography-l’yrolysis Methods . , 131 E. Analysis of Phosphorcwcnt Decay . . 131

V. Rapid Determination of Microbial Viability . . 131 A. lndirect Methods . . 131 B. Direct Mcthoda , . 132

A. Immunofluorescent-Monolayer Techniques . . 132 B. Immuno-Adherence . . 133 C. Radioactive Antibody Method Applied to Bacteriophage T7 133 D. Detection of Virus Activity with Gas Chromatography . 135

VlI. Conclusions and Prospects . . 136 VIII. Acknowledgements . . 137

References . . 137

VI. Rapid Detection and Determination of ViruHes . . 132

Bacterial Exopolysaccharides. I . W. SUTHERLAND

T. Introduction . . 143 I T . Production. . 14fi

B. In Wttslicd Cell Siwpeiihnn . . 148 A. Tn Crowing CuItiiiw . . 14fi

CONTENTS ix 111. Properties .

A. Isolation . B. Purification . C. Composition . D. Structural Features . E. Enzymic Hydrolysis . F. Structures of Rome ExopolyHncchnritl(.n

A. Enzymes and Precursors . B. Cell-Free Synthesis. . C. Control.

TV. Biosyntliesis ,

V. Function of Exopolysaccharides . VI. Unanswered Questions .

VII. Conclusions . References .

VIII. Acknowledgements .

. 149

. 149

. 149

. 150

. 156

. 158

. lf15 1 185 . 185 . 188 . 201 . 200 . 200 . 207 . 208 . 208

Physiology oft he Bdellovi brios. M. P. STARR and J. C.-C. H U A N G

I . Introduction .

uibrio . 11. Isolation, Cultivation, Distribution, and Taxonomy of Bdello-

A. Isolation . B. Culture Media . C. Maintenance of Bdellouibrio Cultures. D. Distribution and Importance in Nature E. A Taxonomic and Terminological Note

TTJ. Structure and Chemical Composition of Bdellovibrio A. Morphology and Ultrastructure . B. Chemical Composition of Bdellowibrio Ccll~,

A. Life Cycle of Host-Dependcnt Bdellovi brios R. Effects of Bdelhibrio on the Host, Cell . C . Mechanisms of Lysis of the HoHt Coll hy Bdellooitwio . D. Kinetics of Host-Dependent. Growth . E. Nutrition of Bdellovibrio . F. Host Specificity .

V. Metabolism of Bdellovibrio .

VII. Bdellotibrio Bacteriophages .

.

. .

.

. IV. Symbiosis Between Host-Dependent BdcllovibrioH and Host tk!lls

Vi . Host-Independent Derivatives of Bdellovibrio .

VIII. Acknowledgements . References . Author Index . Subject Index .

215

218 218 218 219 220 221 222 222 226 228 229 239 24 2 246 246 %4!4 352 254 256 257 257 263 277


Comparative Aspects of Bacterial Lipids

HOWARD GOLDFINE

Departmen4 of Microbiology, School of Medicine, University of Penaaylvania, Philadelphia, Pennsylvania 19104, U.S.A .

I. Introduction . . 1 . 2

A. Apolar Components: Fatty Acids, Alk-1-Enyl Ethers and Alkyl Ethers . . 2

B. Extractable Polar Lipids of Bactoria . . 6 C. Non-Extracteblo Lipids in Bactcriu . . 16 I). Neutral Lipids of Bacturia . . 17 E. Poly-/3-Hydroxybutyric Acid . . 18

111. Uiotiynthosis of Bacterial Lipids . . 19 A. Biosynthesis of the Apolar C'oixipomiits . . 19 B. Biosyntheais of the Complex Lipids . . 28

IV . Bacterial Lipid Compositions . . 35

B. Complex Lipids of Gram-Positive Bactoria . . 42

11. Lipids of Bacteria Compared With Those of Higher Organisms .

A. Lipids of Gram-Negative Bacteria . . . 36

C. Conclusions . . 50 V. Acknowledgements . . 60

References . . 51

I. Introduction

A deoade of intensive work on the lipid componente of the bacterial cell has revealed major differences in the lipids of prokaryotic and eukaryotic organisms and in the lipid compositions of different group of prokaryotic organisms. In this review, I shall attempt to outline them differences, to describe the biosynthetic systems the presence or absence of which lead to the observed composition8, and to atamsa the significance of these differcncea. Baoterial lipid8 have been discuseed extonsively in monographs by Asselineau (1966) and O'Leary (1967). Several review articles concerned with various aspects of bacterial lipids have appeared in therecent past (Kates, 1964,1966; Maofarlane, 1964b; Lennan, 1966;

In the shorthand designations of unsaturated fatty acida and aldehydes, the number preceding the colon ia the chain length. Following the colon in the number of double bonds. In the designation (n-x) for mono-unsaturated fatty acids, the position of the double bond is x rlrrbon atoms from the methyl end of the c h i n . Abbreviation8 used for baoterial phospholipids are given in Fig. 4 (p. 6).

1

9 HOWARD GOLDFINE

Law, 1087; lltawu, l!)ti7 ; Lennarz, 1!)70). A review on tlir nionibriiiies of bacteriit with pnrticular rcferciic.c! to the phosp1loli~)ide hus ulso appeared (01) t l c i i l i itttip el al., 19ti!)c).

11. Lipids of Bacteria Compared With Those of Higher Organisms

A N D ALKYI, ETHERS

I . Fatly Acids

The fatty acids of bacteria are generally 10-20 carbons in length, with 15-19 carbon chctins predominating. These are mainly of four types: straight-chain saturated, straight-chain mono-unsaturated, branched

11. APOLAIt COMlWNENTS : F A T T Y ACIDS, ALK-I-ENYL ETHERS

<')I 3

I is0 fatty acid CH,C:H(CIi2),COOiI

C ' H J

I ankiso fatty acid CH,C€I,I:H(C'H,),COOH

Ci*z / \

CHJ( cl€I2)J( !-.( '(('H,)&OOH I 1

lsctobacillic acid

R 11

CH3(clH2)5( 'H - - C ' H ( C ' i I 2 ) , ~ ( ' 0 O H cis-vacccnic acid

Fro. 1. For~uulao of bruiich~d, cyclopropnno arid mono-unsaturated fatty acids of bllcteria.

chains, predomin;intly iso and unleiso, and oyclopropanc fatty acids (Fig. 1). Several differences from the fatty acyl chains of higher organisms are immediately apparent. With few exceptions, which will be discussed later, the longer chain polyunsaturated fatty acids typical of the lipids of animal tissues are absent, as are the C,, di- and tri- unsaturated acids. The cyclopropane and branchetl-chain fatty acids, on the other hand, are rarely encountered in higher OrguniHm, with tho exception of the cyclopropeno fatty wids of pluntH (Law, 1971). Among the mono-unsaturated futty acids, the 11,-7 series (counting from t l i c methyl group) is the predominant typo. 'l'hus the C, mono-unsaturated fatty acid most commonly found i R cis-vuccenic acid, 18: 1 (Fig. 1) rather than oleic acid, 18: 1 d9 (Scheuerbrandt and Bloch, 1962).Tho C,, mono-unsaturated acid most frequently found is palmitoleic acid, 16: l d9, which i n d s o hhc most common 1O:l isomer in plantR and animals.

COMPARATIVE ASPECTS OF BACTERIAL LIPIDS 3

The cyclopropane fatty acids are widely distributed among both Gram-positive and Gram-negative species, for example in the lactobacilli, streptococci, clostridia, enterobacteria, and Brucellaceae (see Section 111. A. 1. d. p. 26). The predominant cyclopropane fatty acids are cis-9,10-methylenehext~decanoic acid and cis- 11,12-methylene octadecanoic acid (lactobt\,cillic acid), but cis-9,10-methyleneoctadecanoic acid (dihydrosterculic acid) lias also been found (Law, 1971). The iso- nnd anteiso-branched acids predominate in the Bacillaceae and Micro- coccaceae.

The distribution of saturated and mono-unsaturated fatty acids on phosphatidylethanolaniine in several Gram-negative species waa studied by Hildebrand and Law (1964). In Azotobacter agilis, Escherichia coli, and Serratia rnurcedcens the fatty acids linked to C-1 of glycerol were mainly saturated and those linked to C-2 were mainly unsaturated and cyclopropane. Agrobacterium tumefuciens had over 90% unsaturated plus cyclopropnne fatty acids, and there was only a slight degree of difference between the reRidues on C-1 and C-2. Tho only exception to the general rille, which i R also obcycd in higher organisms, that the fatty acids linked to C-1 tend to be more stiturated than those linked to C-2, was Clostridium butyricum, an anaerobic, Gram-variable species that has large amounts of plasmalogens (see p. 4).

Another group of fatty acids common to the Gram-negative bacteria, and not usually found in the lipids of higher organiama, are the 8-hydroxy fatty acids. The most common of these are /%hydroxydecanoic, /3- hydroxylauric, and /?-hydroxymyristic acids which are found in the lipid A (see Section 11. C. p. 16) of Escherichia wli (Burton and Carter, 1964), Proteiis (Nesbitt and Lennarz, 1965), Pseudornonas (Hancock et d., 1970) and other Gram-negative species.

2. Alk-l-E,nyl Ethers

I t has recently beeii shown that a wide variety of anaerobic bacteria contain plasnialogens, which are glycerolipids containing an a,P un-

H,COCH-CH( CHI),CH, I I -

1tCOOCH

H,COPO,CH,CH,NH,

PIG. 2. Formula of an othanolarnino plaamtllogen.

saturated or alk-1-enyl ether on the C-I of glycerol (Fig. 2) (Allison et aZ., 1962; Wegner and Foster, 1963; Goldfine, 1984; Kamio et al., 1969). Thc other carboiiv of thc glycerol backbone of plasmalogcns arc linked

4 110 WAR 1) (1 OLL) 1PI N E

in the usual fibahion to a fatty acid at C-2 and a phosphuto ester at C-3. This structure has been verified for the plasmalogens of a number of higher organisms (Rapport and Norton, 1962) but in only one species of bacteria, Clostridium butyricum (Hagen and Goldfine, 1967). Presum- ably the same structure will be found for the plasmalogens of other anaerobes, but this remains to be confirmed. On acid hydrolysis, plas- malogeris yield long-chain fatty aldehydes. I shall refer to the alk-1-enyl ether-linked ohains as aldehydes for oonvenience despite the fact that the evidence that these chains urisc from long-chain aldehydes during tho formation of plasmalogens is not completely convincing (see Section 111. A. 2. p. 28).

At present it is difficult to generalize about the variety and distribution of aldehyde chains in bacterial plasmalogens. Allison et al. ( 1962), Wegner and Foster (1963), and Katz and Keeney (1964) demonstrated the formation of branched-chain fatty aldehydes with 13-17 carbon atoms in pure cultures of rumen bacteria and in the mixed flora isolated from cow's rumen. These branched-chain aldehydes were shown to be derived from branohed volgtile fatty acids, which are known to be nutritional requirements for this group of organisms (Allison et al., 1962; Wegner and Foster, 1963).

The long-chain aldehydes derived from the plasmalogens of G'. butyricum are similar in composition to the long-chain fatty acids of this organism. The major components are 16:O, 16:1, l7:cyc, 18:0, 18:1, and l9:cyc (Goldfine, 1964; Goldfine and Panos, 1971).

The fatty aldehydes releafled from the plasmalogens of Selenomonas ruminantium have been analysed by Kanegasaki and Taltahashi ( 1968) and Kamio f t nl. (1970a). 'I'hoy cotisiHt of normal Haturated and mono- unsaturated fatty aldehydes, w i t h 12 -18 carbon chain longths prc- dominating. A reqionm t o voliitilc fiktty cLc:ids i~ddccl to tho growth medium was seen in that cells grown on odd-chain volatilo fatty t t c i d H or lactic acid contained predominantly odd-numbered chains, and colls grown on even-numbered volatile fatty acids contained rnuinly even numbered long-chain aldehydes. The long-chain fatty acid compoeition reflected the uddcd volatile fatty acids iii a similar munncr.

3. A l k y l h2her.c.

Long-chain ulcoliols bound to pliospholipids in alkyl ether linkuge huvc been found in the extremely halophilic bacteria. Kates et al. (1966) have niade a thorough study of extretiic hdophiles, which require 1 M sodiutn chloride for growth, and modcrate lidophileu requiring 1 M sotliurn chloride. The latter group Iiad tiorrn:tl fatty acid ester-containing IipidH but the extreme halophiles had very little if any fatty acids in their lipids.

COMPARATTVR ASPECTS OF l3ACTRRTAL IdPTDR 5

Instead, the lipids contained predominantly d i-0-alkyl analogues of phosphatidylglycerophosphate. The major alcohol waa found to be dihydrophytyl alcohol (Pig. 3). This lipid appears to be unique to extreme halophilrs. 0-Alkyl ether lipids are found in varying amoiints in animal tissucs, but these arc usually of thc inono-alkyl, mono-ac,yl typo, have no dihydrophytyl alcohol, and are of the normal sn-glycerol3-phosphate configuration while the di-0-alkyl phosphatides of Halobacterium cuti- rubrum are of the opposite m-glycerol 1-phosphate configuration (Joo and Katcs, 1969).

Small amounts of O-ulkyl, acyl, glycerophosphatides have recently been fonnd iri tinaerobic bacteria (Hagen and Blank, 1970; Kamio et al., 1970b; Kim rt n l . , 1970).

I CH,OPO: -

1 I I CH,CHCH,CH,( CH,CH-(:H,CH,),--OCH

FIQ. 3. Formula of the diether analogue of phosphatidylglyoerol phosphate.

B. EXTRACTABLE POLAR LIPIDS OF BACTERIA

I . Diacyl Phosphoglycerides

As in the lipids of higher organisms, lipids built around a sn-glycerol %phosphate backbone are commonly found and are indeed the most abundant form of polar lipids in most groups of bacteria. Figure 4 illustrates the basic structure of these lipids and the major types of substituents found to be attached to the phosphate of the glycerophosphate backbone. Although moRt of the common glycerophospho- lipids of higher organisms are also found in bacteria there are major differences in their abundances, which will be demribed below. a. Phosphatidylethanolamine is distributed widely among bacteria as it is in the tissues of higher organisms. In Gram-negative bacteria it is frequently the major glycerophospholipid. The exceptions are some of the Gram-negative bacteria that are capable of carrying out a stepwise methylation of phosphatidylethanolamine to yield phosphatidy1-N- niethylethanolamine, phoephatidyldimethylethanolamine, phosphatidylcholine or mixtures of these (Fig. 4). As shown recently by Rmdle P t nl. (lass), the relative amounts of phosphatidylethanolamine

0 HOWARD OOLDFTNE

and phosphatidylcholine in Agrohacterium tumefacieits varies during the growth of batch cultures. In the log phase, phosphatidylethanolamine was predominant, but in the stationary phase the ratio became reversed and phosphatidylcholine predominated. A similar change was reported by Shively and Benson (1967), who found that the percentage of phosphatidylethanolamine decreased from 20 to 3%, while the percentage of phosphatidyl-N-methylethanolamine increased from 36 to 54% between the first and third day in n, culture of Tlriobacillus thio-oxirlans.

0 It

H,COC( CH,),CHj I

I RCOOC

HaCOPOj-X -

X = Name of phospholipid

Phosphatidic acid Phosphatidylethanolamine Phosphatidyl-N-methylethanolamine Phosphatidyl-N,N’-dimethylethanolamine

+ CHWaN(CH,), Phosphatidylcholine (lecithin) CHSCHOHCHZOH Phosphatidylglycerol Phosphatidyl glycerol Cardiolipin (diphosphatidyl glycerol)

~HOH--(CHOH),CHOH Phosphatidylinositol CHICHOHCH, 0-Amino acyl phosphatidylglyccrol

1

I

I 0

RCH-C==O I NH,

From the data amassed in maiiy laboratories, it q)j)(:arti t h u t ull of the Gram-negative organisms of the order Eubactcrialos (Brced el d,, 1967) are capable of synthesizing phosphaticlylethanolaminc. Among these are the large and important groups of enterobacteria, which have been studied in great detail, and the medically important family Brucel- laoeae which includes species of Rrucella and Haemophilus. Unfor- tunately, the lipids of only a few members of this family have been studied with modern analytical techniques. Most members of the other orders of Gram-negative bacteria also appear to be capable of synthesizing phosphatidylethanolarnine, but the data on these other groups are less


completr. The phospholipid compositions of individual species will be describcd in grwter detail in the libst section of this article.

The distribution of pho~pli~tidylcthanol~minc in the Gram-positive organisms is more complex. I t is :I inajor phospholipid constituent of the spore-forming genus Bacillus, but is not found in most of the non-spore- forming Cram-positive groups including tho large groups of Micro- roccaceath, T,il(rtol)ncillacenc, tmd somc of trhc Corynrbacteriacrne (Tktiwa, 1007).

b. l ’hos~~?~a/ i i i~~lr . l~ol i , ,~ , tL niibjor componrnt of the mrmbranes of fungi, plants, and animals is :L ri+Ltively rare component of bacteria (Goldhe and Ellis, 1064; Hageii et nl., 1066; Ikawa, 1967; Randlo et al., 1969). Among the exceptions to this gcw~ra1iz:~tion arc a number of Gram- negative bactcriih iuclutliiig the tLgro1):wtcriiL (Kancclhiro and Marr, 1962; Goldfineand Ellis, l004), Nitrosocystis oceanus, Hyphomicrobiales (Hagen et al., 1966; Goldfine and Hagen, 1988 ; Park and Berger, 1967), Brucelh abortus Bang (‘Chicle et al., 1968), Thiohacillus novellus (Barridge and Shively, 1968), and some of tho photosynthetic bacteria (Wood et al., 1965; Gorchein, 1964; Laseelles and Szilagyi, 1965). The other N- methylated derivatives of phosphatidylethanolamine, phosphatidyl-N- methylethanolamine and phosphatidyl-N,N’-dimethylethanolamine, are also being found in an increasing number of Gram-negative bacteria, either with or without phosphatidylcholine. For example, phosphatidyl- dimethylethanolamine occum in Iiyphoniicrohiurn vulgare in substantial amounts along with phosphatidylcholine (Hagen et al., 1966). Phos- phatidylmethylcthanolamine has been found in Clostridium butyricum (Baumann ~t al., 1965), several species of Thiobacillus (Barridge and Shively, 1988) and in Proteua nulqaris ((:oldfino ~ ~ n d Ellis, 1964; Randlo et al., 1969) in the absrtioe of 1)ho~1)I i i~ t i~~y lc~ io~~~ le , and in Agrobacteriurn tumefuciens, Azotobacter a g i h (Law 4 d., l!J(J3 ; lbndle et ul., l98e), Thiobacillus novellus (Barridge and Shivcly, 1008) nnd Forrobacillue ferro-ozidans (Short et al., 1969), dong with pl~o~phatidylcholine,

Tn animal tissues phosphatidyl incthyletliiLiiolamine and phoflphatidyl- di~~ietl.iylethi~nolamine are rarely found ILH major lipid components. Rather, they appear to be present ill small amounts as metabolic intermediates between phosphatidylethanolamine and phosphatidylcholine in those tissues that contain the phosphatidylethanolamine methylation pathway (Bremer and Grecnberg, 1960; Artom and Lofland, 1960; Gibson et al., 1961). This pathway was first described in rat liver and is now known to occur in Neurosporu (Hall and Nyc, 1959), yeast (Letters, 1966), and protozon (Lust and Diiniel, 1966; Smith and Law, 1970).

c. Pho.~phatidylglycerol is widely distributed among both Gram-positive and Gram-negative lmteria but the proportions of thiR phospholipid

R IIO\VARl) 001,IIWNlC

foiind in bactrrin vwjf widely. Tn general, a higher proportion of the phosphoglycerides of Gram-positivc cells is accounted for by phosphatidylglycerol (or the phosphoglycerides derived from it, cardiolipin and amino-acyl E’hosphrttidylglycorol) than with tho Gram-negative organisms where phosphatidylethano1;Lmine and its methylated forms tend to be the predominant class of phospholipids by R ratio of roughly 2 : 1 to 4 : 1 . Phosphatidylglycerol is n major phosphoglyceride in the spore-forming bacilli and in the stnphylococci that have been studied. The lipid composition of a number of other Gram-positive groups is not as clear, but phosplitLtitlylglyrrro1 ha3 hem found in streptococci, micro- cocci, Sarcina, ant1 pneurnoc~occi. 1 t i H also present i n C‘lostridiuvn but?/ricum and in C1. wcllchii. 1 t8s diHtrihition a~iioiig thc? otthcr dostridilt has not beon rcportcd. d. Cardiolipin or diphosptiatidylRlycro1 oc~iirs in many of thc orgnn- isms in which phosphatidylg1,ycorol oocurs. Since the two lipids are difficult to separate, they have often been reported a8 their sum. It is known that cardiolipin is derived from phosphatidylglycerol in E . coli (Stanacev et al., 1967). As batch cultures of Gram-negative cells go from the logarithmic phase of growth to the stationary phase, the proportion of phosphatidylglycerol tends to fall while that of cardioliph tends to rise (Shively and Benson, 1967; Cronan, 1968; Randle et al., 1969), but not necessarily stoicheiometrically (Randle et al., 1969).

Both crtrdiolipin and phosphatidylglycerol are well known components of the oells of higher organisms (Macfarlane, 1964b). Animal mitochondria have more cardiolipin ( 16% of total phospholipids; Fleischer et al., 1981) than phosphatidylglycerol, whereas phosphatidylglycerol predominates in plant subcellular organelles (Benson, 1964).

e. Amino-acyl phosphatidylglycerol. These derivatives of phosphatidylglycerol appear to be unique to bactrria. The structure of thie clans of compounds WRS elucidated by Macfurlane (1962) in tt study of thc previously ill-defined lipo-amino acids of bacteria. Shc showatl thnt the amino acids were esterifid to one of t ho free hydroxyl groupn of glyac?rol in phosphatidylglycerol in the lipids of Cloelridium welchii and Staphylo- coccus aureus. Since then these lipids have h e n detected in a number of other Gram-positive bacteria including Bacillus subtdis (Bishop et al., 1967; O p d e n K a m p ~ t al., 1969a), B.megaleriumMK 10D (OpdenKamp et al., 1965), B. cereus (Houtsmiiller and van Deenen, 1963; Lang and Lundgren, 1970), B. natlo (Urakami and Umetsni, 1968), Streptococcus faecalis (Vorbeck and Marinetti, 1966; Kocun, 1970) and in Mycopla.qma laidlawii (Koostru, and Smith, 1989). There are very few well-documented reports of the occurrence of this class of compounds in Gram-negative bacteria. The amoiints of this compound in Gram-positive l,nt:tpriu, differ

COMPAltATlVE ASPECTS OF BAC‘TXRIAL LIPIDS 9 from species to species and varies within a given species (see Seation IV. B. p. 42).

Only a limited range of amino acids has been found in this olaas of compounds. The most common is lysine which has been found in Staph. aureus, B. niegateriunt MK 10D, B. subtilis and Strep. faecalis (see references above). Alanylphosphatidylglycerol has been reported in Strep. faecalis, C1. welchii. B$dobacterium bifidum (Exterkate and Veer- kamp, 1960) and B. cereus. The ornithine derivative has been found in B. cereus and tentatively identified in B. natto (Urakami and Umetani, 1968).

The amino acids in this group of compounds are probably linked to the 3’-hydroxyl group of the unacylated glycerol moiety. In confirmation of enzymic studies carried out by Lennarz et al. (1967) on the bioeynthesis of these compounds with deoxy analogues of phosphatidylglyoerol, Molotkovsky and Bergelson ( 1968) produced evidence with chemically synthesized lysylphosphatidylglycerols that the natural aompound haa the amino acid on the 3’-hydroxyl group.

A glucosaminyl derivative of phosphatidylglycerol occurs in B. megaterium (Op den Kamp et al., 1965, 1069b; Gurr et al., 1968) and in I’sewtomnas oiialis Chester (Phizackerley et al., 1966). A compound from B. megaterium has the g1 iicosamine linked glyoosidically a t the 2’- hydroxyl of the glycerol moiety. Other isomers have been reported (Phizaekerley and MacDougall, 1969). f. Phosphatici?ylinositol. This important lipid of higher organisms is not a common phosphoglyceride of bacteria and appears to be absent from Cram-negative bacteria. Among the Gram-positive bacteria, there are two reports of its occurrence in the Micrococcaceae, namely Sarcina lutea (Hustoii et al., 1965) anddlicrococcuslysodeikticus (Macfarlane, 1961a, b), but it appears to be absent from staphylococci or at least from Staph. aureus, the best studied member of tha t genus. Ikawa (1963) did not find inositol in hydrolysates of Lactobacillus casei, L. plantarum, Leuco- nostoc mesenteroides, and Strep. faecalis.

There are groups of bacteria that generally do contain phosphatidyl- iiiositol or lipids containing this structure. The acid-fast mycobacteria, the related propionibacteria and some corynebacteria tire among these groups which will be discussed below (see Section IV. B. 3. p. 48).

g. Minor phosp?wlipid components and biosynthetic intermediates. Phos- phatidic acid is rarely prcsent as more than a few per oent of the total phosphoglyrc.~.idc~ of bacteria, but i f tho puthwuy for the bioHyrlthesiH of phosphatidw i n E . cali is univcrsully preseiit in bacteria (HW Section I l l . B. 1. p. I S ) . tlieii all bwterith that uotitain I)tloHphetidylcttl~t1ol- amine, phosI’hutidylglyccro1, u i d tho otlicr phosphatides derivod frotn

10 HOWART) (2 OLUFIN 1C

them should have trace iiniounts of phosphatidic acid as an intermediate in tlicse patliwitys. KiLtltllc (it (12. ( l!)tiD) recently surveytd stationary- phase cells of eight s l w k s of Grain-iiegative bacteria and found trace arnounts up to 4.1% of their total phospholipids as pliosphatidic acid. Early log-phase cells of 1G. coli growing in a mineral stilts-glycerol medium were rcportrd to JIILVP 10% of thcir totiLl ~~l ios~~liogl~cerides iis

phosphatidic acid, and IC)g-I)tiiLRC c ~ > l l ~ of Proletis tiidgaris were reported by the Sitme mithors to IiiLvC 7% ihospli;itidie actitl. TIWSP findings arc in accord with the c.uiioci~t of pliosptiiLticlie acid as a bioHynthetic intcrmcdiatc.

l’hosphtitidylecrin(. is iitl itit,c.rmcitliiLt,c. b c t w c ~ n ptiosptiatidic acid and phosphatidylcttiariolni~~iito (Fig, 1 1, J). 2!)) iLntl is usiuitlly detected i t i very small ainounts or rriisscd witirdy I I I I I ~ W isotopic tracers tire used. There itre two other ~ ~ ~ I ~ ~ ~ ~ ~ I ~ I ~ I I s - ~ ~ o I I ~ ~ ~ ~ I I ~ I I ~ lilkls of the phospho- glycwiclo type. ‘I’~IPSO i l t*(* ~~l10~~~l1i~t~itl~~lfilyc:crol,hoHp1I;L iintl oytidinc diphosphiktc diglyceritlr, both of IC liicli iirc interniccliittw in the biosynthesis of the rnujor p l~os~~l io l i~kls of E. coli (see Section 111. B. 1.

Moiio-acylphosphoglyceridcn o r lysophosphatides are also generally found in trace amounts. l ~ ~ s o ~ ~ l i ~ s p l i i ~ t i d i ~ acid or mono-acylglycerophosphate is an iiitcrrncdiiLtc it1 the biosynthesiu of pliosphatidic acid in bacteria (see Seutiou III. U. 1. 1). 28). Other lysophosphatides, such as lgsophosphatidyl e t l ia~l~l i l~t i i t i~~, are probably formed as a result of the partial hydrolyeis of tlio ttliljor ] ~ l i ~ ~ s p ~ i o ~ ~ y c e r ~ d e s of the cell.

p. 28).

2. Alk 1-4ny1, l ~ ! j l l ’ l t o . v ~ ~ I i o ! ~ i ! j c . r r i r l ~ s : I ’ l ~ i ~ ~ i ~ ~ ~ i l ~ ~ ~ ~ ~ ~ v ~ , ~

Until rccctitly thcrc: \VCI’C otily ib li.\+ I V I M I ~ ~ S 0 1 t i t ( : ~ ~ I W C I I C C of i ~ l t l ( . liydogenic lipitls in bii(*tct*iiL, 1 J U t it I r i t ( l t)(v.rt 1totc.d tliat hct(!ri t~ th i~ t contained plasinalogc~ns were: ;ill iLtiil(~t~(~b(~s (HCO Sec:tiori I I . A . 2. 1). 3) . This weak gencrtdizatioti W i l s t t i a t l ( ~ rnuc:h utrotigcr i n ii rcwnt, survcy by Kamio et al. (1969) wlio frirtittl m d i u ratios of aldehyde: phouphoruu of 0.14 to 0.43 in triixetl pol)iilat,ioiis of cells ohtainctl from the sheep rumen by differential cctitrifugation of the contents. ‘I’liis oonfirmcd a11

earlier observation of Katz and Kccney (1964). ‘l’ttcay also studied anaerobic bacteria obtained by enric4imcnt of mixed rumm bacteria and mixod soil bacteria growl 011 various energy sourcc’s. ‘l’he range for ttie rumen bacteria enrichrtients WUR 0.19 -0.89 (aldehyde : phosphorus) ; the range for the soil bnctcriitl enriclinicwts was 0.26-1.38 (Knmio et al., 1969). These authors a1w stitdied tlw occurrence of pli~srnulogens in a wide range of p i t r ~ cultures of 11 tiiicrohic biiCtt:ritt and cornpared these lipids with tliosr obtiLinetl from i~ smaller numbw of aerobic or fitcultittivc organism. A~iio~if i the lattcar were t?Sc‘1Lfl10ti10~1(~~~JIuOreYce11Y,

UOMPARATIVE ASPEOTS OF BAUTERIAL LIPIDS 11

E. w2i (grown either aerobically or anaerobically), Corynebacterium sepedonicum, Bacillus subtilis, IChohspirillurn rubrum, end Strepto- myces aureofaciens. None of the aerobic or facultative organisms has plasmalogens, in confirmation of the results obtained in many laboratories (see for cxamplo, Urity and Wilkinson, l!M%; White, 1968; ltundle et al., 1969). I'lrt~rnalogcri~s woru d ~ o ttbsoiit f m i i ttw micro- twrophilio luctio noid btwtoriik, wliic:ti i r i c : l i i t l oc l Strap. fuenuli.u, ller~con*,uloc tnesenteroirlas and two q)ec ic~~ of' Luclobncillus (Kamio et al., 1989). Tho phospholipids of all tho tmaerobw tested, however, did contain plaa- malogens with ratios of aldehyde :phosphorus varying from 0404 to 1.04 (Table 1).

Bacterial &rail1

Bocteroidea ruminicola Bacterdea auccinogenea Cloatridiurn acetobutylicum

Cloetridiurn acetobutylicuni

Cloatridium butyricum Cloatrirlium kairiaiitoi Clontrirlium kair cboi Cloatridiutn prrfrinyem Cloatridium xacclulropcrbutyl.

Deaulfovibrio e.p. Peptoatreptococcus alurleii ii Propionibueteriurn

f reuden reichii Anaerobic culturo Standing culturo

Anaerobic culture Standing ciilturc

- _ _ -

179-121

314-48

acetonic r i m

Propionibacteriuni ahernrutr ii

Aldoliyclo : pl1oupl1orLlK ratio in phospholipid

(molar) lCeference8

0.004 Ihmio el al. (1969) 0.71 Wegner and Foster (1963)

0.72 Kamio et al. (1969)

0.92 Kamio et al. (1969) 0.40 0.8fl Karnio et al. (1969) O*H2 Kamio et al. (1969) 0.04 Kiimio ct al. (1'369)

Bauniann et al. ( 1 965)

0.80 Kemio et al. (1969) 0.09 Kamio et al. (1969) 1.04 Karnio et al. (1909)

0.68 Kamio ct al. ( 1969) 0.60 Kamio et al. (1969)

0.37 Kamio et al. ( 1 969) 0.45 Kamio et al. (1969)

Ruminococcw jlaz*efacieiiu i, 0.56-0'80 Allison et ul. i1962j Selenomonas rum inantium

Lactate-grown 0.50 Karnio et al. ( 1969) Glucoso-grown ' 0.25 Karnio et al. (1969)

Sphaerophorus ridiciclosus , 0.25 P.-0. Hagen (unpublished

Treponema pallitluna (Roiter) 0.14 Veillonelb gazogeries 0.69 Kainio et al. (1969)

data) Meyer and Meyor ( 1 97 1 )

--

I:! l lOWAltD QOLDYINE

The probleni that rcrnuins is to determine the structures of the pol^ g r o u p of these bacterial plasmitlogens. In animal tissues these are generally ethanolaniinc or choline. Only a small amount of information is available on the bacteriti. Wegner and Foster (1963) reported that almost all of the phospholipid in Bacteroidee 8uccinogenes was ethamol- amine phot.lphntitfe, tilid that upproximately 70% of t lw tottd phoH- pliutide WUB ethunolumino plusindogen. Uuurnann ct al. ( 1 YU6) urielyxocl tho three major lipids of Closlridium bulyricurn for plasmulogen content, and found that 78% of the N rnethylethltnolainine phosphutides, 55% of the ethanolarnine phosphatides and 9% of the phospliaticlylglycerol were of the plasmalogen type.

Tn their extensive survey of tlie plusmulogens of unaerobic bacteria, Katnio el al. (1969) subjected thc phospholipids from enrichment cultures and from five pure cultures to thin-layer chromutography on silica gel H in chloroform-methanol- wrLter (62 : 25 : 4) and found, in each extract, a major component (R, 0.45-0.50) which gave positive reactions for fatty aldchyde, phosphate, and amino nitrogen. These data are consistent with thc presence of an cthanolamine plasmslogen, but ure only indicative, since N-methylethanolamine plasmalogen behavcs similarly upon thin- layer chromatography (Baumiitin et nl. , 1965).

.‘i. Alhyl Ether Lipids

As mentioned above (set Section 11. A. 3. p. 4) a unique type of tlialkyl glyccrylpliosphor~yl lipid O C P I I ~ H in the extremely halophilio hoteriu. ‘1’110 prcdotniiia t i t rtwriibcr of t l i i H 1)liospliolipid class in Halo- hac/wiiirtr ( w t i r u h r ~ m hiis twct i foiiitd 1)y Kiitm i ind his associatex to be iin ~ ~ I I ~ L I O ~ ~ I C of [ ~ ~ i ~ ~ ] ~ ’ 1 i ; L t i t ~ y ~ g ~ ~ ~ ~ ~ l ’ O J ~ ~ l ~ ~ H ] ~ h ~ t ~ (Fig. 3, 1). 6 ) . The alcohol attached to C-3 anti 0 - 8 of‘ glycerol ( tho oonfigurtition being opposite t o that of the u~311al plio,.iphofilycerideH) i H dihydropliytol (KatCs et al., 1965). A sirnilar, probably itlrntical, lipid has been found in six other species of extremely halophilic bactcriu (Kutcs el n l . , 1906). Minor lipids in these halophiles iwe the c1ietttc.r aitaloguc of phosphatidylglycrrol, a diether glyaolipid, iirltl its siilpliate ester (KatoH el al., 1967).

Other phosphoriis-cotittliiiiti~ lipids tl ir t t are not of the glycerophospholipid type, s\ieli us lipid -4 of Chwn-ncp~tivc bacterial cell wall^ and polyisoprcnoid phospliiitcs involved in cell-wall synthesifi, will be discussed elsewhere.

CO'MPARATTVE ASPECTS OP JiACTXRTAL LTPTDS 13

tures of two of these bacterial glycolipids. A compilation and review of the distribution and structures of the glycolipids in bacteria has recently appeared (Shaw, 1970). The most common type, which is also commonly found in plants, is a diglycosyl diglyceride, composed of a disaccharide glycosidically linked to C-3 of a diglyceride. According to Shaw and Baddiley ( 1968) five types of disaccharide residue are widely distributed in the glycolipids of Cram-poRitive bacteria. These are : a-glucosyl- gluoosyl, P-gl ucosylgluroa,yl, a-galnctoeylglucosyl, ~-gal~ctoaylgalnctosyl

I H,('OOCR I

FIQ. 6. Structural formulae of 8omo bacterial glyconyl diglyceridon. Top: a - D - gelacotoeyl-( 1 +. 2)-a-D-glucosyl-( 1 -+ :I)-diglycorido, which hm buon detoctud in pneumococci and Isctobacilli. Bottom : a-I,-rnntinonyl-( 1 --z 3)-a-11-rnur1nonyl- (1 -+ 3)-diglycerido, which h w been detucted in Micmcoccuu lywrirleikticua, Micro- bachrium lacticurn and three specie8 of Arthrobacter.

and a-mannosylmannosyl diglyceritles. On further Rtructural investigation, these authors and others have found that the two anomeric centres in the glycosides have the Name configuration, and the a-linked disaccharides containing glucose or galactose have a 1 -+ 2 linkage between the two sugars but the /Clinked disaccharides have a 1 -+ 6 linkage.

A large group of Gram-negative bacteria has been examined for these glycosyl diglycerides (K. Heatherington, unpublished observations cited by Shaw and Baddiley, 1968). None of those examined contained glycosyl diglycerides. Glycolipids have, however, been reported in two species of PsewEomonas (Wilkinson, 1988a) b; 1969). In addition to an a - g h o - pyranosyl-~-2,3-diglyceride there is :L hexuronyl diglyceride in these

14 NOWARD CIOLDPJNE

organisms. This has been identified as 1 -0-a-o-gliiropyranurotiosyl-n- 2,:$-diglyrcritlr in 1’s. di?ni,/?itn (Wilkinson, 1969) and ;L /3-litilwd species in Ps. ruhescerrs (Willtinson, l!M8b), The taxonomy of these pseudo- inonads is, howcvcr, in doubt (Willtinson, 1968b). Glycolipids have also been reported i n photosynthctic bactcria (Wood et al., 1965; Gorchein, 1968c; Rarlunz, l96!,). On the other hand, all of the Gram- positive organisms examined at the University of Newcastle upon Tyne (Shaw and Baddiley, 1968 ; Shaw, 1970) contain glycosyldiglycerides.

The amounts of glycolipid in a givcn species may range from just one to three per cent of the total lipid in the luctobacilli and staphylococci (Brundish et al., 1966; Shaw and Baddiley, 1968) to 34% in a pneumococcus (Brundish et al., 1906) tLnd 40% in Microbacterium Zacticum (Shaw, 1968) and Jlycoplnsma laidlawii 13 (Shaw and Smith, 1967).

Katcs nnd his coworlters ( l!)(i7) denionstrcitcd the prrwncc of a diether glycolipid und its sulphiite cstor i n Ilalobor.teriu?n c/h!kul)rurri rind in other extrctne halophiles (Kntes et al., 1 ! ) ( i G ) and h a w chunLctorizrd the lipid from H . cutiruhrum as it 2,3-di-O-dihydrophytyl-r,-glycerol-l-O- (glucosyl-mannosylgalactosyl) snlphate. Like tho dicther analogue of phosphatidylglyrcrophospliate of theso organisms, the glycolipids have the polar substitucnts on the 0-1 of L-glycerol.

ii. Mycosides. Among the complex mixtirrc of lipids characteristic of the mycobacteria are mycosides. ‘rhe Mycoside B from bovine strains of myaobacteria contains a benzene ring linked to a sugar residue (2-0- methylrhamnose) and to it long-chain hydrocarbon containing hydroxyl groups t o which are esterifird palmitic acid and mycocerosic acids (branched fatty acids of 29-32 carbon atloins) (Asselineau, 1966). It is beyond the scope of this nrtielc to discuss the lipids of rnycobacteria in detail. The interestred reader is rcferred to the monograph of hselineau (1966).

b. Spiiingolipids. Until rcrciktJy it WUH thought that t h i q (*IILIIH of lipid was not synthesized by bactc’riil. ‘I’his rtilo, l ikv l r I O H t , h i l H trow found its exceptions. White n.nd tiis ~ o w o I ~ I ~ ~ ~ ~ I I ( l , i h l 3 w t i iLr t ( J Wlritc*, 1 of!!); White P t al., 196!); ItizztL 411 u l . , 1!)70) Iiilv(* H I I ~ W I I tliat tiiv lipids of‘ Bacteroirles rtzrlani?iolleriicus cot it:ii I 1 iL fwri i ly of spii i t ig~l ipi~ls wlt icli represent over half of the total (1Xtra(:tiL1)1(: lipids. ‘I’lrrw! corn pound^ havr been carefully characterized as ccr;tmide TihosphorylethanolaInine ant1 ceramide phosphorylglyccrol, the mihjor sphingolipid components, and ceramide phosphorylglyccrol phosphate, a minor cornpotlent. It is of interest that the water-soluble moieties resemble the phosphoglyceridc water-soluble moieties of bacteria, rather than pl1oapl1or.ylcholine which is more commonly found in animals. The long-chain bttses of B. melanino- genicus sphingolipids litwe been characterized as 17-methyloctadeca-


sphinganine (63%), n-octadecasphinganine (21%) and 16-methylhexa- decasphinganine (12%) (White et al., 1969). A similar group of sphingolipids have been found in B. ruminicola (Iannoti et al., 1970).

The more complex sphirigolipids common to the tissues of higher organisms, the cerebrosides and gangliosides, have not been found in bacteria. c. Ornithine amides. Laii6elle et al. (1963) found an ornithine-containing lipid in a strain of atypical mycobacteria which yielded an ether-soluble ornithine derivative upon saponification. Infrared spectra indicated the presence of an amide bond. Gorschein ( l964,1968a, b, c) isolated a similar non-saponifiable ornithine-containing lipid from the photosynthetic bacterium, Rhdopseudomonas spheroides. This lipid is thought to have a long-chain alcohol csterificd to the carboxyl group of ornithine and a long-chain fatty acid in amide linkage to the a-amino group of ornithine. Alkali-stable ornithine-containing lipids were also reported in Rhodo- microbium vannielii (Park and Berger, 1967), Rhodospirillum rubrurn

CH3 CH3 CH, I I I

CH,--C=CHCH,(CH,C=CHCH,),CH,C=CH-CH,OPO:-

PIG. 6 . l'ormula of undocappronyl phosphato.

(Depinto, 1967), two other photosynthetic bacteria, and Streptornyces sioyaensis (Kimura and' Otsuka, 1969) where a similar lysine-containing lipid was also found. Prome et al. (1969) have proposed that a fatty acid is linked to the carboxyl group of ornithine through 1,2-propanediol in Brucella melitensis. They reported a similar lipid with an ethylene glycol bridge in Mycobacterium bovis.

d. Glycosyl derivatives of polyisoprenole. In two important series of investigations on the biosynthesis of the peptidoglycans of Gram-positive bacteria and of the 0-antigens of the lipopolysaccharides of the enteric Gram-negative bacteria, Strominger and Robbins and their colleagues characterized a novel class of sugar derivatives of polyispprenols. In the biosynthesis of the peptidoglycans, a phospho-N-acetyl-muramylpentapeptide is transferred from UDP-N-acetyl-muramylpentapeptide to e polyisoprenoid phosphate to form a pyrophosphate linkage. The polyisoprenoid was established as an undecaprenol by mass spectroscopy (Higashi et al., 1967). In the cam of thc biosynthesis of tho lipopolysaccharides, the repeating po1yuacch:Lridcs of the 0-antigen ofh'almnslla typhimmrium werc shown to h! I t u i l t up on a sirnilw, if not identical, undecaprenyl phosphate (Fig. 6) (Wright et al., 1967). Again a pyrophosphate bridge links the polyisoprenol to the polysaccharidc. This type

16 HOWAltD (:OLDNINE

of lipid rcprrseiits only a fraction of a per cent of tlie total extractable lipids of bacteria and was not recognized until its functions were explored. Thorne and Kodicek (1966) hRd, however, isolated a non-phosphoryleted form of a C,, polyisoprenoid alcohol in lactobacilli, where it was showii to become labelled when the cells were fed radioactive moviilonio acid. Since this typo of lipid is involvotl i i r thc biosyiithcsis of tho universtd poptidoglycuii or rigid ltiyer of buatcirial c ~ ~ H , it is implicit that it or IL

similar lipid is present in all btwterid cellw t h u t have ad1 walls. I n the species that coritttiri O-aiitigenic polyHacchrtrides, this lipid appears to play a dual role. Scher et al. (1968) have reported. the involvement of a similar lipid intermediute in the biosynthesis of a mannan of the membrane of Micrococcus lysodeikticzcs. The lipid and hexose of this intermediate are linked through a phosphodiester bond rather than a pyrophosphate link.

These findings htLvc Icd to u search for similar lipid intermediates involved in the biosynthesis of other complex polysaccharides. The results thus far seem to be encouraging. Baddiley and his coworkers (Watkinson et al., 1971) have obtained indirect evidence for the involvement of a similar lipid in teichoic acid biosynthesis in Gram-positive bacteria, and Troy et al. (1971) have reported the involvement of these lipids in capsular polysaccharide biosynthesis in A erobacter aerogenes.

C. N O N - ~ ~ X T ~ C A C ~ ~ ~ A I I I , E Li tws I N BAWIWA

A broad survey of the litcrttture reveals that u portion of the fatty acids of many bacteria can bc relerhsed frorn cells by wid or ulkaline hydrolysis after the cells have been exhaustively extructucl with lipid solvents. In many bacteria, thc mittcrials to which the fatty acids are bound are not known nor is the location of these materittlfl in the cell understood. There is one type of bound fatty acid that is better undor- stood than most, but the structures of these materials are far from clear. This lipid is obtained from the lipopolysaccharide of the cell envelope of Gram-negative bacteria by treatment with dilute acid. It was given the name lipid A by Westphal and Luderitz (1954) in order to distinguish it from the lipid extracted from lipopolysaccharide prior to acid hydrolysis, which was called lipid B and is predominantly phosphatidylethanolamine (Kurokawa et al., 1959). The lipid A component contains phosphate, glueosamine and fatty acids, and the fatty acids are qualitatively different from those found in the glycerophosphatides of Gram- negative bacteria. There is a high proportion of 12 : 0 and 14 : 0 fatty acids and/3-hydroxymyristic acid (Ikawa et al., 1953; Burton and Carter, 1964) in fhe lipid A of I$. coli. The longer chrtiii mturatcd, unsnturRkd rLnd cyclopropune fatty acids, whiolr cve coin inolr to the I’hOHI)hoglycericlttN

COMFARATTVR ARFRCTR OF BACTIRIAT, LTPTDR 27 of E. coli, are present in small amounts. The fatty acids appear to be linked to the amino and hydroxyl groups of glucosamine. The linkages between glucosamines are not clear. The possibilities of glycoside and phosphodiester bonds have been oonddered (Burton and Carter, 1964) and recent work favours the glycosidic linkage (Liideritz, 1970).

Lipid A is distributed widely in Gram-negative bacteria. Material of similar composition was isolated by Nowotny (1961a, b) from a number of strains of Salmonella and from other species of Entero- bacteriaceae, Neisseriaceae and Pseixdomonadaceae. A review of the structure and function of the lipopolysaccharides has recently appeared (Liideritz, 1970).

11. Nb:iifricAi, T,ri>i 1)s oiq BA~:TERIA

Several important generalizations hnvc cmerged from exnmination of the neutral lipids of bacteria and have been noted by previous authors including Kates (1964), Bloch (1966), Asselineau (1966), and O’Leary (1967). Bacteria generally contain either no sterols or amounts very much smaller than are found in the tissues of higher organisms. It is quite clear that bacteria are capable of synthesizing polyisoprenoids. These are found to be widely distributed in prokaryotes in the form of quinone coenzymes, carotenoids, and the recently discovered undecaprenol phosphates needed for peptidoglycan and lipopolysaccharide synthesis (see Section 11. B. 4. p. 12). The missing reactions are those required for forming the ring systems of sterols. These reactions are oxygen-dependent and were evolved at a later period than the time of evolution of the forerunners of present-day bacteria (Goldfine and Bloch, 1963). The presence of trace amounts of sterols in bacteria has recently been re-investigated by Schubert et al. ( 1968). They found sterols premnt as 0.01% of the dry weight of Azotobacter chroococcum and smaller amounts (0.0036%) in Streptomycee olivaceue and (04004%) in I?. coli. A number of other bacteria were investigated and were found to have less than 0.0001% sterol. When one consitlcrs that lipid8 represent anywhere from 3 to 20% of the dry weight of many hctaria, them amounts appear to be of slight significance in terms of membrane structure. That the sterols may play some other role cannot be denied. There is still no conclusive proof of the ability of any bacterium to synthesize a sterol de novo.

The mycoplasmas, a group of bacteria that lack a rigid cell wall, almost universally have a requirement for sterols. Of 31 species recently examined by Razin and Tully (1970), all but three required cholesterol for growth, but none appeared to be capable of synthesizing sterols. These organisms are parasitic and have ready access to sterols, which

18 TTOM'ARD CIOL,T)PTNP:

may function to strengthen the inrmbranes of these wall-less cells (Smith, 1904; Bloch, 1966).

Bloch ( 1965) has outlined thc stcrol-syntlicsiziiifi ciipwity of higher protists. The blue-green dgae do not synthesize sterols (Levin and Bloch, 1964), but till the other algae do (Carter ~t nl., l!%l). The fungi are also generally cnpable of synthrsizing sterols. As one goes up the phylogenetio scale, sterols are universally found, but many insects have lost the ability to syntheaize thcrn (Bloch, 1965).

With the exception of the strictly anaerobic bacteria, the quinone coenzymes, coenzymes Q and vitamins K, and the carotenoids are found in a wide variety of bacteria (Coldfine, 1965; Liaaen-Jensen, 1965).

Another significant differcwce betwcen the lipids of bacteria and those of higher organisms is a qutintitative one. Although bacteria have been widely reported to contain glycerides (Kates, 1904), the amount's of the neutral fats per crll are usually low, and mono- and cliglycerides are reported more often than triglyyrerides. Bacteria do not appear to store glyceridca as energy reserves. A form of lipid, unique to bacteria, poly-/3-hydroxybirtyrie acid, clors however appear to serve this function (see p. 19).

The proportion of neutral lipids, including glycerides, 11 ydrocarbons, yuinoncs, carotonoids, free f&ty iicids and poly-/3-hydroxybutyric acid varies widcly from species to species. One problern in assessing these data is the variation in reporting poly-p-hydroxybutyric acid, which has somewhat different solubility properties from the other neutral lipids and may or may not be included with neutral lipids depending on the isolation procedures used. With the exception of Corynebacterium diphtheriae, Kates' (1964) summary of the data on Eubacteriales and Pseudo- monadales gives the neutral-lipid content of bacteria as anywhere from 3 to more than 50% of t,hr* total lipid, if poly-/3-hydroxybutyric acid i H excluded. Among the species containing large amounts of neutral lipids are Surcina Zufen (>50% of total lipids; Akaahi and Saito, 1900; Hustori and Albro, 1964), Clostridiim ?idc?/ i i ( 3 0 % of total lipid ; Macfarlane, 1962), and a number of the liictic acid bacteria (Ikawa, 1963; Thornc and Kodicek, 1962). The Gram-negative bactrriii generally have smaller amounts ofneutral lipids, usually 5 - 1 5% of total lipid, if poly-p-hydroxybutyric acid is excluded. The distribution and structures of hydrocarbone in bacteria have recently been reviewed ( A h 0 and Dittmcr, 1970).

E. P o I , Y - p - H Y l ) R O X Y r ~ r ~ T Y R r ~ ! AWJ)

As qoted above, although glycerides are generally :L minor oomponen t of bacterial lipids, a storage form of lipid does occur. Poly-/3-hydroxybutyric acid is widely distributed in both Grsm-positive and Gram-

COMPARATIVE ASPECTS np BACTERIAL LIPIDS 19 negabive bacteria (Dawes and Ribbons, 1964). Among the genera in which is has been reported are Bacillus, Azotobacter, Pseudomonw, Rhodopseudomonas, Rhodospirill?m, Spirillim , and Vibrio. The polymers can reach a molecular weight of 250,000 iind we found in cells as granules (Merrick et a,?., 1965; Tmidgrcii cl nl., l!)M). I n somc species ofSpiriZlum ( I ! ! . r r y n s ) , i n nf irror'orr'm Irnlo(i~n%lr.i/ic.n,,H, I L I I ~ i i i IZ. rna!qatvriwrrL in- clusions of~~oly-~-liyci~~xyl,~ltyrcLtc. : L t h i i t i 40 W% of the cell dry weight when the cells are grown with good ttcration on an appropriate carbon source (Hayward et al., 1969; Sierra and Gibbons, 1962; Lemoigne et al., 1949).

In. Biosynthesis of Bacterial Lipids A number of important diffwenccs i n tlic lipid composition of bacteria

and higher orgttnisms were described i n t h e preceding sections of this review. Since ninny bacteria synthesize their own complex lipids as well as the precursors of these lipids, the diflcrenccs in lipids must be the result of differences in biosynthetic pathways. For obvious reasons, studies on the biosynthesis of bacterial lipids have not kept pace with knowledge of their composition and, although many of the biosynthetic pathways are known, there is little comparative data between groups of bacteria. In most cases, we have nothing more than the hope that what is known about Escherichia coli or Staphylococcus aureus will also apply to closely rebted species and the even more fragile assumption that it will apply to more distantly related genera.

A. BTOSYNTFIESTS OF THE APOLAR COMPONENTS

1. Fatty Acids

a. Saturated, stvaight-chain fatty acids. The formation of fatty acids in bacteria has been studied intensively in thc past decade in a number of laboratories. An outline of the biosyntliesis of saturated fatty acids is given in Fig. 7. This pathway begins with the formation of malonyl- CoA by carboxylation of acetyl-CoA in a manner analogous to the reaction found in animal systems (Wakil, 1958; Brady, 1958). Transfer of malonyl and acetyl residues to the free sulphydryl of an acyl carrier protein of molecular weight 9600 is carried out in E. coli by separate transacylases (Alberts et al., 1964). The reactions involve transfer of the acyl moieties from the 4'-phosphopantetheine residue of coenzyme-A to an identical residue on an acyl carrier protein (Majerus et aE., 1964, 1966 ; Sauer et al., 1964). In essence, this process does not differ from fatty-acid synthesis in higher organisms in which acetyl and malonyl reRidues are transferred to similar 4'-phosphopantetheine prosthetic groups on a multi-enzyme complex (Majerus and Vagelos, 1967). The subsequent

b b

20 IlOWARD OOCDFTNW

condensation between acetyl-S-acyl carrier protein and malonyl-S-acyl carrier protein to yield accto-acetyl-S-acyl carrier protein and carbon dioxide, the reduction of the 13-keto acid to a n(-)-P-hydroxy acid, followed by dehydration to givc the a,/? unsaturated aoid, and reduotion t o the saturated fatty mid, all occur with the wyl groups csterified t o the aoyl carrier protcin (Majerus and Vagelos, 1907). All of thesereactioiis are similar to those known to occur in the synthesis of saturated fatty aaids in higher organisms. I n animal tismes and in yeastr, a multi-eneymc complex carries out tho reactions of tho cycle, and the acyl carrier protein is firmly bound to thc complex (Mtbjcrus ant1 Vagelos, 1967; Brindlcy et al., 1969). In bactcria and plants, the acyl carrier proteins arc small proteins and aro easily separated from thc other cneymes of fatty-acid synthesis. Mast of the enzymes involved in fatty acid synthesis in E. coli have beon extensively purified (Majerus and Vagelos, 1967).

The formation of the 16- and 18-carbon saturated, straight-chain fatty acids commonly found in bacteria involves repetition of this cycle (Fig.

Malonyl-S-CoA + ACP-SH + Melonyl-S-ACP i- CoA-SH Aoetyl-S-CoA + ACP-SH P Aoetyl-S-ACP + CoA-SH

Aoetyl-S-ACP + Melonyl-S-ACP + Aoeto-aoetyl-S-ACP + CO, + ACP-SH Aoeto-metyl-S-ACP + TPNH, F? D(-)-fl-Hydroxybutyryl-S-ACP + TPN D(-)-fl-HydroxybutyryI-S-ACP .$ Crotonyl-S-ACP + H,O

Crotonyl-S-ACP + TPNH, e Butyryl-S-ACP + TPN

Prtr. 7. Initial Rteps in the pathway for hiosynthnRis of straight-chain fntty acid8 in bnctorin. My€’ indict~tc~ ncyl cnrricxr protoin.

7) in which the addition of two-carbon units occurR by conden,lirLtion of molecules of malonyl-S-acyl carrier protein with the fully reducctl aoyl- S-acyl carrier protein formed in tho preceding round of condensation and reduction. The saturated products of this cycle are similar in chain length to those produced in higher organisms (Brindley et al., 1969). The factors that control the chain length are not well understood. Free fatty acid8 are formed by the action of thiolcsterases in the fatty-acid RynthetaRe from E. coli during in vitro experiments. The chain-length specificity of such thiolcsterases may play a role in dctermining the lengths of the fatty acids formed. As will be described below (Ree Section 111. B. 1 . p, 28) the fatty acids can be transferred directly from acyl carrier protein to glycerophosphate and mono-acylglycerophosphate to yield phosphatidic acid. The chain-length specificity of these acyl transferamn may also play a key role in determining the chain length of the fatty acids synthesized. In higher organisms, the controlling factor may be the tranefer from the fatty acid syntlietase complex to coenzyme-A (Lynen, 1901). Coenzymc-A appears to be required for the transfer of

COMPARATlVP AtlPEUTS OY BAUTEItIA.fr LII’IDY 21

fatty acids from the synthetase to glycerol 3-phosphate in yeaat (Kuhn and Lynen, 1965). b. Terminally branched fatty acids. Synthesis of saturated, terminally branched fatty acids occurs in the Gram-positive aerobic bacilli and in the Micrococcaceae in which they are the predominant fatty-acid type. Rumen bacteria are also known to contain substantial amounts of branched fatty acids (Allison et al., 1962; Wegner and Foster, 1963). In theory, the problem of their biosynthesis was solved by Horning et al. (196l), who showed that 11 fatty acid synthetasc from rat adipose tissue could utilize branched-chain volatile fatty acyl-CoA derivatives in place of acetyl-CoA as initiators of fatty acid synthesis. For example, initiation with isobutyryl-CoA yielded iso- 16 : 0, and incubation with isovaleryl- CoA yielded iso- 16 : 0 plus iao-17 : 0 as the major products. The branched precursors provided the brmchcd cnd of the fatty acid molecule, and malonyl-CoA provided thc straight chain portion. Lennarz (1 961)

CH3 H3C 0 CH3 I I 11 I

+scz I C2 I

CH,CH,CHCH(NH,)COOH -+ CH3CHaCHCCOOH -+ CHSCH,CHCOSCoA

CH3 CHl

4 CH,CH,CH(CH,),,COOH + CH3CH$H(CHa)a,COOH

FIQ. 8. Outline of the proposed pathway for synthesis of anteieo-C15 and anteko- Cl, fatty acids from isoleucine by bacteria.

demonstrated the formation of arbteiso-16:O and anteiso-17: 0 (Fig. 8) from isoleucine by cells of Micrococcus ~?yeodeikticus. Presumably the coenzyme-A ester of 2-methylbutyrate, which ia postulated to be the chrtin initiator, wag formed by transamination and deoarboxylation. Similar findings have been reported on the formation of anteiso-branched fatty acids in rumen bacteria (Allison et al., 1962). The ieo-branched, odd-numbered fatty acids are formed in bacilli from isovaleric acid or leucine and the iso-branched, even-numbered fatty acids from isobutyrate or valine (Fig. 1, p. 2; Kaneda, 1903). Initiation of fatty-acid synthesis by these branched-chain acyl-CoA derivatives rather than acetyl-CoA poses a problem of specificity. Are the branched precursors favoured over acetyl-CoA as a result of greater availability, or as a result of enzyme specificity? The answer is still not completely clear. The work of Kaneda (1903, 1966, 1967) with bacilli provided evidence that the availability of precursors determined the types and amounts of saturated fatty acids produced by these organisms. He showed that feeding butyrafa to N. subtilis inmusod thc amount of rnyristic and palmitic acids synthesized rolative to the branched-chaiii fatty mid@, whereug

22 IlOWAltD UOLDh'INE

feeding isobutyrate increased the relative amounts of isomyrbtic and isopalmitic acids, and feeding 2-methylbutyrate increased the relative amounts of anteiso-15 : 0 and anteiso-17 : 0. These findings are in accord with the chain-elongation mechanism shown above (Fig. 8). The fatty acid and fatty aldehyde compositions of the lipids of Selenomonas ruminantiurn are also under the influcnce of volatile fatty acids addod to the medium. When grown on gliicoso, this organism requires fatty acids of 3-10 carbon clmin length (liamio ot al., 1970a). Kancgasaki and Numa (1970) investigatcd the fatty-ucidsynthctase from AS. ruminantiurn, in wilro, und have showii that tlic syiithctasc is much more active with C, to C, acyl-CoA derivatives acl priniers than it is with acetyl-CoA or propionyl-CoA, and that thc relutivo effectiveness is related to the Michaelis constants of the synthetase for these substrates. They suggest that the fatty-acid synthetase may lack the acetyl-CoA: acyl carrier protein transacylase found in E . coli (Albert8 et al., 1964), which waa shown to transncylate poorly with propionyl-CoA, butyryl-CoA, hexanoyl-CoA and octanoyl-CoA (Williamson and Wakil, 1966), and that instead it has a transucylase specific for longer chain acyl-CoA derivatives. No experiments with acetyl-acyl carrier protein were done, however.

A similar study on the fatty-acid synthetase system of B. subtilis was carried out by Butterworth and Bloch (1970), who showed that the enzymes utilized branched-chain acyl-CoAs in preference to acetyl- CoA as primers for long-chain fatty acid synthesis. However, when acetyl-acyl carrier protein wus provided, normal straight-chain fatty acids were produced. The specificity of the acyl-CoA : acyl carrier protein transacylase, therefore, seem to bc a determining factor in directing the cells toward the almost exclusive production of branched-chain fatty acids. The products obtained from the enzymic incubations were exactly as predioted by the scheme shown in Fig. 8 when isobutyryl-CoA, isovaleryl-CoA and 2-msthylbutyryl-CoA were used as primers.

o. Mono-unsaturated fatty acids. Fundamental differences in the biosynthesis of mono-unsaturated futty acids in most, but not all, bacteria on the one hand and in higher organisms on the other, were revealed by work done in the past two decades in a number of laboratories. These studies have been described in considerable detail in several reviews and monographs (Bloch et al., 1961; Hofmann, 1963; O'Leary, 1967; Bloch, 1969).

Derivation of mono-unsaturated fatty acids from saturated fatty acids in animal tissues had long been suspected (Schoenheimer and Rittenberg, 1936). This pathway was first confirmed by Bloomfield ant1 Bloch (1960) who demonstrated a TPNH- ant1 oxygen-dcpondont dusaturation of thc

(!OMPAlL41'IVE ASPECTS Olr UAUTEltIAL LIPIUH 23

coenzyme-A esters of palmitate and stearate to palmitoleate and oleate, respectively, using extracts from yeast.

A similar desaturation involving TPNH, and molecular oxygen ha8 since been demonstrated in a variety of organisms including Myco- bacterium phlei (Lennarz et al., 1962 ; Scheuerbrandt and Bloch, 1962; Fulco and Bloch, 1964), Corynebacterium diphtheriae, Micrococcus lysodeikticus, and Bacillus megaterium (Fulco et al., 1964). Bloch (1964) has also reported that desaturation of preformed fatty acids occurs in J'treptomyces venezuelae, the blue-green alga Anabena variabilis and the protozoan Tetrahymena pyriformis. It also occurs in rat liver (Bernhard et al., 1959; Imai, 1961), and in Euglena (Nagai and Bloch, 1966). Using extracts of Euglena, Nagai and Bloch (1968) showed that preparations from dark-grown cells deseturated stearyl-CoA to yield oleyl-CoA but did not desaturate stearyl-acyl carrier protein. On the other hand, preparations from light-grown, photo-auxotrophic cells desaturated stearyl-acyl carrier protein but not stearyl-CoA. The oxygen-dependent desaturation usually leads to the formation of the A9 mono-unsaturated fatty acids (Bloch et al., 1961). An interesting exception occurs in B. megaterium KM which desaturatcs 18:O and 16:O fatty acids to the corresponding d5 compounds. This reaction is more extensive in cells growing a t 23" than at 30" (Fulco et al., 1964). Fulco (1967, 1969, 1970) has subsequently demonstrated that this temperature-dependent desaturation is characteristic of a number of species of bacilli. It should be recalled that these organisms have mainly terminally branched fatty acids a t their optimal growth temperatures. Bacillus Eicheniformis is capable of desaturating 16:0 to yield 1 6 : l A s , 16: l A9, and 16: l A L o . A t 20" it also is capable of desaturating a t the 5,6 position and thus can produce di-enoic acids, eg . 16: 2 at lower temperatures (Fulco, 1969, 1970). The capacity to desaturate at the 9,lO position is present in several other bacilli (Fu~co, 1967). These findings represent an exception to the rule that bacteria do not synthesize polyunsaturated fatty acids. This rule should perhaps be amended to state that bacteria do not produce the types of polyunsaturated fatty acids characteristic of plants and animals, fuch as linoleic, linolenic and arachidonic acids.

The major pathway for formation of mono-unsaturated fatty acids in bacteria does not depend on molecular oxygen (Goldfine and Bloch, 196l), does not involve desaturation of preformed saturated fatty acids (Hofmann et al., 1959; Bloch et al., 1961), and usually results in the formation of the n-7 series ,of mono-unsaturated fatty acids, i.e. 16: 1 A9 and 18: 1 d" (cis-vaccenic acid) or a mixture of the n-7 series and the n-9 series (Kateg, 1964). This pathway has often been called the "anaerobic pathway" because it does not require molecular oxygen. Dcspite this name it should be emphasized that i t also occurs in aerobic

L

24 HOWARD GOLDFINE

and facultative bacteria (Blochet al., 1961). A key feature of this pathway is the introduction of the double bond in the fatty acid during the process of chain elongation. Nutritional experiments by Hofmann and his coworkers on the replacement of biotin by modium chain-length and long-

OH 0 I It

CHJ(CH~)SCH,CHCH~C - R-ACP I

I)(-) B-hytlroxydocanoyl-S-ACP

II 0 J.

CH~(CH2),CH-CH(CH2),CS-ACP 11 CH,(CH,),CH2CH2CH2C-S-ACP

Pelmitoloic acid (ACP ostor)

1 4%

3. I t CH>(CHZ)&HzCH(CH2)&- 8- ACT

cis-vecoenic acid (ACP cnter) stcclric acid (ACl’cstor)

FIG. 9. Terminal steps in the synthesis of mono-unsaturatod and saturatcd fatty acids in Eeoher&ohia cola’. ACP indicates acyl carricr protoin.

chain mono-unsaturatcd fatty acids ill lactobacilli hiit1 strongly ~ u g - gested the existence of such a pathway (Hofmann el al., 1969). Expcri- ments by Bloch and his coworkers (1901), first with isotopically labclled precursors and whole cells and then with enzyme preparations, demonstrated that the formation of mono-unsaturated fatty acids was linked to the synthesis of saturated fatty acids, and that the former branched off from the latter at the C, or C,, stage of fatty acid synthesi8 (Pig. 9).

COMPARATKVE ASPRCTS OF BACTERIAL LIPIDS 95

The branch-point reaction of this pathway in E. coli is the dehydration of the D(-)-j3-liydroxydecanoate intermediate to yield cis-#l,y-unsaturated decenoate, which is not subsequently reduced, as are the trans-a,p- unmturated intermediates formed before and after this step. Subsequent work with crude and purified enzymes has substantiated this pathway (Lennarz ~t al., 1962a; Norris et al., 1964). The intermediates are now known to be acylthio-caters of MI iicyl clnrricr protoiii rathor than coenzyme-A. Ihrther ovidoncc for t h i H pithway has recently come from work with an unsaturated fatty acid auxotrophic mutant of E. coli that lacks the key j3-hydroxydpcanoyl thio-ester dehydrase and is therefore unable to form unsaturated fatty acids but is capable of synthesizing the saturated fatty acids (Silbert and Vugelos, 1967).

Uepending on the chain length of the j3-hydroxyacyl-acyl carrier protein a t which j3,y dehydration occurs, a variety of unsaturated fatty acids can be produced. For example, j3,y dehydration of both 8-hydroxy- decanoyl-acyl carrier protein and j3-hydroxydodecanoyl-acyl carrier protein and subsequent chain elongation provides the mixture of 16 : 1 A’, 16:1 A9, 18 : l A 9 and 1 8 : l A” found in Cloatridium butyricum (Scheuerbrandt et al., 1961) and presumably provides mixtures of 18: 1 A 9 and 18: 1 A” found in some strains of E. coli (Bloch, 1970). Many of the mono-unsaturated Fatty acids predicted by this scheme have been found in bacteria (O’Leary, 1967). Bishop and Still (1963) have also found 16: 1 A s , 18: 1 A * , and 18: 1 acids inSerratia mrceacena. They postulate the formation of these unusual mono-enes by the retention of a,j3 double bonds at the C,, and C,, level of chain elongation. There is as yet no enzymic evidence for this proposal.

Evidence for the existence of the “anaerobic pathway” for mono- unsaturated fatty-acid biosynthesis has been largely obtained by analysis of the positional isoniers of the mono-unsaturated fatty acids of bacteria or by isotopic labelling experiments with whole cells. Most of the bacteria that are believed to have this pathway are of the orders Pseudomonadales and Eubacteriales. There are, however, bacteria of these orders that make very little or no mono-unsaturated fatty acids. For example in the aerobic bacilli and a number of Micrococcaceae, the terminally branched acids are the predominant types. When these organisms form mono-unsaturated fatty acids, it is by an oxygen-dependent mechanism (see p. 23). The few species of corynebacteria and mycobacteria that have been studied also have an oxygen-dependent desaturation mechanism that forms d9 mono-enoic fatty acids (Fulco and Bloch, 1964; Fulco et al., 1964). As one goes up the evolutionary scale, the aerobic desaturation mechanism predominates a8 has been outlined above.

The 8-hydroxy CIo, C,, and C,, fatty acids found in the wll-wall “lipid A” of Gram-negative bacteria are also provided by the normal

"(i T I O M \ i t 1) (1 ni,n FT s I?

nnacrohir pathway for Haturntrd fatt8y-ncid synt~lrc~nis. /3-FLy(\roxy- stearic arid and othcr jl-hydroxy long-chain acids linvc brcii found in aniide linkage in the ornithinc-containing lipids of Briicella melitensis, Mycobacterium bovis (Prome et al., 1969) and Ti~iobacillus thio-ozidans (Knoche and Shively, 1969).

d. CycZopropane falt?) acids. Biosyntlicsis of cyclopropsrie fatty wids has bcrn recently rcvicwcd by LLW (197 1). Thcso c~ornpounds are dcrivctl from the corresponding mono-~msatrirated fthtty acids by :ddition of ii

C, unit ticross tlic tloublc bond. Liu and Hofinsnn ( 1962) dcmonstratcd

Fro. 10. Syntho~is of n cyclopropnnr ftit t,y-arid roRicliio on pliosphntictylethanot- amine.

the derivation of the C, unit from methionine in the formation of lactobacillic acid from its precursor, cis .vaccenic acid. O'Leary (1962) provided evidence that S-adenosylmethionine was the active donor of the C, unit and this was confirmed by Zalkin et a2. (1963) who showed that a C, unit derived from S-adenosylmothionine was utilized in the formation of cyclopropane fatty acids catalysed by extracts from S'erratin marceecens and Clostridium butyricum, and that the C, u n i t W&H incorporated into mono-unsaturated fatty acid moietics of phm phatidylethanolamine. Working with apurified enzyme from CZ. butyricum, Chung and Law (1964) provided strong evidence that the actual acceptor was a fatty-acid residue bound to a phospholipid (Fig. 10). Subsequent work has shown that aldehyde residues in plasmalogens (Chung and Goldfine, 1966) and alcohol residues in alkyl ether lipids (Thomas and TAW, 1966)

COMPARATIVE ARPECTR OF RAOTERTAL Lll'TDR 27

could also serve as acceptors of the C, unit to form the corresponding cyclopropane aldehydes and cyclopropane alcohols. Cyclopropane aldehydes (Goldfine, 1964) and cyclopropane alcohols (Day et al., 1970) in addition to cyclopropane fatty acids have been found in the lipids of CZ. butyricum. Various phospholipids have been shown to be potential precursors of cyclopropane fatty acids provided they contain unsaturated fatty acids (Thoinas and Law, 1966). Phosphatidylglycerol, phosphatidic acid, curdiolipin and phosphatidylserine as well as phosphatidylethanol- amiiic were active precursors in vitro in the presence of the cyclopropane synthetase from CZ, butyricum. Cronan (1968) has since shown that growing cells of E. coli form cyclopropane fatty acids on phosphatidylglyccrol and cardiolipin in addition to phosphatidylethanolamine. Cyclo- propane fittty-acid synthetase has also been domonstrated in extracts of AtroOacter aerogen,es and Lactobacillus arabinosus (O'Loary, 1965).

Cyclopropanc ftitty acids have been found in a variety of bacteria. The Gram-negative species include many members of the Entero- bacteriaceae, including E . coli (Dauchy and Asselineau, 1960 ; Kaneshiro and Marr, 196l), A . aerogenes (O'Leary, 1962), SaZmonella typhimurium (Gray, 1962), Serratia marcescens (Bishop and Still, 1963; Kates et al., 1964) and the Brucellaceae, including Pasteurella pestis (Asselineau, 1961), Brucella abortus and B. melitensis (Thiele et al., 1969). Among the Gram-positive bacteria, cyclopropane fatty acids have been found in a number of the Lactobacillnceac, including Lactobacillus acidophilus, L. arabinosus, L . casei, L. delbrueckii (Hofmann, 1963), Streptococcus lactis and S. cremoris (MacLeod and Brown, 1963). The most commonly found cyclopropane fatty acids are cis- 1 1,12-methylene-octadecanoic acid (lactobacillic acid), which is derived from cis-vaccenic acid (Hofmann, 1963), and cis-9,lO-rnetliylenehexadecanoic acid, which is derived from palmitoleic acid (Krylcshiro and Marr, 1961). C'is-9,lO-Methylene- octadecanoic acid (dihydrosterculic acid) has also been reported (Gray, 1962; Goldfine and Panos, 1971). The latter authors, in a study of the positional isomers of the mono-enoic and cyclopropane fatty acids and aldehydes of Cl. butyricum, have provided evidence for a strong preference of the cyclopropane synthetase for thc n-7 serieR of mono-enes. Cis-O,l0-Methylenchexadecanoic acid waa tho major 17-carbon cyclopropane fatty acid formed despite a preponderance of the 16: 1 A7 over 16: 1 d9 precursors. A similar preference was seen in the cyclopropane aldehydes. A similar sequence of reactions may be involved in the formation of cyclopropene fatty acids in higher plants (Hooper and Law, 1966). Cyclopropane fatty-acid synthetases do not appear to be preRent in animal tissues.

The mid-chain, branched fatty acid, tuberculostearic acid ( 1 O-methyl stearic acid) is formed from oleic acid in an analogous manner. Lennan

28 lTO\VARD CIOCDPTNR

et al. (1 9G2b) clcmonstrated the formation of this compound in Myco- bacterium phlei from oleic acid and the rncthyl group of methionine. A similar reaction was shown to occur in M . tubercuEosis (Ledercr, 1964). In two recent papers, Akamatsu m d IALW (1968,1970) presentedevidence for the formation of tubcrculostearic acid on an oleyl residua of a phospholipid. The inctiiyl donor was 8-adenosylmethionine, and phosphatidylglycerol, pliosphRtidylinosito1, and phosphatidylethanolamine all served as acceptors. In cmdc extracts, the tuberculostearate formed enaymically on endogenous lipids was found in both phosphatidyl- ethariolamiiie and an oligomannoside of pliosphatidylinositol. 1 0-Methyl- enestearyl (phospholipid) is an intrrmediate in this reaction (Jauregui- berry ef al., I96G).

2. Alk- I-Enfyl IC/ I tP1 .R

' 1'11 0 11 lcloli ydogon ic (;I1 ai n M of' 1 )I iLSr11 i~log~iis ~LI'P t 1 I o I I g I 1 t to 1 dcri vcd froin long-rlidn fiit,ty tL(*ids. Siiioc~ tllw biosyiithcsis of tlic! d k - I -ctiyl c 4 h r bond of btmtcria has not bwii ilcliicved in vitro, the 1)roposcd routc is bused almost entirely on cxperiments with whole cclln. 1,abellcd long- chain fatty acids were incorporated into the alk-1-cnyl ethcr chains of plasinalogens in Clostridium, butyricum (Baumann el al., 1965). Kinetic experiments with [ 14C]acetate were also consistent with tho concept that the alk- I-enyl ethers arc derived from the corresponding fatty acids (Hagen and fJolclfine, 1967). Compositional data on tho chain lengths and positional isomers of the tinsaturated and cyclopropune fatty acids and aldehydes also lend support to this hypothesis (Goldfine and Panos, 1971). The major unresolved probloni is the stage a t which the fatty acid is converted to the corresponding aldchyde. Sonic evidence points to a conversion of the cstcr bond of ti ~li~~cylpl~ospholipid to an alk-I-enyl ether (Baumann et al . , 1OG5), but recent work suggests that the fatty acid or their activated tiiioester tlc~ivti tives undergo rcduction prior to the formation of the vinyl cther linkage (Hagen and Goldfine, 1967). Day f t al. (1970) have dcmonstrutrd thc crizymic reduction of JJalrnityl-(hA to palmitaldehyde and cotyl iiloohol b y ctxtrrtcts prcpiired frorrr 6'1. butyricum. The biosyntlmis of ~ ~ l i ~ ~ m ~ ~ l o g ( ~ n s i n I):wtclri:i II:IH ~ V I I

reviewed recently (Coldtinc and Hagen, 1!371).

B. BIOYYNTITESTS OF TIIE COMPLEX LTPITIS

1. Pho.qhglycorides

a. I ' h o s ~ ~ l u t l i d ~ / 1 ~ ~ l I t u r ~ ~ ~ l ~ m i n ~ , ~ ~ h ( ~ ~ ~ ~ J I t ~ ~ t . l d ? y l ~ l ? j c e r o l and cardiolipin. T h c t assembly of the diacylpliosptio~lyccri~l~~~ begins in bacteria, as i t does in higher organisms, with a twd-step acylation of glycerol %phosphate

COMPARATIVE ASPECTS 01 BACTERIAL LIPIDS 39

to yield sequentially lysophosphatidic acid (mono-acylglycerol 3-phosphate) and phosphatidic acid (Fig. 11) (Van den Bosch and Vagelos, 1970; Ray et at., 1970; Hechemy and Goldfine, 1971). In experiments with particulate enzymes derived from Escherichia coli, both acyl-CoA and acyl-acyl carrier protein derivatives have been shown to serve as acyl donors (Pieringer et al., 1967 ; Ailhaud and Vagelos, 1966; Kit0 and ?izer, 1969; Van den Bosch and Vagelos, 1970). The glycerol 3-phosphate

Phosl)het itl~lrthniiolaiiii~ii~ (hdiolipin t CMP

FIQ. 11. Pathways for phospholipid synthosie iri E8cherkhk coli.

acyltransfcrase of Cl. bwtpricum wtis shown to hc highly q m i f i c : for t h e acyl-S-acyl carrier protein derivatives ((:oldfine, 1966 ; Uoldfinc el al., 1967). Since 3. coli synthesizes fatty iLcidH de novo as the acyl-8-scyl carrier protein derivatives, and no trarimcylation between acyl carrier protein and Co-ASH has been demonstrated, it is reasonable to aswme that the fatty acids synthesized de n,ovo are transferred directly from acyl carrier protein to glycerol 3-phosphate. The recent work of Van den Bosch and Vagelos ( 1!170) lias shown that tr;uirtacylation from clayl-acyl carrier protein by E. coli ctnzymcs i,r rrwre specific for the placcment of unsaturated fatty acids on the (3-2 of 1 -niono-acyl glycerol 3-phosphate

30 110 WAlt D (I OLDBINIC

than transacylation from acyl-CoA. I n earlier reports on the specific acylation of glycerol 3-phosphate by palmityl-S-acyl carrier protein, catalysed by membrane particles from C1. 6utyricum, the reaction proceeded only as far as lysophosphatidic acid (Goldfine et al., 1967). Recen t work (H. Coldfine and C. 1’. Ailhaud, unpublished observations) has demonstrated the formation of phosphatidic mid with acyl-S-acyl carrier protein derivatives as the acyl donors.

Exogenous fatty acids can be used by E. coli for both phospholipid synthesis and energy production, via /3-oxidation. lt is now clear that these exogenous fatty acids becomc activated by >L fatty acyl-CoA synthetase and not as tho acyl carricr protein dorivi~tivcn (Overath (4 nl. , 1969; Samuol and Aill~;~ud, I!M9; Samuel et al., 1!17i)), ‘I’hiis trans- ucylation from coonzymo-A catws to glyCerophOsphiLt~! p v i t l c s a mcch- anism for utilizing exogcnous fatty acids directly for 1)hospholipid synthesis. Samuel and Ailhaud ( 1909) were unable to demonstratc activation of exogenous fatty acids by Cl. 6utyricum extracts with either coenzyme- A or acyl carrier protein, despite the ability of those cells to utilize exogenous long-chain fatty acids for lipid synthesis (Goldfine and Bloch,

In higher organisnis, the acyl-CoA derivatives have long been re- garded as the acyl donors in the formation of phosphatidic acid (Korn- berg and Pricer, 1953) and indirect evidence for two enzymes has been obtained (Lands and Hart, 1965). Kuhn and Lynen (1965) demonstrated an obligatory requirement for coenzyme-A in the acylation of glycerol 3-phosphate by fatty acyl thio-esters of the yeast fatty-acid synthetase, in a reaction catalysed by a particulate preparation from yeast.

From phosphatidic acid, phospholipid synthesis in E. coli proceeds by way of CDP-diglyceride (Fig. 11) which is formed by the reaction of phospliatidic acid with CTP (Carter, 1968; McCamen and Finnerty, 1908). An analogous reaction wan previously dcrnon~tratcd i n guinca-pig liver (Carter ant1 Iienncdy, 1WXi). Kwincdy i L n d l i i H coworkwH showr*d that two pathways of phospholipid biosynthesis bmnch off’ from CiJ1’- diglyceride in E. coli. One pathway leads first to j’hospiiutidyleeririo by reaction of CDP-diglyceride with free L-serine (Kanfcr and Kennedy, 1904). Phosphatidylserine is then rapidly deoarboxylatcd to yield phosphatidylethanolamine ; thus, the steady-state pool of phosphatidylserine in E . coli is always very small (Fig. 11). A ~imilar Htqumcc of reactions occurs in two species of bacilli (Lcnnarz, 1970; Patterson and Lennarz, 1971), and in Micrococcus ceriJcnns (Makula and Finnerty, 1970).

The other branch of phospliolipid synthesis in E . coli lcads first to phosphatidylglycorophosphate hy reaction of CDI’-diglyceride with glycerol 3-phosphate (Kanfer and Kennedy, 1964 ; Chung r~nd Kcnnedy,

1901).

COMPARATIVE ASPECTS O F BACTERIAL LTPIDR 31 19678, b). Phosphatidylglycerophosphate is then rapidly converted to phosphatidylglycerol by n specific phosphatase (Fig. 11). Evidence for these reactions in two species of bacilli has also been obtained (Lennarz, 1970; Patterson and Lcnnarz, 1971). Stanacev et al. (1967) showed that a reaction betwccri phosphatidylglycerol and another molecule of CDP- diglyceride catalysed by particles from E . coli leads to the formation of cardiolipin (diphosphatidylglycerol). These two sequences of reactions account for all of the known phosphoglyeerides in E’sclberichia coli. The formation of phosphatidylglycerol and cardiolipin by a similar route has been postriluted for Micwcocciis cerijicuns (Makula and Finnerty, 1970).

In buctcriir, cthimoliLminr, is clcrived from serine by the sequence of reactions describccl iibove, No cvidcnce for a reaction involving CDP- ethnnolumino h i ~ s bccn found in bectcria, although many groups of bacteria contuin ~~hosphiititlylotllunolamino. T t uppoars that the reaotion dcxoribctl by Konncdy and Weiss (1956) in which a 1,2-diglyceride reacts

AM0

Phosphaticlylcthanolnmino ---+ PhosphtLtidyl-N-methylethanolamine (1) AMo --+ Phosphatidyl-N,N’-dimethylethanolamine (2) AM8

+ Phosphatidylcholine (3)

Fro. 12. Reactions leading to phosphatidylcholine synthesis in Gram-negative bacteria. AMe indicates S-adenosylmethionino.

with CDP-ethanolamine to form phosphatidylethanolamine, did not arise a t the prokaryotic stage of evolution.

Formation of phosphatidylglycerophosphate from CDP-diglyceride and glycerol 3-phosphate and the subsequent removal of inorganic phosphate that occur in E . coli wcre also found in animal tissues (Kiyasu et a,!., 1963). Guinea-pig liver mitochondria arc apparently capable of synthesizing cardiolipin by the pathway that operates in E. coli, according to a recent report by Strtnaccv and Ihviilson (1971).

b. Phosphatidyl- N - methl/letlmnolarnine, phosphatidyl- N,N’ -dirneIhyl- ethanolamine and 2~hosl)l~afi~ylch~oline. As discussed in Section 11. B. 1. (p. 5), phosphatidylcholine and the N-methylated phosphatidyl- ethanolamines are found in a number of Gram-negative organisms. Their biosyiithesis in bacteria, in every organism SQ far stud$d, is through a stepwise rnethylation of phosphatidylethanolamine (Fig. 12), a pathway demonstrated earlier by Uremer and Greenberg (1900), Arton and Lofland (1960) and Gibson et al. (1901) in the rnamrnuliun liver, and by Hall and Nyc (1959) in Neurospora. The same pathway also occur8 in yeast (Letters, 1966; Waechter el al., 1969) and in protozoa (Lust and Daniel, 1966; Smith and TARW, 1970). Studies with growing cells of CZ.

32 IlOWARD QOLDFTNE

6~dyric?ma (Goldfine, 1962), Agrohacterium tumefacielzs ('Law et ul., 1963), two otlicr speoirs of agrobactcria, I'roteus vulgaris (Goldfine and Ellis, 1964), Hyphornir.robiurn, Nitmsoc?jslis oceanus (Hagen PI al., 1986 ; Gold- fine and Nngrn, 1 %%), Aficrococcus ceriJican,s (Makuln : ~ i i d Finncrty, 1970), and Rho~opsrudon,oiias spheroidtv (Gorchein et el. , I !Mi%) hiLvr demonstrtited the utilization of the inethyl group of mc~thioninc i n thr formation of N-nrcthylatcd cthunolamine phosphatides. 'l'hc enzymic formation of thesc! conipounds has becn studied in two spccies of bacteria. l'aneshiro and Law (1964) obtained a soluble enzyme that catalysed the first mc:thylation of phosphatic3yletha~iolatnine and a particulate fraction that cittalysed tho conversioii of phosphatidylmethylcthatiolaminc to phosphatidyldirricthylct~lrniiol~~mitie nntl ptiosphaticlylcholirie from cxtracts of A . tumefucien,s. 'l'hc rnrtliyl donor in each reaction was S- adenosylmethionine. Work with cholinc-dcficicnt mutants of Neurospora suggested that a single enzyme catalyses steps (2) and (3) (Fig. 12) (Scarborough and Nyc, 1967). Extracts of Hyphomicrobium sp. also catalyse steps ( l ) , (2) and (3) (Fig. 12). This organism is somewhat unusual in that phosphatidyldimethylethanolaminc is present in the cell in quantities equalling those of phosphatidylcholine (Hagen et al., 1966 ; Goldfine and Hagen, 1968). The regulation of these reactions has not been studied in bacteria, but the recent studies of Lester and his coworkers (Waechter et al., 19G9; Steiner and Lester, 1970) suggest that the methylation pathway is repressed in yeast when the organism is grown with adequate choline. 1 t should be notcd, however, that ycltst has both thc mcthylation pathway and thc enzymes for forming phosphatidylcholine via CD L'-eholine, whilc 110 such scavcrigiiig pathway has bccn demonstrated in bactcria, despite at least two attempts (Sherr and Law, 1966; Shieh and Spears, 1967). I t seems clear that the methylation pathway evolved in the prolwyotic organisms, but thc cvolutionury development of animals has led to a Imrtirtl loss of this pathway, and thus to a nutritional depcndetrcr on cholinc.

c. 0-Amino Acyl P ? ~ o ~ ~ ~ ? ~ a t i d y l y l ~ c o r o l ~ ~ . The biosynthesis of thrse oorn- pounds was studied by Lennurz and his colleagues with several Uram- positive bacteria. A particulate fraction obtained from h'tuphylococcus a u r a s catalysed the transfer of lysine from lysyl- t-RNA to phosphatidylglycerol (Lcnnarz etal., 1966; 1967). Lysyl-t-RNAfrom several organisms served as the lysine donor. Among the natural lipids tested, only phosphatidylglycerol served as an acceptor of the lysyl group. Synthetic 2'-deoxy-, but not 3'-deoxy-, phosphatidylglycerol served as an acceptor, suggesting that the amino arid normally becomes attachcd to the 3'-hydroxyl group of phosphatidylgllyccrol. Spccificity for the t-RNA carrier was also shown. Studiw o n tho nlnnylphospttntidyl~lycsrol


synthetase of Clostridiurn welchii showed that ala-t-RNAaia, but not ala-t-RNACYS was active. Tlactyl-t-ltNAa'a and N-acetyl-ala-t-RNAaia were also inactive. Enzyiiiic degradation of t-RNA inactivated the carrier (Gould et al., 1908). Extracts of Bacillus cereus, B. megaterium and Streptococcus faecalis also catalysed formation of lysylphoaphatidyl- glycerol. With the last preparation , arginylphosphatidylglycerol formation was also observed (Gould and Lennarz, 1967). Koostra and Smith ( 1969) demonstrated the formation of L-alanylphosphatidylglycerol by extracts of Mycoplusmu Eaidluwii from L-ala-t-RNA. Synthesis of D- alanylphosphatidylglycerol was also observed, but the mechanism of activation did not appear to require t-RNA.

d. Phosl3hatidylinositol. Information is lacking on the route of formation of phosphatidylinositol in the limited range of bacteria that synthesize this phospholipid or compounds containing it. In animal tissues it is formed by a reaction analogous to thc! bactcrial pathways leading to phosphatidylethanolaminc and pho~phatidylglycerol, i .e. reaction of frce iriositol with CUP-diglyccride (Pardus and Kennedy, 1960).

e . Plasmalogens. The detailed mechanism of formation of these l-alk- l'-enyl-2-acyl-glycerophosphatides in anaerobic bacteria and in animal tissues has not been elucidated. As mcntioned in Section 111. A. 2. (p. 2 8 ) , tho alk-l-enyl ether chains in bacteria are derived from long-chain fatty acids. Experiments in which the kinetics of incorporation of 32Pi into the diacylphosphatides and plasmalogens of Clostridium butyricum were measured provided evidence that the diacylphosphatides were precursors of the corresponding plasmalogens (Baumann et al., 1965 ; Goldfine and Hagen, 1971). When [ 1-3H-1-14C]palmitaldehyde was fed to this organism, much of it was oxidized to palmitic acid which was then incorporated into both acyl and alk-l-enyl chains of the phospholipids ; however, some was incorporated into the alk- 1-enyl chains without prior oxidation (Hagen and Goldfine, 1967). The enzymic reduction of palmityl-CoA to palmitaldehyde and cetyl alcohol in extracts of Cl. butyricqhm has recently been tlrmonstratcd ( h y et al., 1970). Bascd on thesc and other cxperimcnts, (ioltlfiiie and Hagen (197 I ) l l ihve proposed that the 1 -linked fatty-acid chains of the diac:ylp)tospl~atides arc replaced by chains at the oxidation level of aldehydes or possibly alcohols, but no direct demonstration of this pathway has been accomplished. This is somewhat different from the pathway proposed for animal tissues based on the experiments of rz number of workers (Thompson, 1968; Wood and Healy, 1970; Hajra, 1970; Wykle et al., 1970) in which 1-alkyl glycerol 3-phosphate is formed by reaction of I -ucyl dihydroxyacctone phosphate and a long-chain fatty alcohol. 1 -Alkyl-2-acyl-glycerol

34 1tOWARI) (1OLI)IQIN E

3-phosphate is thcn formed, and the plasrnalogen is forined subsequciitly either before or after the addition to the lipid of a water-soluble base, e.g. ethanolamine. In view of the large evolutionary gtxp between the anaerobic bacteria and the animnls, i t is conceivnblc thnt different mechanisms for the formation of pltimalogcns may havc evolvcd (Cold- fine and Hagen, 1971).

f. alycolipids. It is beyond the scopc of this review to discuss the extensive work on the biosynthcsis of the lipopolysaccharidcs of Gram- negative bacteria. An cxcellont review has rccently appeared (Liiderite, 1970). The formation of glycosyl diglycerides was shown by Lennarz (1964), Lennarz and Talamo (l966), and Kaufmann et al. (1965) to involve the transfer of a sugar from its appropriate nucleotide derivative to a diglyoeride. The system from Micrococcus lysodeikticus studied by Lennarz and Talamo (1966) rcsulted in the sequential formation of an a-D-manllOsJd (1 --f 3) diglyceride and a dimannosyl diglyceride from GDP-mannose and a 1,2-diglyceride, In the studies of Kaufman et al. (1965) on a Pneumococcus sp., the stepwise formation of a galactosyl- glucosyl diglyceride was dcnioristratcd in tho presence of a cell-free preparation and the appropriate UDP-sugars. Pieringer ( 1968) reported the formation of a-n-glucopyranoRy1 diglyccride and 2-O-a-n-glucopyranosyl-a-D-glucopyranosyl diglyccridc from U IIP-glucose and 1,Z- diglycerides catalyscd by a pttrticulate preparation from Btreptococcus faecalis. A similar mechanisni presumably exists in plants. Synthesis of galactolipids by spinach chloroplasts was studied by Neufeld and Hall ( 1964) who showed that galactose from UDP-galactose was transferred to endogenous acceptors.

g. Polyisoprenoids. The buildi~~g ~JI{JCI<~ for ~iolyinopr(:~i"icl RynthoniR arc similar to those needed for sterol biosynthesis, which does not occur in bacteria. Bloch and his colleagues (Kandutsch et ul., 1984; Allen el at., 1967) studied the formation of gernnylgeraniol, a C,, terpcnoid, from isopentenyl pyrophosphate, which was catalysed by enzymes from Micro- coccus Zy~odeikticus. Polyisoprenoids up to C,, in length werc also formed by enzymes from this organism. Similar reactions arc prcsumably involved in the formation of carotcnoids, quinone coenzymes and the ixndecaprenylphosphate sugar carriers (Fig. 6, p. 15) nceded for O-antigen (Wright et at., 1967), peptidoglycan (Higashi et ul., 1067), mannan (Scher et al., 1968), and capsular polysaccharide ('l'roy et al., 1971) biosynthesis. The related buctopronol, isolatcd by 'l'hornc und Kodicek (1966) from lactohacilli, is a l ~ o tlorivctl from mavalorlic ncid. Gough et 01. ( 1970) have recently chsractcrizcd u sirtiilur cornpound from Luclobacillus plantaruni .

COMPAHA'PIVE A8PECTS OP BACTEItlAL LIPIDS 35

IV. Bacterial Lipid Compositions

During the past five yeam, quantitative data on the lipids of a number of important groups of bacteria have become available. Earlier observations have been confirmed, extended and corrected. These efforts have been aided by a greater awareness of the types of lipids that may be found in bacteria, along with the application of a variety of recent techniques for the identification and quantitative analysis of complex lipids. Especially important hag been the rcalization that chromatographic comparisons of intact lipids, though valuable, are not sufficient for the identification of complex lipids. Nor is the examination of total hydrolysates of purified lipids sufficient for identification, since a number of these compounds yield identical hydrolysis products, albeit in different proportions. The increasing use of the deacylation procedures pioneered by Dawson ( 1960), followcd by puper- or thin-layer chromatography of the water-soluble products, or more recently by anion-exchange chromatography as developed by R. L. Lester and by Wells and Dittmer (1966), has aided considerably in the analysis of complex bacterial lipids. Several reviews and monographs cover the information available up to 1966 (Kates, 1064; Asselineau, 1066; Ikawa, 1967; O'Leary, 1967), and the reader is referred to these for data from the earlier literature. In the tables and discussion that follow, I have attempted to select from the recent literature the most complete data available for a given species. In some cases similar data may be found either in the older or more recent literature, but repetition has been avoided in order that the outlines may stand out more clearly. The classifiontion of bacteria given by Breed et al. (1057) is used throughout.

A. 1,rrrus OF CRAM-NEGATIVE UACTRRJA

1. complex Lipids

Tables 2 and 3 present the compositions of the complex lipid8 of Grarn- negative bacteria of tlie Ortlcr8 I'scudo monad a h , H yphom icrohiRleH, and Eubacteriales. It is upparent from thcw data that no out- standing quantitative or qualitativc gcneralizations can be rnttde that would enable us to distinguish the Pseudomonadales and Gram-negative Eubacteriales on the basid of their phosphatide compositions. Organisms from both orders usually contain phosphatidylethanolamine, phos- yhatidylglycerol, and cardiolipin (diphosphatidylglycerol). Bacteria in tlie Pseudonionadaleg have somewhat more phosphatidylglycerol plus cardiolipin, average 3.1% (range 17-44%), than members of the Eubac- teriules, average 26% (range 20-33%). If tlie family of lipids derived from phosphatidylethaiiolamine, including phosphatidylethanolamine,

W a

!CBLE 2. Compositions of Lipids from Gram-Negative Bacteria of the Orders Pseudomonsdttles and Hyphomicrobiales

Phm- Phos- phatidyl-

x phatidyl- N-methyl- Phos- Phos- 0

References 0 ? ethanol- ethanol- phatidyl- phatidj-I- Cardio-

c amine amine choline glycerol lipin Others

PSEUDOMONAD ALES

Thiorhodaceaa

Athiorhodecoae Chromatiurn Strain D

RWpseudomonas sphemides Rhodopseudoiiwnas capordata RhodospiriUuna rubruii,d Rhodoapirilluna cap*icldiisd

Nitrosocystis oceati its

Nitrosornoium e i i r o p m

Thiobacillus mapolitnii i ~ *

Thkbacil1tt.s th ioparirx

Nitrob~teracoac~

Thiobscteriaceae

55"

41 4G 19 31

42-45 20-27

G 5

39 5

19 36 13 41

10 5 9 1

3 c 2 8 + C l 7 +

11-15 17-23

24 11

P C

b Steiner et d. (1970b) 2 M

C Gorchein (1968a, b) Steiner et aZ. (1970a) Hhyama (1968) Ehayama (1968)

Hagen et al. (1966) Hagen et al. (1966)

Barridge and Shively

Barridge and Shively (1968)

(1968)

ThiobaciUua intetnaediid

Thiobacdiua thkwxidans (log-

Thiobaciuzls thwoxiduna (stationary -phase)

T11iobdus novellime

P k )

Pseudomonadaceae

Sidempsaceaa Pseudomo~s aeruginosa

Fewobadlua fewooxiduns

HTPHOXICEOBIALES Hyphomierobium vulgare XQ521 23

R1iodomicrobiu.m tan niellii 4-5

2-20 10-26 Barridge and Shively 55-60 12-15 (1968)

(1967)

(1967)

(1968)

37 7 c Shively and Benson

27 16 c Shively and Benson

20 36

4 53

Barridge and Shively 23-27 3-11 33-37 2 4 3 0 5-7

69 1-4 15 9.4 Randle et al. (1969)

20 42 1.5 23 13 Short et al. (1969)

29 10

27 10

I GoldfineandHagen 6 til

(1968) 0

(1967) %I

c Park and Berger 1 0

m * Values BW e x p m d 88 per cent of lipid phosphorus.

Clucosyldiglywride, 8-10% of tot.&] lipid; d e r amounta of other glycolipids. -thine-containing lipids.

Rangee for different media. Ph~hatidyldimethylethanolamine, 36%.

’ -4s per cent of total lipids.

TABLE 3. Compositions of Lipids from Gram-Negative Bacteria of the Order Eubacteriales

Azotobacteraceae Azotobacter agilia (log-phase) Awtobacter agilis (stationary-

Azotobacter ainelandii Phase)

R h i z o b i m AgroWtium ttcmefackns (log-

&dacteriuin tumefaciens

Chromobacteriu m v iolace it iii Enterobacteriaceac

Escherichiear

Phase)

(stationary -phase)

Escherichkz c d i (log-phRsed) Escherichia coli (stationary-

Enterobacter aerogellev phased)

ATCC 13018 Serratieae

Proteeae Sewatia marcescens

Proteru aiclgaria (log-plinsc) Probus vulgaris (stntionary-

Ph=e) Salmonelleae

Salmonella typh iintcrirrrri

Brucella ubortits Bnng 11 19 Haemopkilzrs parainfftwia:at

Brucellaceae

~

Phm- Phos- phatidyl-

phatidyl- N-methyl- Phos- Phos- ethanol- ethanol- phatidyl- phatidyl- Cardio- amine amine choline glycerol lipin

64'~' 5 1 27 2-4

5 2.4 13 23 53 t 7 - t

45 14 7 29

18 16 28 13 19

18 4.6 i 7

i6 77

20 1-1 11 6.6

74 21 3.2

06 14 17

63 1.0 63 12.1

17 9.9 5.8 14.8

7 8 18 3.2

,27+ 37 16 5.8 0.4 0.4 18 3 - 1

l b

cs P

References Others

Randle et al. (1969)

Randle et al. (1969) C Jurtshuk and Schlech

(1969)


Randle et al. (1969) ? > Randle et al. (1969) 2

3 3

3

r m 4

Randle et al. (1969) s Randle et al. (1969) z


d -->


Randle et al. (1969) Randle el al. (1969)

Ames (1968)

13.5 Thiele el al. (1968) White (1968)

(I Values are esprtx-xd as per cent of lipid piiosphorus. Vnidentifmt.

* Includes phosphrttidyldiinethylethanola~nine. Cells g o n n on glucose-minimnl media.

COMPARATIVR AWRCTR OF RACTERTAlr TATl'TDS no phosphatidylinonomethylethanoltrmiiie and phosphatidylclioline are added, the organisms in Pseudomonadales have an average of 66% (range 5.5-78%) compared with an average of 72% for the Eubacteriales (range 64-79%). When these major biosynthetic families of phospholipids in the two orders are compared, the similarities are probably more significant than the small differences in phosphatide compositions noted above.

Within each order there are organisms that can carry out the three- step methylation of phosphatidylethanolamine that yields phosphatidylcholine, organisms that can carry out only the first methylation and organisms that appear to be incapable of N-methylating phosphatidyl- ethanolamino. Even within some of the families of the Order Pseudo- monadalcs there are species differences in biosynthetic capacities. Among the Athiorhodacese, for example, two species of Rhoabpeeudo- ~ T L O ? Z I L R contain ~~houptii~titlylcholin~ but two Hpecies of Rhdospirillum (lo not. Among tho 'I'hiobacteriaceao oiie HptwieR of Z'hiobacillua s t o p a t pho~,phutidylet~~clnolnmino, thrco tit ~)hosl~hatidylmethylethanol- amine, and one Rynthcsizes phosphatidylcholine. In this regard, there is conflicting evidence on P s e u h n a s aeruginoaa. Randle et al. (IS0S) and Sinha and Gaby (1964) found small amounts of phosphatidylcholine, but Hancock and Meadow (1969) and Goldfine and Ellis (1964) did not. Among the Eubacteriales, some organisms in the families Azoto- bacteraceae and Rhizobiaceae are capable of forming phosphatidylcholine (Table 3). In addition to Agrobacterium tumefaciem, phosphatidylcholine has also been found in A . radiobacter, A. rhizogenea (Goldhe and Ellis, 1964), and Rhizobium japonicum (Bunn et al., 1970). Most of the organisms in the Enterobacteriaceae have a relatively simple phospholipid composition consisting of phosphatidylethanolamine, phosphatidylglycerol and cardiolipin. The only exception to date is Prolew vulgaris, which synthesizes phosphatidylmethylethanolamine (Goldfine and Ellis, 1964; Randle et al., 1969). Hagen et al. (1966) pointed out that a number of the organismn that contain phosphatidylcholine also have complex intracytoplasmic membrane syHtcmu ; for example, Hypho- microbium vulgare, Nitrosocysti~ ocaanim, A zolobmtar agilis, and the photosynthetic bactwi a Ithodom icrobium, vannielii, IChodopeuhonas spheroidas, R. capsulata and 12. paluslris (Wood et at., 1906). They postulated that the unique size andlor charge of the polar head group of phosphatidylcholine may facilitate folding of the oell membrane. However, this correlation is not universal. Agrobacterium tumefaciens (Kurkdjian et al., 1966) and Thiobacillua nouellus (Caeseele and Lees, 1969) synthesize phosphatidylcholine but do not possess complex intracytoplasmic membrane structures. There are other organisms which contain complex intracytoplasmic membrane systems but do not have

40 TTOWARn OOLDFTNE

phosphatidylcholine. Included in this group are some of the photosynthetic bacteria, including 1ihodoj)seudomonas gelulinosa, Riiodo- spirillum rubrurn, and Chromatiurn strain D, and Nitrosornonas europaea. Another view of the distlribution of phosphatidylcholinc in bacteria was suggested by Ikawa ( 1 907), who pointed out that most of thc organism that have ~~lios~~lititid,ylrlioliiic U ~ R O possess efficicnt rlcct~ron-transport, systems. 'L 'h iH woiiltl hying togcthcr '1'. noarllua wid t h c t photosynthetic bacteria as well I L ~ N . oceavius, the hyphomicrobia and Azotobucler agilis. The exact function of phosphatidylcholine is not clear, but these relationships, as well as its known presence in tlie intracytoplasmic membranes of higher organisms, suggest an important role for this lipid in membrane structure and function. The evidence from the comparative lipid compositions of bacteria favours the idea that phosphatidylcholine biosynthesis evolved more recently than the biosynthesis of phosphatidylglycerol and phosphatidylethanolamine.

The polar lipids of several species of Gram-negative bacteria have been found to contain ornithinc. Among these are Rhodopseudomonas spheroides, Rhodospirillurn rubr.um, T'hiobacillus thio-oxidans, Rhodo- microbiurn vanniellii (Table 2), Pspudobmonas rubescene (Wilkinson, 1968a) and Brucella melitensis (Prome Pt ul., 1969). In all organisms except Rhodomicrobium vannielii, t h o purified ornithine-containing lipid does not contain phosphoru~, and a fatty acid in amide linkage is usually found. The detailed structure is still unclear (see Section 11. B. 4c.

For several (Xrum-ncgativc orgaiii~ins, data on the lipid composition of cells in the logarithmic untl ntiLtiol1tLry phams of growth in batch cultures are given in Tables 2 and 3 (p. 36) and illustrated in Pig. 13. Data for Azotobacter a!@s, A grobncterium tumefacien.9, I$. coli and Proteus idgaris are taken from Itandlc el al. (1969) and data on Il'hiobacillus tkio-ozitlans from Shively and Benson ( 1967). Changes in the phospholipid composition of these organisms during different stage8 of growth arc evident, and it is clear that no single set of compo~itional data for an organism is likely to hold up under all conditions. AA batch cultures of Gram-negative cells become older, tho proportion of cardiolipin in phosphatidylglycerol plus cardiolipin increases, while that of phosphatidylglycerol decreases. The sum of the two is not always constant, for example in E . coli and A. agilis. It i R known that tjoth phosphatidylglycerol (Kanfer arid Kennedy, 1963) and cardiolipin (Kanemasa et al., 1967) undergo turnovcr in E . coli. Another observation from Tables 2 and 3 is the increased methylation of phosphatidylethanolamine in ageing cultures. In A. tumefmiens, the proportion of' phosphatidylcholine increases markedly in stationary-phase cell8, while that of ~)Iio~phntitlyl~~t~i;Lnnli\miiic~ d c w w s t ~ 8 . l ' h c . oont,prlt of p}bos-

p. 16).

COMPARATIVE ASPECTS OF BACTERIAL LIPIDS

Chanaes in PhosDholiDid Composition

T hio baci It us t thio-oxidansa 3 -

41

Agpobactepfum

t u mefa c i en s b

c

I 5 [L

PG I PG

St a p h ylococcus I Baci It us

aureus megaterium d

FIG. 13. Examples of the alteration of the phospholipid compositions of bacteria during growth of batch cultures. Opon bars indicate the composition of bacteria from logarithmic-phase cultures, and stippled bars of bacteria from stationary- phwe, or presporulation-phase cultures with Bacillw megaterium. CL, indicates ctwdiolipin ; GlcNPG, glucosaminephosphatidylglycerol ; IysPG, lysylphosphatidylglycerol ; PC, phosphatidylclioline ; PE, phosphatidylethanolaminc ; PG, phosphatidylglycerol; PME, phonphatidylmonomethylethanolamine. References: a, Shively and Benson (1967); b, Randlo et al . (1969); c, Houtsmuller and Van Deenen (1966) ; d, Bertsch et al. (1969).

phatidylmethylethanolamine increases in both I'roteux aulpzria and T. thio-oxidans and the proportion of phosphatidylethanolamine uithor remains constant (P. vulguris) or clecrcmcs (1'. thio-oxidun~; gig-. 13).

42 1 I0 WA R U (1 0 L I) 1" IN E

Glycolipids are not usually found in Gram-negative bacteria. One exception noted in Table 2 is Claromatium Strain D. Glycolipids have also been found in sevcral other photosynthetic bacteria (Constanto- poulos and Bloch, 1967; Radunz, 1969), and Wilkinson (1968a, b, 1969) has found glycolipids containing glucuronic acid in two spccics of Pseudomonas (sco Section IT. B. 4.11. 12).

2. Fatty Acids of the L'ram-Negative Bacteria

There are abundant duta on the fatty acids of this group of organisms (Kates, 1964 ; Asselineau, 1966; O'Leary, 1967). The major saturatcd fatty acid is usually 16:0, with lesser amounts of 14:O and 18:O. The major unsaturated fatty acids arc 16 : 1 A 9 and 18 : 1 A". Cyclopropane fatty acids are frequently encountered in the lipids of the Gram-negative bacteria of the order Eubactcriales and these are usually mixturcs of 17 : cyc-9,lO and 19: cyc- 11,12. Among the Pseudomonadales, the cyclopropane acids have not been found in photosynthetic bacteria (Wood et al., 1965), in Pseudomonas aerwginosa or in Ps. diminuta (Wilkinson, 1969); however, Thiele et al. (1969) reported the presence of a 17:cyc fatty acid in Hydrogenomonas eutropha.

B. COMPLEX LIPIDS OF GRAM-POSITIVE BACTERIA On the basis of their lipid compositions, the Gram-positive bacteria

divide into two well-recognized groups. The first is tho group that docs not form endospores, including the lactic acid bacteria and the Micro- coccaceae (Table 4). These orguniwns gctnerally contain phospholipids of the phosphatidylglycerol family, which hcrc inoludc~ 0-amino acyl phosphatidylglycerol as well as cartliolipin, but thcy do not aonttriri t h c phospholipids of the p1~0,qphatidyIcthanolarnine biovynthetio family. 'I'hc second major group is the endospore-forming Bacillaccac. 'I'hc complox lipids of the aerobic members of this family have been intensively studied and are characterized by the presence of phosphatidylcthanolamine in addition to phosphatidylglycerol and tho lipids derived from it. Othcr groupa of Gram-positive organisms will be discusscd separately.

1. Lactic Acid Bacteria and the Micrococcaceae

These organisms do not appear to contain the phosphatidylethanolaniine family of lipids. The major cotnponents are phosphatidylglycerol, cardiolipin and the 0-amino acyl derivatives of phosphatidylglycerol. In addition, glycoeyl diglycerides tnsy represent from a few per cent up to 36% by weight of the total lipid (Table 4). As can he seen, thc

TABLE 4. Bacterial Lipid Compositions. Gram-Positive Bacteria and Spirochaetales

0-Amino- Phos- acyl

phatidyl- Phos- Phos- phos- Phos- ethanol- phatidyl- phatidyl- phatidyl- Cardio- phatidyl- amine choline glycerol glycerol lipin inositol Other References

__ EUBACRZEIAISS Micrococcaceae n

Mimococcw lysodeikticun 72' 1 13 Macfarlane (1961a, b) Micl.ococcwr cerijicans 47-87 12-31 1-15 MakulaandFinnerty +

Staph ylococ~ls at i remC 10-60 18-80' 0-20 f GouldandLennarz 2 - (1970) t

0 HOI-Nc (1970)

/

Houtsmuller and Van Deenen ( 1965)

Staphy2.ococclca aicrezis 6.7 1G-18e 11-11 f Joyceetal. (1970) 3 Barcina lutea 1 14 17 5.5 G Huston et d. (1965) @

0 Lactobacillaceae % Streptococceae m

Brundish et al. (1965, $ 4 & kz 6 Marinetti (1965) P

Sanhs Mota et al. E ( 1970) 21 z

Diplococcccs About 25 About 50

A'treptococcus faecal& (Pneumococcus) 1967)

Major Major' Minor f Vorbeckand

li Exterkateand Veerkamp (1969)

8-6-19 About 3 42-32 ( a W

+ + Brennan and Ballou (1968)

Shaw and Dinglinger Ip

Pmttey and Ballou (1969) cs

(1968)

Bifidobacterium

bijiditm Bi#obacterdtc ni

Pmpionibncteriaceuc Propioiaibactericirn

shtrmanii

TABLE 4-ce4tm.d

0-Amino- kP rp

Phos- my1 phatidyl- Phos- Phos- phos- Phos- ethanol- phatidyl- phatidyl- phatidyl- C d o - phatidyl- anline choline glycerol glycerol lipin inositol Other References

Corynebacteriaceae Lkteria nionocyto9e H es Alierobaclerium

thermosphaet zon ArtIirobarter simpler

(stationary phase) Bacillaceae

Bacillus cereus

Bacillus: cereit.9

Bacillus liche?iifori)c is

BaciU u8 mgateritr QM B1551

Bacillus naegatcriton MKlOD, pH 5

Bacillus megateriitn? MKlOD, pH 7

Brt~ilZz~ natto (lop-plinsrt

Bacilli18 stibtilis B a d 1 ti,?

stearothennopli iIus Clostridiwn butyrictrni

ATCC 4342

Major -!-

Major -

About 85

- 35 8 (om)

23-32 1-5(ala) 5-25

About GO 2 (lys) 2-1’

49-69 10-2

5-10 -I-

35-45 8-14 (IJw)

27 34

13 10 (1YS) 38 22-30 46-55

26P -?-

+-Major lys, ala-

f Carroll et al. ( 1968) *, Shaw and Stead

(1970) Yano et al. (1970)

x O

Houtsmullerand $ Lang and Lundgren

Morman and White

van Deenen (1963)

(1970) 0

(1 970) 2 7-8” Bertsch et al. (1969) M

30-35” Op den Kamp et al. (1965)

Op den Kamp et al. (1965)

Urakami and Umetani (1968)

Bishop et al. (1967) Card et al. ( 1969)

38’ Baumann et a€. (1965)

Macfarlane (1962)

SPIROCH AETALES

Treponemataceae

Kazan 5q Trepvnema pallidurn 5-10 30-40

Treponema palluluni ReitcrQ 4 1 s 7

Trepone ma z udzeraeq 31

Leptospira canicola 60-63 Leptoapira ~ o c 80-90 5-10

4

16

1-5

44-55' Johnson et al. (1970a)

-75' SIeyer and Meyer (1971)

37' SIeyer and Meyer

Stern et al. (1969) (1971) 9

8 Johnson et al. * a

9 (1970b)

3

d 4 Values are expressed as per cent of iipid phosphorus except where otherwise noted.

Ranges for different media or growth conditions. * Dimannosyldiglyceride (Lennan and Talarno, 1966).

' Traces to 9% of phosphatidylglperophosphate. More than 50% of total lipid by veight is neutral lipid.

' Dig1ucosT;ldiglyceride and "phosphatidylglucose". See Fisher (1970) and Shaw et al. (1950) for recent work on the structure Of this

5 d n

Lysylphosphatidylgl~ce~l.

w glycolipid. * @ Lipo-amino acids. including perhaps 0-aminoacylphosphetidylglycerol and other uncbracterized polar lipids. Over 70% of total lipids 3 - - - - -

by weight are neutral lipids. Up to 30% of lipids by wright is ~~l~ctosyl~lucogvldiglyceride. ' Lysylphosphat idylgl-cerol and nlaii~-lptiaspl~~tidylglycerl. ' Mainly phosphatidJ-linositol nuinnoside aiid wylated inositol mannoside.

P

3 E d

r H

r, =! x

Glycolipid. Acylated glucose. 3%50% Meiitml lipid und sun:ill mnount of diglucoayldiglyceride.

Diglyrosvltliplyc.rridr acwuiits for 15" ,, of lipid weight. 38% Phos)~hnti~iyltiirth~l~~th~iiiolaiiiine of which 78% is plasmalogen; 55% of phosphatidylethanolamine and 9% of phosphatidylglycerol

" Glucosamiuyl phosphnt id>-lglyc.erol.

are also of thc pfastnnlogcii forin. q Per rent of totnl lipid by weight. ' Monogalact oxy ltiplyceridr . ' 20% of ph~~s~~htltid?-lrholiiil. is iii plasmalogen form.

" Cells rcwtnin 30°C by weight r > f iieiitml lipids. P Glucosyldiplyerride. ;7

4 (j ITOWARD CIOLDPTNE

proportions of thr phosl)holipids tend to vary greatly from one species to another and i t ia now becoming increasingly apparent that the proportions play vasy withiii a given species. In this regard, Staphylo- coccus aureus has been tho most carefiilly studied. Hontsmuller and van Deenen (1965) Confirmed the wide variations in the proportions of phosphatidylglycerol and i tls lyxyl derivative at different stages of growth that were cwlicr obsrrvrd by MnnfiLrltLne ( I 9 0 4 ~ ) . These changrs (Fig. 13, 1). 41) wrrd ibscribd to c:trarigw in t ho plt vduo of the growth nicdiuin, which could bo iiitluctd iWiw by f‘ernwntation of glucoso or 1)y artificid I I I ~ I L I ~ R (Hoiitr~niiillcr i ~ i i t l V:LII l)rcnc~ii, I !NX). I,,yxylphos- phrttitlylfilycrrol W ~ H foun(1 Lo ~)redoiiiiiit~tc a t lowcr. pfi viLliies antl ptiospt~ut,itlyl~lyccrol w t ~ s l)rrdotnin;uit whcn the rntxliutn was near neutrality. Ciirdiolipin a p p e a i ~ I rnain1,y I L ~ or around pH 4! f . Uould and h n n a r z (1870) sliowcd thitt these change8 in the proportions of phosphatidylglycerol and lyaylphosphatidylglycerol were mainly the result of n large decrease in the absolute amount of phosphatidylglycerol per cell, concomitant with n sni:~ll increase in lysylphosphatidylglycerol. Comparing growing and resting cells at pH 7 and pH 5 , they observed the largest changes a t the lower p€I value in resting cells. Joyce et aZ. (1970) saw little change in the proportions of the three major phospholipids of Staph,. aurem tluring exponential growth in temperature shift-down experiments.

The phospholipid composition of Micrococcus cerijcans is obviously anomalous (Table 4) . The classification of this organism has been re- evaluated (Baumann et al., 1968), antl its phospholipid composition also suggests that i t may be more closely related to Cram-negative bacteria. I ts fatty acid composition, consisting largely of normal saturated and unsaturated fatty acids rather than the branchod is0 and antciao fatty acids usually found in tho Micrococcacoae, also f4LtfifieHtt” mcmhcrship in a different group (Mitkula and I h i c r t y , 1968). ‘l’hcsc cells Rtain Cram- negative (Finnerty et d., 1902).

Surcinu Zuten was found to contain relatively large proportions of neutral lipids, conRisting mainly of hydrocarbon8 and glycerides (Huston and Albro, 1964). ‘rho hydrocurbon composition of thiR qxxies and that of other bacteria was recently reviewed by Albro and Dittmer (1970).

The coccal members of the Fanlily Lactobacillaceae that have been studied resemble the Micrococcaceae in having large amounts of phos- phntidylglycerol, amino aeyl-phosphatidylglycerol, and cardiolipin. The lipids of Pneumococcus I-R144 were studied by Ishizuka and Yamakavra, (1968) who confirmed the findings of Brundish el al. (1967) with another strain o€ Pneumococcus (Table 4). Diglycosyldiglycerides are found in both groups of organisms (Shaw, 1970). Unfortunately, the phospholipid composition of organisms in the genus LactobaciZZus is still not clear. A


large proportion of the total lipid in several species of lactobacilli was found to be “bound”, i.c. not extractable without prior hydrolysis (Asselineau, 1966). The nature of these bound lipids is unknown. Ikawa ( 1963) found no ethanolamine, serine, choline, or inositol in hydrolysates of the phospholipids of L. plantarum and L. casei. Instead, the nitrogen- containing products of hydrolysis were found to be amino acids. Thorne (1964), however, reported the presence of phosphatidylethanolamine, phosphatidylcholine, and cardiolipin in L. casei. These conflicting results have not been resolved. No evidence for the biosynthesis of phosphatidylcholine in thcse organisms was obtained by growing L. casei and L. plantarurn in medium containing [‘4C]-CH,-methionine (Goldfine and

Exterkute et al. (1871) havc recently analysed the extractable phospholipids of riinc Lactobacillus straina and compared them with a number of Bifidobacteriu,m strains of human, iriuect and bovine origins. All of the Lactobacillus strains contained a large proportion (&i-83%) of J2P bound to lipid in phosphatidylglycerol, and from 3 to 16% in cardiolipin. Seven species contained 3-32% of lipid-bound I2P in lysylphosphatidylglycerol, but Lactobacillus helveticus and L. delbrueckii did not synthesize this compound. The phospholipid compositions of some Bifidobacterium bifidum strains of human origin are given in Table 4 (p. 43). Cardiolipin and phosphatidylglycerol were also found in the bovine and bee strains, but alanylphosphatidylglycerol was not present in the three bee strains and one of the two bovine strains.

miis, 1 ~ 4 ) .

2. Bacillaceae

This group of spore-forming bacteria, uspc(:ially t h o aerobic hacilli, has been analysctl extenHively during t h o past five years. Unlike the lactic acid bacteria and the Micrococcaceac, membera of the genus Bacillus all synthesize phosphatidylethunolumine, which usually com- prises 20-40% of the total phospholipids. The average is conderably lower than tha t for components of the phosphatidylcthanolamine biosynthetic family in Gram-negative organisms. Another important difference is the absence from the aerobic bacilli of the methylated members of the phosphatidylethanolamine family that are found in a wide variety of Gram-negative bacteria. Conversely, the phosphatidylglycerol family occupies a more prominant place in the bacilli, and the O-amino acyl esters of phosphatidylglycerol aro found in many species. A glucosamine- containing derivative of phosphatidylglycerol has been found in B. megaterium (Table 4 ) . Major variations in the phospholipid composition of B. megalerium MI< 1011 wcre reportcd by Op den Kamp el al. (1 965). A large decrease in the proportion of phosphatidylglycerol WUH countcr-

48 HOWAllI) QOLDFINP

balanced by the nppewance of the glucosaminyl derivative of phosphatidylglyccrol when cells were grown on a peptone-yeast extract medium that was supplemented with 2% glucose and 0.2% ammonium sulphate. These supplements resulted in a lower pH value (6 versus 7) at the time of harvesting the cells. A smaller change in the phospholipid composition, in the same direction, was achieved by artificially lowering the pH value of the medium with hydrochloric acid. Changes in the phospholipid composition of B. megaterium QM B1551 as cultures go from log to early stationary phase (Bertsch et al., 1969) are shown in Fig. 13 (p. 41).

The phospholipids of a. large niirnbcr of clostridiiil Rpeciee were examined by Kainio ct al. (I%;!)) tint1 many were found to contain plasmalogens (Ttiblc 1, p. 11) . 'I'lie phospholipid compositions of only two spccics h a w bccn studied in tlepth. Baurnann st a1. (1965) found that tho diacyl and placJmalogwi forms of phosphatidylethunolamine and phosphatidylmethylctl~anolamine were major constituents of phospholipids from C1. tJutyricum along with substantial amounts of phosphatidylglycerol mainly in the diacyl form. Cardiolipin was found in stationary- phase cells. The complex lipids of C1. welchii (perfringens) were studied by Macfarlane ( 1962) who demonstrated the presence of phosphatidylglycerol, lysyl- and alanylphosphatidylglycerol, and cardiolipin as the major components. This species has little plasmalogen (Kamio et al., 1969). Some species of clostridia appear to have 70-90% plasmalogen according to these authors (Table 1, p. 11). Radioactive N-methylated derivatives of phosphatidylethanolamine were not found in C1. histolyticum, C1. propionicum, C1. acetobutylicum and Cl. tetanornorphum when these species were grown on [ 14C]CH,-methionine (Goldfine and Ellis, 1964). Clostridium butyricum seems to be unusual among clostridis in this regard, but more species will have to be examined before general conclusions can be drawn.

3. Corynebuctarin a.rd l'ropionibacbria

The lipids of Corynebacterium diphlheriaa have bccri stutlictl iritcn8ivcly in a numbcr of laboratories and tho results haw bcen summarized by Asselineau (1 960). Free fatty acid8 ( 10 : 0, 16 : l) , corynomyuolic acid and corynomyoolenio acid rcprcscnt much of thc free lipids. Thc liittcr two are branched C,, fatty acids related to the mycolio acids of'mycohautcria. Neutral glycerides and esters of ethylene glycol have also bcen found. The phosphatides represent less than half of the total free lipids. From strain P.W. 8 ofC'or.ynebad. rliphtheriaf, (iornes et al. (l!Njfi) isoli~tcd threc phosphoglycolipids which contnincd 4 0 -50% fatty acids and 19--20% hexoses. On hydrolysis, niannosc and iiiositol were found in addition to


glycerol. GnwjnPbacterium xerosis and C. diphtheriae were shown to contain acylated diniaiinophosphoiiiositides and phosphoinositides (Brennan, 1968 ; Brennan and Lehane, 1969). Similar acylated dimannophosphoinositides also occur in the mycobacteria (Pangborn and McKinney, 1966; Brennan and Ballou, 1967) and in the propionibacteriu Brennan et al. (1970) have recently identified mono-acylglucose as a major lipid in Corynebact. diphtheriae and Mycobacterium smegmatis. The major fatty acid esterified to glucose is corynomycolic acid. Corynebacterium diphtherine docs not contain phosphatidylethanolamine or phosphatidylcholine. Thc lipids of ‘several other organisms classified as Coryne- bacteriaceac havc bcen stutlicd and appear to more closely resemble t h e lipids of other Cram-positive organisms. In both Listeria monocylopnes and Arthrobaclw simpZ~r, ~~lio~~~h~itidylglycerol is reported to be a rnajor lipid. 1 t, iH 11 IHO found ill Mir*r~~/)ar./~rirc?tt lk~rmo.~~kaclurn. (furdioiipin is found i i i J,. ~ t r o ~ o r . ~ ~ l o g c ~ r ~ ~ ~ c~nd M . th~rmo~~~~) l~~ac tum and tlic 1;itter is also reported to contuiq phosphatidylethunolamine (Table 4 ) . Stanier et al. (1970) have discussed the indefinite nature of the relationship of other genera of Corynebacteriaceae to Corynebact. diphtheriae.

In a number of ways the propionibacteria are also related to the mycobacteria (Brennan and Ballou, 1968) and this is reflected in their phospholipid composition. Propionibacterium shermanii has phosphatidylinositol, phosphatidylmyoinositolmonomannoside, phosphatidylglycerol, and phosphatidylglycerophosphate (Brennan and Ballou, 1968 ; Prottey and Ballou, 1968). Similar lipids were found in P . freudenreichii (LanBelle and Asselineau, 1968). In addition to these phospholipids, Prottey and Ballou (1968) and Shaw and Dinglinger (1968) found an acylated inositol mannosidc in I-’. shermanii, in which it rcprctscnts 40% of tho total lipid. Shiiw und U)inglingcr ( 190!)) domonxtrt~tc:d smdlw amounts of thc same glycolipitl in fivc othor I’roI’ionibiLotctrirl.

The broader subject of the lipids of the rnycobacteria is hcyontl ttic scope of this discussion. Lipids are prexcnt iii largo amounts in thew organisms and have a complcxity which is unicpc to tho acid-fast bacteria. The interesfcd reader will find much material on this subject in the monograph by Asselineau (1966) and in a review by Lederer ( 1967).

4 . Spirochaetales

Recent work on the medically important Treponemataceae has begun to clarify their complex lipid composition. Meyer and Meyer (1971) examined the lipids of Treponema pallidum Reitcr, and have reported the presence of 26% monogalactosyldiglyceride, 41 % phosphatidylcholine and smaller amounts of phosphatidylglycerol and cardiolipin (Table 4). One-fifth of the phospliatidylcholine is in the plasmalogen

.3 1 TTOWAR n CI OTDPTNlC

form. 1 , a r p ;I mounts of the galuctosyldiglyceride, a similar phosphatidylcholine content and 5-10% phosphatidylethanolamine were found in the Kazan 6 strain (Johnson et al., 1 9 7 0 ~ ) . Similar amounts of the galacto- syldiglyceride were found by these authors in other strains of T. pallidu,m. Treponema zuelseme, a free-living spirochete, has mono- glucosyldiglyceride, phosphatidylglycerol and cardiolipin as its major lipid components (Table 4). The two spccics of Leptospira that have been studied rcscmble the c1 rain -negixti ve bacteria in hnving phosphatidyl- cthanolttrnino I L R their tniijor ~~hosphoiipid. In ntldition, small amounts of ~~1iospRtit,iclylglyc~e~~ol nntl cmlioli pin wcrc found in thc patoc strain (Johnson et al., 1970b).

(!. Cjow4rmrow

Thc study of bilctcrid niemhrane lipids has advanccd beyond the descriptivc and uiiulytical stage, cLnd research on the biosynthesis of the component parts and on the assembly of the complex lipids is moving rapidly forward, Research on bacterial lipids has begun t o focus on such questions as their location in functional membrane units, the relationship of individual lipid spccies to vrtrious enzymes, multi-enzyme complexes and transport systems. The regulation of membrane-lipid biosynthesis a t the enzyme level is receiving more attention in bacteria, and the isolittion of a variety of mutants with lesions in the synthesis of lipids promises t o lead to a better understuriding of the genetics and control of lipid biosynthesis. Studies of such cell functions as wall synthesis, 0-antigen synthesis and the traiisport of sugars have revealed several roles for individual spccies of lipids. These exciting area8 of research have barely been touched in this review in order to limit its size and scope.

Despite the new w ~ t ~ l t h of data on complex hactcrial lipids, i t i H obvious that many important groups of orgunims h a w rcwivcd little or no attention. From the data prescntcd in ‘I’thics 2, R :tnd 4 and discussed in this section, it swms certain that knowlcdge of lipid composition will add much to xtut1ic.r on the relationships between cell chemistry and taxonomy. 1 t has long h e n recognizrd that ccll diversity among the bacteria tends to incrctise towards the periphery of the cell, and the membranc lipids clearly offer :t fertile ground for the study of eompitrative biochemistry.

V. Acknowledgements

The research in the author’s laboratory described herein was supported by grants from the National Institute of Allergy and Infectious Diseases (AI-06079, AI-08903). I should like to thank Dr. N. C . Johnston for helpful criticism and cditorial nclvice.

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58 HOWARD QOLDFINE

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106.

The Pathways of Nitrogen Fixation

JOHN R. BENEMANN

Department of Chernistry, University of California#, Sun Diego, La Jolla, California, 92037, IJ.8.A.

ILlld

R. C. VALENTINE

lhparlm E ti I oJ’ Hiocltm mistry, Uniaereity of California, Berkeley , California, 194720, U . S . A .

1. I l l t l ~ ~ ~ d l l c ~ t l l ~ l l . A. N I trogci i.1’1 xi iig M I C ~ I J -organ i n l n s

15. 13ioclicmistry of Nitrogcvi Pixatloll 11. Elertron Doriora .

A. Pyruvntp . B. Forrnatn . C. Nicotirinmidc Nuclcwtldcs . D. Hydrogen . E. Photosyntlietic 14:lactron Doiiora

111. Electron Carriers. . A. Ft~rrodoxiiiu . B. Plavodoxiris . C . Coupling Factors of Azotobacter.

IT. Nitrognnsse . A. Isolation mid l’ropwt i ( w . B. Mechurii.trn of Acttoil .

v, ( ‘ 0 1 1 C ~ l l ~ l ~ J l l ~ t l I l ( l I “ l l t l l ~ c ’ OlltlOOk . \‘I Arkriowl~.tlgc,riic.rits .

Rcfercr1cw .

. 59

. 60

. 64

. 70

. 70

. 72

. 73

. 70

. 76

. 78

. 80 ’ 83 . 83 . HH . HW . It I . IJJ . 07 . IJN

I. Introduction

Biological nitrogen fixa tion is the enzymic red u (*tion of atm ospheri c nitrogen to ammonia. Beside8 the obvious scientific interest of nitrogen fixation as a fimdumental biochemical reaction, it is of great ecological and agricultural importance, since it is the most important source of the iiietabolizable nitrogen needed by all living organisms. Nitrogen fixation is ratalysed by nitrogenase, which requires energy in the form of AT€’

Thir article is dedicated to H. A. Barker, arid the Van Niel Rchool of microbiology. 59

60 JOHN It. BENEMANN AND R. C. VALENTINE

aiid u biologically strong reductant for the formation of ammonia. The flow of electrons (the electron-transport chain) from an electron donating- substrate to molecular nitrogen is referred to here as the pathway of nitrogen fixlition. Nitrogen fixation differe from the moro fuiniliar patliwsys of carbon and nitm)gon inottlbolisrn in that it ix pri tiitwily t i i i

elcct,ron-tr~~nsport chain coupled to mirnorrin synthosis. How t h various physiological groups of nitrogen-fixing micro-organinms gcmerate the sti-oiigly reducing electrons required for reduction of molecular nitrogen and the nature of the electron carriers and enzymes which handle these electrons is the main subject of this review. We will also give a general review of tho field of nitrogen fixation with emphasis on recent develop- mcnts. For previous rcvicws on tho biochemistry of nitrogen fixation we Hardy and Knight (11)88), Hardy and Burns (11M8), Burris (1909) and Uenemann and Valentine (l97O).

A. Sitrogen- Fidng Micro-organisms Nitrogen fixation is a property found only among the prokaryotic

micro-organisms, namely bacteria, and bluegreen algae. No higher or eulraryotic organisms have been shown to fix nitrogen (Millbank, 1909). There are many, taxonomically quite different, types of free-living and symbiotic bacteria snd blue-green algae able to fix nitrogen. Almost every physiological group of prokaryotic micro-organisms has a t least some representatives in the still expanding list of nitrogen fixers.

The first scientific proof of nitrogen fixation oame in the 19th century when it was shown that peas, soybeans, and other leguminous plants were capablo of growing in the absence of any nitrogen source besides air when their roots bore characteristic nodules. The root nodules appeared to contain bacteria, and were produced by inoculating the plants with soil. A bacterium able to nodulate legurninow plants was isolated in pure culture by Beijerinck (1888) and later named Rhizobiurn japonicum. Many upecies of Rhizobin have since been isolated and, in n11 cases, nitrogen fixation is a strictly symbiotic process ; the bacterium is unable to fix nitrogen without the plant. The process of noduletion involves profound physiologicd changes in the bacteria (which become “bacteroids”) and root tissue (such as the leghaemoglobinn produced which colour the nodules pink), but little is understood about the biochemical basis of this symbiosis. For review#, see Stewart (1966, 1967), Bergersen (1969, 1971) and Dilworth and Parker (1969).

Xany different types of plants besides the legumes are capable of forming nitrogen-fixing root nodules (for reviews, see Stewart, 1966, 1907: Becking, 1970a; Silver, 1969; Bond, 1967). Often them plant8 are found to grow in adverse environments and to pioneer tho estahhhmont of a more varied vegetation. The endophyta reRponnihlo for nitrogen

THE PATHWAYS OF NITROQEN FIXATION 61

fixation in non-leguminous plants have not yet been isolated, apparently because they are difficult to grow in the absence of the plant. The nodulating bacteria appear to be actinomycetes and/or streptomycetes, and have recently heen classified in one genus named Frankia (Becking, 1970b).

Free-living nitrogen-fixing bacteria have been known since Winogradski (1893) isolated the strict anacrobc ('lostridiurvi pusteurianum arid Reijerinck (1901) showed that several Azotohucter species fix nitrogen aerobically. Clostridium pusteurianurri and ,4. vinelandii have been t h e suhjccts of much of the research, bnt,h physiological and hiochernicd, in iiitrogcw fixirtioir l )c~rrrtxc they (wily grown ur1dt.r 1ill)oratory (witlit ions, ihtt (1 ~ ~ I N o I)(*('iLtIHC for iLlmost SO .y('arH thchy wvrt' the. only Iciiowii gtwrrL o f frw-living l)wt(*riib trI)l(* to fix nitrogcn. Nitrogcw tixibtiotl, itndcr wiwrobi(* conditions, I1.y specie^ of the farrrltativu tinaerobes Kkhsiplln and Uacillus was not, confirmed until the 1 !)5Os, although earlier reports existed (see Stewart, 1966, for references).

These free-living bacteria are heterotrophs which must obtain carbohydrates (or other carbon and energy sources) directly or indirectly from plants. Since carbohydrates are usually in short supply in che environments (soil, water) where these bacteria are normally found, their contribution to the total nitrogen fixation in the biosphere has been considered minor. Indeed, the use for several decades of Azotohacter in Russian agriculture has had at best a marginal effect on crop yields (Mishustin, 1970). However, many of these bacteria are often found in close association with plants (see Stewart, 1969, for a review). Azoto- bacter (and related Beijerinckia) spp. are found on root and leaf surfaces of tropical plants. Clostridia can fix large amounts of nitrogen in water- logged soils supplemented with straw. KlehsiPZla species are the endophytes of nitrogen-fixing leaf nodules of many tropical plants (stbe Silver, 1969) ilnd 1)ougllt.r Fir (#JoneH, ]!)TO) r t r d haw e v ~ n t w n fotrtd i n the guts of animals and man whcrc thcy fix riitrogcm (Ihyq*rsm it,nrl Hipsely, 1 !170). Thus the hetcrotrophi(* nitrogcm fixers should not tit.

discounted when estimating nitrogen fixation in the hiosphrre. Some nitrogen-fixing bacteria do not use carbohydrates for growth.

For example, some methane-oxidizing bacteria (Davis et nl . , 1964; FYhittenbury et nl. , 1070) are capable of fixing nitrogen aerobically, and Desulfovihrio vulgaris can grow, and fix nitrogen, by uulphate reducetion with organic acids (Sisler and Zobell, 1!)5 1 ; Riederer-Henderson and n'ilson, 1970). More widespread and numerous are t h e photosynthetic bacteria able to grow on light and carbon dioxide in the presence of a reductant such as molecular hydrogen, hydrogen sulphide or some organic compounds. Kamen and Gest (1949) first showed that Rhodo- spirilluni rubrum fixed nitrogen, and most photosynthetic. bacteria

02 JOHN R. HENEMANN A N D It . C . V A I A J ~ N T I N R

tested h i i w this ability (Lindstrom et al . , 1H5O). The ecological iniport- ancc’ of nitrogrn fixation by tliesc types of bacteria remains to be estab- 1 ishcd.

’I’hc. I)luc*-grccn iilgac iire t81ic> most widespread and versatile group of iiitrogcn fixers. l‘iicy are strict F’hoto-iiiitotro~hs able to grow and fix nitrogen under niiiriy differcwt I often extreme, environmental conditions, including the Artbtic, oce~ins, lakes, hot springs, and especiitlly in rice paddies. where they are of‘ great iigricultural importance (see Stewart, 1!)70, for II review). Filarnentous nitrogen-fixing blue-green algae contain lict,erocyt~,q, I)cculiarly large t hick-walled, empty looking cells occurring a t int~rrvals i n the filaments of thc algae.. The cwrrelution between heterocyst, ocwirrencv cind development and nitrogen fixation led to the throry t,Iiibt hctcrocyntn tirc the site of ni trogcri fixation in them blue- grwn i~Igii(1 (see 1). (i7 for IL discussion). Like nitrogen-fixing bacteria, the bliic-grwii r d g w arc often found iii Hymbiotic associations such iLs with fungi to form lictwns and with rnaiiy plantn (Holm-Hannen, I ! N i H : SilvcNtc5r and Sin i th , l!)f;!j).

‘ I ’ h c x lid. of n i trogcn- fi x i rig rn icw)-orgtinisms is not yct coin plete, sinc*r ncw nunies arc beiiig idtied (Wyatt and Silvery, I !Mi!) ; Pcderov and Kdiniskaya, 1 M I ) and old ones removed (Hill and Postgate, 1960; Patrcjko arid Wilson, 1968 ; Millbank, 1969). Thus, besides Azotobacter, the genera of free-living aerobic nitrogen-fixing heterotropha now includes Derria, Heijerinckia, illycohacterium and Axomonas. However, there tire still only two species of the large genus Bacillus known to fix nitrogen (13. pol~yl/m,y.ca and B. macerans). Better assays for nitrogen fixation are encouraging the isolation and study in both laboratory and field of an increasing number of nitrogen-fixing micro-organisms. Some represeiitative nitrogen fixers, selected because they have been used in

T H E PATH\VAYA OF NITHOQEN FIXATIOK 03

Pltl. I

64 JOHN i t . UENlZMANN A N D It. C. V A b h ; N T I N E

biochemical investigations, UPC listed in Table I. Figure 1 illustrthtcs some free-living nitrogeii fixers mid symbiotic assochtions.

B. Biochemistry of Nitrogen Fixation Before reproducible cell-free bacterial extracts capable of fixing

nitrogen were prepared in 1960 (Carnahan el al., I Q ~ O R , b), gtudien using whole cells and nodulated plants had established t h o following facts (Wilson, 1969): (1) Molybdenum and iron are specifically requirod for nitrogen fixation; vanadium is able to replace molybdenum in some cases (Becking, 1962); (2) ammonia appears to be the firnt product of nitrogen fixation ; (3) R relatiomhip oxistn between nitrogen fixation and hydrogen metabolism ; and (4) R large number of aorn~ioiincl~ (Huch HH

oxygen, ammonia, hydrogen, cyanide, nitrow oxidc!) Hl)wifioull,y inhibit nitrogen fixution (Bradbccr and Wilson, 1 9 N : j ) .

In 1 iJ60, Carnahan and hin coworkers achieved reprocluaihlc! nitrogen fixation in cell-free extracts of Clostridium pmteurianurn hy nupple- menting the extracts with high concentrations of pyruvate. Pyruvate WRS shown t o have a dual function. It was an electron donor providing a source of reductant (electrons) and also a source of' ATP (Hardy and D'Eustachio, 1964; Mortenson, 1964). The electronFi tlerived from Iiyru-

THE PATHWAYS OF NITROGEN FIXATION 65

vate were not used directly in the nitrogen reduction reaction but were passed through an intermediate “electron carrier” which later was isolated and named ferredoxin (Mortenson et al., 1962). Other electron carriers such as flavodoxin (Knight et al., 1966) and the artificial dye methyl viologen (D’Eustachio and Hardy, 1964) were also shown to function in this reaction. The ATP generated from pyruvate through acetyl phosphate proved to be an cssential (and previously unsuspected) substrate in the nitrogen fixation reaction. The requirements for ATP and a suitable strong reductant for nitrogen fixation in cell-free extracts has since been found in till extracts tested (see Hardy and Knight, 1968, for a review). Sin(*(! ATP in high c~onecnt~rutionn (above 10 mM) inhihitn nitrogctn fixtLtion ((‘t~ri~til it~n, I !)fW), i t t ix iinritJl.v gcvwrattd in t h c b I L H H I L ~

vtwsol from tL(vt,yI plionphittc! or (*rtuthith Ihosphrktch (Hrirtly t i t i d

I )’Kuetaohio, 1 $)f14 : Mortenson, 1!)64). Nitrogen fixation in cell-free extracts does not depend on the presence

of pyruvate; indeed pyruvate did not support nitrogen fixation in extracts of aerobic, symbiotic or some photosynthetic organisms, due to their lack of enzymes that catalyse a pyruvate phosphoroclastic reaction (see Section 1I.A. p. 70). In the presence of ATP any substrate, natural or artificial, which can supply the reducing electrons required is able to drive nitrogen fixation. Thus, substrates able to reduce ferredoxin (or flavodoxin or methyl viologen) such as hydrogen (D’Eustachio and Hardy, 1964), nicotinamide nucleotides (D’Eustachio and Hardy, 1964; Benemanii et al., 1971), and even illuminated chloroplasts (Yoch and Arnon, 1970), can serve as electron donors in nitrogen fixation. Bulen et al. (1965) found that even the electron carriers could be dispensed with if sodium dithionite (an inorganic reducing agent) was used as a direct reductant of nitrogenase. The use of dithionite has now become the method of choice in the study of cdl-free nitrogen fixation.

The enzyme system preNent in cvll-frcv* cxtruc*tn whivh c t in &dyw the reduction of nitrogen to ammonia i n thc j)r(w!nw of the rcduc:tc~nt (which is oxidized) and A‘I’I’ (which i n hy(lrolysct1 to AJJJ’) in vdI (*d nitrogenase. Nitrogenase binds molecular nitrogen arid cwnvwtn it into ammonia in a series of redox reactions requiring six eloctronx and ahout 15 moles of ATP. Nitrogenase, which will be described in detail later, has very similar properties in all organisms where it has been studied. It is the only protein which is known to be unique to the nitrogcn fixlition pathway ; the others (such as the electron carricrn) have other func:tionx outside the pathway and, unlike nitrogenwe, their hionynthc+k in usually not repressed by the‘ presence of ammonia or other ni trogcnouH compounds. A large num er of reactions, besides nitrogen reduction,

inorganic phosphate (Hardy and Knight, 1966; Kennedy et al., 1968) in are catalysed by nitrogena !3 e. h e enzyme hydrolyNes ATP to ADP and

06 J O H N R. BENEMANN AND R. C. VALENTINE

a reaction which is dependent on tlie prescnce and oxidation of reductant. However, some ATP hydrolysis (tan be independent of reductant, especially ut.low pH values (Jeng et aZ., 1970). Reductant is oxidized by nitrogenase only during ATP hydrolysis, and ATP-independent reductant oxidation is not known. Nitrogenase reduces, to varying extents, tiocrtylene, cyanidcb, nitroils oxide, azidc, and rnaiiy other Rmall (Rtraricdly unhindrrcd) tri pic! hondcd moleoulus (HCC Hardy and Knight, IOOH, for a rcvicw). I n thc! iLhRence of ~t reducible Hubstrate, nitrogeniiso continues to hydrolyse A'J'P and oxidize reductant (at unditninished rates), and releaseR electrons in the form of hydrogen (Burns and Bulen, 1965). This ATP-dependent hydrogen evolution is found also when nitrogen fixation is inhibited by carbon monoxide, as a side rcaetion during substrate reduction, and in whole cells or nodules incubated under an inert otmosphore. Finally, nitrogenose catalyzes a I), - H, exchange reaction which occurs only during nitrogen reduction (not during hydrogen evolution) (Hoch et al., 1960; Jackson et al., 1968; Kelly, 1968; Turner and Bergerson, 1969).

Assays for nitrogenase take advantage of the reactions which i t catalyses, and the most important ones are: (1) ammonia formation (Mortenson, 1961); (2) lsN, incorporation (Burris and Wilson, 1957); (3) acetylene reduction (Dilworth, 1966; Hardy et al., 1988; Schollhorn and Burris, 1967) ; and (4) ATP-dependent hydrogen evolution (Burns, 1965). The lsN2 incorporation assay introdbced by Burris and Wilson almost 30 years ago is the most rigorous method, but also the most difficult itnd laborious. The ucetylcnc assay, which takes advantage of tho f w t that nitrogenasc ruduces acetylene to ethylene, involves a simplo and very sensitive gas chromatographic determination and has become the method of choice for in vitro, in vivo and even in situ assays for nitrogenase activity.

Nitrogen fixation by broken-cell preparations (cell-free extracts) i R a strictly anaerobic process. This is not surprising considering that: ( I ) the strong reductants (reduced ferredoxin, dithionite) used in thin process are easily oxidized in air; (2) nitrogenase itwlf is irreversibly inhibited by oxygen : (3) oxygen immediately and irreversibly inhibits whole-cell anaerobic nitrogen fixation ; (4) competitive inhibition of nitrogen fixation in whole cells of A zotobacter vinelundii has been observed (Parker and Scott, 1960), and other aerobic nitrogen fixern also fix nitrogen better at oxygen tensions below atmospheric (Dalton and Postgate, 1969a; Bergersen, 1969; 8tewart and Pearson, 1970; Biggins and Postgate, 1960; Drozd and Postgate, 1970a) indicating that nitrogen fixation is also inhibited by oxygen in aerobes. How then do strict aerobic and oxygen evolving (blue-green algae) organisms protcct the nitrogen-fixation reaction from oxygen? The availtlhlc cvidencc

THE PATHU’AYH OF NlTROl>I1:N FIXATION ti5

indicates that, in these organisms, oxygen is excluded from the actual site of nitrogen fixation. Thus Dalton and Postgate (1969a) concluded from studies of continuous cultures of Azotobacter chroococcum that nitrogen fixation is protected in ,4zotobacter by a respiratory system which “scavenges” oxygen from the neighbourhood of the nitrogen- fixing site. Their conclusions were supported by Yates (1 870a), who found that inhibition of oxygen uptake also inhibited nitrogeii fixat ion, and by Drozd and Postgate (1970b), who showed that the organism is able to vary its respiratory activity (depending on PO,) so as to prevent oxygen from reaching the nitrogen fixation site. Nitrogenase activities in cell-free extracts of Azotohacter and Mycobacterium,flavum are present in particulate (sedimentsble) form and are the only instances of nitrogenases being relatively stable in air (for a matter of hours instead of minutes) (Bulen et nl., 1964; Biggins and Postgate, 1969). Purification of Azotobacf~r nitrogenase results in its bccoming as sensitive to oxygen US the nitrog:rtitiscvi from othrr orgenims ( LZiiIcn and IleComtch, 1!166: Kclly. I !1fi !h). ‘I’lic~ nitrog!cwLsc* of lzotob r is thus prcncwt i n wll-frc~e cxxtrwtx ILH p r t of IL ItLrg(1 c*ornplcx (poxsi hly c~)rrtt~ining ot11c.r proteins bcnidcs thcl iiitrogwtixe protcinn) w t r i c 4 i is irrovcrsibly tlissoviatcd during purification ant1 which apparently gives the nitrogenase its unusual properties of oxygen (and heat) resistance. However, if the nitrogenase was obtained by osmotic lysis of the ($ells (instead of using the French pressure (*ell), wtivity was lost about twiec as fast on exposure to oxygen ~ n d it was not sedimentable or awociatcd with membrane fragments (Oppenheim rt nl., 1!)70). Therefore, the mechanism of the reversibility of oxygen inhibition of nitrogenase in whole cells of Azotobacter is yet to be estublislicd.

Other nitrogen fixers living under aerobic conditions (rhizobia, blue-green algae) do not manifest oxygen-resistant nitrogenase activity in cell-free extracts ; therefore oxygen scavenging, dependent on a high rate of oxygen consumption, cannot be their method of aerobic nitrogen fixation. They must have evolved other methods of oxygen protection. Legliaemoglobin could have a function in protecting nitrogen fixation in bacteriods, which have been shown to rccjuirc. oxygon for nitrogrn fixation (Hergrrsen, 1 !)ti!)). The c * c ~ l l rnwnlmncs found in root, n o c l i i l ~ w and the ccll wall of hetcwoysts of Miw-grccn ulgtw ( l h r r t t i r i d U’olk, 1970; Kale d al., I!)’?()) might play II rolc in protecting t lw nitrogttritw in these organisms which also fix nitrogen t m k r aerobic coriditionn.

The hetcrocysts of blue-green algae were proposed as thc sites of nitrogen fixation (Fay et al., 1968; Stewart et al., 1969; Pringsheim, 1968) because all nitrogen-fixing blue-green algae were thought to he heterocystous and because there was a definite correlation between heterocyst formation axid nitrogen fixation (Fogg, 1948), both being

68 JOHN R . BENEMAXN AXD R. C. VALENTINE

inhibited by amtnonia a,nd appearing in nitrogemlimited cultures (Stewart et al., 1968; Neilson et nl . , 1971). Heterocysts are thought not to evolve oxygen, but to contain a reducing environment (Stewart et al., 1969), an excellent place for the nitrogenase to be located. However, non-hetcrocytous bluc?-green algae were discovered to fix nitrogen (Wyatt, tmd Silvery, I !If+!): Stowart, ~ n d Lox, 1970; Huystead c:t aZ., 1!)70; ltippktk r t u l . , l!)7l). A l ~ ~ , C ; ~ i i i t h r i i t t l 1Cvunn (1!)70, 1971) found that, in ooll-froc cxtraots of }~ritit!robioiill,y grown Anaboena cylindrica, thc nitmgentuio ww not annociiLtcd with hotcrocysts, which let1 them to c:onclutlc that- tho hc~tnrocynts woro not, t h o Hito of nitrogen fixation. ‘l’h~wforc, 41 rolc fbr lrc!tc!rocc,yst,x it) tiitropti fixation in not yct (!&at)- I is1 ~ ( 1 .

‘ l ’ I t ( ! rcgiiltbt,ioit i ~ , r i c l t i i ( h i h o l i ( ! ~ * ~ i i t , t ~ ~ l of‘ nitmgwr 1ixath)ri in i~i i

iiit(!;vst,iiig i L i d i i i i l )or l t tbi i t j , I ) i i t , rc!li~tiv(!l,y litth! d d i o d , tLHI)(!Ct of th in rc!ac!t,ioii. It hw t )wn kiiown fibr t b long tirnc! thu t tidtlitiori of nitrogon- oontrbining winpounds (cnpx:itrlly immonitt) to culturcn of nitrogen- fixing organinmn inh ib i tn nitrogcn fixation (Wilmn el aZ., 1943; Kamen and ( h t , 11)4!1) indicating that nitrogcnosc was un inducible cnzyme whosc syrithcsis is represscd by ammonia (Strandberg and Wilson, 1968; Munson and Burris, 1‘361b; Daesch and Mortenson, 1968). Indeed, nitrogen-fixing organisms show typical diauxic growth curves (indicating induction of an enzyme) when grown on limiting ammonia (Yoch and Pengra, 1968). This explains the absence of nitrogenase from ammonia- grown cc l ln (Bulcn et al., 1964). Nitrogenase does not appear in the cells until ammonia is exhaufited (Strcwdbcrg and Wilson, 1!M8 ; Daesch and MortonRon, 1!)88). Even thc ammonitL produced by the nitrogen-fixation I)roonnn is cnoicgh to rcpresn synthosis of nitrogcnnne, UH wen from tho inarc!uned nitrogciionc lovc!ls of cdls growti in (:ontiiiiioiis cultitrtrn or1 limiting ammonia in thc uhxc:rioc: of rriolcc:ular rritrogc!ti ( M I J I I ~ ~ I I rbritl

Burris, l !M!b; I)aonch arid Alortc!nnoit, I!)BH). I n tLilditioit to t ) i o n , y i i t t i ~ ! t i ~ ~ control, nitrogonunc is also fi!c*d h : k iri hihitd, tin i i t /Ih./~ldo.uyiirilli~~~~, ruhrurn, which immotliutctly s top iiitrogcn fixtLtioti or ttic: r d t ~ t c d c!vol~r- t ion of molccula’r hydrogen upon nddition o f timrnotrin ( ( h n t d al., 1 !GO, 1962; Schick, 1971a). Nitrogan fixution I-)y wholc d~ of Axo/dbaclar in only dowly inhibited (ovcr s w w d Itoirrs) hy rLddition of amrnoriirl (Hardy el nl . , I!)IIH; Bcnemann. I !j70) indic:ating n c m c indirc!r:t (tmd unknown) regulation mechanism. Nitrogen fixation l)y Olon~ridiu7n, pastaurimurn, however, is not inhitlited by ammonia, hut furthor nitrogenase biosynthefiis ifi stopped (I)aesch and Blortenwn, 1 !)AX). In photosynthetic bacteria (Schick, 197 Ib) and blue-green algae (Cox and Fay, 1969) nitrogen fixation is dependent also on the availability of carbon skeletons (and thus on photosynthesis) ; in their absence, ammonia accumm ulates and inhibits nitrogen fixation. ?‘he hiochcm ical

basis for these regulatory mechanisms is not understood, since neither ammonia nor metabolites derived from it are specific inhibitors of nitrogenase in cell-free extracts (Carnahan P.! a,!., 1960b, Benemann, 1970). It is therefore possible that the feedback inhibition is in some other protein of the nitrogen-fixation pathway. Regulation of symbiotic nitrogen fixation is even less understood, although it is well known that in the presence of sufficient metabolizable nitrogen, root nodules will either not form or be “ineffective” (non-fixing). Bergersen (1969) recently showed that ammonia does not inhibit, nitrogen fixation by bacteroids.

The cell provides nitrogenase with t i Rteady supply of ATP whose soiir(lr is tlia d i i l t i r ATl’ pool. Using purificd nitrogenam, about 12 to 15 mole8 of A‘l’l’ tire hydrolyscd per mole of molecular nitrogen reduced (Rulen and l,rComtc, 1966; Winter and RiirriR, 1968; Kcnnedy ct al., 11)8B). Thin is u remarkably high vtrlire, trnd would make nitrogon fixtition an extremely inefficient proems. The large amount of ATP hydrolysed could be an artifact prodiiced by the alteration of nitrogenase during cell breakage and isolation. Unfortunately carbohydrate balance studies comparing cells grown on molecular nitrogen and ammonia have not resolved this issue, since contradictory results have been obtained. Twenty moles of ATP are reportedly required per mole of molecular nitrogen reduced in Cloetridium pasteurianum cells (Daesch and Mortenson, 1968) while only four moles per mole of molecular nitrogen in Azotobacter chroococcum cells (Dalton and Postgate, 196913). Therefore the question of the amount of ATP required for nitrogen fixation by the native nitrogenase functioning in vivo is still unresolved. The role of ATP in nitrogen fixation is discussed in Section IV (p. 92). An interesting observation is that ADP is an inhibitor of nitrogenase action. This inhibition might well represent a feedback control of nitrogen fixation under conditions of A‘I‘P starvation (Mountafit ant1 Mortc:nwn, 1 !Ni7).

The supply of reductant reciuircd for nitrogcm fixtition w r r w n from relatively few cellular metabolites (‘‘(!Icctron tlonom”), such 118

pyruvate. They do not donate electrons tliroctly to nitrogonanc: ; ritt,ht.r they are dehydrogenated by specific dehydrogcnasos or rcducttlwn t i r i d

their electrons are trapped by specialized elcctron-carrying protcincl, t h e ferredoxins and flavodoxins. These electron carriers arc redox protein8 which are able to reduce nitrogenase and are the actual reductantw of nitrogenase. Thus ah electron-transport chain linking the reducing power of the electron donors to nitrogenase is estsbliwhed. Xitrogcn- fixing organisms differ in the electron donors and electron carrier8 used in this chain, but the pathways of nitrogen fixation in all nitrogcn-fixing organisms can be summarized as an electron transport chain as shown in Fig. 2. This picture of nitrogen fixation comes from a study of a variety

cellular Electron Electron energy

(pynrvate, roductaeo

”- Dehydrogen- - carriers - donors - or -% Nitrogenaae (ferredoxin or flavodaxin)

BOUNX

NADPH2) light]


Strong reductants were produced during pyruvate metabolism by extracts of CE. pasteurianum, as was evident from the evolution of molecular hydrogen and the additional observation that some low- potential redox dyes (such as methyl viologen) were reduced by this process (Mortlock et al., 1959). That the phosphoroclastic reaction was the driving force in nitrogen fixation by the pyruvate-supplemented clostridial extracts became obvious when the same cofactor requirements (TPP, coenzyme A, ADP, Mg2 I-, Pi and ferredoxin) werc found for both reactions (Munson et al., 1065). The reaction sequence is complex involving a number of steps catalysed by several enzymes (Pig. 3). First

12NHJI *u Nitrogonase

FIU. 3. Tho piiosphoroglustic reaction in nitrogcw fixation in clostridiu. Prl irrclicutarr ferredoxin.

pyruvate is decarboxylated by the TPP-containing pyruvate dchydro- genase. The acetylated dehydrogenase then passe8 the acetyl moiety to coenzyme-A while the electrons are used to reduce fcrredoxin. The acetyl group is next transferred to inorganic phosphate forming acetyl phosphate and regenerating coenzyme-A. Finally acetyl kinase generates ATP from ADP and acetyl phosphate. The decarboxylation step is the key step in the phoaphoqoclastic reaction, since the release of the COO- group as carbon dioxide gas provides the driving force for ATP formation and the reduction of ferredoxin. The phosphoroclastic reaction is not specific for pyruvate ; a-ketobutyrate is also a substrate and was found to drive nitrogen fixation, although at half the rate of pyruvate (Carnahan et al., 1060b).

4

72 .TORN R. UENEMANN AND It . C . VALENTINE

Both rcquircnients of nitrogenase (reduced ferredoxin and ATP) are met by a single substrate (pyruvatc or a-ketobutyrate) through the phosphoroclastic reaction. It should bc noted that the ratio of ATP to reduced ferredoxin formed in this reaction is unity, while that needed for nitrogen fixation is about five, The excess reduced ferredoxin is used to make molecular hydrogen, thus accounting for the formation of this gas observed during nitrogen fixation, and for the high levels of pyruvate needed. Indeed, addition of acetyl phosphate (or other suitable ATP generators) decreased tho pyruvate rcquircmcnt in nitrogen fixation. l’yruvatc will ulso support nitrogc!n fixation by whole cells and extract8 of many orgunisins bcsidcs Cl. pcistPurianurn, (sco Hardy tmd Knight, 1968; Pischer and Wilson, IIV70).

‘l’lic historical position ofpyruvatc us thc first known olcotron donor of nitrogcm fixation and its high activity may have impeded the search for alternative clcctron donors in nitrogen fixation. Thus although molecular hydrogen and nicotinamidc nuclcotides were shown to be effective (but less so than pyruvate) electron donors in nitrogen fixation by extracts of Cl. pusteuriawum (U’Eustachio and Hardy, 1964), they were thought to have 110 real physiologicd significance. But, under certain conditions of cell growth, these substrates might well take a leading role in the pathway of nitrogen fixation, as is discussed below.

B. ii’wmnte

Formato is a common fermeiitntion product, derived from pyruvate, among the facultative and strictly unarrobic bacteria. It can be a strong reducing agent, and WHB first shown to drive nitrogen fixation (because of thc prcsoncc of u formatc : fiwedoxin oxidorcductssc) in cell-frce extracts of 6’1, pusteurinnum (MOrteIiHOII, 1‘366).

In our laborutory we showod tht~t formatc could mpport nitrogen fixation in two organisms, namely Klebsiella pneumoniue und B ~ i l l u e polymyxu (U. C. Yoch and 1%. C. Valentine, unpublished data), both of which are known to metabolize formate. Activity W ~ H found to bc highest (approaching that of pyruvatc) in freshly propared extracts, and rapidly deteriorated with ageing of the extracts. This might be an explanation for the low activities observed by Fischcr and Wilson (1970) for this reaction. Formate dehydrogenasc and pyruvatc dchydro- genase activities were clearly separate enzymes in thcRe organisms. For example, an extract of Klebsiellu frozen under argon for 48 hours lost its formate reaction while the pyruvate-driven nitrogen fixation was affected but little. The activities were approximately equal in fresh extracts. In extracts of Bucillas, formate dehydrogenase was sedimentable (14ci,OOO g for 2 hours) and was easily separated from the pyruvate

THB PATHWAYS OF NITROGEN FIXATION 73

system which W&E soluble. The mechanisms of formate-driven nitrogen fixation in these organisms are not known.

C . Nicotinamide Nueleotides

Until recently, no electron donors were known which were involved in nitrogen fixation by aerobic or symbiotic organisms. The lack of a clastic-type reaction in such species suggested that donors other than pyruvate were supplied by these cells. With the recent isolation in our laboratory and in other laboratories, of electron carriers &om Azotobacter vinelandii and soybean root nodule-extracts (Rhizobia) able to reduce nitrogenase (see Section 1II.A. p. 78), the way opened for the investigation of the pathways of nitrogen fixation in these organisms. Our experiments indicated that NADPH, was the electron donor in nitrogen fixation by A. vinelundii (Bonemann et al., 1971c) and that it plays a major role in other nitrogen-fixing organisms (D. C. Yoch and R. C. Valentine, unpublished observations). Strictly defined, NADPH, is an electron carrier, sinoe it transfers electrons from a donor to an acceptor, but for tho purpose of this review we will consider it an electron donor in the category of formate or pyruvate.

There is a major objection in considering the nicotinamide nucleotides as potential electron donors in nitrogen fixation. The redox potential of the nicotinamide nucleotides is -0.32 V, while that of ferredoxin (at least the clostridial type) is -0.42 V (Tagawa and Arnon, 1962; Sobel and Lovenberg, 1966). Therefore, transfer of electrons between nicotinamide nucleotides and ferredoxin should be in favour of nicotinamide nucleotide reduction, not ferredoxin reduction. Since reduced ferredoxin is the actual reductant of nitrogenase (NADH, or NADPH, being unable to reduce nitrogenase by themselves), this seemed to present a thermodynamic barrier to the involvement of nicotinamide nucleotides in nitrogen fixation, unless one postulated some kind of energy (ATP)- driven reaction similar t o the reverse electron flow in photosynthetic bacteria (see Gest, 1972). However, the redox potentials in question are only halfway potentials ; therefore if the ratio of oxidized :reduced species is increased, the effective potential is lowered. Thus a ratio of 100 : 1 NADPH, : NADP would give an effective potential of -0.35 V. Indeed, ratios of over 1OO:l (NADPH:NADP) are not uncommon in living cells (Veech et ul., 1969). Secondly, the actual potential of the reductant of nitrogenase is not yet known ; it could well be higher than the potential of clostridial ferredoxin. Indeed, flavodoxin with a redox potential above -0.40 V (Mayhew et al., 19698) is an effective reductant of nitrogenase albeit less so than ferrcdoxin (Knight and Hardy, 1966). There are several lines of experimental evidence to indicate that

74 .JOIIN R. DENICMANN AND R . C . VALENTINF:

Iiicotillalnide n~Icleotides are able to reduce ferredoxin both i n vitro and in vivo. In tlic ct1ianol-fi.rrnentitlg bacterium, C l o s t r ~ ~ i ~ ~ i ~ Z ~ ~ v e r i , ATP can only be produced if oxcess electrons from nicotinarnidc nucleotides are removcd by frrredoxin and disposed of as rnolccular hydrogen by the ferr~doxin-linl~e~ Iiydrogenase enzyme. In vitro, this reaction was readily reversible {although subject to regulatory control) depending on the partial pressure of molecular hydrogen and required no additional ATP source (Thauer et al., 1969, 1971 ; Jungermann et al., 1969, 1971). Even in sucrose-fermenting clostridia, some NADH, appears to reduce ferredoxin and be liberated as molecular hydrogen ~ ~ ~ o r t c n s o n , 1968). Thorrforc, as long as there is an activc reduced nicotinrzmide nucleotide- generuting system which lccep the. roduced : cixidizr!d ratio high, NADPH, (or NRl)IZ,) could bo rzn cffcctivc electron tionor in nitrogcn fixation. Thig was clearly (~(?moIistrated by a simple cxpcrirnent in which NAIIP H,, gcncrntcd k~y glucosc 6-photlphute dchydrogcnesc and linked to oloatriditd fcrredoxili by the spinach chloroplast NADP- fermdoxin reductilse, roduecd nitrogenase from A. vincZandi~ with an e ~ e j ~ n c y of about 40% of tho maximal ~ i t ~ ~ i o n i t e reduction rate (Uenemann et al., 1971~). Although the Bystem was non-physiological in that purified cnzymes from many different sources were used, the results clearly showed that there was no thermodynamic barrier to NADPH, bcing an electron donor in nitrogen fixation.

The role of NADPH, in nitrogen fixation by A . vinelandii was discovered only after its electron curriers, azotoflavin and azotobacter ferredoxin, wcre isolated (see Section 1II.A. p. 78). A cell-free extract, contaiiiing both electron curriers in ~ ~ ~ c ~ e n t eonceiitrations, exhibited nitrogenase activity when supplied with various substrates. Dialysis showed that NADP (but not NAD) WUR a required cofactor ant1 all active substrates were known to bc NAIW linked. Addition of NAT)PH *, either in s ~ ~ ~ ~ s t r a t ( ~ ~ ~ ~ i ~ h r i t ~ t i ( ~ s or ~K-I 1)ih't of' a ~ ~ ~ n ( i ~ a t i n ~ ~ y ~ t ~ r i i (frcirn glucofie 6-phorjphatr) resulted i n twrul)ar~tivdy high iwtivitiw. I:cdurtd nicotinarnido adcninc dinucleotitlr (or NAI)H,-linkcd m c t d ~ o l i t r ~ ) W ~ L H

inactive. The ,substnnccs rnorjt activc: in generating NA IIPH irr rdi-Srt*c: extracts of A . v i n ~ ~ ~ ~ ~ ~ ( ~ i i wcw i s o ~ i t r ~ ~ t ( ~ (jsr)~itrtit(~ c1r:hyrlrngr:ni~~r: makes up about 1% of the fiolublc! cell protein; C f i u ~ g :Ind l+'rsnzc?n, 1969), mulate, nnd glucose 6-phosphate (Brnemunn ct al., 1!)7 lc). Probably no single metabolite has tin exclnsivc role in gcnwtbtirig t h e NADPH, irscd far nitro~eii fixation by A z~~~~actcr.

We have recently extended the investigation of NADPH, as eIcrtron donor in nitrogen fixation to other organisms (Table 2), and found that i t is an effective electron donor in extracts of all organisms studied, except for the soybean root nodules where the lability of the system made such experiments difficult. The finding of NADPH,-clriven


nitrogen fixation in extracts of so many different organisms makes it a most important electron donor of nitrogen fixation.

Prior to this work, NADH, had been implicated in nitrogen fixation by several laboratories. D’Eustachio and Hardy (1964) found that NADH, would drive molecular nitrogen fixation in extracts of C1. pasteurianum with about one-third the activity of pyruvnte. Klucas and Evans (1968) reported low rates of acetylene raduotion in eoybonn root nodulc and extracts of A . vinelandii supplemented with NADH, as electron donor and the dyes methyl- or benzyl viologen as electron carriers. The use of

TABLE 2. Nicotinamicie Nnctootitirs RR Eloctron Donor8 for Nitrogen Fixation

Organixrri -~

Clostridium

Klebeiella

Bacillua polymyxa

Azotobacter

Rhizobium

Anabaena

pmteurianum

pwurnoniae

vinelandii

japonicum

cylindrica

Eloctrori n o I 101’

NADPHz NADHl NADPHZ

NADPHz

NADPHZ NADH, NADH,

NADPHl

Nitrogenaso activity

(xnpmolos ammonia

or othylono cvolvcxl/rniri ./ mg. protoin)

1.0 2.0 0.91

- _ _ _ _ _ _

1.4

0.36 0 0.18

0.21

ltofo roncc:

D’Eustachio and Hardy (1964) __ .__ -

H. Nagatani and R. C. Valontine, (unpubiishod obsorvations)

D. C. Yoch and R. C. Valentino (iinpublished observations)

Bsnemann et al. (1071)

Klucas and Evans (1968) H. Botho aridIIt. C. Valontirio

(~~npiihli~hed ohsnrvations)

artificial electron carriers (dyes), which are known to interact with many enzymes non-specifically, raises the possibility that these reactions were not physiological. Indeed, the recently isolated bacteroid ferredoxin could not substitute for the dyes in these reactions (D. C. Yoch and R. C. Valentine, unpublished results; P. Wong, H. J. Evans, R. Klucas and S. Russel, unpublished observations), and recent reports from Evand laboratory suggest that the bacteroid nitrogen-fixation pathway is very similar to the one found in A . vinelandii. Yates and Daniel (1970) reported that NADH, was the electron donor in nitrogen fixation by A . vinelandii ; however, the activities they obtlerved could be accounted for by the presence of NADY in the crude extracts used (M. 0. Yatm, personal communication).

76 JOHN R. BENEMANN AND I t . C. VALENTINE

D. Nydrogeit MolccdiLr hydrogcn is R common fermentation product of many

anacrobic btictcria and could be an electron donor in nitrogen fixation as a rcsult of activity of the rcversible ferredoxin-linked hydrogenase reaction. Indeed, molecular hydrogen was an effcctivc electron donor in cell-free extracts of clostridia (Mortenson, 1964) and was even used (together with a crude nitrogcnase-free clostridial extract) in the early studics with Azotobacter extracts (Bulen et al., 1964).

Several aspects of molecular hydrogen as an electron donor must be Considered : (1) molecular hydrogen is an inhibitor of nitrogen fixation ; and (2) the gas is a by-product of nitrogen fixation itself; oven in the presence of molrcular nitrogrn a significant amount of ATP-dcpendent evolution of molraulrir hydrogen often occurs as a side reaction. Rut thcsc findings poso no scrious obstacle to molecular hydrogen bcing considrrcd as tin clcctron donor in nitrogen fixation Rince nitrogenasc inhibition is significant only a t high partial pressures of molecular hydrogen, whilc uptake and rc-use of this gas would be an efficient method of recycling a waste product of the nitrogenasc reaction. This could also account for tho failure to observe molecular hydrogen evolution during in vivo nitrogen fixation by many organisms.

The Azotobacter hydrogenase has the unusual property of only taking up hydrogen, being unable to evolve it (Hyndman et al., 1953; Peck et al., 1956). It is associated with the cell membrane (Cota-Robles et al., 1958) and is preferentially synthesized during growth on molecular nitrogen, indiiction of nitrogen fixation causing a stimulation in hydrogenase synthesis (Green and Wilson, 1953; Lee and Wilson, 1943). ThiR clearly indicates n rclationship between hydrogenase and nitrogenase in this aerobic nitrogen fixer, but thc nature of this relationship remains to be rluciduted. Photosynthetic bactcria can grow and fix nitrogen with molcctilur hydrogen us thc elcctron donor ; the hydrogenam appcurs to bc similar to the Azotobuctcr cnzymc.

E. Photosynthetic Electron Donors The nature and origin of the elcctron donor8 in nitrogtm fixation IJY

the blue-grcep algae and the photosynthctic bacteria arc r iot yet estub- lished. The depcndence of nitrogen fixation on photosynthesix observed in long-term studics of whole cells led t o suggestions that photosynthesis was directly linked to nitrogen fixation in blue-green algae (Fay and Fogg, 1962; Fogg and Than-Tun, 1958) and photosynthetic bacteria (Arnon et al., 1961 ; Pratt and Frenkcl, 1959). A direct way of gcnerating reductant for nitrogcnnsc in thew organisms might be by n photochemical reduction of fcrredoxin a8 occurs in chloroplasts (Arnon,

TFIE PATHWAYS OF NITROGEN FIXATION 77

1967). However, such a reaction has not been shown in photosynthetic bacteria, and more recent studies suggest that photosynthesis is not directly coupled to nitrogen fixation since nitrogenase activity of whole cells continues in the dark (Fogg, 1961; Schick, 1971a; Cox, 1966). The available evidence indicates that, in the photosynthetic nitrogen- fixers, the same electron donors drive nitrogen fixation as in other organisms.

In photosynthetic bacteria, attempts to link in vitro the photosynthetic apparatus, located in the chromatophores, and nitrogen fixation have had only limited success. Yoch and Arnon (1970) using Chromatium extracts were able to provide the A W requirement for nitrogen fixation through photophosphorylation by illuminated chromatophorcc;,; however, the reductant had to be providcd through others moans. Onc euch method was the u8e of illuminated chloroplast fragments (hcatcd to avoid evolution of molecular oxygen) which, in the presence of ferredoxin or other suitable electron carriers, are able to provide the reductant requirement of nitrogenase (Yoch and Amon, 1970; Benemann et al., 1969), indicating that a ferredoxin-linked photoreduction of molecular nitrogen is at least theoretically posaible. The only known case of photosynthetic ferredoxin reduction in photosynthetic bacteria was reported (at low rates) in carbon dioxide fixation by cell-free extracts of Chloro- bium thiosulfatsZlhilum (Evans and Buchanan, 1966 ; Buchanan and Evans, 1966). Recently Evans and Smith (1971) have been able to couple the photosynthetic apparatus of another green sulphur bacterium, C h ~ o r o p s e ~ m n a s ethylicum, to nitrogenase in a ferredoxin-mediated reaction. However, in the purple bacteria, the mechanism of ferredoxin reduction is not known, but pyruvate can drive nitrogen fixation in cell-free extracts of Chromatium (Bennett et al., 1964). Thus the question of the relation between photosynthesis and the reductant requirement for nitrogen fixation in photosynthetic bacteria is not yet ucttled.

Evidence against a role for direct photoreduction of ferrcdoxin i H

strongest for the blue-greenalgae. Nitrogen fixation in A mbaenucylindrim takes place in the dark (after light has been turned off) (Cox, I966), and some blue-green algae can slowly grow and fix nitrogen heterotrophically on sucrose in the dark (Fay, 1965; Watanabe and Yamamoto, 1967). Also, the absence of photosystem I1 in heterocysts (deduced from the absence of oxygen evolution, carbon dioxide fixation and phycocyanin) (Fay, 1969; Wolk and Simon, 1969; Fay and Walsby, 1966; Wolk, 1968) would rule out generation in these cells of a photosynthetic reductant. The action spectra of nitrogen fixation by Anubaenu cylincErica (Fay, 1970) indicates that the primary involvement of light in this pathway is due to cyclic photophosphorylation (photosystem I) and that the direct contribution of photosystem I1 is, at best, minor. Recently

78 JOlTN R. BENEMANN AND R. C. VAIJENTINE

it was found that NADPH,, generated from glucose 6-phosphutc by an endogenous 'dehydrogenase, functions as un effective dcctron donor in nitrogen fixation by A . cylindricn (Uothe, 1970). Earlier work had indicated that decarboxylation of pyruvate would support nitrogen fixation in whole cells or extracts of this organism (Cox and Pay, S9G9;

In conclusion, thcn, it qqwarN t h i r t , in thr! photosyrithc!tic nitrogon fixers, the photosynthotic system is not directly involved in the gcneration of rcductant for iiitrogonase with the possible exception of green sulphur bacteria. However, photosynthesis, through photophosphorylation, does providc tho A'I'P requirement of nitrogenasc and, through carbon dioxide fixation, the carbon slrcletons needed for ammonia incorporation. I n tho absence of suitable ammonia acceptors, nitrogen fixation is quickly inhibited due to buildup of ammonia (Cox and Fay, 1969; Schick, 197Sb), thus ticcounting for some of the dependence of nitrogen fixation on photosynthesis. We believe that the electron donors which drive nitrogen fixation in these organisms appear to be the same as those found in other nitrogen-fixing bacteria. However, further studies are required to provc this contention, and definitcly cstablish the relationship betwcon photosynthoeis and nitrogcn lixrrtion, two of the rrio~t importatit biologictrl proce~os.

cox, 1966).

ILL Electron Carriers

The natural reductants of nitrogcnase are electron carriers of the ferredoxin and flavodoxin type. These electron carriers have been found in bacteria, bluc-grcon algae, and plants, and have many functions in cellular metabolism in addition to nitrogen fixation. The ferredoxins and flavodoxins of nitrogen-fixing organisms can be easily purified as coloured acidic proteins which bind to DEAE-ccllulose. Their common characteristics are a low-rcdox potential (below iiicotinamide nucleotides) and the reversibility of their reduction and oxidation. They have no catalytic function of their own ; they act only in transferring electrons from one enzyme to another. Among bacteria these electron carriers had, until recently, been isolated only from strictly anaerobic and photosynthetic bacteria (Valentine, 1964). During thc last year, work in our laboratory (and in others) led to the diwovery of fcrredoxin- and flavodoxin-type elcctron carriers in fwultativc anaerobic, aorobic, and Rymbiotic nitrogcn-fixing bactorith. Altho iqt i thc stiidy i d ohrtructr?rizclr tion of these new clcctron carricm i8 not yc:t f i n i H h d , t h y oxtc!nd thc concept of low-potential electron carrier8 to all nitrogen-fixing organiHms and bacterial types. A summary of'the properties of the fcrredoxine and flavodoxins from nitrogen-fixing organisms is presented in Table 3.

TABLE. 3. Properties of Electron Carriers used for Nitrogen Fixation

Ferredoxin Flavodoxin

Visible NADP Spectra Reduc- Activity

(chloro- Mole- Maxi- tion by Mole- cular Iron and mum chloro- cular co- plast- e

2 Organism weight (sulphide) (nm.) plasts References Name weight enzyme NADP) References

C1ostridiu.m

r Awtobacter pa8teurianum 6,000 8(8) 390

vinelanrlii 14,500 8(8 ) 375-415

Rhizobiton about shoulder .japoiiicu?i& 10.000 0 300-400

Aiiabaena 330, 420 cylindrku 10,000 2(1) 463

Baeil1U.S pZym yxa 11,000 4-5(+-5) 385 -

Chromtitcm 5.600 8(8) 385

Desulf ocibrio

Lovenberg et al. Flavo-

D. C. Yoch (personal h o b - + (1963) doxin 14.600 FXN

+ communication) flavin 31.000 FMN

Burton et al. (1970); Rhizo- - D.C.YochandR.C. fiavin i: 9

Valentine (unpublished observations)

Yamanaka et al. Phyto-

Shethna (19704 : + (1969) &\in - FBm + - D.C.YochandR.C. - - -

Valentine (unpublished observation)

(personal communication)

+ B. B. Buchanan - - -

Lsishlev et d. (1969) Flavo-

Knight and Hardy 3

Benemann et d. + (1966) $ - (1969); Hinkson

and Bulen (1967) D. C. Yoch and 5

- R. C. Valentine

i;l

- - 5

(unpublished observations) 2

s

E? x + Smillie (1965)

Dubourdieu and gisas 6,500 4(4) 400 notcited doxin 16,000 FMN notcited LeGall (1970) -.l

E3

80 JOHN R. BENEMANN AND R. C. VALENTINE

A. Ferredoxins Ferredoxins have been isolated from plants, blue-green algae, and

many bacteria. They can be best defined on the basis of their prosthetic group and rcdox properties. They are iron-sulphur (Fe-S) proteins with a redox potential of about -0.40 V. and are able to undergo reversible oxidation and reduction. The non-haem iron and “labile” sulphide are released upon acidification and are present in approximately equivalent nmoants. Dcfinitions basod on their elcctron-carrier properties in a airnplc spcbcific onzymic aystcm such as the phosphoroclastic reaction, tho N R 111’ rcduction by illumiiiutod chloroplasts, or cvcn their ability to rcdiioo nitrog(wusc, two lirnitcd by the enzymic nature of these reac- tiom sincc fcrrcdoxin-linked cnxymcs often do riot interact with ferredoxins of different spocies. However, activity in at least one of the characteristic reactions of ferredoxin, such as reduction of nitrogenase, hydrogenasc, or NADP-ferredoxin reductase, is essential to establish a newly isolated iron-sulphur protein as a ferredoxin. Not all iron- sulphur proteins are ferredoxins, and even the iron-containing protein of nitrogenase does not fall into this classification. The isolation, properties and functions of ferredoxins have been extensively reviewed (Valentine, 1964; Buchanan and Arnon, 1070; Hall and Evans, 1969; Malkin and Rabinowitz, 1967). This discussion will emphasize recent developments of interest to nitrogen fixation.

Bacterial ferredoxin was discovered by its ability to link pyruvate oxidation to hydrogen evolution in extracts of CI. pasteurianum (Mortenson et al., 1962). Clostridial ferredoxin was first seen as a dark brown band whioh adsorbed to a DEAE-cellulose column and could be eluted at high concontratioris of Halt. Tho protein thus obtained was a required cofactor in the pyruvato phosphoroclantic roaction. That fcrrcdoxin transported clcctrom from pyruvato or hydrogcn to nitrogenasc waa shown independently by D’Euatachio and Hardy (1984) and by Mortenson (1964). Ferredoxins participate in a largo number of bioohemical reactions, nitrogen fixation being only one of them (see Benemann and Valentine, 1971 ; Buchanan and Arnon, 1970). Clostridial ferredoxin is a small (mol. wt. 6,000) acidic protein containing eight iron and eight sulphide atoms per moleculc. It has a characteristic adsorption spectrum with maxima at 280, 300, and 380 nm. All ferredoxins can be reduced by dithionite (with a decreafic of abeorbance a t 380 nm.) and completely re-oxidized in air. Clostridial ferredoxin is typical of the ferredoxins isolated from anuerobic bacteria. If iron and sulphide are removed (Lovenbcrg d nl., 1963), the remaining ferredoxin apoprotein is biologically inactive. Malkin and Rabinowitz (1966) have been able to reconstituto native ferredoxin from the apoprotcin by addition of’


mercaptoethanol, iron, and sulphide, This is also the way ferredoxin biosynthesis occurs; the iron is incorporated after the apoprotein is completely synthesized and released from the polysomes (Trakatellis and Schwartz, 1968). Reconstitution experiments are now routine (Hong and Rabinowitz, 1967; Devanathan et al., 1969).

The ferredoxins isolated from the various nitrogen-fixing organisms (Table 3) differ in many properties including molecular weight, iron and sulphide contents, redox properties, and biological activity. Some of the recently isolated ferredoxins have no activity in the clostridial phosphoroclastic reaction, a reaction which until recently was thought to be characteristic of the bacterial ferredoxins. However, all ferredoxim isolated from the nitrogen-fixing organisms reacted with their homologous nitrogenases and in most experiments showed reactivity with other nitrogenascs. Several of the new ferredoxins were isolated by their activities in the coupled chloroplast-nitrogenase assay developed by Yoch and Arnon (1970) and first used in the isolation of an electron carrier by Benemann et al. (1969). In this assay, illuminated washed chloroplast fragments reduce the electron carrier which then serves as a reductant for nitrogenase. Azotobacter ferredoxin (Yoch et al., 1969) and Rhizobium ferredoxin (Yoch et at., 1970) (from soybean root nodules) were first discovered by this method.

Azotobacter ferredoxin was isolated from A . vinelandii by the normal procedures of DEAE-ceIlulose chromatography (Yoch et al., 1969). It reduced both nitrogenase or NADPH, in the presence of illuminated chloroplasts, but had only low activity in the clostridial phosphoroclastic reaction. It has been crystallized, and contains eight atoms of iron and eight sulphidc residues and has a molecular weight of 14,500 (D. C. Yoch, personal communication), higher than any othor fcrrecloxin previously known, I t undergoes reversible reduction arid re-oxidation ; however, re-oxidation in air seemed to occur in two steps. About 600/, re-oxidation took less than a minute while complete re-oxidation proceeded only slowly and took about an hour. This sluggish re-oxidation of Azotobacter ferredoxin appears to be an adaptation which enables this aerobic organism to conserve, in the presence of oxygen, the strong reducing power needed for nitrogen fixation. Azotobacter ferredoxin is distinct from other iron-sulphur proteins which have been previously purified fiom A . vinelandii (Shethna et al., 1966, 1968). Sbethna (1970a) confirmed the presence of Azotobacter ferredoxin, and Yates (1970b) reported finding a similar protein in Azotobacter chroococcum. Whether or not Azotobacter ferredoxin is a new type of ferredoxin (Yoch et al., 1969; Buchanan and Arnon, 1970) remains to be confirmed by further work; however, it represents the first example of such an electron carrier from an aerobic nitrogen-fixing organism.

82 JOTIN lt. IIRNEMANN A N l ) lt. (!, VALRNTrNIS

Rhizobiiini ferredoxin, isolutcd from soybean root nodulc I);interoids, was more difficult to purify sincc i t iws srnsitivc to osygcn (Yoch et al., 1070). This redox protein is an csuniplt. of a ferrcdoxin which was not active in NADP reduction by illuniinated chloroplasts, although it worked in the coupled chloroplAst-nitrogeriuso t~ssay (with both Rhizo- bium and Azotobacter nitrogenascs ; Yoch el nl., 1970). Apparently the spinach chloroplast NRDP-ferredoxin rcductaso cwinot react with this electron carrier, accounting for the lack of NAUPH, reduction (Burton et al., 1970). It was also inactive in the phosphoroclastic reaction. It contains non-hacm iron and sulphido (Burton et al., 1970), but no data on its physicnl properties itre yet uvuilablc.

I+’crrcdoxinn h u v ~ tilso bctrn iHolatcd from Nevcml typrr of photo- ~ y n t h c ~ t i c ! nitrogcn 1ixwH. ‘l’tiosc! from bluc!-grc~c~ii tilgacb iirc wry rrimilar to the pltmt-typo fcwcxloxiil, cwituining two atoms of iron, one of sulphide and LL molecular weight of about 10,000 ( Yamanaka et al., 1!)60; Rmillic, l!lti!j). This similarity probably rcflects a similarity bctwocn tho ~~I~oto~ynt l ic t ic syHtcms of blue-grccn algue and plants. The fcrrcdoxins of tho grccn j)hoto~yiithotic hctcr ia ((jhkorobium q)ccies) rcscmblo thow isolated frorn clostridiu in physical properties and amino-acid scqucnccs (Buchnnan et nl . , I!G!l). Tho ferrcdoxin of the purple photosynthctic biLctcriiim Chromatium is also similar, containing about eight atoms of iron and eight sulphidc groups with a molecular weight of 9,000 (Bachofen and Arnon, 1966). Two distinct membrane- bound ferredoxins, one similar to plant-type ferrcdoxin, the other (absont during aerobic growth) siniilar to clostridiul fcrredoxin, have been found in Iihodospirillurn rubrum (K. T . Shanmugam, personal communication).

Perredoxin hus also been recently isolated from thc facultative anaerobe, Baci$lus polylnyxa, by mcthods ~ i m i h r f a tlic~se U H C ~ for Azotobacter and Rhizbbia fcrredoxins (Shcthna, 1970h ; 1). C. Y(J& and R. C. Valentine, unpublishcd data). This ferrcdoxin was dirrtinct from the clostridial type but showed similar biological activity. The clcctron carrier@) from Klebsiellu pneumoniac, which links to nitrogcnase in this organism, has two unique propcrtieH which hevc mado isolation difficult. Unlike all other fcrredoxins and flavodoxinH it docs not l~irid tightly to DEAE-cellulose and it iH not rcducc:d in orutlo extracts ~J.Y illuminc~tcd chloroplasts. h r t h c r work irr nccchd to c:luc:itlritc: t h Iintltrc: o f tha electron carriors in t h i s nitrogon fixvr.

Clostridial-type f~:rrotloxins c’ i~11 trutlHfw u p to t w o r:lcotrcJnH wittl IL

reducing potential o f about -400 rnV ( l h n f i ~1 nl . , I!)fiH; M;iylrt:w et al., 1969b; Nisenstcin und Wang, l!jfi!j) whilc ~ ~ l r ~ t - t y l ~ ~ fi:rrcdoxins (containing only two iron-sulphur groups) transfcr only cJnc ( , I , , cvtron (Whatley et al., 1963). ‘The naturo of the prosthetic iron-sulphur F O U ~ H


of ferredoxins, which give these proteins their characteristic properties, has been the subject of many investigations (see review by Malkin and Rabinowitz, 1967). The evidence points to each iron atom being bonded to two cysteine sulphur groups and two labile sulphides in a tetrahedral arrangement. Physical measurements indicate thc presence of two distinct types of irons in clostridial ferredoxins, which led Bloomstrom et aZ. (1964) to propose a structure in which the iron atoms were linked in a single linear array by alternating bridges of cysteine sulphur and labile sulphide (the central and terminal irons having different environments). However, alterntLtive arrangements are possible (such as two clrixtcm of iron groups) and X-rey diffraction analysis will have to be undertaken to provide the final answers. The mechanism of electron transfer by ferredoxins (and other iron-sulphur proteins) also remains to be elucidated. A number of artificial non-haem iron proteins with ferredoxin-like absorption spectra have been synthesized by treating proteins with S2- and Fe2+ (Suzuki and Kimura, 1967 ; Lovenberg and McCarthy, 1968), but they lacked biological activity. The protein itself is not even required in models, since mercaptoethanol, iron and sulphide, a t pH 9, form n soluble complex whose spectrum was similar to those of the non-haem iron proteins (Yang and Huennekens, 1970). Whether biologically active models can be made remains to be seen.

With the discovery of ferredoxins in aerobic, facultative, and symbiotic organisms, the already extensive work on ferredoxins will undoubtedly expand in the near future. The evolutionary history of some of the new ferredoxins in ,relation to that established (see Matsubara et aE., 1968; Hall et al., 1971 ; Buchanan and Arnon, 1970) for the anaerobic and photosyntbetic ferredoxins will bc of special interest. The presence of ferredoxins in all types of nitrogen-fixing organisms makes it clear that they are indeed'an important part of the pathway of nitrogen fixation.

B , Flavodoxins Several years after the discovery of ferredoxins, another electron

carrier able to function in the phosphoroclastic reaction was isolated from Cl. pasteurianum (Knight et al., 1960). This electron carrier was a flavoprotein which was found only in cells grown in media containing a low concentration of iron. It was named flavodoxin sincc it could replace ferredoxin in vitro in a large number of reactions. I t apparently alrro replaces ferredoxin in vivo, during conditiorui of iron Htarvation, &H

suggested by the almost complete abRencc of ferredoxin from c e h grown under these conditions. Clostridial flavodoxin (molecular weight, 14,000) contains one flavin mononuclcotidc (FMN) group but no iron or sulphide (Knight and Hardy, 1966, 1967). The FMN group i R bound to

84 JOIIN R. I3ENEMANN AND R. C. VALENTINE

tho single cyst,ciiic residue in tlic molccule and coniplcxed with one or two of the four tryptophan residues (McCormick, 1070). Flsvodoxin was similar in physicul and catalytic properties to a flavoprotein named phytoflavin, which had been previously isolated from bluc-green algae (Smillie, 1966) grown on media containing a low concentration of iron (Trebst and Bothe, 1966). Flavodoxins have recently been isolated from other anaerobic bacteria ( Peptostreptococcus elsdenii ; Mayhew and Maasey, 1969 ; and two Desulfovibrio species ; Dubourdieu and LeGall, 1970) and a closely related protein, iiamed azotoflavin, from A zotobacter vinelandii (Benemann et al., 1969). Flavodoxins can be defined (by criteria very similar to thosc usccl for f(~rcdoxins) as flavoprotcins which function as low rcdox potcntid (bclow thnt of riieotinatnide nuclcotidcs) chloc!t,rori ctwricw i n a t least ono of’ tho biochcmical reactions of fcrredoxins. ‘I’huH w o t o h v i r i uric1 tho known fli~vodoxins form a ncw group of electron aarriors with similar biological activity to the ferredoxinH. We propose the general name “flavodoxin” for this class of electron carrier.

On a molar basis, clostridial fluvodoxin is only about 30% as effective as ferredoxin in the pyruvate phosphoroclastic reaction, NADP roduction by hydrogen, or nitrogen fixation reactions (sce Hardy and Knight, 1968). This, coupled with a higher molecular weight, malres flavodoxins much less efficient electron carriers than the ferrcdoxins. The reasons for this could be due to a difference in the specificity of the carrier in these reactions, but more likely to its redox properties. The results of Mayhew ct al. (196%) showed that flavodoxin from Peptostreptococcus elsdenii accepted two electrons a t different oxidation levels. Addition of one reducing equivalent of sodium dithionite (or NADPH in the presence of NADP-ferredoxin rcductase) generates a new species of flavoprotein, the flavin semiquinone. This reduction is almoHt instantaneous and can

easily observed sincc? t h r coloiir of thc fhvodoxin c:hr~ngcs from yellow to blue. This btcp i n tlio rctluc.tioii of fli~votloxin ~ ; L H a j)ot(*rititLI of -0.1 16 V at p H 7. Further rcduotion with two rcduc:iiig (:quivulcrits of dithionite gave the colourlcw fully rcduc~d scmiyuiiiortc. ‘I’his seuortd step in the reduction of flavodoxiti is probably t h v more important jn reactions in which this protein substitutes for bacterial ferredoxin because the oxidation-reduction potential for this second Ntep was -0.373 V, at pH 7 , somewhat above that of fcrrctloxin (-0.42 V) but below that of the NADP/NADPH2 couple (-0.32 V). This could account for the lower reactivity of flavodoxin when substituted for ferredoxin as electron carrier. But differences in specificity can, of course, not be excluded.

Azotobacter vinelandii contains a flavoprotein of unusual redox properties (Hinkson and Bulcn, 1967; Shethna et al., 1964) which


Benemann et al. (1969) found to function as reductant of nitrogenase in the coupled chloroplast-nitrogenase assay, and named “azotoflavin”. The most unusual property of azotoflavin was its oxidation- reduction behaviour. It was reduced only slowly by excess sodium dithionite to give a strikingly blue-coloured protein, the semiquinone flavoprotein. A very large excess (100-fold) of dithionite for several hours was required for reduction to the colourless fully reduced form. Re-oxidation in air was also very slow; the semiquinone form was extremely resistant, taking over 12 hours for complete oxidation to the yellow oxidized form. These properties are distinctly different from the behaviour of the flavodoxins which are easily reduced and oxidized. The flavin prosthetic group, although easily released from azotoflavin (Hinkson, 1968), appears to be complexed in this protein in such a way that the reduced species are not accessible to oxygen. The resistance of reduced azotoflavin to oxidation by air may be a distinctive adaptive advantage for transporting high-energy electrons to nitrogenase in the highly aerobic cellular environment of Azotobacter. At first (Benemann et aE., 1969) it was thought that azotoflavin was unrelated to flavodoxin because of: (1) its unusual redox properties (stable semiquinone); (2) virtual inability to replace ferredoxin in the clostridal phosphoroclastic reaction or in the reduction of NADP by chloroplasts; (3) the high molecular weight (31,000) of azotoflavin; and (4) the presence of azotoflavin, together with azotobacter ferredoxin in cells grown in media containing either high or low concentrations of iron. However, these differences are not crucial to the electron-carrier nature of the protein, and azotoflavin, although quite distinct from flavodoxin in both chemical and biological properties, can be grouped along with phytoflavin in the flavodoxin class of electron carriers.

Flavodoxins have now been found in most groups of nitrogen-fixing organisms (anaerobes, aerobes, and photosynthetic bacteria; Table 3, 9. 7$), and we may expect in the future to find more examples of these electron carriers. Thus, a flavoprotcin with olcctron carrier propctrtiee appears to bo present in extrncts from Roybcan root nodulcs (1). C. Yoch and $3,. C. Valentine, unpublished data) and a flavoprotein from E’. cola’ (Knappe et al., 1969) has flavodoxin-like properties (D. C. Yoch, personal communication).

C. Coupling Fuctors of Azotobacter Although both Azotoflavin and Azotobacter ferredoxin could function

independently and separately as reductants of nitrogenase when reduced artificially with illuminated chloroplasts, this was not the cam when endogenous substrateR, such I ~ R NADPH,, were the electron donor. Both Azotobacter ferredoxin and azotoflavin were needed in the

86 JOIIN It. DENEMANN A N U I t . C’. VALICNTINE

trttnsfcr of electrons from NADPH, to nitrogenase using a crude extract of A . vinelandii. This was most clearly shown when the electron carriers were removed from the crude extract by absorption on DEAE-cellulose ; both were required to restore NAI)PH,-driven nitrogenase activity. This indicated that the two Azotohacter electron carriers acted together in an electron cliairi extending from NADPH, to nitrogenase (Benemann et al., 1 9 7 1 ~ ) .

Two other factors (called “coupling factors”) present in the crude extracts were also needed in the complex Azotobacter pathway of nitrogen fixation, and will be discussed briefly. When NADPH, is the electron donor, the first coupling factor is NADP-fcrredoxin rcductase which cataly~cs reverd)lc cloctron trimsfcr betwcrri fcrredoxin und NADP (Shin t ir id Arnon, 1965). J t hi188 only 1,ern i~oli~tod nnd orystallizc?d from spintt(~h ehloropli~st~ (Sliin E t u l . , 1963) whcrc! it prticipates in tho photosynthrtic NADI’ rcdiictiori by illuminutc~cl chloroplasts (Keister et al., INN). ‘I’hc spinnch enzyme is a, flavoprotcin, and was shown to form a coniplcx with spinach fcrrcdoxin and also NAD1’ (Nelson and Ncuman, 1969; Shin and San Pietro, 1968; Foust et al., 1969). Spinach NAD1’-ferrcdoxin reductase is able to replace an enzyme present in A , vinrlandii (removed by DEAE-cellulose treatment) which wits required for the transfer of electrons from NADPH, to nitrogenuse (Renemann el al., 1 9 7 1 ~ ) . Although itj remains to be isolated and characterized, it is logicnl to asswnc that the Axotohacter and spinach eiizymcs :trr u t lcast functionidly dmilar. Azotoflavin, Azoto- bacter ferredoxi n and NAT)P-ferredoxin rcdactilse were not sufficient for the transfer of electrons from NADI’H, to nitrogenasc. A soluble heat-labile factor present in :t centrifuged (nitrogcnasc-free) ex tract was also required. Although no furthcr information about this coupling factor is presently known, its function is most liltely thc transfcr of electrons bctwcrii the two Azolohuclpr rl(v:tron cnrricw, Hinw tlwy do not interact Hp017tLLllC?OLlhly. ‘~’~illH, i L c:lonOr look tLt th(! A%rhhr/dPr HyHtPWl hes shown that the p t h w : i y is r n itah inoro compic.x tlisri :Lnticipt,id ; :I proposed scheme, indirxting tho funotiond g r o u p involved, is givcbn in Pig. 4. Even the order of the c h h ix not yet definite; howovc:r, the fiwt that spinach NADP-ferredoxin reductase is unablo to transfvr elcotrons to azotoflttvin would put Azotobirt*tcr fcrredoxin nrxt to NADPH with ilzotofltLvin being the actrial reductant of nitrogvnafio. Although this pathway shows d l componcnts working indc1pendcntly of rach othrr, they might well be complexed and arranged in sornr functional s t r u c t ~ ~ r e in the cell. Indeed, preliminary evidmce indicatcs that Azotobacter ferrcdoxin and azotofavin are present in the A zotobact~r cell complexed to each other and possibly to other factors of the pathway (J. R. Bcnemann, unpublished dnta). IJI an aerobic environment the extremely

1 Glucose 6-phosphate Isocitrate B5ialSte a-Kotoglutarete

B

0 NADP azoto&avin

FIG. 4. The pathway of nitrogen fisation Azdobncter vi.n&tzdii.

88 JOHN R. IIENEMANN ANI) It. C. VALENTINE

reductive process of nitrogen fixation may best be accomplished by an enzyme-carrier complex.

IV. Nitrogcnasc

For many years, the term nitrogenase had been uscd to describe thc hypothetical enzyme involved in the reduction of molecular nitrogen to ammonia. This central catalyst of the nitrogen-fixation pathway tiirncd out to be a complex of two metal-containing proteins which arc surprisingly similar. in all the organisms from which i t has been isolated. Howcvcr, the actual mecliaiiisms of the reactions catalysed by nitrogcnase have not yet been elucidated.

A. Isolation and Properties The first two nitrogenascs investigated in detail were those of Clost-

ridium pasteurianurn and Azotobacter vinelandii. From carly work with coll-frec cxtracts, it appeared that tlinso two nitrogcnasoa, although thoy outslysed the stirne rcantions, werc quitc differcnt. Hcnt, cold, and oxygon ( I ~ L a i d Ijurris, 1965 ; Carnahan et al., 1960a) quickly inactivated nitrogcnusr iidivity in cxtracts of GI. pasteurianum but not A . vinelandii (Rulon et nl., 1964). ‘Fhr Azotobacter enzyme was particulate (it could be scdimentod at 144,000 g for 0 hours) while the clostridial enzymc was soluble. Ilowcvcr, during purification Azotobacter nitrogenase lost its partioalatc character, and the solubilized Azotobacter nitrogenme was just as air-, heat-, and cold-sensitive as thc clostridial enzyme (Bulen and LeComte, 1966). The nature of the Azotobacter nitrogenasc complex which appears to give it its unique properties is not understood.

Purification of iiitrogeiiaso progressed slowly until the introduction of sodium dithionitt as an artificial reductant (Bulen et al., 1965) simplified the assay system for riitrogenttse by eliminating the iiced for an electron carric.r and ptiosphoro~1:i~ti~ system. Not surprisingly, similar purification schciiics wrrc dcvc~lopcd almost simultaneously by Mortenson (1966) for iiitrogenase from C‘Z. pasteicrianum and by Bulen and T Ae C omtc (1066) for the crizymc from A . vinelandii. The moat significant finding was that nitrogcnase wits m d e up of two different protcinH whioh interacted to form the active enzymo. Tho basic technique in thc pilrificfb tion involvccl precipitation of nuclcic acids in the crude oxtracts with protaminc sirlphatc (a basic protcin) followed by precipitation of the nitrogenase by further idditions of protamine sulphate. The nitrogenase was resuspended with cellulose phosphatc (which removed the protamirle sulphate) arid chromatographed on DEAE-ccllulosc~. This uhromato- graphy resulted in q ) w a t i o n of t h e nitrogerltLsc itctivity into two


protein fractions, easily visible as two brown bands on the DEAE- cellulose column (Fig. 5) . Each fraction was further purified by ammonium-sulphate fractioriation and Sephadex chromatography. Anaer- obic procedures had to be used throughout due to the oxygen sensitivity of the componcntd These two proteins were found to contain non-haem iron and labile sulphide (in approximately equivalent amounts) ; they therefore both belong in the family of ion-sulphur proteins. The larger nitrogenase protein also contained molybdenum (Fig. 6), a finding

s e

- .-

BUFFER 0.15 M -NaCI 0.25M-NoCI 0.35 M-NoCI c 0

u-

-1 150

FRACTION NUMBER

FIU. 6. Soparation of tho two nitrogonano protcirin frorrl Azotobactar vineluntlii on DEAE collulovo (Bnlon et al., 1966). A intlicatos ooritcrnt of protoin; (1, Of iron; 0 of molybdenum.

which explains the long known molybdenum requirement of nitrogen- fixing organisms. Purification of nitrogenase has been extended to other organisms (Detroy et al., 1968; Klucas et aE., 1908; Kelly, 1969b) and by modified purification procedures. Thus, Vandecasteele and Burris (1970) have eliminated the protamine sulphate step completely and chromatographed a crude extract of Cl. pasteurianum directly on DEAE- cellulose, followed by further purification of each fraction. With Azoto- bacter (Kelly et al., 1967), heat treatment (60'; 10 min.) can be used to remove a large amount of contaminating protein. Nitrogen-fixing extracts of soybean root nodules were obtained, and nitrogenase purified,

90 JOHN R. BENEMANN ANT, R. C . VALENTINE

by adding polyvinyl pyrrolidone to remove interfering phenolic compounds (Koch et al., 1067).

Tho two protein fractions obtained by these purification procedures arc eiizymically inert in any of the reactions catalysed by nitrogenase ; they have to be recombined for activity in the many different assays for this enzyme. The names “Fe-protein” (for the smaller protein) and “MoFe-protein” (for the larger one) seem to be most popular now, although other names are still being used. The proteins have not been purificd to com plctc homogoncity in most etmw, but their gcwtral IiropcAcN h v ( ! 1)rcti ( ~ ~ t u b l i ~ h c d . Ilooently Nal<o~ und Mortcnson ( 1!)7 I ) foiintl that tlw I+-protc.iri of‘ tlic nitrogcwrLHc! of 0 1 , pmlw&murrb c:oiitrLins r~boitt four 1tiI)ilc iroti-Hulphid(~ group, i h t i t l i H r n t h u p of two, r~l)l)r~wntly ithi nl,i ( ’ i d , H I I 1) 11 I I i t H . ‘ 1’h: I?(,- 1 irotci ri fro i n A , w k & d i i han riot yvt twc.ti H t i d i ( : d t~ c:loHcbly, but the datiL civnilahlc (13ulcn et al., L!)fifi) ugrcw with cwlior report8 of’ the olostridiul Vc-protein (Moustafa and Mortcnson, 19fN ; Vandccustcele and Burris, 1970) suggesting that they are similar. Tho spectrum is of the Fe-S-protein typc, and although tho protein can bc reduced by dithionite (however rcportedly not by fcrrcdoxin; Moustafa and Mortenson, l969), i t is not rcversibly re- oxidized by oxygen. The Fe-protcxin is cold-labile and extremely oxygen- sensitive, but can be stored in liquid nitrogen. The MoFe-protein from A . vinelandii has been crystallized as white crystals from a low ionic- strength Holution, by Burns et al. (1070). It uppcars to be a protein of about 270,000 molecular weight containing two molecules of molybdenum, 34-38 molecules of iron, und 26-28 molecules of S2-. The protein can be dissociated into more than one type of subunit. Similar values for metal content (and minim um molecular weight) have hccn published for impurc proparatioris of t h c b clostridial protcin (Mortcnwn et d., 1967 ; Vandrcwtcclt: and Burris, 1970) .

The Fc- and MoPo-proteiris are prtwnt only in W I I H ~ J Y J W I I uriclt:r nitrogen-fixing contlition~; growth in t l i ~ ;irc:xc+rw of i m r r i o r i i i i pr(:vcrit~ their synthesis. In all CUHCH, hoth ttiv I{’c.-protc:in tirid thc: Mob’c!-protcin were required for reduction of molcoular nitmgan, fiubfitrete analoguw (acotylenc, cyanide, azide), A‘I’P-dcprndent evolution of ndecular hydrogen, or reductant-dcpendent Al’Yasc activity (Kennedy et al., 1968). The molar ratios of the two protein8 nccdcd in thc: vuriom reac- tiona are not yet definite, hut a ratio of 2 : I Fc: MoFe proteinH (ctiloulated on a molecular weight of 150,000 for ttic MoE’c-protein) has hem found to give highest activity (Vandecasteelc and Burris, 1970; Kdly, 1l)Ol)a).

Several workers (Mortenson, 1966; Taylor, 1969) reported a third componcnt (found also in cells grown on ammonia) which W ~ H part of the nitrogcnase C1. pasteurianum; howcver, Jeng et al. (1969) refutctd these claims. Kajiyama et al. (1960) claimed that the nitrogenltse of A .

THE PATHWAYS OF NITROUEN FIXATION 91 vinelandii was made up of three proteins (one a ZnFe-protein), but their cxperiments could not bc reproduced independently (M. Kelly, private communication).

Isolation of the two proteins which make up nitrogenase from Azoto- bucter and Cloetridium and the finding of similar proteins in other bacteria raised the possibility of cross-reacting the Fe- and MoFe- proteins from the different organisms. The first results with Azotobacter and Cloetridium proteins proved negative (see Burris, 1969). The Fe- protein of one showed no activity in the prescnce of t h e MoFc-protein of the other. Howcvcr, tho protoilis from Kbb8ieZZn pneumniae and BaciZZw polymyxu (lid (:rosR-roiu:t with c!ooh other, and cvcn with Azotobucter and Cloetridium protrin8 to a limited oxtcnt ( Detroy et aZ., 1968; Dahlen et a!., 1909). Thcse rexults wcre confirmed by Kclly (1969b) who also found that the cross rcactions were independent of the assay used for nitrogenast? activity. Cross reactions between the iiitro- genaae proteins of blue-green algae and photosynthetic bacteria have also been carried out (Biggins et al., 1971 ; Smith et al., 1971). These experiments strikingly demonstrated the homology both in structure and function which exists between the nitrogenases of the various organisms. They indicate a good deal of functional and chemical similarity between the nitrogenase proteins of even distantly related organisms, a situation perhaps dictated by stringent structural and catalytic requirements for nitrogen reduction. This idea is supported by the fact that the catalytic activity of all nitrogenases is very similar.

B. Mechanism of Action The question of how nitrogenaso reduces molecular nitrogen to

ammonia (the mechanism of action of nitrogenam) is ccntral to thc study of nitrogen fixation. There have bcon two main approaches in the qwst to discover the mechanisms of nitrogen fixation by nitrogcnasm, namely the study of nitrogenase itself and the reactions it catttlyws, and the study of model systems involving inorganic complexes of molecular nitrogen.

Nitrogenase catalyses the transfer of electrons from a suitable rcductant (which is oxidized) to a variety of electron acceptors (N2, H+, CN-) in a reaction which is absolutely dependent on the hydrolysis of ATP to ADP and inorganic phosphate. It also catalyses other reactions, including ATP hydrolysis independent of reductant oxidation and molecular nitrogen-dependent H,-D, exchange. It is quite obvious that the function of nitrogenase is the reduction of molecular nitrogen ; all other reactions are secondary and are the result of the structural and catalytic properties of the enzyme. However, the study of these secondary reactions can yield valuable clues, and any propoeed mechanisms must


ticcount for them. Beforo u detuilctl mcoliuiiim of nitrogcn fixation by riitrogcnase can be worked out , the functions of ATP, molybdenum, and iron will hnve to be understood; the order of the reaction between substrate, reductant, ATP, and the two nitrogenase proteins known; and the activc sites for the various reactions determined. Much work remains to be donc, but recent experiments have added to our knowledge in this area.

The role of ATP has been one of the most puzzling questions. From thcrmodynamic considerations, ATP should not be required since the overall reaction: 3H, + N, + NH, is slightly exothermic a t physiological conditions and thus, assuming perfect catalysis by nitrogenase, only a reductant of redox potential about that of molecular hydrogen (such as ferredoxin) would be required. However, this argument does not take into account the energy of activation of the reaction, which in the absence of catalysis, is very high due to the great strength of the triple bond in molecular nitrogen. Although this energy of activation could theoretically be overcomc completely by a perfect catalyst, practically it is not too surprising that nitrogenase uses an energy source such as ATP to help drive the reaction. The following facts have been recently established about the role of ATP: (1) the rate of ATP hydrolysis by nitrogenase is independent of the substrate reduced but dependent on the presence of reductant; in the absence of a reductant, nitrogenase still hydrolyses some ATP, especially at low pH values (about 5) where the requirement for a reductant disappears (Bui and Mortenson, 1969) ; (2) the stoichiometry of ATP utilization by nitrogenase is complex; the ratio of the amount of ATP hydrolysed for each electron pair transferred to an electron acceptor (ATP/2e- ratio) is dependent on pH value (Winter and Burris, 1968) and temperature (Hadfield and Bulen, 1969), and values ranging from 4 to 5 have been obtained. If allowances are made for the ATP hydrolysed, which does not result in product formation (that is, no electron transfer), the ATP/Ze- ratio is decreased to only two (Jeng et al., 1970; Hadfield and Bulen, 1969; Kelly, 1969a); (3) both proteins can bind ATP (Biggins and Kelly, 1970; Bui and Mortenson, 1968). Four different roles for ATP in the nitrogenase reaction have been proposed: (i) as a proton Bource for nitrogenase (Brintzinger, 1966) ; (ii) as an “electron activator” (Mortenson, 1964 ; Hardy and Knight, 1968) ; (iii) as a source of hydrated electrons (Bui and Mortenson, 1969); arid (iv) as an inducer of conformational change (Bulen et al., 1965). Which of these four proposals comcs cloRest to representing the actual mechanism of action of ATP is not yet clear.

Nitrogenase appears to function in a stepwise fashion adding two electron4 a t each step to the substrate. The evidence for this is that all substrates are reduced to products which contain an added even

THE PATHWAYS OB' NITROGEN FIXATION 93

number of electrons. Thus acetylene is reduced to ethylene in a two- electron step, nitrogen to ammonia in three two-electron steps, and isooyanide to a complex mixture of products in as many aa six two- electron steps (see Hardy and Knight, 1968). Intermediates of nitrogen reduction (diimide and hydrazine) have never been observed (Burris d al., I966), presumably because they are strongly enzyme-bound, However, substrate analogues are often reduced to varying degrees, the intermediates being able to dissociate from the enzyme. It is clear that there must bc several distinct sites on the nitrogenase enzyme which involve ATP hydrolysis, reductant oxidation, and substrate reduction, since thesc processes are affected by different inhibitors. Thus carbon monoxide stops all substrate reduction, but ATP hydrolysis continues undiminished and molecular hydrogen is evolved (Hardy et al., 1966; Burns and Bulen, 1965).

The nature of the substrate-binding site, where molecular nitrogen is reduced to ammonia, is at the centre of the study of the nitrogenam reaction, with the key questions being the nature of the metal(s) and ligands at this site and the mechanism of action. A large amount of work has been, and continues to be, done on the inorganic chemistry of complexes of molecular nitrogen (mostly of transition metals) with the hope that these would result in model systems of nitrogenwe which would answer these questions (see reviews by Calderazzo, 1969; Kuchynka, 1969; Hardy and Knight, 1968; Allen and Bottomley, 1968; Van Tamelen, 1971). These inorganic models, which in some cases exhibited nitrogen-fixing capabilities, were not very fruitful in the solution of the nitrogenase problem. On the basis of theoretical considerations, many workers considered iron to be the key metal in the nitrogen binding and reduction site, with molybdenum playing only an incidental role (Chatt, 1969; Hardy and Knight, 1968). Rome recent Rtudies have suggested molybdenum as the primary metal involved in binding and reduction of molecular nitrogen. Chatt et al. (1969) have mado comploxea of molybdenum and molecular nitrogen. Shrauzer and Rchle~inger (1 970) developed a simple model system of nitrogemfie involving molybdenum-thiol complexes which catalysed (at pH 9 to 10, room temperature, and several atmospheres pressure) reduction of molecular nitrogen with dithionite or sodium borohydride as reductant. Activities were low (Schrauzer et al., 1971); however, the model had the uniquo property of reducing all of the substrate analogues of nitrogenase tested (acetylene, azide, nitrous oxide) with similar stereospecificities. Also, ATP stimulated the acetylene-reduction activity of the model system (Schrauzer and Doemeny, 1971). Newton et al. (1971) reported, without giving experimental details, that simple organic-iron complexes can reduce some molecular nitrogen at physiological conditions in the

94 JOHN R. BENEMANN AND R. U. VALENTINE

presence of potassium borohydride and the absence of molybdenum. Shilov et al. (1971) reduced I5N2 to lSN-hydrazine in strongly alkaline solutions with either molybdenum or vanadium as catalyst. Hill and Richards (1971) have confumed all three model systems and were able to improve on the yield of thc Schrauzcr model over a thousand-fold by use of 2-aminoethancthiol as ligarid of molybdenum. With thcsc latost results, i t would appear that nitrogenasa modol chemistry is going to contribute significantly to tho study of tho mechanisms of thirr cnayme.

A moro direct approach to tho qucstion of tho motal in the active site of nitrogcnasc W ~ R taltcn by McKcnne et al. (1070) who prepared a vunadirim-containing nitrogenaso (obtained by growing A. vinelandii on modis lacking molybdcnurn but containing vanadium). The “vanadium nitrogenasc” differed in substrate-binding affinity and reduction kinetics from the “molybdenum-nitrogenase”, findings which were confirmed by Burns et al. (1971). Further investigations revealed that the enzymic activity of “vanadium-nitrogenase” could be attributed to the presence of contaminating molybdenum ; the vanadium present in the eneyme appeared to stabilize the Azotobucter nitrogenase complex allowing the small amounts of molybdenum to function (Benemann et al., 1971b). This accounted for the ability of vanadium to replace the molybdenum requirement of some nitrogen-fixing strain8 of Azotobacter. These cxpcriments with “vnnndiiim-nitrogcnase” gave the first direct ovidcnno of a rolo for molyhdcnum in the active sito of nitrogenasc (MoKcnna et d., 1070; 1311rtis et id., 1!)71). That iron is a180 involved in tho Itctivo site of nitrogenasc? is suggostod from the work of Ward et al. (1971) who found that tho rcductivc activity of t h o MoFe-protein of C1. pasteurianum nitrogonase W ~ R lo& upon rcmovnl of Nix iron atomH while the ATPaso activity romtiincd intact. No dctailcd mcchaniHm of nitrogenase action which can account for all of tho known data and observations has been proposed. It is obvious that, dospito tho largc amount of work done in the past decade, we do not yet understand how this enzyme reduces molecular nitrogen.

V. Conclusions and Future Outlook

Table 4 gives a summary of the biochemical pathways of nitrogen fixation in micro-organisms. The pathway is divided into three parts: (i) the electron donors (derived from the metabolic pathways of the cells) ; (ii) the electron carriers (fcrrcdoxin orland flavodoxins) ; and (iii) nitrogcnasc (made u p of tlic Fc- and MoFe-protcin8). Thc morrt Ntriking aspect is thc unity of the pathways which underlio thc moro npparcnt physiological diwmity of t h c vwioiin i i i ~ t , ~ i K f ! i i - f i x i r l ~ mic:ro-rJrKlulinrrlH.

TABLE 4. Summary of the Pathways of Nitrogen Fixation in Micro-organisms

References to the nitrogenase proteins a s Organisin Electron donors Carriers

Cloatrkiium pteurknum Pyruvate, H,, NADH2, NADPH, Ferredoxin or flavodoxin Kkbsiellu pneumonke Pyruvata, formate, NADPH,, H, ? Detroy et d. (1968); Kelly (1969b) 8 Bacdlus polymyxa Pyruvate, formate, NADPH, Ferredoxin Detroy et aZ. (1968); Kelly (1969b) 5 Azdobacter vinelandii NADPH, Ferredoxin and azotofbvin Bulen and LeComte (1966) ; Burns et al. 3

0

Rhizobiurn japonicum NADH,( 9 ) Ferredoxin Klucas et d. (1968)

‘Nortenson et al. (1967)

(1970) €2

Anabaena. cylindrku Pyruvate, NADPH, Ferredoxin (phytofisvin 3) Smith et al. (1971) l3unhpirillum rubrum Pyrurate, H, ( 0 ) Ferredoxin Smit.hetal. (1951)

06 JOHN R. BENEMANN AND R. 0. VALENTINE

The basic components of the pathway arc vcry similar in all organisms. I n the case of the aerobic nitrogen-fixing Azotobacter (and probably others) the pathway involves two electron carriers in a complex sequence of reactions (Benemann et al., 1971). The question marks in Table 4 will have t o be answered by further research. It should be noted that thc. pathways of nitrogen fixation were clucidated by studies with cell-free cxtracts ; physiological studics with whole cells are needcd to confirm the results, especially with regard to the electron donors used.

It is hazardous to predict the future of scientific research, but recent advances point the way to developments in our understanding of nitrogen fixation which can be expected in the coming years. The mechanism of nitrogen fixation will be one of the most exciting areas. Now that pure nitrogenase proteins have been obtained (Burns et aZ., 1970; Nakos and Mortenson, 1971), the physical and chemical characterization of this interesting enzyme can proceed. The sequence of interaction between ATP, reductant, substrate, and nitrogenase proteins still needs to be elucidated, as docs the location and structure of the various sites (ATP hydrolysis, reductant oxidation, substrate binding) on nitrogenase. This, together with careful kinetic studies and development of model systems, should result in the solution of the question of the mechanism of biological nitrogen fixation. It is likely that a detailed mechanism will be elucidated before all of these data arc accumulated.

The development of the acetylene assay has led to a resurgence of physiological and ecological research in nitrogen fixation. Studies on the distribution and activities of nitrogen-fixing micro-organisms are becoming inorc numerous and quantitative (for examples ace Stewart et al., 1967, Brezonik and Harper, 1969; Howard et aZ., 1970; Silver and Mague, 1970; Horne and Fogg, 1970; Bunt et aE., 1970) and should soon give us a more detailed picture of the true extent and importance of this microbial reaction in nature. Tho biochemical relationships of ~ymbiotic nitrogen fixation present a fertile area of research for biochemists. A recent report by Holsten et al. (1970) that soybean root-cell tissue cultures are able to form nitrogen-fixing symbiotic associations with Rhizobium could provide the wedge needed for entry into this problem.

The use of modern genetic techniques in nitrogen fixation is just beginning. The study of the biochcmical genetics of nitrogen fixation could yield important basic knowledge regarding the pathway, mechanism and regulation of this process. Biochemical mutants blocked in the nitrogen-fixation pathway have been obtained from A . vinelandii (Fischer and Brill, 1969; Sorger and Trofimenkoff, 1970 ; Bcncmann eta,?., 1971a), CZ. pasteurianum (Simon and Ijrill, 1971), arid Klebsiella pneumoniae (Streicher et a,?., 1971). Strcichcr et al. (1971) wcrc able to devclop a genetic system in K . pneumoniae, wing the transducing phage P;,

I

l


and achieved the transfer of nitrogenase genes from one strain to another. Mapping of the nitrogenase genes is now possible. In symbiotic nitrogen fixation, both plant and host bacterium carry genetic inform& tion relevant to this process, making the genetics of the system complex (see review by Nutman, 1969). The acetylene =say has been used (Schwinghamer et aZ., 1970) to study nitrogen fixation by Rhizobium mutants.

The potential contribution of research in nitrogen fixation to the welfare of mankind is immense. The major nutritional problem associated with the exponentially growing world population is a deficiency in protein supplies, especially in technologically underdeveloped countries where the use of synthetic fertilizers is limited because of their expense. Although production of chemical fertilizers is rapidly expanding, it appears unlikely that major improvcmcnts will be made in their production, since the basic processes havc remained unchanged for over 50 years. Also, chemical fertilizers are threatening to pollute thc onviron- ment by upsetting the biological nitrogen cycle (ace Dclwichc, 1070, for u, review). Considering that one acre of nitrogen-fixing soybeans produces over thirty times as much protein as one acre used for cattle raising (Dawson, 1970), it is obvious that a solution to the problem of world protein supply could come from practical applications of research on biological nitrogen fixation. The past record in this area is already impressive. Seeds of leguminous plants are now routinely inoculated with specific strains of Rhizobium which are available commercially. Molybdenum is added to deficient soils to facilitate nitrogen fixation. Future applications could be the development, through genetic techniques, of hardier, more active strains of nitrogen-fixing organisms (symbiotic and free living) which could be used as cheap non-polluting sources of nitrogenous fertilizers. Understanding of tho biochemical relationships in symbiotic nitrogen fixation might allow the cxtension of this process to non-leguminous agricultural crops. The recent report of nitrogen-fixing KZebsieZZa in human and animal intestines (Bergcrsen and Hipsely, 1970) and the availability of a genetic system in theso organisms (Streicher et aZ., 1971) raises the fascinating prospect of solving the protein supply problem directly. Howevcr, scientific and tcchnological advances alone cannot provide thc ultimate solution to tho problems they created. That will have to come from political and moral adjurdmente of our societies.

VI. Acknowledgements We wish to thank M. D. Kamen for assistance and encouragement

and R. Y. Stanier and D. C. Yoch for many helpful discuwionA. Unpublished experiments quoted in this review were carried out in


cooperation with D. C. Yoch of D. I. Arnon’s laboratory at University of California at Berkeley and H. Nagatmi of the Biochemistry Department. We wish to thank tho National Institutes of Health (Grant No. 1-F02 CM37, 78401 to ,J. R . Renemana, and HD-01262 to M. D. Knmen) and the National Science Foundation (GB-7033X to M . 11. Kumen, aiitl GB-7036 to R. C. Valentine) for support.

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Rapid Detection and Assessment of Sparse Microbial Po pu I at ions

R. E. STRANGE

Microbiobgical Research Establishment, Porton, Nr. &?isbury, Wilbhire, England

I. Introduction . 11. Principles of Rapid Miorobial Assessment Methods .

A. Physical Properties of Microbes . B. Chemical Composition of Microbes . C. Miorobial Growth and Motabolism . D. Bwterial Enzymes . E. Immunological Propertios of Microbes .

111. Rapid Broad-Speotrum Mothod8 . . A. Luminol Chomiluminosconco . B. Determination of ATP . C. Staining Methods . D. Physical Methods . E. Methods Depending on Growth and Metabolism . A. Immunofluorescence-Membrane Filtration Techniques . B. Radioactive Antibody Technique . C. Analysis of Bmterial Growth Products. . D. Gas Chromatography-Pyrolysis Methods . E. Analysis of Phosphoroscont Docay .

V. Rapid Determination of Microbial Viability . A. Indirect Methods . B. Direct Methods . A. Immunofluorescent-Monolayer Tcchniquos . B. Immuno-Adherence . C. Radioactive Antibody Mothod Applied to Bacteriophage T7 D. Detection of Virus Activity with Gas Chromatogrsphy .

IV. Rapid Specific Identification Methods .

VI. Rapid Detection and Determination of Viruses

VII. Conclusions and Prospects . VIII. Acknowledgements .

Referenoes .

. 10s

. 107

. 108

. 109 . 111

. 113

. 114

. 118

. 118

. 120

. 121

. 124

. 126

. 126

. 126

. 121

. 130

. 131

. 1 3 1

. 131

. 131

. 132

. 132

. 132

. 133

. 133 , 135 , 136 , 137 . 137

I. Introduction

The significant words in the title of t h k rcview arc “rapid” and “sparse”. If the concentration of microbes in the sample is high enough, and/or sufficient time is available for a detailed examination to he made,

1 OK

106 R. E. STRANGE

their detcction, specific identification and quantitative assessment (i.0. dctermination of total number or mass of microbcs/unit volume of sample and/or viability) present no problems to the microbiologist. On the other hand, immediate or rapid detection or specific identification of a sparse microbial population is much more difficult and requires the use of specially devcloped methods; it is with these mcthods, and thcir physiological basis, that this review is mainly concerned.

If a sparse population of microbes is to be detected and determined within a few minutes i t might be possible initially to increase the microbial concentration in a sample by centrifugation or membrane filtration ; but augmentation of thc populntion by growth (on which most conventional tcchniqucs drpcnd) must be excluded. Sincc, (luring uriimpcdcd growth in a suitable culture medium, bacteria with a moan gcneration time of 30 min. could incrcasc 10’~-fold during 24 hours it is not surprising that, in general, tho sensitivity of rapid methods does not approach that of thc more conventional time-consuming techniques.

Many of the known chemical, physical and physiological characteristics of microbes have been exploited for rapid microbial detection or identification purposes. Methods based on widely differing principles, capable of detecting within a few minutes 103-104 bacteria (and, in some cases, yeasts) have been reported (Oleniacz et al., 1966; Mitz, 1969; Strange et al., 1971). Tn this conncxion, a much sought after capability is the rapid detection of microbes in the atmosphere, and automatic monitoring systems ham been developed for this purpose (Nelson et al., 1962; Olcniacz et al., 1966; Mitz, 1969). As Oleniacz et al. (1966) statc, there are certainly a number of potential applications for a reliable miorobial detection devicc (monitoring of the atmosphere, the air in dairy and other industries, “clean” rooms and opcrating theatrm). There may bc groundrc for belicving t h r h t monitoring of thc eir for t h c presence of bacteria, arid othcr rnicro-organinma of Himiltir or lrtrgcr nizc., is a t least fcasiblo but this is not thc casc: with virustw ‘I’ho r q h l tlctooticm and identification of viruacs is usually whicvod with immunofliiorcsccnac! techniques, but with extremely small viral populutiona tt propagation step that takes several hours is necessary before detcction is possible.

A major problem with rapid, highly sensitive, microbial detection methods (and with sub-micro analysis in general) iR interference by extraneous material in the sample. An assay may bc accurate and rc- producible when i t is applied to a small number of purified microbes suspended in a bland medium, but may give meaningless results in the presence of extraneous particulate material and/or certain soluble substances. The use of a highly specific assay does not nccessarily resolve the problem of interference. For example, if 103-104 bacteria/ml. of sample are to he detected, the equivalent hacterial dry weight ranges

SPARSIP WUROBIAL POPULATIONS 107 around g./ml. ; even if the concentration of extraneous matter in the sample is only g/ml., significant interference will occur unless the response of the bacteria is above 1000-fold that of the extraneous matter (on a dry weight basis). If readout depends on microscopic examination, microbes might be distinguished from interfering matter but, if response is determined by other means, interpretation of the results depends on the “blank value” of the assay. Provision of suitable blank samples presents no problems when samples of washed bacterial suspension are assayed, but can be difficult or impossible with, for example, samples from natural environments.

It is convenient to divide detection methods into two main classes. “Broad spectrum” methods detect and/or estimate microbes in general, or a taxonomic group of microbes (bacteria, yeasts, fungi, viruses), without specifically identifying them ; “specific identification” methods detect and idcntify microbes in one step. Broad-spectrum methods have potential application in situations where the identity of microbes likely to be present is known or not of immediate importance. Specific identification methods at present available vary very much with respect to the time taken to complete them, and their overall capability. Methods based on immunological principles are rapid and sensitive but their application to the detection of microbes depends on having a specific reagent for each of the species or even strains of microbes that may be present. Methods depending on other principles are generally less sensitive and, although more rapid than conventional techniques, they are too slow to be adapted for monitoring purposes.

In general, the rapid methods for bacterial and viral assessment discussed in this review are far less precise than the longer conventional methods, and several give results only in terms of the order of magnitude of the number of microbes present. A few are established methods based on proven principles (e.g. immunofluorescence) for which adequate published methodology, sensitivity and performance data are available ; others are either “in the experimental stage” or based on principles of questionable validity.

II. Principles of Rapid Microbial Assessment Methods Properties of microbe8 that have been utilized in rapid aHscHsment

methods include their charactcristic Hieo and mass rangcq chomicrtl composition, growth characteristics, metabolic activities, specific enzymic activities and immunological activities. For practical purposes, the only rapid viral detection methods available are based exclusively on immunological properties although other approaches have been proposed.

108 R. E. STRANGE

Consideration of the actual amount of biomass, mass of a particular component, or physiological activity in a sparse microbial population, makes it clear why rapid assessment poses difficult analytical problems.

A. PHYSICAL PROPERTIES OF MICROBES

The size ranges of the different taxonomic groups of microbes overlap (for cxamplo, the larger virusca arc similar in size to the Rmallest bacteria) but dimcnsiona of most individual bactcrial cells are 1-3 pm. and of virus particles, 20-200 nm. Similarly, valucs in terms of equivalent dry weight are extremely variable but the ranges 10-i3-10-12 and 10-L7-10-'6 g. are represcntative of many individual bacterial cells and viral particles, respectively. The shape, dimensions and dry weight equivalent of bacteria vary not only in different species but also in the same species or strain depending on growth conditions and past history. I n spite of the large variations, ranges of values for bacterial dimensions and mass are sufficiently characteristic to be used as the basis of assessment. The concentration, size distribution pattern and volumes of small particles in liquid suspension can be rapidly dctermined with the Coulter counter (Coulter Electronics Inc., Hialeah, Florida, U.S.A.) (8ec Iiubitschek, 1969). Similar information for air samples can be obtained with the Royco particle counter (Royco Instruments, Inc., Menlo Park, Cali- fornia, U.S.A.).

Extremely sparse microbial populations must be concentrated before assay and, in the case of bacteria and larger organisms, this can usually be done either by centrifugation or membrane filtration. Membrane filtration tcchniqucs art: ptirticularly umful for concentrating small numbcrs of tniorohcs (oonttLinc.tl in ltkrgc! volumcR of liquitk) on to Hinall arean whwc tho rnic0roh:H cur1 hc Htihinnd, or tillowctl to multiply, urid tho t i examined microncopicully (me Mulvany, 1U69). Sptmnc viral populetionn can bc concentrated by ultraccritrifugcttion, electrophorcsis (Bier et d., 1965), treatment with polyethylene glycol to remove water (Clivcr, 1965a), ultrafiltration through an aluminium alginate gel soluble in sodium citrate (Gartner, 1965), fractionation in aqueous two phase polymer systems (Albertsson, 1958; Philipson et nl., 1900; Schmidt, 1968; Shuval et al., 1969), gauze samplers (Liu et al., 1971) or membrane chromatography (Cliver, 1965b).

Interference by extraneous particulate matter is a major problem in many rapid assessment methods and it may be necesgary to fractionate the sample before analysis. Particles collected from air samples are fractionated on a size basis with a liquid pre-impinger (May and Druett, 1953) that removcs particles of >4 pm. diameter, a multistage liquid impinger (May, 1966) that separates particles into size ranges of >6, 3-6

SPARSE MICROBIAL POPULATIONS 109

and (3 pm. diameter or through filters of graded pore size. Liquid samples are fractionated by membrane filtration, differential or gradient centrifugation, liquid two phasc polynicr systems (Albertsson, 1058 ; Philipson et al., 1960: Tiselius et nE., 1!)63; Schmidt, 1968; Schuval ~t al., 1969) or magnetically stubilizcd dectrophorcsis (Koliii, 1960). TWO phase ayueoiix polynicr sgstcins ~ L W pirticulurly i i w f i t l for purifying microbos and virus c i ~ i i be cloiieriit~:Lt,c,tl 111) t>o loo-fold i i i oil(’ stop with the method (Philipson ~t aE., 1960). If meinbranc filtration is adopted for fractionation purposes it is important to realize that even though bacteria and viruses have smaller diameters than the mean pore size of the filter they may still be retained. Rao and Labzoffsky (1969) found that more than 50% of poliovirus type 1 (Mahoney strain) were absorbed by Millipore membrane filters (HA; 0.45 pm.) when suspensions also containing a certain concentr&ion of electrolytes were filtered through the filters and prefilter pads (AP25). The technique was used to detect low concentrations of viruses in large volumes of water. Cliver (1965h) reported that 99% or more of enterovirus in deionized water, tapwater or saline phosphi~tc buffer failed to pass through Millipore membranes with pore sizes of‘ 0.45 pm. Absorption of virus by membrane filters was strongly inhibited by serum or whey protein in ttrc input suspension. Sinlilidy, significant iiumhor of /lacillus sublilis spores arc retairicd whcii salinc phosphatc buffer Buspensions cootaining 103-104 spores/ml. are filtercd (and washed) by suction through Millipore membrane filters of 3 pm. pore sizc (i.c. much largcr than t h o spore diameter).

B. CHEMICAL COMPOSIT~ON OF MICROBES

Broad-spectrum methods depending on analysis for a particular constituent common to bacteria in general have been reported. With few exceptions, the various constituents of bacteria are present in other life forms and this type of method may give a response with other biological material present in the sample. Unless samples are free from, or contain, a known amount of interfering material, bacteria must be separated before analysis.

The chemical composition of bacteria varies markcdly in different species, or with growth conditions and past history in tho same spccics, so that quantitative compositional data arc of littlc valuc: without reference to these factors. In d l cases, the biomass i H Iargcly ;iccountod for by protein, RNA, DNA, lipid and carbohydrate but, with ttic exception of DNA, the relative concentrations of thcso macromolccules vary widely. Compositional data for Aerohucter aerogeenes grown t ( J thc! stationary phase in a defined maniiitol-dts modiam and tryptonc- glucose medium are given in Table 1.

110 R . E. STRANGE

If a microbial detection method based on analysis for the whole biomass or a major constituent(s) is required to detect lo4 A . aerogenes cells, the respectivc amounts of substances that must give a measurable response are shown in Table 1. If lo4 bacteria are concentrated into a sufficiently small volume iind arr not obscured by cxtrmeous tntltter, thcsc? smtdl tunourits of mcrtc~rid ( V L I I lw cletJcc:tcttl niicroHc~oi)ic!wIly nftcbr stuiiiiiig o r tr~rrtriwiit witli I i i H t o c : l r c . i n i c a I r ~ v q . y i h hiit iiot by t l i v microsccdc iiicthode iiscd in biochcniical analysis. Only methods with the scnsitivity and precision to mcasiire nanogram to picogram quantities can be applied, for t:ximpl(~ giis ohromiitogruphy, nitiss spectroscopy and rtldiochemical techniques.

TABLE 1. Biomass or Wciyht of Constituent in Equivalent Dried Weight of lo4 %abionary-Phase Aerobacter aerogenes NCTC 418 Calls Grown in a Mannitol- Limited Defined Medium and Tryptone-Glucose Medium. Data from Strange et al. (1901) and Strange et al. (1903).

Grams component/104 cells

Biomass Protein ItNA DNA C'mlmlr ydmto Lipid' ATPb

Tryptoric-glucose medium

2.4 x 10-9 1.4 x 10-9 2.9 x 10-10 9.0 Y 10-11

3.6 -4.8 x 10-'0 1.8 x 10-10

C

Extractud with ehloroforrtr-methanfll (:I : 1, v/v). In fully aerated bactorici. Not determined.

Gas chromatography has been applied to the analysis of the pyrolyuis products of bacteria with results that suggest this is an acceptable method for rapidly and specifically identifying bacterial species (Oyama, 1963 ; Garner and Gennaro, 1965 ; Reiner, 1965, 1967). Specific identification of small numbers of bacteria involving pyrolysis and product analysis with gas chromatography and mass spectroscopy w ~ s proposed for rapid biological detection purposes by Mitz (1969).

Other constituents of bacteria comprise a wide spectrum of substances most of which individually account for (1% of the equivident bacterial dry weight. Certain of these constituents can be accuratcly measured in extremely low concentration8 atid anulyscs for thcm have bCCrl proposed

SPARSE MIOROBIAL POPULATIONS 111

~,EI rapid microbial detection methods. Examples are ATP and hltematin, compounds which are apparently universally pkeserit in bacteria and that can be detected at, a minimum concentration of about lo-" g./ml. in each case. Even if these compounds each account for only 0.1% of the equivalent bacterial dry weight, analysis should allow detection of a minimum of 104-105 bacteriti/ml.

The major constituents of viruses are protoins, nucleic acids (RNA or DNA), lipids and carbohydrates; estimates of the relative amounts of these components in Semliki Forest virus and bacteriophage T7 are

TABLE 2. Biomass or Weight of Conetituent in Single Dried Particles of Phage T7 and Semliki Forest Virus (SFV). Data for Somliki Forest Virus are from D. Titmuss, K. H. Grin- etcad niid -4'. D. Oram (unpublishod work)

Grams component/particle ( ~ 1 0 1 ~ ) __ . _ _ - ____ ._ - -

I'hcige T7 SFV _ _ . . ._ _ _ - __._-I__

Biomtws 1.9 3.7 Protoin a 0.91 2 4 RNA 0.08" 0.2 DNA 0.64 - Lipid b 0.84 Carbohydrate -_ 0.29

a Probably due to host cell contamination and/or nterference by other phage constituents in assay.

Not determined.

given in Table 2. An extremely large number of virus particles is required to provide a sufficient rnws of material for analysis and this, together with the likelihood that virus preparations will contain interfering host cell material, eliniinatcs the chemical analysis approach to viral detection except with relatively concentrated virus suspensions.

C. MJCRO~IAL Urtowwi AND METABOLISM

Expression of the ability to reproduce i H ono of hhe mmt rclishlo indicators of the presence of microbes and nttcmpts have been made to modify classical microbiological methods, based on growth, to give results in a shorter time than usual. Provided that the microbes in the sample are viable and results are not required in leas than a few hours, growth methods are to be preferred.

112 n. E. STRANGE

The varioiis methods avdlable for determining microbial growth were analyscd by Merclr and Oyama (1968) who were concerned with the detection of growth in the search for extraterrestrial life. The methods were determination of dry weight, “silting index” (developed by Millipore Corp., Bcdford, Mass., and depending on the principle that particles plug membrane filter pores imd dccrease the rate of flow of liquid), electronic particle counting (Coulter counter; sce Kubitschck, 1969), monitoring for metabolic activity (rate of substrate consumption, rate of product formation, changes in pH value, redox potential changes), chemical analysis of thr pmticlcs (e.g. nitrogen determination; see Ferrari et al. 1965), optical tcchniqucs applied to a surface (Glaser and Wattenburg, 1966) iiiid optical techniques applied to a liquid (proposed by Dr. Wolf Visliiiiac; scc Quimby, 1961). Merek and Oyama (1968) consider optical ruo~iitoring in i~ system frre of interfering particles is the be& of these nicthotls for t heir p i q ) o s r , ;md they discuss the problems of formulating 811 it iI,bhl (:[I 1 t wc ni CCI i i ~ .

M(wiirc*nirnt of thct light, S(‘iLt,tCr(’d when xin;dl prtir les itre exposcd tJo i~ Iwam of light, ix iL sonxitniv(~ mcdiod for thvir dct,cxtion c~nd detcrminii- tion, i~iid tlrv prinvi1)lc~ hi1x I ) o r , t i ilscrl i n iLiltort1iLtd microbiid dntcction illStririncbiit,s. ‘ 1 ’ 1 ~ wnsor inc.lir(lrs iLIi opticid tixscbrn1)ly t h u t I)(wnits only light soiLtt(wd by piLrt~ic+lw t,o 1)rocliice IL signal i n ii phototubc connccted to a ptilso rcwwdcr and/or a digital rcadout systrm. Zn the “Wolf Trap” (invented by Dr. Wolf Vishniac, University of Rochester, New York ; see Mitz, 1969) captured microbes we cultured in liquid medium and in the “capillary tube scanner instrument” (Bowman et al., 1967) microcolonies arc formed in gelled nutrient agar held in capillary tubes; in both instruments, growth is measured with a scattered light sensor.

Bacterial growth in the presence of carbon-energy sources like glucose results in the production of organic acids that affect the pH value of the medium, and measurement of changes in pH value in appropriately formulated growth medium is u potential method for detecting bacteria. I n addition to scattered light sensors, the Wolf Trap is provided with pH value probes and growth can be monitored with cithcr one or h t h types of sensor (Mite, 1969).

Products of biLct(*riitl rnr4nholism include ~ , R C N (carbon tlioxirlr?, hydrogen sulphidc, methane, moloc:illt~r hydrogcn ant1 timmonia) nnd

ion of gas cvolution is anothc~ potential growth monitoring nnrJ/or microhiid dctJuction rrtrthod. With :L H I m w mic.robial rmI)ilI:Lt,ion, h o w - ever, the amount of gas cvolvcd is c.xtrc!mc:ly srnull ( l o 7 fast growing E’. coli cells produce aboiit 1 p l . (:0,/hr):ind canonly br tJct&(:d with, for examplc, radiochemicd t ~ ~ ~ ~ h ~ i i q i i ~ ~ s . Scirntists of the N;itional Acronau- tics and S~)actx Administration in the U.S. have developed an automated detection dcvice hasctl on this principle called “The Qullivcr” (see


Mitz, 1969) that monitors the l4CO, evolved from a small number of bacteria growing in a mcdium containing a I4C-labelled substrate.

During bacterial growth, metabolic products that vary qualitatively and quantitativr1.V with the particular species or strain of microbe and the growth conditions arc releasrd into the eulturc mcdium. An in- triguing discovery made conccrning products r(+ascd undcr ii given set of growth conditions was that they wcre qidatitivcly and quantitatively distinctive of a particular bacterial spccies (Hcnis et al., 1966). A highly sensitive gas chromatographic technique was used to analyse the growth prodiicts cxtractccl from the medium, and scvcral workers hsve eonfirmed that tlic protiles (“Hignatm~x”, “fingorprints”) iirv sui1icic:ntly distinctive to be used for s1w‘:ificalIy idcntifying ba(;twiii (Moor(: el d., I96fi ; Moort., 1 !M7; Mitruka iilld Alcxandcr, 1967; 1968; l!b69). The minimum readout time depends on thr species, initial concentration of 1)actcria and the sensitivity of the gas chromatographic detector ; in certain circumstances results can be obtained in 2-4 hours.

Detection of metabolites elaborated by virus-infccted cells during the course of infection in tissue culture or host animal was reported as a means of rapidly detecting viral infections (Mitruka et aZ., 1968; 1969).

D. BACTERIAL ENZYMES

Growth and metabolism of bacteria depend upon numerous endogenous enzymes, the activitiw of which can bc specifically and quantitatively determined in whole bacteria, hornogenates and/or extracts with simple, highly sensitive methods. Many of thcsc enzymes arc apparently present in all bactcria although thore 1niL.y be xomc c1iff~:renocs in the physical and chemical structure of thc csnzyme molecul(~x in tliff(:rcn t bacterial specics. In principle i t is fcaclible to dctrret hhsrii i , with an assay for a particular common cnzymc: but in most C;LH(!H the urnount of enzyme present, rate of substrate brcnltdown p r ~JiLCtrArillm s n d wnsi- tivity of the assay are too low to allow dctcct ion ofa few bactcaria. Scicn- tists of the National -4eronautics and Space Administration (we Mitz, 1969) have considered enzyme activities that might be exploited for rapid microbial detection purposes and selected two that can be assayed rapidly and have high “turnover numbers” (molecules substrate de- composed/bacterium/tnin.) with their respective substrates. One is phosphatase (the activity of which is used as an index of bacterial action in milk) and the assay depends on hydrolysis of p-nitrophenyl phosphate with release of p-nitrophenol that is measured spectrophotometrically ; alternatively a fluorescent organic phosphate can be used which releases a highly fluorescent product that can be measured fiuorimetrically. URe

114 R . E. BTRANQE

of t lw lnttc>r substrate allowed detection of 10' b;ictc\ria within a few niiiiutcb. Tlio secoiid useful enzyme is bacterial esterasc(s) which Mitz (196!,) and his ciollc:~guc~ (Dr. Gordon Nlmchwrd) founcl in 12 represcntative bacterii\l specics ; the> tissi~y depends oil hydrolysis of phenyl acetate with rcleiise of acetic wid t1i:rt can be mcasitrcd by ciutoinutic titration with standard potiissium hydroxide to niaintuin thv pH vdue of the reaction mixture at 7.4. Tlio inininiuni number of bacteria detectable with the esteruse assay varied from lo3 to lo6.

I!:. TMlfIINOLOGIC'AL PROPERTIES OF MICI~OBEB 1. Bacteria

Inimnnological metliods are indispensuble for the specific identification of microbes. Simple agglutination tests with oiie or more antisera and appropriate controls often suffice to identify spccifically a Iiomogeneous bacterial population. A relatively dense bacterial suspension ( lo8- 109 bactcrialml.) is required, but bacteria in liquid samples can be (?ollcelitriitcd by centrifugation or membrane filtration and this, com- biiied with microscopic observution of agglutination, idlows identification of bacteria i l l uri initial conc:ciitration of about 10J/ml. However, with sparscr populations, sonie otliw indicator of an immune reaction must be employed.

Bacteria usually have scvcral distinct immunospccific surface com- poiients (e.g. 0, K and I1 antigens) and 011 exposure to homologous antiserum each cornbiiics with its specific antibody to an extent depending or1 tho amounts of respcctive antigens present, their accessibility and thc dissociation constants of tho immune complexes. Surface adsorption of specific antibodies prccwlcs bacterial agglutination that in dilute bacterial suspensions occurs a t a slow ratc becausc of the low cell collision rate. A solution to the problem of detecting antibody specifically attached to bacteria in liquid suspension, clinical apccimcns and tissuc sections was found by Dr. Albert Coons and his colleagues (Cloons et al., 1941; Cooiis et al., 1942; Coons, 1961) who invented the fluorescent antibody technique. The numerous successful applications and modifications of the method arc adequately reviewed elsewhere (e.g. see Nairn, 1964; Goldnian, 1968) ; only broad principles and a few pertinent applications are mcntioried in this rrview.

Ultraviolet microscopy of samplcs treated with fluorcwxnt antibody, followed by washing with alkaline buffcr, shows homologous hactoria as strongly fluorescent partidw wtwreas hetcrologous hoteria are unfltuined. The sensitivity attained is extremely high dop(:nding or1 orlcB ability to firid the stained particles and the cxtont of iritcy-fercnce duo to autofluorescing mntcrial (and/or non-spccific uptako of the trtggc!d


antibody by supporting media and extraneous particlee or substances in the sainples). Ininiune globulins and other proteins in crude antibody preprations am 111i~croin01e~ules of rrlativcly high molecular weight (1.6 x 105-106 daltons) that tend to adhere to many types of material and complete elimination of background fluorescence may not be attained. Background fluorescence may be decreased by counter- staining preparations with Evan’s blue dye (White and Kellogg, 1965).

In attempts to improve the reproducibility of standard immunofluorescent techniques, emphasis is now being placed on standardization of rmgents, methodology and instruments (Nairn, 1968 ; Holborow, 1970; Beutncr, 1971). Important factors include the quality of antisera, globulin fraction or purified antibody, optimum intensity of labelling with fluorochrome (Spendlove, 1966; Brighton, 1966; Beutner et al., 1968) illumination (Johnson and Dollhopf, 1968 ; Tomlinson, 1970; Johnson, 1970), and design of optioal equipment (Lidwell et al., 1967).

Immunofluorescence c&n be quantitatively determined with automatic microscopic scanning techniques (Mansberg and Kusnetz, 1966) and microfluorime’tric techniques were reported by Goldman (1967), Pearse and Rost (1969) and Ploem (1970). The fluorescent antibody test for syphilis has been automated (Coffey et al., 1971).

Immunofluorescent methods are applied to heat- or chemically-fixed samples on microscope slides or to microbes filtered on tonon-fluorescent membrane filters (Danielsson, 1965 ; Danielsson and Laurell, 1965 ; Guthrie and Reeder, 1969). Increasing the size of the bacterial population by a short growth step before fluorescent antibody staining greatly iiiureases sensitivity (Danielsson & Laurell, 1965).

“Ilircct” or “indircct” irnmunofluorescence techniques can be used for identifying bacteria. In the direct incthod, tho sample is treated with fluorochrome-labelled antibody spccific for the bactcria being looked for ; in the indirect method, the sample is treated with unlabelled antibacterial serum, and after wishing, with fhorochrome-labelled antibody against the whole serum or serum globulin fraction of the animal in which the antibacterial serum was produced. An advantage of the latter method is that only one labelled antibody reagent is required to detect each of any number of different bacterial species provided that antiserum against each of them is prepared in the same species of animal.

Radioisotopes of iodine have also been used for labelling antibody proteins (Miles and Hales, 1968) and a ‘ZSI-labelled antibody-membrane filtration method was reported for the rapid specific detection and determination of small numbers of vegetative bacteria and bacterial spores (Strange et al., 197 1). As with immunofluorescence techniques, the major sensitivity-limiting factor in the radioassay is variable non- specific uptake of labelled antibody by material8 wed in t h e amay and

1 l(i R . R . STRANCIE

extraneous particulate mather in samples ; nevcrthcless, srnsitivity compared fibvowably with that i~ttninrd with otlirr rapid nwthods.

Viral particles have surfarc antigcns that arc det(wt,abIe irnmuno- logiciilly. Coiiccritriitctl poliovirii~ prcpwiitions (Smith r / al . , 1!)5(1) wid 1)Iii~gv pirtic+*s (Jrriie i ~ i i d Avcyyo, I !&ti) both tlocc!ulatc! in tha prcscmx of homologous antiserum. Howcvcr tho large number of viral particles rcquired to produce a disccriiablc prccipitate usually cxcludes the USC of fioaculutiori terrts for dcteetion ;ind identification pirposes. A concentration of about 10” phage ‘l’7 particles/ml. is required to produce a visible precipitate with antiphage serum. The sensitivity of flocculation tests for viruses is incrcnsed by adsorbing either viruses or antiviral globulinson to various kinds of larger particles (for details, see Kwapinski, 1965). Segrc (1957) used ion-exchange resin particles coated with specific virus antibodies for drtecting relatively small numbers of hog cholera and vesicular stomatitis viruscs ; the method was modified from that of Evans and Hnines ( 1854) who cmployed ion-exchange resin particles coated with polysucchiwidc mtigrns.

Under certciin conditions, viruscs cause agglutination of erythrocytes, a phenomcwon first shown with infiurnza virus (Hirst, 11)41 ; McClelland and Harc, l!Nl). Scott rt d. (1!)57) uscd tho passivc haemagglutination test to detect viral antil)otly by wiiting tsnnic acid-trcated erythrocytes with virus iuitigcin. hi i t l i tuid (lourtncy (1965) detected virusea with tanned crythrocytc>s coated with unti-viral globiilin and markedly incrcascd thc sensitivity of t h c h 1)askiVo ~iaomugglutination assay. In studies dcsigned to dcmonstrate phagocytosis of virulent Illreponema

pallidurn, in uitro, observations on control preparations led t o the discovery of a11 immunologically specific adherence reaction between normal human erythrocytes and treponemes sensitized with antibody from syphilis seruni (Nelson, 1953). The reaction required a heat-labile substance in normal serum, presumably complement ; erythrocytes played a specific role since, when they were replaced by human platelets, charcoal, magnesium silicate particles or cells of Candida albicans, no adherence reaction was observcd. It was found that the reaction occurred with several other bacterial spccies and tho phenomenon was called “ttw imniunc adhcreiirc rcaction”. h’c+wrr ( 1953) sugge!~ttd tttc. rcviction of erythrocyte8 and micrn-nr~ai,isrtts c~oulcl tjc usctl to tlc+*c:t tairouletirlg antibody.

Since Xelson’s ( I 953) discovcry, thv irnmuno ~dhctrc~nc:c: rc:rtction h l l H

bcxm shown to occur with yeast cf~lls, Httirch grt~~iulcs, ric*kt:ttsi+h, s(jV(:raI viruscsandprotcins (Ncl~oniintJ Woodwortti, l!j.57), I t ltas boet i 11Hfjd. t&+ II


sensitive method for titrating animal virus antigens and antibodies (It0 and Tagaya, 1966; Nishicka et al., 1967) and a small-scale adaptation of the principle was used for the detection of Moloney virus on monolayer cells (Tachibana and Klein, 1970).

Immunofluorescence methods (see Nairn, 1964 ; Goldman, 1968; Carter, 1971) are extensively used for the rapid detection and identification of viruses-indeed no other similarly rapid and reliable methods are available. There are three main ways in which immunofluorescence can be used in virus identification. First, serum virus antibody can be detected with indirect immunofluorescence or by neutralization tests based on fluorescent cell-counting assays. Second, viral antigen may be detected in smcars nnd impressions from cxiitlatw and timiles and 10s~ commonly in cryostat tissue sections. Third, virctl antigotis miiy ho datccted after inoculation and incubation of tissuc culture monolayers on covcrslips or slides.

The aim in immunofluorcscent dctection methods is to achieve the highest sensitivity, and methods employing cell monolayers facilitate this by providing conditions that augment the initial viral population by replication. Sensitivity for a particular virus depends on many factors of which susceptibility of the tissue cells to virus infection is of particular importance. Depending on the type of virus and nature of the sample, pretreatment such as ultrasonics or repeated freeze-thaw cycles may be necessary to release viruses from cells, or cell debris, before applying the sample to the monolayer. The rate of adsorbtion by and penetration of virus into a cell monolayer is increased by a centrifugation technique first described by Hahon and Nakamura (1964). Adsorption of psittacosis virus was carried out with a centrifugal force of 500 g using centri- fuge adaptors for coverslips. More recently Hahon and his colleagues described the use of centrifugal forces up to 30,000 g for othcr viruses (Hahon, 1965, 1966, 1969; Hahon and Cool<(:, 1!)66; Hahon and hank in^, 1970). As the rate of virus-cell contact is virtually intlopcndcnt of inoculum volume, a relatively largc volurnct of ~ctmplc ctm hc tiswl, dlowing detection of very low conccntrationH of virus.

The total period of monolayer incubation from inoctilittion to fixation is called the detection time and consistx of two phiLReH: that of adsorption and penetration of virus, and that during which thc virus replicates to detectable levels. Infected cells are demonstrated by staining with fluorescent antibody and counted with the microscope at relatively low magnification, using ultraviolet radiation or blue light. Under appropriate conditions, a linear proportionality exists between the concentration of virus in the inoculuni and the number of fluorescent cells. The mean count is multiplied by the field factor (i.e. area of the coverslip monolayer divided by the area of field for a given microscope objective

118 It. E. STRANGE

and ocular system) to give the number of fluorescent cells on the entire monolayer. The value multiplied by dilution and volume factors provides the titre of the virus in some convenient units.

The fluorescent antibody-cell monolayer method is sensitive, rapid and reliable. In many cases, readout is possible within less than 24 hours and, in the case of foot and mouth disease virus, the assay is completed within 4 hours (Mohanty and Cottral, 1970).

The feasibility of rapidly detecting virus particle8 in liquid suspension with '251-labcllcri homologous itntibody was invcstigatcd with phage T7 as thc test particlo (R. El. Strange, unpublished observation). A major tcchnical problem W ~ R thc rapid and quantitative separation of virus particles after attachment of lebclled antibody and, although tentative solutions were found for phage T7, the mcthods may not be applicable to viruses in general. Since the uptake of antibody by a microbial particle depends on its sizc, the theoretical sensitivity of the radioassay in terms of the minimum number of particles detectable is much lower than with bacteria.

111. Rapid Broad-Spectrum Methods

A. LUMINOL CHEMILUMINESCENCE

The catalytic effect of haematin on the chemiluminescence of luminol has been discussed by White (1961) and this principle can be used to determine extrcmply small amounts (minimum lo-'' g.) of various haematin compounds (Neufeld et al., 1965). Bacteria and yeasts contain haem compounds (0.g. catalase) and Pisano et al. (1965) reported that cell-free cxtrtlcts of these microbcs activrbtc! luminol.

Luminol (5-amino-2,3-dihydro- I ,4 ~~hthnlaziric!dionc) in thr! pwcncc of alkali hydrogen peroxide (Whitrb, I !MI) or sodium prboreto (Wrlen- son, 1987) and an activating agent (sodium hypochlorjto, potassium ferricyanide or a transition mctal) produces photon8 (White et al., 1964). If the reaction is allowed to take placc in a dark chambcr, the c m i t t d light can be measured photonictrically.

Oleniacz et al. (1966) described an automated luminol system for the detection of small numbers of bacteria and yeasts based on a Technicon Autoanalyser (Technicon Corporation, Ardsley, New York). Samples of 1 1 representative species of bacteria and two species of yeasts all activated luminol to give a light emission peak that decaycd in about 100 seconds. In all cases, the light signal minus the assay blank value was directly proportional to microbial concentration. Of the Oram-positive bactaria tested, Bacillus stearothernaophilus gave tlic most light cmisuiori per cell and about 600 cells could tw dfatrlcted ; Micrococcus Zpodeikticus

RPABSE MICROBIAL POPULATIONS 119

and Harcinu lutea gave the smallest response, the minimum number of cells detectable beiag greater than lo4. The responge of Gram-negative bacteria also varied with spccics but 5 x lo4 or fewer of the species tested (Enterobacteriaciae, Serratia marcescem, Pseudomom jluorescens, Neisseria catarrhnlis) were detected. Sacchnromyces cerevisiae gave more light per cell than C'andida albimns but, u'gctin, less than lo4 cells of each could be drtected. Thus, in gcncral, thc automated luminol syRtem was scnsitivc to 103-104 ccllv of cnah microbial species tcstcd. Oleniacz et a2. (1966) suggrst thtbt, if the system is attltchcd to an efficient air oollector, thc detection of biological aerosols is feasible.

0 0 II

Luininol J NM2

Aminiiphthalato

The ,simplicity of' t lw procodum ;md the: high scrisitivity and short rcadout tinic arc foutures of the luminol chcmiluininosccncc mcthod that make it worth considcring for rapid microbial dctcbction purposw. A lem attractive feature is that the reaction is activated by sub&ancc!s other than hltem compounds and intcrfereiice can be a serious problem. Our own laboratory tests with a manual mrthod confirm that haematin compounds and washed suspensioiis of various bacterial species in distilled water can be determined with the sensitivity clairned by Neufeld et nl. (1965) and by Oleniacz et al. (1966). However, it was our experience that : (a) assay blank values determined by mixing the luminol reagent with distilled water varied markedly with different batches of water from the same still; (b) the reproducibility of the signal with the same amount of haemin or bacteria was poor from day to day and often from one series of test and blank determinations to the next ; and (c) if bacteria were suspended i n solutions of sodium chloride or phoaphiitt?, blank values were extremely high n l ~ d small nurnbars of hacteria could not be clctt-T!ted.

120 R. E. STRANUE

Oleniacz et a2. (1966) stat8e that in their automated luminol system, the effects of chemical contaminants in samples that activate luminol could be eliminated by splitting the sample stream with a microbial filter and monitoring the sample before and after filtration. Howcver, this pro- crdnrc coii Id not eli mi 11 nto i ii tcrtiwv iv by ox trtm!otis piwt ici I lntc ma ttcr ill H ~ L I ~ ~ ~ I C H hhut, is r i4 , thwi I Ity trIw iiii(troljitd filtrr.

0lciiiiic.x ti/ r r l . ( l!Hi7) itivctnt~igirtwl tlw c d k t : t of i i o i ~ - l ~ i o l ~ ~ i i ~ i i i ~ ~ ~ ~ ~ ~ ~ ~ ~ t ~ micro-orpilienis on otliw e hrin i Iii niint!swnt compounds ttcwiden luminol. Intact cells and cell-free extracts of Serratia marcescens activated lucigenin (10, I0’-dimethyl-0,9’-biacridylium nitrate) luminescence in alcoholic solvents in the absence of either added hydrogen peroxide or alkali. Peak light transmission was attained three seconds after initiation of the reaction followed by rapid decay to a low constant light level. Sensitivity of the reaction in the context of bacterial detection was not reported.

€3. DETERMINATION OF ATP

Adenosine triphosphate is apparently present in all living organisms and i t can be determined with the firefly luminescence technique (Mc- Elroy, 1947 ; Strehlcr and Totter, 1052). Light emission when the enzyme luciferrtse reacts with liiciferin (both in fircfly tails), Mgz+ and ATP is directly proportional to the amount of ATP prcsent. Tho chemistry of the rcw4on i, ILR followe (MoI4:lroy et al., I!lfj!); 11; indicates liiciferin ; I,H 2, lucifcmsc) :

I n its simplest form, tho mctliod involves adding a crude buffered extract of firefly tails, containing mrignesiiim ions, to a xamplc contained in a light-proof chamber adjacent to a phototube, and meawring the peak light emission \i ith a photometer. Certain extraneoux rcactione (e.g. involving ATP production in the reaction mixture) are largely eliminated if purified lueiferase and luciferin are used instead of crude extracts of firefly tails.

The “760 Luminescelice Biomrter” (E. I. DuPont de Nemours & Co. Inc., Wilmington, Del. 19898) is based on firefly luminescence and is claimed to detect down to lo-” g. ATP per 1 0 pl. sample. Extraction of bacteria with butan-1-01 is rccommc~ndrtd and an M ~ U C O U H phanct i.r introduwd to purtitiori t l i c b hydrophilic ATP. A samplo ( 1 0 1 ~ 1 . ) of tlio aqiicow p l i ~ ~ is i i i j ( v 4 d into t l i c . Itic.ifi:rin-lucifi,r}~s(~ rrwt,iotl rtiixturc

SPARSE MICROBIAL POPITLATIONS 121

and the resulting light flash is autoniatically converted to concentrtbtion of ATP or micro-organisms. Assuming (as DuPoiit state) that bacterid ATP falls within a range of 2.2 to 10.3 x g. per cell, detection of ATP from 100-500 bacteria is feusible. However, because an extraction process has to be iised. H larger nunibcr of bnctcria is rcquirtd tind about 2 x 104 is probably a realistic estiniiitSe of thr niininiuni iiiinilwr

of bacteria dctectihlc. Another automated firefly luminescence assay is being dcveloped a t

the Goddard Space Flight Centre of the National Aeronautics and Space Administration (see Mitz, 1969) and it is stated that the instrument will detect lo5 bacteria within a few minutes.

The fact that the bacterial ATP content varies not only in different species, but also in the same species with the physiological state and environment of the bacteria (Strange et al., 1963), means that with bacteria from, for cxample, natural environments, the minimum number required to give a detectable response in the firefly luminescence assay may be comiderably higher than is the case with freshly grown bacteria in the laboratory.

C. STAININQ METHODS

1. Partichrorue Instrthment

1)otection of bavtcriu by sttiining with dyes, followed by microscopic examination, is routine microbiological practice and this principlc was used by Nelson et al. (1962) in their rapid detection device, the “Parti- chrome” (“particle ” and “colour”) instrument designed to detect airborne microbes. With this apparatus, samples of air are drawn in (17.5 l./min.) and the particles deposited by impaction on to a moving tape (Cronar from E. I. DuPont de Nemours & Co., Inc., Wilmington, Del. U.S.A.) coated with a thin film of immersion oil. Impaction of particles larger than 5 pm. is prevented by means of a cyclone-type separator placed ahead of the impactor. The deposited microbes are treated successively with HCl (1.9% aqueous) a t 60’ for 1 min., water, ethyl violet (1% in 0.5% aqueous Triton X-100, 8 5 O , 45 sec.), watcr and, after drying, with nitrobenziw~ for 5 scconcle. ‘I’hc trcatcd tap : is then scanncd with white light through tin oil-immcwion lcris and thc tranH- mitted light beam is split. The two bcwns we dirccted towards ptioto- tubes made sensitive, by means of filtcrs, to blue and green light, rwpec- tively. A scanning pattern is obtained by rotation of a mirror HyHtom placed after a zirconium arc lamp used as tho light source. When white light, passes through an unBtainc.d particle, both thc bluc and green

122 R . E. RTltANQE

componeiit,s are absorbed and the output of both phototubes is de- areased; if light passrs through a bluc-stained part,iclc, the output of the green phototpbc is decrruscd more than that of the bluo phototube and, therefore, blue particlcs may be counted in the presence of other particles. Pulses from tlic photomultipliers are tlnalysed with a signal analyscr (base-line straightener, amplificr, discriminator and anti-coincidence circuit). Nelson et al. (1962) state that low “blue counts” were obtained with oarbon and dust particles but the maximum sensitivity of the intltrument is not clear from their report.

2. IIio~Yensor

’I’hc. HioSonsor, l i k c t tho l’artichromc instrument, is an ilutomlnted dctwtor bwod on s t thing micro-orpniams and observing them elec- troniodly with microscopical optics (Whittick et al., 1!M7). It was dcvcloped by scicritists of the National Aeronautics and Space Ad- ministration for space biology, and Mitz (1969) suggests that the use of a fluorescent dye for staining could improve detection smsitivity ; sensitivity attained with the present staining method was not reported.

3. Fluorescin Isothiocpanate Staining

The use of fluorescein isothiocyaiiate in a direct staining method for microbial dctection was reported by Pita1 et al. (1966). I n principle, the method provides broad-spectrum detection capability in contrast to the high specificity of immunofluorescent staining. Heat-fixed smears of bacteria and proteins either alone or in the presence of atmospheric debris and soil were stained with the dye, washed with alkaline buffer (pH 0.6) and examined microscopically with ultraviolet radiation. A stablcb and a1)parcntly spwific linl<iLgc: formed with jirotviri, ; m l non- I)rotr.iii SlJbStitll(W w ( w r d i l y (lc!sL:Liii(d. A I I utnhc:rof IjwLt~ri~i I K ~ M ~ V K ,

hihmstcr kidney cclln, whont gc’rrri, i L l i d ( y g dhui~ i in W ( ~ I X - tlr.tr~c:t,wl t t i ~ t

sensitivity datri arc not givt~ii. I’itiLI P t (12. (I!)fX) mention t,wu I J O t ( b I l t i d sources of error, namcly, reaction of fiuorcscoin iwthiocyanato with now protein substanccs of unusual c h e m i d configuration, arid ernission of green autofluorescence by non-protein mbstancea. Tho method is presented as a “possible life-detcction technique”.

4 . Membrane Filtration-S‘tainirhg Methods

The detection and counting of bacteria in very low concentrations by microscopical observation is difficult, tedious and often inaccurate. Methods for concentrating sparse bacterial populations include cvapora- tion underreduced pressure (Kuzentsov and Karsinkin, 1931 ; Collins and Kipling, 1957), centrifugation and membrane filtration. Uaing the latter

SPARSE WUROBIAL POPULATIONS 123

method, Cholodny (1928, 1929) quantitativtdy tlnalyscd bactrriii in natural waters by trmsferring t h rwidiic rctiLi11rd by IL II\(xn11)riLtI(b liltor to a slide and exaniiniiig it, niicrosropic;rlly. iitisiiitiov (1938) iuiprovcd Cholodny's technique by directly observing bitcteriiL filtrrcd on to a membrane filter after the filter was rendered transparent with immersion oil. Erlich (1965) investigated several methods for conccntmting bacterial suspensions, and considered membrane filtration hiid advan tnges over sedimentation or vacuum distillation. He developed a quantitative membrane filtration-staining method that involved staining bacteria on the membrane with Victoria pure blue dye, removing excess stain under suction, and microscopical examination. Better contrast was obtained by prestaining membrane filters with basic fuchsin. The bacteria present in 60 microscopic fields (xX00) were counted and the total converted to number in unit volume of original sample by means of a simple formula. Earlier workers who reported membrane filter-staining techniques for counting sparse bacterial populations include Tietz (1949), Jannasch (1953) and Richards and Krabek (1954). Jannasch (1958) describes the use of the technique to study planctonic bacteria.

Eckrr and Lockhart (1959) described a rapid membrane filter method for direct count8 of micro-organisms in small samples. Millipor(? mcm- branes (0.46 pm. pore size; 1 in. diamctcr) are divided into 12 individual filtering areas (2.5 mm. diameter) by pressing with (t die hiiving a series of circular indentations smeared with 10% paraffin in petroleum jelly. The membrane filter is placed on the Hintered glass of a Millipore micro- analysis filter holder and accurately measured volumes (50-100 pl.) of sample (or dilutions of it) are measured with a microlitre pipette on to the marked areas, The samples are filtered under reduced pressure (5-10 mm. Hg). Samples are diluted in 0.85% (w/v) sodium chloride containing 1% (w/v) picric acid to fix the bacteria but, if undiluted sample is used, bacteria are fixed on the membrane filter by adding a drop of saline- picric acid solution. The bacteria are stained with acid fuchsin (0.1% w/v, in H,O, pH 3.0; 1 min.) and the excess stain sucked through under reduced pressure (50-60 mm. Hg). The stained membranes are taped to glass slides and dried (37'; 15-20 min.). A drop of immersion oil is placed on the dried filter and the bacteria, counted under the microscope. To calculate bacterialml. original sample (n), the following formula in uncd :

conversion factor x dilution factor x cclls counted n = _--____ _ _ _ - ___.

number of ficlds counted The conversion factor doperids on the size of the ficld at thc mag11ificatior1 used. If a calibrated Whipple ocular is usctd and thc grid arc:& is t, [L k en as the standard field, then C is equal to the filtering area divided by the area of the Whipple field a t the magnification used.

124 R. E. STRANGE

A rapid membrane filtrittioii staining method for dcterniining the number of viable microbes in food and food processing equipment was reported by Wintrr et al. (1!171). Bacteria are rinsed from food or swab samples with sterile diluent and concentrated by filtration on to membrane filters. The filters are incubated on suitable mcdia for 4 hours a t 30°, heated a t 105” for 5 minutcs iilld stained. Comparison of counts on the dried membranes (rendered triulsparent) with thosc of the standard plate count method showed u correlation coefficient of 0.906.

TI. PHYSJCAL METHODS

1. Ho?yco i’article Countw

‘ I ’ h i M inritrunic*itt (Icoyco I iiNtrirnicwtH, In(:., M i v ~ l o l’ihrk, (h~i for t l i~~ , [J.S.A.) rnc~iL~iirc~R quwitity and tliimc!tc*r o f micrornc~tr.ci-rii~c,d particlw prcrrent in the air or othcr gasmi arid can ba progrmirricd to count all particles within one or more size ranges giving a separate total for each range or all particle8 larger than any selected range. Full operating instructions are given in the manufacturer’s “Operating and Service Manual”. One of fifteen individual channels that cover a band from 0.3 pm. to 10 pm. and above can be selected to measure particles within a particular size and 30,000 particleslmin. can be counted with a coincidence loss of <lo% using a sample flow rate of 0.01 cu. ft./min. The air sample is passed through a light beam where the measured particles scatter light on to a phototube, the pulses from which are analysed, sorted and counted electronically according to the particle size selected for the count. The instrument is calibrated with uniformly sized polystyrene htex particles (graded from 0.5 to 5 p m . ; Dow Chcmical Com- pany, Physical ltescttrch Laboratory, Midland, Michigan, U.S.A.) disseminated with un acrorsol gcnrnctor considing of‘ an iitomirrcr thiit form8 a fine mist of’ difltillcd wutw in which tho partidm m! mrric.d. The mist is pts8rd down R drycbr till)(. to givc! i i l i r w (IiHIwrHioii o f thc: calibration purticlcs that t h m go t,o thc: cwurltcr. l ~ ~ x ; w i l i l w of potoritiid uses of the instrument arc monitoring o f “(*loan roo1nx” and dctc:ctiort of biological aerosols resulting from leaks in culturc apparatue.

2. C‘oultey Counter

Rapid and accurate determination8 of cell volumea and c~onccntration of micro-organisms in liquid suspension car1 be made with the Coultcr counter (Coulter Electronics Inc., Hialeah, Florida, U.S.A.). The principle, theory of operation and applications of the inatrument are discussed by Kubitschek (1969). Sensitivity of detection depends on the cell volumes of the particles ; with bacteria having cell volumes greater

' SPARS1 MIUROBIAL POPULATIONS 125

than 1-2 pm'., bacterial concentrations down to several hundred cells/ ml. can be determined but, with smaller cell volumes, the background noise levels are relatively larger and the necessity to increase sensitivity of the instrument increases the detection of foreign particles that are undetected at lower sensitivity settings.

E. METHODS DEPENDING ON GROWTH AND METABOLISM 1. Wolf Trap

This instrument was invented by Dr. Wolf Vishniac for operation on the Martian surface. It consists of a culture tube containing nutrient medium that is automatically monitored for changes in acidity and/or turbidity with pH-value probes and scattered light sensors respectively. Samples are collected and delivered with water and nutrients into five culture tubes. In actual tests, an inoculum of 10-20 bacteria grew to 103-104 in a few hours to give a signal dctectably greater than background. Some details of tho instrument and sensor systems are described by Mitz (1969).

2. Gulliver

Thc device (Mitz, 1969) iiutomatically mcasures radioactive gases evolved during microbial metabolism of radioactive substrates. A medium was developed in which detectable levels of I4CO, were evolved by the metabolic activity of representative bacteria, streptomycetes, fungi and algae within minutes to several hours. Mixed microbial populations give rise to several population curves that can be distinguished with time.

The early detection of bacterial growth with 14C-labelled glucose was described by Dcland and Wagner (1969) who developed an automated radiometric assay (Deland and Wagner, 1970). However, Washington and Yu (1971) evaluated the production of I4CO2 as an index of bacterial growth and were unable to detect I4CO2 from blood cultures within 6 hours with inoculum sizes ranging from 4 to 4250 colony-forming units.

3. Uptake of 32P-Labelled Phosphate

Macleod el al. (1966) reported a membrane-filtration mrthod for tho rapid detection of small numbera of viahlc bacteria bawd on their ahility to take up I2P as orthophosphate. A modium containing potuwium chloride, magnesium sulphate, glucom arid 3 2 PO:- waH inoculatotl with a small number of bacteria and iiicubated with shaking at 37" in parellel with controls without bactcria. After incubation (usually 1 hour), samples ( 1 ml.) were filtered and washed on stcrilo Milliporc membrane!

126 R. E. STRANGE

filters (HA; 0.46 pm.). The membrane filters were placed on planchets, air dried nnd retbined radioactivity was determined with a thin ond window Geiger counter attached to a Picker scaler.

Macleod et al. (1966) claimed that, under optimum conditions, as few as 23 vinblc cnlls/ml. wcw tlctrotcd, hiit, this olaiiri WRR lttter withdrawn (Mcwltwd t i d,, 1970). N c w r t h o h w , H o v t w I frltoiistiriid Im!l,oi*ia oftlifiwrrtt Npwioa ( w i lw ticdmttd with tho rnothod. An iniportnnt qucation i H tho oxtent of iiitorfcruncc that would bc encountered if, for example, samples of natural water were analysed with the technique.

IV. Rapid Speciflo Identification Methoda

A. IMMUNOPLUORESOENCE-MEMBRANE FILTRATION TEUHNIQWES

Danielsson (1965) reported a methodological study of a membrane Glter-immunofluorescence method for the speuifb identification of bacteria in tapwater or broth cultures within 1 hour. Samples are Gltered through non-fluorescent black Millipore membrane filter (HAB(P)G047) and circular pieces (12.6 mm. diameter) cut out with a metal die. The bacteria are stained with fluorescent antibody for 46-60 minutes, washed, mounted and examined with a Zeiss fluorescence microscope. By means of the formula x = NR2/20r2 (N, number of bacteria/20 fields ; R = 20 or 40 mm. depending on the diameter of the membrane surfaces used; r, diameter of field of vision, 0.32 mm. ; with 40 mm. diameter filtering surface, x = 781.25 N; with 20 mm. diameter filtering surface, x = 196.31 N) tho conccritration of bactaria specifically reacting with antibody can be dctermiricd. ‘rho technique eardly allowed quantitative detcrmiriation of bacteria proscnt in a minimum concentration of 105/l. but, with 10’ bacteria/l., the method wai time consuming. Danielsson and Laurel1 (1966) described the application of this method to tho detection of small numbers of bacteria in water. When bacteria, separated from a sample by filtration through a membrane filter, were eluted by the method of Miller (1963) that involves a simple washing procedure with glass beads or a magnetic stirrer, the lower limit of sensitivity for direct fluorescence microscopy was about 6000 bacteriall. water. By direct staining of bacteria on non-fluorescent membranes with fluorescent antibody, the lower limit was about 1000 bacteria/l. water; if this technique was combined with an enrichment procedure, it was posriblc to demonstrate 2-60 bacteria/l. water within 4-6 houri.

Closs (1968) reported a membrane filter-immunofluorcHconcc tcchnique that is claimed to ovcrcorw: ccrtain diHadvuntageH of J)imiehRon and Ltturcll’~~ (1 966) two-Ntvl) tmhniqus. Mambranc filtarH (blectk SartoriurJ, MFfio) on to which t h t : bwtoriu were colleotud wore cultured on ;L filter sottkcd with growth r n d i u t r i ooiittiiniiig Nuorctsccnt antibody


conjugate. Specifically stained microcolonies developed within 2-4 hours and were detected with the fluorescence microscope. Haemophilua influenax, Proteus rettgeri and Pastcurella haemo1,yticn wcro detcctod in this way. Sensitivity limitations are discussed und possiblo USPS of tho technique mentioned (Closs, 1968).

Guthrie and Reeder (1969) reported a further modification of the membrane filter-fluorcscenco techniquc. Watcr from ~1 srndl pond wt~s filtered through a Millipore membrane filter (HABG 047) that was then cultured on Trypticase Soy agar at 35". Colonies appeared after 5 hours but incubation ww continued for 12 hours. The membrane was overlayed with 1-2 ml. pooled normal rabbit serum for 6 minutes at 20". The serum was removed by suction and the membrane overlayed with fluorescent rabbit antiserum for 5-20 minutes. The antiserum was sucked through, and the membrane washed with 10-15 ml. phosphate- buffered saline. The stained membrane was overlayed with mounting fluid and examined with a dissecting microscope (magnification x 10) using visible light to determine the total count and ultraviolet radiation to determine fluorescent colonies. According to the authors, adaptation of the technique for use with lower magnification takes somewhat longer than the method of Danielsson and Laurel1 (1965) but has the advantage that all colonies on the entire filter can be counted. Critical factors are : (i) washing filters during staining by filtration under reduced pressure ; (ii) using glycerol-containing mounting fluid to prevent drying, while enhancing fluorescence ; and (iii) rapid counting of colonies once the preparations are exposed to ultraviolet radiation (because bleaching occurs).

The rapid detection of a small number of airborne bacteria with a membrane filter-fluorescent antibody method was described by Jost and Fey (1971). Calibrated air samples from aerosols of Serratia mclrces- cena were drawn through (10 l,/min.) a Millipore aerosol filter holder fitted with a non-fluorescent membrane filter, and the organisms were rapidly identified with Danielsson's (1965) method using a high power incident light ultraviolet microscope.

B. RADIOACTIVE ANTIBODY TECHNIQUE Analysis of data that have been obtained for antibody uptake/

bacterial cell, maximum l 2 'I or l 3 'I-labelling intensity of antibody protein commensurate with retention of immunological activity, and the efficiency of radioactivity counting instruments with 12sI, indicate that extremely high sensitivity is theoretically attainable with microbial deteation methods baaed on the use of '2sI-labelled homologous antibodies. In practice, however, determination of a few bacteria is poNHiblc

128 R . E. STRANGE

only if, after separation of the Ia\)ellcd immune oomplex, the variation in the level of non-specifically attached radioactivity is less than the radioactivity of the imtnuntl complex. If rtsidunl noti-speciitically attached radioactivity (dctcrmined by tcsting “bltink” suinples without bacteria) is high coinpr(d with thc radiociotivity of tho immunc coin- plex, even relutivcly sintill vuriutioiis will Hwiiinp tho signal from the complcx. Strango et ul. (1971) largcly resolved thc problctn of high erratic assay blank values with a radioactively-labelled antibody- membrane filtration tcchnique and were able to detect and determine specific bacteria and bacterial spores (down to a minimum of 500-1000 organisms) within 8-10 minutes. This order of sensitivity was only obtained with radiolabelled immuno-purified antibodies having extremely high specific immunological activity.

1. Zmmuno-Puri$ed Antibodies

When bacteria were incubated a t 25’-37’ with 1251- or 1311-labelled homologous antisera or crude salt-precipitated antiserum globulin fractions for 6-15 minutes, and the resulting immune cornploxes separated by filtering and washing on a mcmbrane filter, u radioactive signal higher than the assay blank (110 bactcriu) valuc was not obtained with less tlim ubout lo5 bacteria i n the sample. lodination of such crude antibody preparations results in products containing a large proportion of labelled non-irnmune protcins, more of which is taken up non- specifically by’ a rnembrarie filter than the amount of labelled antibody that combines spdica l ly with a few bacteria. The amount of antibacterial globulin in a high-titre antiserum may account for only 1-2% of the total protciti which means that up to 99% of the isotope used for labelling is wasted and relatively large amounts of isotope must be used to obtain antibody of sufficiently high specific radioactivity for rapid detection purposes.

Methods investigated for purifying antibody proteins included electrophoresis, isoelectric focusing and immunopurification. Immuno- purification provided the bent products for the radio-axsay and pre- parative methods, based on the previous work of Dr. B. 7’. Tozor and Dr. A. P. MacLennan (unpublished), were described by Strange el al. (1971).

2. Radioactive Iodination of Antibacterial (llobulinu

Purified antibodies werc iodinatcd with carrier-free i 2 s I or 1 3 1 1 (obtained from the Radiochemical Ccntrv, Amcrsham, I ~ I J C ~ H , England) using the chloramine-T method (Hunter and Greenwood, 1962 ; Bocci, 1964; Glover et al., 1967) as described by Strimge et at?. (1971). Optirnum

SPARSE WCROBUL POPULATIONS 129

specific radioactivity of antibody protein for radio-assay purposes was 15-30 pCi./pg. protein. Although a higher labelling intensity increased the signal from a given number of bacteria, the assay blank value also increased and no improvement in sensitivity was obtained.

3. Radio-meay

The simple assay procedure consists of incubating samples of bacterial suspension (0-1-1-0 ml.) and saline buffer suspending medium alone, with labelled antibody reagent (50-100 pl. containing 10-26 pg. 12sI- labelled antibody protein and 0.1 6 ml. clarified normal rabbit serumlml.) for the selectod rcaction time a t 25"-30". Reaction mixturos are diluted with snlinc phosphato buffer (0.11 M-sodium chlorido and 0-02 M - sodium pliosphato buffor, pH 7.7) containing 1% Brij 36 (polyoxyethy- lcno lauryl ether ; British Drug Houses Ltd., Poole, Dorset, England) and rapidly filtered by suction through black or white Millipore membrane filters (0.75 in. diameter) held in special stainless steel filter jigs (Strange et al., 1971). After a standard washing procedure with saline-phosphate containing Brij 36, the washed membrane filter is placed between discs of linen tracing paper and a central area 8% less than the filtration area is accurately cut out, with a compound blanking punch, and collected into a glass vial (3.5 x 1 cm.). Test and blank samples should be tested at least in duplicate. The radioactivity of the membrane discs is measured with a conventional well-type sodium iodide scintillation counter and scaler, or liquid scintillation counting can be used (Ashcroft, 1970).

The method allows the specific detection and determination of a minimum of 500-1000 washed bacteria, or bacterial spores, contained in 0.1 ml. samples, to be obtained within 8-10 minutes. Of course, very small bacteria (e.g. species of Brucella) tend to give a lower signal per cell than larger bacteria, but, provided that the assay blank value i R relatively low, and reproducible, less than lo4 cells of all Rpccies aro detectable with a significant signal to blank ratio.

The relationship between number of bacteria (BOO-I Os) and radioactive signal is usually linear, but somctimes tho signal per bsctcrium decreases progressively as thc numbcr of bactcria in the sumplo i R increased. Non-linear relationships arc presumed to be due to the immunological heterogeneity of antibody molecules in certain purified antibody preparations.

The sensitivity and accuracy of the assay decrcase if samples contain particulate matter that non-specifically attaches antibody and is retained by a membrane filter. This type of interference is decreased by pretreating samples with olarified normal rabbit serum (0.1 to 0.2 ml./ml. sample) for a few minutes before assay.

130 R. E. STRANGE

C. ANALYSIS OF BACTERIAL GROWTH PRODUCT^

Dotention and identification of bacteria by examination of their growth products with gas chromatography has bcen reported by sevcral workcrs (Henis et ( 7 L , 1966; Moore et al., 1966; Rricbn, 1967;MosstiiidI~ewis, 1987; Mitruk;L i b n ( l Alcxandcr, l!)(i7 ; 1!)6r( ; 3 969). Mitrulrr~ ~ l d Alcxandcr (1968) werc able to dctcct a small number of brwtcria of scveral different specics within 2-12 hours dcpending on the species. A modified Proom and Knight (1966) liquid mcdium (10 ml.) was inoculated with a washed suflpcnsion of bacteria (0.1 ml.; 4-810 x 10* cells) and incubated a t 35’. The eulturc was ncidified (6 M-HCI and 0.2 M-HCl-KCI buffer, pH 2.0) and centrifuged (2000g; 16 min.). The supernatant liquid (5 ml.) was saturated with anhydrous sodium sulphate and extracted with cthyl ethcr (3 x 10 ml,). The combined extracts were acidified (pH 2-0) and concentrated to 6 ml. on a rotary evaporator a t room temperature. The concentrate was Raturated with anhydrous sodium sulphate and samples (3 pl.) injected into a dual channel gas chromatograph fitted with two dctectors (electron capture, “ECD” ; flame ionization, “FID”) each of which received the equivalent of 1.5 p1. of the input. Eluted compounds were ident,ified by comparing their retention times and “Q valucs” (area of ECD pcak)/(area of PID peak) with those of authentic standard compounds. The sensitivity limit was the quantity of metabolite or number of viable bacteria giving a peak of 10 mm2. area with appropriate settings of the instrument. With inocula of less than lo4 cclls/ml., the presencc of Proteus vulgaris, Streptococcus faecalis, Strep. liquffaciens, Ihcherichia coli 13, llacillus cereus and l3. popilliae was detcctcti within 2-4 hours but a 7-12 hours growth period was necessary to dctect products formed by Srrrntin marcescen.9, AProbacter aerogene.9, h’. coli K 1 2, ,Staphylococcus auwus and ,Salmonella hgphimurium. Motabo- ljtcs formed by thc eq~i ival(~i t of Irxs than a single ccdl of 11. cereuR, Strep. fnccalis, P. vulgaris or Id. coli 13 wvre fitmcd with the 15CD; thc 1~11) W ~ H generally lcss sensitive. Letters wcrc assigned to the gas-liquid chromatography peaks (“Bacterial signatures”; Henis et al., 1966) in the order of their retention times to provide a “formula” for each bacterial species.

Gas chromatographic analysis of bacterial growth productu provides a relatively rapid means of specifically identifying bacteria and, indeed, there is evidence that different strains of a single species can be differen- tiatcd. In certain genera of anaerobic bacteria, the fermentation pattern8 do not differ between species in each genus but the patterns may be useful to establish the identity of the genus with certainty (Moore, 1967). Mixed bacterial species in a sample may be difficult to deal with but Fuller (1967) suggests that “ . . . perhaps new media that bring about


the generation of unique dead-end metabolites, or application of sophisticated cross-correlational techniques to a maze of peaks may provide a path to the answer”.

D. GAS CHROMATOGRAPHY-PYROLYSIS METHODS Identification of bacteria by means of “pyrograms”, that is profiles of

the products separated from pyrolysed bacteria with gas-liquid chromatography, has been reported (Garner and Gennaro, 1965 ; Reiner, 1965, 1967). Pellets of freeze-dried killed bacteria are heated in an inert atmosphorc 011 a nickcl filamont at 850’ for 10 seconds in a pyrolysis modulo attachcd to a gas chromatograph, and pctiks repremnting the eliited productr;l two recorded. I)iffm!ncca in tho pyrogramR of different biictoritL1 HpccicH aro mainly qwntitiitivc, not qualitative, and Itcinor (1967) Btates that scveral pap(w dctlling with gas-liquid chromatographic analyscs of microbial extracts or metabolic products either ignore quantitative aspects or they simply lack rigorous data. According to Reiner (1967), pyrolysis-gas-liquid chromatography is not a rapid detection-identification method but it allows unequivocal identification of bacteria within a considerably shorter time than is possible with conventional techniques.

In contrast, Mitz (1969) proposes pyrolysis coupled with gw-liquid chromatography and mass spectroscopy for the rapid detection of minute amounts of bacterial components (protein, carbohydrates, lipids, nucleic acids). The “projected characteristics” of potential instrumented systems are compactness, and analyses every few minutes with sensitivity and specificity to detect a few hundred micro-organisms.

E. ANALYSIS OF PHOSPHORESCENT DECAY

When proteins or aromatic amino aoicls arc irradintud with rrltraviolet radiation, they exhibit fluorc8ctmw und j)hOsfl’l.~riroHoc:tlcr: ( Hot:rrrnthn and Balekjian, 1966). Adelmtin el a2. ( I 967) irradiatcd intiiut living or killed cells of five bacterial strains, and found that the gencrtll forms of tho total and phosphorescent emisrrion curves were differont, if not unique, from one species or strain to another. They proposed analysis of phosphorescent decay curves as a means of identifying bacteria.

V. Rapid Determination of Microbial Viability A. INDIRECT METHODS

Methods for determining microbial viability not depending on cell multiplication were reviewed by Postgate et al. (1961) and Postgate (1967). They include vital staining (Gillilaiid, 1959), differential staining

132 R. E. STRANGE

(Struggrr, 1948; Wade and Morgan, 1954), reduction of redox dyes (Grecnburg et al., I%%), leakage of purines (Koch, 1959), changes in refractive index (Barer et al., 1953; Fikhman, 1959a, b), changes in extinction values of btbcterin in solutions of salts compared with extinction values in distilled water (Mager et al., 1966) and growth without division (Valentine i ~ i d Jh~i~dfi~4d, I 0534). None of these method8 has been acccptcd by niicw)l)iolo~~ i s ts 21s I L vdid itltoriii~tiva to the more lengthy but rr1i:hlo prorcdurtbn iiivolving iniorobial growth, although irll of tticm may give usoiiil in forrnirtion ronccrning microbial structure and function.

B. DLRECT METHODS A comprehensive survey of microbial microculture methods is given

by Qucsnel (1969). Microcultures on cellophan irrigated with nutrients (Powcll, 1'356) or thin layers of' nutrient agar (Postgate et ale, 1961) will give rcsults (in terms of' the percentage of viable bacteria in apopulation) within 2-2.5 hours with fibst-growing bacteria. The simple elegant slide culture method of Postgate et al. (1961) has found wide acceptance as a rapid rdiable nltmnative to conventional plate count methods for determining viability. A small loopful of a bacterial suspension containing about 5 x lo7 cells/ml. is required as the inoculum, but less dense suspensions are easily concentrated by centrifugation or membrane filtration. The method may give inaccurate results with populations of bacteria having n wide range of generation times (0.g. certain stressed populations) tmd cannot be used with filamentous bacteria (e.g. certain I~ucillus spccic~s). The nicthotl cmnot, be used to determine the total number of t)act(*riii or thr n i imhcr of' viable bacteria in a population.

Mrrnl)riiti(b filtration tcchri i~~tm ~)rovidc i t mt:iinH o f tlotcwnining fhn viability of sptme microbial prq)uIiLtionH in m;Lny CILHPH within ii Hhortc!r time than is possible with conventional viable cell plute counts (Mulvany, 1969).

VI. Rapid Detection and Determination of Viruses

A. IMMTJNOFLUORESCENT-MONOLAYER TECHNIQUES

Early quantitative methods based on counting fluorcscent cells after viral infection, and fluorescent antibody staining were reported by Deibel and Hotchkin (1959) and Rapp et al. (1958) but the first fully characterized assay was described by Wheelock and Tamm (1961) for Newcastle disease virus. Application of the principle to the detection and determination of a 'large variety of viruses and rickettsiae has been reported by many virologists (see Carter, 1971). The sensitivity of the fluorescent antibody-monolayer technique with vaccinia and variola


virus was investigated by Carter (1966). Coverslip monolayers of HeLa cells were infected with varying concentriitioiis of virus m d the rnini- mum incubation time required for the clppcwaiicc’ of foci dctecttiblc with fluorescent antibody was determined. Infcctioii with from %lo6 pltique- forming units of vncainin or variola vinisc~s/tiil, ww tlrt,cwt~ahle within 16 hours docrcmhg to 4 hoiir~, a i i d 14 IioiirH (iwrwwiiig to I0 I i o i i r H ,

respectively. As mentioned above, centrifugal forcc may be used to promote

adsorption of virus on to coverslip monolayers (Hahon and Nakamura, 1964). Under these conditions, adsorption is highly efficient and rapid. Plastic chambers for the cultivation of tissue cells on microscope slides were reported by Sattar and Westwood (1967a). Fluorescent cells are counted in a random 5-10% sample of the monolayer and the use of a simple counting grid and pointer system to select a uniform pattern and number of randomized fields (Sattar and Westwood, 1967b) improves the accuracy of results.

B. IMMUNO-ADHERENCE The “immuno-adherence phenomenon’’ and its applications in virology

were diiscussed in Section 11, E, (p. 11 4). An “immuno-adherence assay” depending on the agglutination of antibody-coated erythrocytes by viruses (Smith and Courtney, 1965) WUB reported by Mitz (1969). Erythrocytes sensitized with antiserum, in the prescncc of a small number of homologous viruses, formed dimers in numbers depending on the viral concentration. The present laboratory method is claimed to detect as few as lo-’ viruses/ml. in a few minutes, ultimate sensitivity depending on the device to count the number of erythrocyte dimers formed. Mitz (1969) suggests the possibility of adapting antibody- coated materials as a staining reagent in an instrument similar to the Biosensor or Partichrome instrument. He also suggests the development of broad-spectra antisera to overcome the limitation of the method, i.e. the requirement for a large number of specific antisera.

C. RADIOACTIVE ANTIBODY METHOD APPLIED TO BACTERIOPHAGE T7 Bacteria can be rapidly detected and determined with homologous

12sI- or 1311-labelled homologous antibodies (Strange et aZ., 1971), and in principle there is no reason why the method cannot be applied to viruses. However, the relatively small size of virus particles presents additional problems that are not easily resolved. The amount of antibody taken up by a virus particle relative to that token up by a bactorium iH one to several orders of magnitude lower and thiR affect8 the thcoretical attainable sensitivity in terms of the minimum number of virus particlee detectable. Then there is the aroblem of raaidlv aeaaratinn labelled

134 R. E. STRANGE

virus-immune coniplcx from excess labelled antibody. The feasibility of detecting viruses with radiolabelled antibodies was studicd using bacteriophage T7 (60 Y 80 nm. diameter) as a model particle in the Virus size rango (R. E. Strange, unpublislicd work).

Purified antiphage T7 globulin was obtainod by adnorbing high-titrc antiphage scrum with purificd phage 'l'7, separating m d washing tho agglutinate by centrifugation and dissociating the complex with 0.1 M hydrochloric acid-1% (w/v) sodium chloride solution (pH 1.2) a t 2". IZcleased soluble antibody was separated by centrifugation, neutralized, concentrated by pressure dialysis and fractionated with ammonium sulphate. The purified protein fraction (precipitated a t O-IiOYo saturation of arnmoiiium sulphate) was dialysed and iodinatcd with lZ51 to give a product with a specific radioactivity of 30-40 pCi./pg. protein.

The problem of separating labelled phage immune complex after reaction of phage suspension with 1251-labelled antibody was resolved in three ways. (a) Phage was adsorbed on to host bacterial cells (Escher- ichin coli MltE 160) for I6 minutcs a t 37" and the phage-cell complex reactad with 1251-labelled antiphage globulin (exhaustively pre-adsorbed with E. coli MRE 160). The labelled immune complex could then be easily separated by filtration and washing on a Millipore membrane filter (0-45 pm.). (b) A suspension of phage particles was rcacted with 1251-labelled antiphage globulin and the reaction mixture filtered and washed on a Millipore mernbrano filter (0.45 pm.) impregnated with hont-fixed (SO", 10-15 min.) purified rtntibody. This "imrnunofiltration tcclrnique" retained 12sI-labcllcd phagc immune coniplcx on the morn- brane filter but allowcd excess labelled antibody to pass through. (0) Phage suspensions in dietilled water were freeze-thawed Revera1 times and the broken phage reacted with 12sI-labellcd antiphage globulin. On filtration, and washing reaction mixtures through Millipore membranes (0.22 pm.), 1251-labelled immune complex was retained but excess labelled antibody filtered through. Tho method was apparently specific since '2SI-labclled heterologous antibodios were not taken u p by broken phage.

Results obtaincd with tho three assay procedures are shown in Table 3. In each case the radioactive signal givcn by the immune complox minus the mean assay blank value was proportional to the phage concentration. The highest sensitivity was obtained with the broken phage method mainly due to the fact that the assay blank value was relatively low; a minimum of 6 x lo4 to los plaque-forming units (about 2.5 to 5 x lo5 total phage particles according to elhctron microscope counts kindly done by our colleague Rlr. J. Harris) were detected.

Thus, particles in the virus size range can bc rapidly and quantitatively detected with the rudiolabelled antibody method, and w n ~ i -

NIIIIII)(V i b f 1 2 3 plaqun-foriniiig (Adsorptior I on to (lmmuno-

units host cells) filt,ration) (Broken phage)

Mr&d I . H i u n l d o (0.1 nil) twatrd with /hrhrric.hirc roli MItE IfiOrolln (fIOpg.tlry wt.; 56 1'1.) for I6 min. nt 37' : ~ ~ ~ l - l i ~ l ~ i ~ l l ~ i ~ l antihotly (0.5 pg.; HO pCi./pg, Iirotoiii) W ~ H adtlrwl arid aftow 16 min. at 26" tho mixtiiro W ~ L H filtoirrcl and wwhod through a Milliporii (HA) mom twano.

Method 9 . Samplo ( 0 . 1 ml.) troatncl with 1231-labrllod antibody (0.6 pg.: 40 pCi./pg. protein) for I6 min. a t 26"; tho reaction mixture W&B filterod and washed through a Millipore (HA) mrmbrane, the centre impregnated with 20 pg. of heat-fixed purified antibody.

Method 3: Sample (0.1 ml.) of phage T7 broken by six freeze-thaw oycles in distilled water wm treated with saline phmphato buffer (0.1 rnl.) and 12sI.labelled antibody (0.6 pg.; 40 pCi./pg. protein) for 16 min. at. 26". The reaction mixture was filtered and wmhed through a Millipore (as) membrane. In oarh cmo, the radioactivity of the punched out memhranr filtration area W ~ R measured.

tivitry, on a particle mass basis at least, is extremely high. In principle, one or other of the methods would appear to be applicable to the detection of animal viruses but this has not yet been tested.

I). DETECTION OF VIRUS ACTIVITY WITH GAS CHROMATO(:RAI*HY

Application of gas Chromatography to the detection of viral infections wlts reported by Mitruka el al. (1968, 1969). Sample8 of serum from infco- ted animals or tissue cultures were acidified, driod unclor v~~o i i i i r n , d i H - solved in pyridine and treated with hc:xarnc!thyltlisilclailnc! r m l tri- methylchlorosilanc. Microlitrc samplw wcro thon oxernincd hy gtw chromatography with electron capture or flame-ionization (1ctc:atorH. Gas chromatographs of serum extractn of dog8 inoculated with canine infectious hepatitis showed two metabolites not present in the serum from uninoculated animals. Chromatographs of extracts of tissue cultures of dog kidney cells inoculated with canine hepatitis, herpes, distemper or parainfluenza virus each showed two or more different metabolites not present in uninocylated cultiircs. Certain unique metabolites

6

VII. Conclusions and Prospects

Iktwtioii o f a few microbes in a few minutes is a difficult problem for which a gerierul solution has not yet been found. Methods designed to have this capability may give entirely satisfactory results with washed microbes i n the labortitory but fail with samples from natural or other cnviroiimrnts. I1:xtlremely fast readout requires the use of near instantaneous reactions suc~h EM liimiiiol chemiluminescence or firefly lumin- wwence? but neitlwr reaction is specifically activated by microbes or PVCW a cwrnponent tl iut is unique to microbes. A microbiologist would be iitlwis(* to rrport thc presence of microbes in a, sample of iinknown corn position solc~ly on the basisof a positive respnw with either method. I t is iircvititblc t l i i L t the muximuni schnsitivity clairned for detection 1nr.t hods is the. threshold rcsponscb of' the react,ioii involvcd dcxtermincd i i i d c r - optiiriutn c.onditions, h u t iii I)rwtiw sensitivity dclwnds on the i*clliit i vo cwiicvnt r:it,ioii of iiit,cdbrinp iiiiLt,(lriitI i i i t~hv wnpl r . Mic*rogriLm c ~ o r t c ~ c ~ t t t i ~ i t t ~ i o t i ~ of' c~xtrtiwoiin rriitttchr wi th i t slwilic. r(wtivity hclow t I l iLt of' t h b rnic.robid cwml)oiin(l hirig tlrtcc*tcrl rriity still interfcre i t i

t tic. prwwiw of on1.y nitnogrmi to piwgram umoirnts of thst c~ornl~ouiid. ('wttiiii t)roatl-sl)(~(~triirii rn~~thods (*a11 thcrcforc? only t)c yqdicd i f it is known t l i i t t smiplcs arc' frcar from or wiitain u constant itmoulit of interfering tntkttw, or following fractionation of Ramples to scpiLrttte the niirrotws. l 'h r inc~lusion of rbutomat ic samy)lc.-frartionation drviws iii fwt, r c w h t dc tors biised on broad-spec.trum principlcv would tit lcitst partially r c ~ e the intc~rf'ermcc~ prohlcm.

€'roblems assoriated with rapid specific idcntification mothods (I(*- pending on immunological principles differ from those with broad- spectrum met hods. The microbial c+omponents detected are usually unique to a particular species aiitl extraneous biological mcttrrial in samplw will iiot s~)iv~ifically r c w t with th(2 reagent. Howovcar, intcsr- ference is wuwd by adhrrcwce unt l ocdusion of t h v rcvtgwit t).v mcttc~rittls i i s d in the assay and extraiieoiis partirlas in s m t 1hs, rind this may p a -

vent qualitative or clumtitativc ttss(wrnwlt of' IL small riumt)cq of microhcs. Appliccttion of such rnc.tlio& t,o mic*rohial tlc

stricttcd hcwirscc. t tic& rnic.ro-or~iiriisms lik(1l.y to t j ( h prwltit, i r i HiLlrtpIVh

rnu& h a pr(4irtctl so t tiat t l i t . I I ~ W S S I I . ~ ~ r(*iLgc~tif,s ;b rv ;~vniIi,,ttlp. It, krns h w nllgg(.stcd thtitl i t i c ) t l o v c ~ l o ~ ~ r i i ~ ~ t of i i t i t i hcn I i r Ix witJi (J(*c*rc.ctLxc*(l specific+ity that r ( w t wit ti u c*omporic.rit I I I I ~ V ~ ? ~ S ; L I I , ~ prcwrit, i t 1 k)uc.tcq-ia or in viruses would rtvmvcl this rcbstriction, hut suitable antibodies are not ut present available.

SPARSF. MTCROBTAT, POPULATIONS 137

Tf the requirenieiit is not for immediate detection, and results within several hours are acceptable, more precise and reliable methods, depending on microbial growth and replication, are available. The large amplification of a population resulting from a short period of growth shifts down several orders of magnitude the sensitivity required of an analytical method ; for example, a population of lo3 viable Wecherichin roli ccll~ will i i i ( w t w ( b to tholit I O5 ( Y > I I S diiring unrrstrirtcd growth i n cwltuw nicd i i in i for d)o i i t :I hoiir~. ‘I’ll(* It~r.g(*r tlw poj)ulation, t tw nrriallcr trIw problcrii of‘ iiitorfi*rtwoe by extratwous rnibtcrirtl initially prcwnt in t h c h mrnpl(>. ‘I’hc intaresting discwciry that t h c growth products of bac*teria grown under controlled cotiditionH are characteristic of the bacterial species (Henis et d., 1966) is an important advance in the context of rapidly detecting and specifically identifying microbea.

Con~iderable effort is being expended on automating microbial cletection mcthods, for example, for use in aerosol detection (Nelson el ul., I M E : Oleniacz ~1 nl., 1966) or spacie remarch (we Mitz, 1969) tirid several working detectors me now i n cxistcnce. It is clear that the relevant engineering problems have been largely resolved but it remainfi to be seen whether the biological principles involved will prove sat isfaCtory in practice.

VIII. Acknowledgements

1 gratefully ackeowledge help and advice from my colleagues, Mr. c*. 13. Cartcr, Mr. K. L. Martin and Dr. D. W. Tempest.

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141 NI’AIIYE MIl’ltORlAL l’~l’ULAl’lOhY

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Bacterial Exo pol ysacchar ides

I. W . SUTHERLAND Department of Oeneral Microhiology, Univereily of Edinbwgh,

fldinburgh, Rcotland

1. Iiitroductinti . . 143 11. Production . . 146

A. In Growing Cultures . . 146 €3. In Wsehed Cell Suspensions . , 148

111. Properties . . 149 A. holetion . . 149 B. Purification . . 140 C. Composition . . 130 D. Struoturd FestureN . 156 E. Enzymic HydrdyNiH . . 168 k'. Btriicturrw of 8onw ~~xn~~~~lyatloclitrriclim . . 165

Iv. Jji<>RyilthOHiN . . lH5 A. Eiizyintw aid l'rwiiiwm . , In5 13. Cell-Fm SynthcmiR . . IHM C. Control . , 201

V. Function of ExopolyacMcharirloR . . 206

V111. AcknowlodgoinentR . . 20H RefctrenceR . . 20H

VJ. Uirenawerd Questions . . 206 VIJ, CoriclriaionR . . 207

I. Introduction

The bacterial cell resembles cells of higher organisms and of othcr micro-organisms in forming a number of polyseccharides. These are either components of cell structures, such RS the teichoic acidrs and lipopolysaccharides which form integral components of prokaryotic cell walls, or they may provide mechanisms for storing carbon or energy in polymeric form. In addition, numerous aclls8ynthesize polyntlcc.haridcs which lie outside the cell wall or are found secreted into the environmcnt. These polymem form the subject of thv pr(wnt Itrtiolo. lhq~!ndi~ig oti

their Rtructural roletionship to t h c t)wtoritd cell, thcy hrrvct hwn vILrioiwI,y termed dime, c:apRular or rnic*roacq)niilrtr rH)lynJi(:c:tirtriden. 'I'hv tiiLrri('

exopolysaccharide provide# u gcnorcll tcrrn for dl t h n c forrnn of' h h w i d polysaccharide found outwith the c c ~ l l wall and will t)c U H C ~ in thin context .

113

1.14 I . \ \ . Sl~ ' l ' l l l ~ ; l l l , . \N l l

BACTERIAL EXOPOLYSACCHARIDES 146

Bacterial exopolysaccharides have been the subject of numerous studies over a period of many years (see reviews by Wilkinson, 1958; Stacey and Barker, 1980; Luderitz et al., 1968). Some studies, such as those on D i p h w pneumoniae, have beeii primarily concerned with the role of the exspolysaccharide in bacterial virulence for the host animal and with the use of immunochemistry LLB a tool in the identification of strains. Other studies on soil bacteria have been more concerned with the possible role of the exopolysaccharide in soil fertility and in microbial survival. Despite much expenditure of effort, little is known about the in vivo role of these polymers and few attempts have been made to correlate the large amount of information obtained on their chemistry and biology.

In the laboratory, it is clear that exopolysaccharides are not eesentiel for bacterial growth and survival. Non-mucoid mutants, unable to form exopolysaccharides, are readily isolated. They occur spontaneously or aftem mutagenesis and can be readily recognized by their altered colonial appearance (Figs. la and b). In mucoid cultures, capsules or slime a n be removed physically or enzymically without adverse effect on bacterial growth. Further incubation l e d s to synthesis of new exopolysaccharide.

Because of their highly hydrophilic nature and their chemistry, most methods of staining exopolysaccharides are of only limited application. Capsules can be observed using light microscopy either in unstained preparations examined directly or by use of phase-contrast illumination. Exopolysaccharides are best detected by negative staining techniques. The India Ink method (Duguid, 1961) has the advantageof distinguishing between cells or cultures which form a discrete capsule (Fig. 2) and those which excrete an amorphous polysaccharide into the environment where it is observed in the form of slime, unattached to the bacteria. Use has also been made of the “Quellung” reaction, often wrongly termed the “capsule-swelling” technique. Tn this, deposition of a precipitate of

Fro. 2. Diagrammatic ropreseritatiorl of a bacterial cell with capule arid slime.

140 I. W. SII’PIIERLAVD

homologous antibody a t the periphery of the capsule permits its visual- ization on microscopic examinat ion (Cruickshank, 1966). It is also unfort~uiate that electron niicwscopy and freeze etching techniques are of limited application with regard to bacterial exopolysaccharide~. As a result, iittlc informntioti on thcir I~ons“i)lc ititeriiid strurt8iircbn in wsilsblr.

In cultures of cupsulrtte c*clln. rcotiw n l i n i c is obtained clue to its gradual release from the capsules. This niutcrial, like the slime obtained from non-capsulate mutants, is chemically indistinguishable from capsular material. Production of either capsules or dime is found in many species of Gram-positive and Gram-negative bacteria. It is assumed, in the absence of evidence to the cotitrary, that these polymers are physically identical and that polystlccharides produced under different growth techniques arc constant in six0 arid othcr properties. Whether this assumption is justified mus t await improved techniques and further experimentation.

The purpose of the present review is to discuss some of the results obtained from studies on the structure and synthesis of exopolysaccharides in order to present a model for their biosynthesis within the bacterial cell.

11. Production

Although most, but not all, muvoid bacteria produced some exopolysaccharidc under all cultural conditionrc, the growth environment is very important for riitlxi niul c’xol)olys}ic’cih~ri~(~ produ(:tion. The in- fiuence of environmriitul ronditionr; has hcen Htudicvl in nirrritwr of bacterial species using both growing rultirrcs snd ItItcrnutivc*ly, (mi-

ditions under which the cells can pwh”. polysac*charid(w hut do n o t grow. Most exopolysacrharide-synthesizing bacteria arc either aerobes or facultative anaerobrs, and polysaccharide production is normally highest when limitation of oxygen is not imposed. Perhaps, BN a result of this, more polymer is frequently excreted in solid media than is obtained from comparable amounts of cells grown in liquid media. Studies on Kkbsiella aerogenes (Wilkinson el al., 1855) established that the composition of the exopolyRaccharide was indepcndent of the carbon and energy source provided for growth and polymcr nynthcsiH. ThiR i H probably true for most if not i i l l t i c . t ~ c ~ r . o ~ ) o l y n ~ L ~ ~ ( ~ t i ~ ~ ~ i ( J ~ ~ ~ fout l t l ILH

hac4x*risl nlirncn and ~ I L ~ S I I ~ ( ~ H . Wticw, howcwr, tnow 1 hit11 o t i ( ’ c.xopoly- Hucchuride is IiJrrtid, wriatiotls i n 1 tw IWOl)(J ’ . t i ( J l lN of’ t h c b cliffi~rclrlt polytners Hyiittitwiz(d rrisy o ~ w i r . I I I cwritrwt, HyIlttlc.Hin of H. r i i i i r l twr o f


homopolysaccharidrs, such as leviins and dextrans, requires the provision of a specific. nubntriite, thus rc4ccbtJing the involvement of highly specific enzymcs tirt in& on oligon;ic.rliuridc cwbon soiirws.

In defined ti l tdii l , c?ropolyNllccliiiritfci prodii(4,ion w t k x st intiiltitrd by nutrient limittition in tlic 1irwciiw of PX(*(W cwrboliytIriit~(l (Ihigiiid r ~ n t l Wilkinson, I!K:I: Wilkinnon F / (11.. I ! M ) . I,iniitrit ion of' t Ii(t c ~ s i ~ l ~ o i t iind enrrgy ~oiirw r w i I I t w 1 i I I i n i i i i n i i i I 1 i( )Iysiicvl t tiritlrt 1 )n M I i t ( 4 io I I . I k- ticicncy of ititrogcw, I)ltospIioriiH, or n i i l ~ h u r w u r w s in tlw l ) r (wnc(~ of' etirbohytlratt., ;dl k d I o i n c w t i m d rxoI)olyNuocharitlc production by striiins of K. aero!~ewrs or Eschrrirhin coli until a maximum was reached. This vtLluc8 was not, vxcecded even when the rulturcs still rtpparc~ntly possessed sutficient cwbohydratc- arid oxygen to permit further synthesis. The maxiinrim value, as incasiircd by the polysaccharide : cell nitrogen ratio, depcndvd on the limiting nutrient. It was highest for phosphate lirnithon (48) and lowcr for nitrogen (29) m d sulphate (17) limitations. Deprivation of potassium limited growth in a manner similar to deprivation of other nutrients, but was much less cffective in stitnulating polysaccharide syiitheRis (Duguid and Wilkinson, 1954). This may have been due to restricted uptake of the cwbohydratc sub- Htrate, tts potassium limitation ulsoresults in cells with a very low content of intracellular glycogen even though the other growth conditions were expected to favour polymcrharide synthesis (Dicks and Tempest, 1967). The cause of the lowrrd syntherJiR of I)olysric,c.liaridc WUN attributed to an tintagonism hrtwccw pottLssiu in iiittl aim m o l t i t i in ions.

For K . ueroyrtws in licliiitl mcvli i i , t h r N I / P o f ' ~ ~ o l ~ v s t ~ ~ o h ~ r i t 1 ~ ~ 1)rotluction W ~ L R greatest during th(1 (bxponcntitd I i l i t w of' growth and gradually dwreased thcrcrLftc1r (I)ugrritl untl Wilkinson. i!Ni). ' l ' h f h maxirnal amount of expolysacchLtridc was, howcwr, ac~cu~nuliitt~tl aftchr c * c 4 multiplication hat1 c4Teectivcly ceased (aftcr 14 4x hr. growth). 'I 'h i f i wiis

also reflected in t l i c b incrcwed tliumc+v of' t tic. ( ' i l l)hli l(*s diiririg t t i c . lii1c.r

stngev of growth. Although IL high carbon :ititrogen ratio iri thc: m c d i i i r r i fbcourcd

exopolysac.charide production in niuc*oi t l strain8 of ( 'hronio/iac.ti,riun, violuceuni (C'orpe, 1961) in a niannw similar to that ohxcwwl for K . aerogenes, this was not true for cellu1oc;c ~)rodurtion by '4 wtobuctrr acetigeuunt (Du Iman. 1959). Both growth and cellulose synthwis w ( w only slightly affected by changing the glucose concentrations in a defined medium. Cellulose synt liesis was st i mu lat ed under these wndi t ions h y addition of acetate, citrate or surcinate. .-Ilthough supplementtition with ethanol increased the cell yield. it did not affect cellulose synthesis. Results obtained with on(' bactc.riiil species sliould only tw c*xtrnpolritc4 with care when c*onsideriiig othclr xtruinn, hiit t ti(& ~ P H I I O I I S V of' c.xopolJ,- sac*c.htLritlr Iwodiic*tioii t i ) /Zhi:obit(nt r / i d ; / o / i t o t I i ( * i i i t rog!c.ri c ~ o r r c ~ c ~ ~ r t ViLt i o t I

148 T, W. Rl~TllERLAND

of the medium resembled that of K. awogenes and E . coli (Dudman, 1964). Where the Rhizobiunc sp. showed a markedly different response from the enteric bacteria was in the effect, of aeration. Both growth and polysaccheride productioii were favoured, undor the conditions employed, by low ecrut4iou. I t is prohhlc; tliot3 siiiiilur cioiiditions, i.c. oarbohydrute ~ X C C B R cuid low wrnt ioii togctlivr with low cioiic:entrcitioti8 of nitrogrii, ciiv fouiid ill i n m y of thc iiciturd c!iivirotitiiciit,s in wliic!li production of large amouritr of cxopolysrtcchuride prcnent an effluent problem. The micro-organisms responsible for this are mainly strains of Sphaerotilus natane and related species.

In his studies using C. violaceurn, Corpe (1964) observed that the presence of Ca2' strongly stimulated polysrtccheride production. When Fez+ was omitted from the culture media, increased polysaccharide synthesis was detected despite decrease in bacterial growth. Both growth and polymer productioii required the presence of Mg2+ ions which could i i o t h i rr~~li i (vd by other divciletit, (bution8 wdi as Mil2 '

Studies on exopol~rsaccharide synthesk in nutrient media showed that the polymers continued to be excreted some time after growth and cell division had ceased. T t was thus logical to usc non-nutrient suspending liquids (washed suspciisions) to determine some of the parameters necessary for optimal polysaccharide production. Using K. aerogenes type 54 (strain A3 (Sl)), Wilkinson and Stark (lO50) obtained uniform produation of exopolysaccharide over a period of 4 hours in washed cell suspensions. Under optimal conditions, about 0.76% of the available carbohydrate was converted to cxopolysaccharide euch hour. A further 0.35% of the of the glucose was utilized to form intritcellular polysaccharide (glycogen). Such high vonversion ratcn were only obtained in aerated cell suspeiiNions with exww utilizable carbohydrate in the presence of K+, Mg2 ' and Ca2 +. The greatent decrcase in euch level8 followed the exclusion of oxygen or the ornienion of K I . Uning a sirnilar slime-forming mutant of K. oeroyenex, Norvul ( I !jfb!j) ohtainod nimilur results and also showrd that t h o rut(! of polyHltnohltridc HynthoHiH in washed cell suspeneion by log-phanc and ntationrtry-phunt! n c ? l l ~ grown in several different media was very similar (Fig. 5). Tho abilitg to synthcsiec! extracellular polysaccharide was lost' from older aulturen (48 hr.) in synthetio media and was very low in cells grown in mcxliu lacking rt

utilizable cerbohydratc:. Prodiiction of' (?X~Jp(~lyHU(:r:haritl(: WBH in&&- pendent of glycogen-H?.nt,henizing (!&phiIit.y, ILN Iog-phw (:(!I18 c:orituiric-tl little, if any, glycogc~~i ( K w ~ ~ ~ ( J w rl d., I ! M # ; Norvd, I w j ) ,

111. Properties ,I. ISOLATION

The isolation of cixoi)olysac.ciharide prrsents few problems when it is secreted as an extracellular slime. The lack of physical attachment between polysaccharide and cell enables differential centrifugation to be employed. The main problem i n such preparations tends to be the high viscosity of the slime solutions which hinders deposition of the cells. Different slime preparations vary greatly in their intrinsic viscosities and no firm rule8 for rrntrif‘iigal speeds required to achieve adequate separation can be given. Thv polymers are rrcovered from the super- natari t! fluids b y rttldition of acvtmc~ or c b t h snol . I f the cxopolysarcharide is i n t h r forni o S z L c ~ ~ ~ p s i i l ~ ~ it, t i i i i s t l w t l ( 4 i w I i c * t l froiii t h r c*rlls. Again it irs t l i f icwl t to gc~iic~ri~lim~, ILS ( L i L p s i i l w i w ’ r n i i ( * l i iiiorc rcwlily rrrnovtvl from somo striliiis thrLii froni o t h ~ s ( h i t I(* stirring or tnixitig in a hornogenizrr may sufice or morc (Irwti(* proc*cdiir(vi m r i y hrtvr to bc c1mploycd. Soiling has frequently brrn used, ti+ I l i ~ treatmrnt with rlilutc alkali. Such methods inevitablv lead to j)rotluc*tion of rt polymcv- cwntaining various contaminants arid some drgrti(1ation mrty ~ S { J o w u r .

R . ~ ’ C - I U F I ( * A T I ( J X

Tlic renioviil of’ ~~s t i~ i i i i r~us mattrr Srom c.xor)olysac:chtLriclo pr(’pt“i- tions, whether of’ dime or (liipstllilr origin, presentrr several prob1r:ms some of which are again due to the high viscosity of the polymer8 in aqueous solut~ions. The deproteinization technique of Sevag ( 1934) htm been widely applied and it’ tin be reasonably successful provided that dilute (<O-Fi%, w/v) solutions of the polysiiccharide are used. Fractional precipitation with organic solvents has been of only limited value and gives poor separation from other linear polyniers which may be present, These may incliide nwleir tt(id8 if the cells Iir~vr untlergonr lysis.

150 1. \V. SIITIIIRT~AND

II~eiiioviiI of' c h i t Iwr i i i iolr i t . il(*idPi or pwtci+i iPi j)rol)ihly b(*sfj cicwttiplishrrl by enzyiiiic digestion mid 11 twwsNioii of t.reutinents with dcoxyribo- nuclease, ribonuclease, try p i n slid proiiuse may be required. After mild heating to dcNtxoy the eiizyme~, the polysacchsrides arc recovoretl from t,hc saprrnutant fltiida itftcr Centrifugation. UNC (mi NO bc* rnstlt* of tho ability of acidic polysacohsrides to combine with quaternary ammonium salts (Scott, lQ66). The complexes thus formed precipitate and can be separated from soluble neutral material. Recovery of the polysaooharides is obtained in strong salt solutions. It is also possible to apply the phenol-water extraction procedure developed for the extraction of lipopolysaccharides from the cell walls of Gram-negative bacteria (Westphal et al., 1962). On addition of the crude material in aqueous Nolution to 90% (w/v) phcnol at 60" and subsequent centrifugation at O", the oxopolysmcharides are found in the upper aqueous phase of the biphasic system. Nucleic acids will also be found in this layer if they were present in the original mixture. An alternative approach is found in the use ofdimethyl sulphoxide, a dipolar aprotic solvent showing selective solubility for polysaccharidos. Seleotive absorption of either the polyNecchurides or of contsminatiiig material on ion-exchange absorbents has found relstivcly little application in the purification of bacterial oxopolysecoharides although it proved successful during the purification of polysaccharides from other source8 (Jermyn, 1962). I t waR NO employed by Guy el aE. (1967) to separate the capsular polystwchiwide of 1). pneurnoniae typo 1 from contsminsting C substance (mothw polysucoheride) and from polyglutamic acid. Use of a sodium chloride gradient in 0.1 Bf-sodium acetate (pH 7.0) with DEAE-Sephadex as absorbent, produced pure polysaccharide. On a micro scale, as in the study of polymer produced by cell-froe synthesis, precipitation with specific antisera provided an elegant method for recovering the p l y - saccharide formed (Smith et al., 1960).

c. C'OMPOSITION

1. 8 U g U T 8

Several different t y p of nugar midue have bmn detected in exo- polysaccharideH, but tho oommonctnt mono~uodhltride~ ltru undouhtcidly the ~OXONCA, ' D - ~ ~ I I C ~ N C , n-gltlactoHc!, and u-mnnnonc!. In cddition, t h o mcthylpcntosw fuco,se end rhumnosc hew frcrq trcintly hocm rq~ort(xl. Thcy provida an intcrenting cxltm plo of r,-iHom(m of HugltrH found in Nature, as opposed to tho more uniiltl D-formr;r. IA!NH commonly found have bcen the pentoses ribono, ltrubinonc and xylo~e, while tho 3,A- dideoxyhexoses appear to bc confined to thc 1i~)lJolynttccharidcs of Gram-negative bacterial cell walls (Liideritz et al., 1888b). AIHO normally

TABLE 1. Specific Enzymic Methods for the Aescty of Monoswcharidea Found in Bacterial Exopolysaccharidm

Enzyme Method Reference

D-GIUCOS~ Glucose osidave Colorimetric Hugget and Nixon (1957) galactose Galactose oxidam Colorimetric Amaral et al. (1966)

D -M&IUIOS~ Blannose isomerase Colorimetric for fructose Pderoni and Doudoroff (1956) L-Fucose Fucosn epirnerase Colorimetric for keto sugars L - R h u o s e Rhainnose isomerase Colorimetric for keto sugars Domagk and Zech (1966) D-Glucosamine Glucosamine phosphate Boetylase Colorimetric Liideritz et al. (1964) ~-Galt~tosamine Galactose oxidase Colorimetric Sempere et al. (1965)

Galactose dehydrogenase NAD reduction Wallenfels and K m (1966)

162 I. W. StlTffERLAND

abscnt appcar to be the lieptoses and octa-osrs spin foniid associated with 1ipopolysrLrchttrides. Howcvcr, exopolysacchtwidrs do contain both amino sugars itnd sugar acids, either separately or togctlicr in the same polymer. The nmirio sugars, in the S-acetyltited form, arc usu~lly D-glucosiminc, D-gidactosaminc or D-mnnnosaininr. A41thougli I)-

glucuronic a i d hits bccn most frtqueiitly nototl, i~-gcLliLCtiiroiii(: r~:id, D - ~ B I ~ I ~ I I ~ O I I ~ C iwid and ~,-giiIuronic acid liiivv rilso brcm found. ‘l’hcsc sugar acids coiltribute to the net nogative charge coininonly found in cxopolysacchrtrides. The role of uronic acids in this respect may also be filled by metliyluronic acids (Humphrey, 1959). It is certain that other sugars and sugar derivatives are components of these polymers, and that careful re-examination of many such preparations will reveal further monomers some of which may rescinble or be identical with t h r unusual amino sugars found during re-appraisal of some bacterial lipopolysaccharides (Volk et al., 1970; Liideritz et al., 1968b).

Most sugars in hydrolysatcs of exopolysaccharides have been identi- ficd iiiitiully by paper- or thin-layer chromatography. In many cases, providd witable solvcnt syrJtems wcrc used, these gave a satisfactory aiitl p:rliaps tho simplcst m(wts of idcntifiouQion. I t should br rcmcrri- berrd thikt tlicsc tochniqii(!s (lo not distinguiali bctwcvn D- and L- isomers. It is clcsiritblc to churactcrizc the sugars cithcr by preparation of suitublc deritatives or hy specific quantitative enzymic mcthoda. Many of these enzymic assays arc both oxtremely rapid und scnsitivc thus being very valuable in stm(1ic.s of oligomccharidcs where only small amounts of tlic produut muy bc avdablc. In some of the :tsseys (Table 1) the enzymic reaction can bc followed directly, but in others tlie spccific enzyme product i,s determined by non-spccific colorimetric procedures. It is unfortunate that, in many studies of polysaccharidc comI)osition, only group-spccific colorimetric trsts have bcen used. An alternative method of sugar characterization ttnd assay involves the preparation of trimethylsilyl ethers and the identification of thrse volatile compounds by gas-liquid chromatography.

2. Non-S’ugur Components

Two typcs of non-sugar componcnts arr found in bacterial cxopolysaccharide#, namcly organic and inorgtLnic*. ‘h orgcinic siibHtitii(mt8 are organic acid# or, in u fi:w ~Joly~ncc~li:Lrirlrs, mothyl groups. ‘ I ’h simplest of thew, and at thr mmr time t h o most recently tliscovcrtd, arc formyl residues. Thew liatl bwn dctcctcd cimlicr in t ho N-forrr1ybtc.d iLiitigr:ns isolated from f2ruceZlu melitensis (Milw u r d l’iric, I !wI) a,nd had U ~ H O been found UR purt of 11 glyaoprotein from ovorri i i c w i t l (Mtmttidl arid Neubcrger, I !IN)). In 1)cbct(!riid c~xoj~olyst ic~c~t~uri~~~!~, forrrititc: W:LN churao-


terized in the slime from a K . aerogenes type 54 strain but is now also known to occur in Iilebsiella type 2 exopolysaccharides (Sutherland, 1971b). The formyl residues in these polymers are attached to either a neutral sugar or to glucuronic acid, and the absence of amino sugars precludes N-formylation. Formate is therefore most probably linked as an ester, attached to one of thc sovcral available frco hydroxyl groups on a sugar residue. The presence of formyl residues confers characteristic chromatographic properties on oligosaccharides in which they are present, while having little effect on the electrophoretic mobility (Sutherland and Wilkinson, 1968, Sutherland, 1971b).

Acetate has long been known as a component of exopolysaccharides, and O-acetyl residues arc probably among the most widespread non- carbohydratc modifications found on ncutral or acidic sugars. They are

FIG. 4. Linkage of pyruvnte to glucoso as a ketal in a polysaccharide from Xanthornonua oampestrk. From Sloneker and Orentas (1062a).

found in polysaccharides from diverse organisms such as D. pneumoniae, Rhizobium raclicicolum, Azotobacter vinelandii and IC. aerogenes. As with O-formyl residues, acetylation affects the chromatographic mobility of oligosaccharides derived from the exopolysaccharides by enzymic hydrolyuis. They also cause sufficient alteration to their charge : mas8 ratio to retard them slightly when Hrrhjr~ctcd to p p c r ohtrophorcnis. In thoso polymers containing amino wgws, ,li-rwc:tyl group i m consistently found.

Yyruvato was first found in a I )~~l ,ys}~(~(~t~~r idc in agar (HiraHo, 1967) but was subsequently detected in capsular polysaccharidw from Xanthomonas campestris (Slonekcr and Orcntas, 1962a). The pyruvate in these polymers is linked as a ketal to glucose (Fig. 4). Thc carhoxylic acid group is thus free and contributes to tho overall charge on thc: polymer as well as being available for the possible binding of salts. Pyruvate is widely found in exopolysaccharides end has been found in many ofthe organiums in the polysaccharidcu of which acetate is present. Pyruvylgalactose appears to be the most frequently encountered residue, but pyruvylglucose has also been identified (Sloneker and

154 I . W. SUTIIlCllLhND

Orcntas, 19Wb). Unlikc 0-ucetyl groups which arc oxtrcmely labile to alkali and to acid treatment and are consequently never found in oligosaccharides derived from partial acid hydrolysis of thc polysaccharides, pyruvylated sugar residues are relatively stable to acid or alkaline hydrolysis. It is thus simpler to obtain pyruvylated sugars or pyruvylated oligosaccharides by partial acid hydrolysis (0.25 M H,SO, a t 100" for 20-30 min.) of the polymers. High yields of such derivatives may also be obtaincd by the autohydrolysis of the polysaccharide

TABLE 2. Properties of Pyruvylatod Sugars Isolated from Klebsiella Poly- saccharides

Mobility rolativo to glucuronic acid i i i

PyrrlvyllLtotl Pyridiniriin acotato : ltgla in Solvont

Typo Stralrl sllglw 13 C plf 5.3

1 1s 18 A 1 1803

2 22 8 8s

30 889 69 7824

1 w coli K12

Eecherkhia

Salmonella typhirnurium 395M2

Glucoso Galactoso Galactose Galactose Galactoso Galactose

Galactoso

Galactose

0.81 2.20

1.00 1.80 0.68 1.65 0.68 1.64 0.93 1.88 0.68 1.64 0.68 1.64

1.02 1-95

0.68 1-04

0.81

0.97 0.98 0.85 0.87 0.87 0.87

0.92

0.87

Solvent R : butan-1-01 :acetic acid : wator (4 : 1 :6, v/v/v). Solvont C : othyl mtato : acetic acid : formio acid :water (18 : 3 : 1 : 4, v/v/v/v).

solutions in the frce acid form for 10-16 hours a t 100'. A H wcllaH pyruvylated galactosc and glucose, pyruvylfueosc~ iu thought to oceur (I. W. Sutherlancl, unpublished results) in the polysacchti,rido of one Kleh~ic l ln scrotype. Pyruvyl sugars h a w high chromatogrephic mobilities and, on paper eloctrophoresis in pyridine-acetic acid bufferft, havc a mobility close to that of glucuronic or gulacturonic acid ('Fable 2). Thcsc proporties may have led to confuFiiori in the examination of partial acid hydrolysates of polymers in which pyruvate was not originally known to be present. The properties of pyruvylated oligosaccharides also differ considerably from the corresponding compounds from which pyruvate is absent (Table 3).

BACTERIAL EXOPOLYSACUHARIDES 155 The fourth type of non-carbohydrate organic substituent to be

detected was succinate. To date, this has been found in a single exopolysaccharide produced by a strain of Alcaligenes faecalis var. myxo- genes (Harada, 1965). The polymer contained glucose as the principal mgar along with smaller amounts of galactose (Misaki et al., 1969). Uronic acids and amino sugars were absent, so the polyrncr diffwcd from most othcrs in whidr nay1 siigiirs htivc bmn fountl i d which

Mobility relative to

Structuro Substi tuent pH 5.3) A B C

GAL -talc A +GAL - 0.64 0.13 0.19 0-08 Gal-+GlcA +Gal Pyruvate 0.87 0.73 0.28 0.34 Gal+GlcA+Gal-+Fuc Pyruvate 0.70 - 0.32 0.63 Gal+GlcA+Gal-+Fuc Pyruvate 0.04 - 0.09 0.42

t Glc

Glo+Gal-+Gal - 0 0.20 0.11 0.09 Glc+Gal +Gal Pyruvnte 0.41 0.28 0.23 0.16

Solvents : A. ethyl wetate : pyrirline : acotic wid :water (6 : 6: 1 : 3) ; 13. butsnol :acetic acid : watur (4 : 1 : 5 ) ; C . wetic acid :formic acid :othyl ncot.nto : w a h r (3 : 1 : 18: 4).

contain various charged siqprs, ‘I’hc: H u c v i r i i h : W:LH probnhly linkcd ILH an ester to free hydroxyl groups of glucosc: moictics. The succinyl groupn resembled O-acetyl groups in being labile to alkali (0.1 N NaOH at 70” for 2.5 hr.) and to mild acid treatment (Saito el al., 1970). Succinylated fragments were not isolated.

It is probable that other non-carbohydrate components will he found in exopolysaccharides, since Lindberg and his colleagues have recently shown that in colanic acid, a heteropolysaccharido secreted by a numbor of species of Enterobacteriaeeae, various non-sugar substituents were present. These included methylene and ethylidene groups identified by mass spectrometry (Garegg et al., 1971a, b).

3. Inorganic Substituents

Crude preparations of exopolysaccharides almost all contain a considerable amount of salts. It is sometimes difficult to determine whether

156 I. W. SUTHERLAND

these form an essential part of the polymer or whether they are non- specifically absorbed to it. Phosphate is certainly a component of one group of cnpsular polysaccharitles from D. pneumoniae. These are exemplified by the specific polysaccliaride of type XVIIIA Pneumo- coccus which contains D-galactose, D-glucose, rhamnose, iV-acetyl-D- glucosamine, glycerol and phosphate in the approximate molar ratio 2 : 3 : 6 : 1 : 1 : 1 (Heidrlbrrgcr ~t al., 1!!64). This type of polymer bears 81

resaniblanoo to tho teiahoic. acids for i t id i n tho wll w;tIls of I T I ~ U I ~ Cram- positiva hcterial sprics. The prcwnoc of other iriorpmio (:ompounds rctnains iinprovcn. 1 t dors S(WIII surprising that H U l f h ~ t C , which is Sound in a iirimbcr of polysaocti:iridcs tlc.rived froni the higher ulgue and from mammalian timues (l’rrcival and McDowoll, 1067), hits not so far been found in burtcrial exopolysucchariclcs.

I). STRUCTURAL FEATURES

A number of features define the structure of a polysaccharidc. These are : (i) the monosaccharides present ; (ii) the type of linkage by which each monosaccharide is joined to the adjacent sugar residues; (iii) the sequence of the oligosaccharides formed from the specifically linked monosaccharides ; (iv) the presrnce of non-carbohydrate substituents as estere or ketals or ester-linked phosphate ; and (v) the molecular weight. This combination of properties may provide a number of problems when attempts are made to elucidate the structure of bacterial exopolysaccharides. Structural studies arc also rendered difficult by the lack of well-charmt~rized cnzymrs !)rcdting down the cxopolysactcharides. It is thus mow difficult to d~~t (wninc whether rc:peating unit,s occur in such polymers and whcthcr vuriiitions occur within such units.

I . Mr4iL?Jlalion

One method for structural c1ctc:rmination rcquirc:s tho rnc4hyl:ttion of all free hydroxyl groups in t h e polymcr. The mcthyl:-ltc!d polys:tc*rhw ide is then hydrolysed and t h o mcthylr-lted Hugars identified, thc prrHctnae of free hydroxyl groups indicating the nature of tho glycosidic boridH in the native polysaccharide. From the identification of the methyl sugars and determination of the relative amounts, it should be possible to deduce the amount and type of the glycosidic linkages in the polysaccharide. Unfortunately, earlier work using this technique was affected by several problems which resulted in incomplete methylation and consequent wrong deductions as to polysaccharide structure. In particular, the frequent references to exopolysaccharides as “highly branched structures” derive from these artifacts and should be rcgarded with some caution. The problems in tho methylation of polymers

BACTERIAL EXOPOLY SACCHARIDES 167

containing nronic acid residues or amino sugars are much greater than in neutral polysaccharidcs which liiok thc highly acid-rcsistnnt aldobiouronic arid linkagcis. ‘l’lwsc~ (WI bi. ovcwonw by ConvrrNioii of the uronic actid rcsidncs to tho c*orrcsponding neutral sugrire (carboxyl reduction) prior to methylation. One of the major problems in the methylittion procedure, that of incomplete methylation, has been overcome through the development of an improved methylating agent by Hnkamori (1964). He employcd the strongly basic methylsulphymyl carbanion to give efficient alkoxidc formation in complex polysaccharides. The procedure was applied to studies on Klebsiella exopolysaccharides with much more satisfactory results than those obtained by earlier workers (Sandford and Conrad, 1966). The methylated polymer should then be hydrolywd under conditions where demethylation and destruc- tion of methylatcd sugars is minimal. A further development is seen in the formation of acetates from methylated sugars and their characterization through dombined gas-liquid chromatography and mass spectrometry. This method was initially applied to lipopolyaaccharides from bacterial cell walls (Bjorndal et al., 1967) but is also applicable to studies on exopolysaccharides.

2. Periodate Oxidation and Carboxyl Reduction I’criodatc oxidation can provide a considerable amount of information

about exopolysaecbaride structure and, when used under conditions where “over-oxidation” is prcven tcd, has been a valuable technique. It has also been followed by reduction and partial acid hydrolysis. Not all bacterial exopolysaccharides, however, possess structures which are oxidized by periodate (Barkcr et al., 1963a). Further information in uronic acid-containing polymrrs can be obtained by esterification of the carboxylic acid and subsequent reduction. The neutral polymer 80

obtained can then be examinrd without the problem8 associated with the acid-stable aldobiouronic acid. When the native polymer contained glucuronic acid, and glucose was already present, it can be distinguished from that formed on carboxyl reduction by the use of sodium borotritide as reductant (Sutherland, 1970b). This is equally true for other uronic acids and the corresponding neutral sugars. Determination of the specific activity of fragments obtained on partial acid hydrolysis then indicated their origin.

Partial acid hydrolysis has been widely applied to the study of exopolysaccharide structure. It has provided much useful information but suffers also from certain drawbacks. Where uronic acids or amino Hugam are present, the remltant glycosidic bondrr to the adjaccnt noutral- sugar residues resist hydrolytic condition8 which clcavc all othc!r glycosidic bond8 in tho polymer. This i~ crrpccially true when furanosidic

158 I. W. SUTIIERLAND

bonds im’ prmt.nt but is idso sccn in highly aritl-lihilc fucosidic, inmino- sidic or ribosidic linkages. If is thus difficult to dcvista hydrolysis conditions yiclding significant aniounts of oligost~ccharidcs larger thaii the aldobiouronic acids or thc corrcspoiiding amino sugar-containing disaccharidcs. A similar problem is seen when the D-galactopyranosyl 1+3 D-glucose configuration is present (Osborn et al., 1964). The yield of fragments from partial acid hydrolysatcs can however be increased considerably through usc of polystyrene sulphonic acid (Painter and Morgan, 1961) or through Controlled continuous removal of the oligosaccharides as they are formed (Galanos et al., 1969).

Biological mcthods of structural dctermination have been surprisingly slow to piin acceptance. This may in part bc due to the small numbcr of cnzymcs known to hydrolysc exopolysaccharides (see p. 159) and to the enormous volumc of work rieccHsary to obtain the basic information rcquirctf prior to t h c me of the imrnunoahemicul qproach. However thc piiinstiiking work of‘ Hrid(~1bcrgc~r and his collciqpcs is iccn in the incrriwingly frequent iiHc of irnrnunochemical mcthods as an adjunct t o other techniques in the dctermination of exopolysaccharidc structure (e.g. Heidelbcger el al., 1970).

E. ENZYMIC HYDROLYSIS

Relatively few enzymcs that hydrolyse exopolysaccharides have been isolated and fewer still have been characterized. One of the first examples cited was ari cnzynie obtained from a soil bacillus capable of growth on the capsular polysaccharide of D.pneumoniae type 111 (Dubos and Avery, 1931). The enzyme destroyed the capsular structure, and resulted in complete loss of the immunochemical activity of the polysaccharidc. It was however inactive against serologically related polysaccharides such as that from type VIII Pncumococcus. Subsequently a soil organism, to which the name Bacillus palustris was given, was found, depending on the polysaccharide used as inducer, to cxcrcte spccific crizymcs that hydrolyse either typc XI1 or t y p VLI I pneumocoacul p01ysacc:hi~r- ides (Shaw arid Bicklcx, 1950; Sicklox t m i Shaw, 1!4!jO; Campbr4 a r i d Pappenheirner, 1966). The bactcrid Htruin producing tbc r:nzymc:H WLLH subsequently identified as a strain of fj. circukuns (‘1’. Gibson, prHorial communication). A thorough investigrhtion of thcsc two cnzymc:x revealed that each wgs highly spcific both with rogard to its induction and to its substratc (Torriani and l’appenheimer, 1962). In both casex, induction only occurred when the substrate polymer or it8 hydrolyHiH products wcre added to the mcdium. Substrate activity did extcnd to chemically related substances such as oxidized cellulose in the case of the cnzymc active against type VIII polysaccharide. The mode of action


of the two enzymes was shown to differ, although each actcd L ~ H an exo- enzyme removing the repeating units from the polymer chain (Bccker and Pappenheimer, 1966). The enzyme active on type I11 pneumococcal polysaccharide was a hydrolase that splits the o-~-D-g~ucosy~ 1 -+4 bonds in the substratc. The xecond enzyme was a lyase or eliminclse that catalyses formation of products contiiiriing terminal 4,5 unsuturuted glucuronic acid residues. Them were recognized by their absorption maxima at 230 nm.

Another inducible polysaccharide-hydrolysing enzyme was obtained from an organism identified as K . aerogenes (Barker et al., 1964). The substrate was the capsular polysaccharide from type XIV D . pneumoniae. Initially the enzyme released galactose and a triwaccharide which was characterizod 8s O-~-D-glucopyranosyl-( 1 -A)-O-(%acet- arnido-2-deoxy-)-P-u-glucopyranosyl-( 1 -z 3)-n-galactopyr~tnos(i. Pro- longed cxposum of thc tristLctcharido to tho KZebsieZZu cclls Ird to induction of a P-ghico~idaw which rcmovctl the krminal non-roducing glucosyl rcsidue, releasing a disaccharide.

Another successful attempt to hydrolyse a bacterial exopolyxi~ccharidc with enzyme(s) from heterologous bactcria was reported by Lesley (1961). The polysaccharide from Xanthomonas phaseoli was degraded by an inducible enzyme formed by a Bacillus species isolated from soil. This enzyme resembled the enzyme active against D . pneumoniae type VIII polysaccharide (Hecker and Pappenheimer, 1966) in its mode of action. The products contained terminal 4,5 unsaturated glucuronic acid residues. The oligosaccharide obtaincd in highest yield was a trisaccharide composed of 4,5 unsaturated glucuronic acid, mannose and glucose which probably derived from the repeating unit of corresponding structure in the original polysaccharide. Thc climinasc type of action observed for this enzyme also resembled the mode of mtion of bacterial and other hyaluronidases examined by Linkcr rtnd his colleagues (1956) and by Ludowcig et al. (1961).

The rclativo lack of fiuccess in firding polysucahnrith! hytlrol;wcw from conventional xourws led to oxamination of anothcr I m x i h l ( * xystvm, namely phago-infected 1)aoteriLt. A crud(: cnzymc prvparation aspthlc o f removing capsules from K . p,neumoniae typc 1 wag obtainod hy Hum- phries (1948). A similar system for K . pneumoniae typc 2 wax examinod by Park (1956) and Adems and Park (1956). Although a c:onnidcrablo amount of knowledge relatin to enzyme synthosis and phage specificity was obtained, hydrolysis products were not characterized from eithcr polysaccharide. This was also true for some other systems such as those for Azotobacter sp. (Ehrlund and Wyss, 1962) in which phage-infected bacteria were a source of enzpmes affecting tho capsular or slime polysaccharides of the host bacteria. Some of these are listed in Table 4.

Ei

TABLE 4. Phage-induced Enzymes that Hydrolyse Exopolysaccharides

Bacteria Phage Products characterized Reference

Klebsiella priezononiae type 1 KMsiella pnctitiaotiim t>-pc 2 Klebsiella pteiinaoniac type 'I

h-kbsklla aerogenca t>pe 3-1 Eecheririiia coli \ Aerobacter cloacae J Awbbacter sp. Pseudomotins aerugiriosa Pseudotnottas pictida Alcaligenw fciecalis

I

- Hurnphries (1948) 3 Tetrasaccharide Watson (1966). C

-3 Sutherland (1972a) 3

F31, F39 Tetrasaccharidea. Octasaccharide Sutherland ( 1967) 5

ffi I<-P - Park (1956)

z

F1, F12 Hexasaccharide

A22 - 2 -

A6 TrisRec harides

Sutherland and Wilkinson (1963), Sutherland (1972b)

Eklund and Wyss (1962) Bartell et crl. (1966, 1968) Chakrabarty efd. (1967) Mare and Smit (1969)

z - "

BAOTERIAL EXOPOLYSACCHARIDEB 101

The Azotobacler system (Eklund and Wyss, 1902) produced phage- induced enzymes which caused a rapid drop in the viscosity of polysaccharide solutions. Although hydrolysis products were not isolated, certain characteristics of the enzyme system, such as pH optimum, were determined using viscosity measurcrnents. Experiments to determine whether the polysaccharide depolymerase was also associated with the phage particles indicated that only 0.1% of the enzyme produced by phage-infected bacteria was actually incorporated into the viral particles. Later work (Barker et al., 1968) showed that the enzymes induced by different phages infecting the same host bacteria had certain physical differences, indicating that their synthesis was under the

TABLE 5. Host Range of Bacteriophages that Iiiduce Synthesis of Polysscoharaees Acting on Colanic Acid

Growth on

&’scher&chia Aerobacter ooli cloacae

strains strains

Other Phage Originalhost 523 S53 S63C K12 Others 5020 Others

Escherichk coli F1 853 + + + + - f - F5,26,27 S53 + + + + - - - F12,13,14 5020 - - -

Aerobacter cloacae - - + -

+ indicates confluent lysis; -, no lyeia; f, variable response.

control of the phagc and not the bacterial gcnome. Liberation of reducing material by the depolymerascs from tho A. vinelandii polysaccharide was observed, and it wan recently reportd (Pikc and Wyuu, 1971) that despite the marked drop in the vi8conity of the ~)dysacohericlc Holutionu no noticeable decrease in the molcculrn Him of the Nubstrthj occurrod. Enzyme digests containcd fragmonts of difimn t uizn, rc:ur:mbling tho original polysaccharidc in their carbohydrate compoHition but lacking acetate. This suggest8 that thc: enzyrncu are t?xo-onzyrnw that rcwwva material from the cnds of the polymer cheinu. The known irrcgiilarity in the substrate molecules (see p. 179) mey account for the failure to produce higher yields of oligoseccharides, as tends to occur in tho hydrolysis of exopolysaccharides comprised of repeating units.

From earlier work in our own laboratory (Sutherland and Wilkinson, 1965), it was clear that, where a polysaccharide of similar composition was excreted by a number of different bacterial strains, there was no

I62 I. W. SUTIIERLAND

correlation between the ability of a phage to infect a bacterial strain and the ability of thc enzyme produced after phage infection to “depolymer- ize” the polysaccharide. Enzynic produced in one strain of bacterium could act on polysaccharids from ariothcr bactcrial strain which was phage resistant. Typical results for E. coli and other strains that produce the exopdysaccharide “colanic acid” are shown in Table 5 . Al- though hydrolysis products were not obtained, improved techniques have recently led to the isolation of the repeating unit from preparations of E . coli and other “coltmic acid” (rctutherlund, 1072b). The rc- pciitirig unit of this cxopolyeucchciridc is II cornplcx hoxamccharide of approxirncito molcculier weight 1100 (Suthcrland, I (369 ; Lawson et nl. , I !%a). Tho fuilurcx to isolntc oligosnccharidcs initially may havc? l w n tluo to L l i o Him ant1 corifigiirntioii of tht : hydrolysis prodiictH. Unlikc muny othw pdysucchnridt! hydrolascs including thoso for slime poiy- saccharidw from Klebsiella typo 54 (Sutherland, 1967) the colanic acid hydrolases only relcesed 30-35% of the polymer as oligosaccharides. This may be due to irregularities in the substrate structure. A t least two hydrolysis products have been identified from E. coli K12 colanic acid, one of which has the same structure as the repeating unit of the polymer. The enzymes are active against colanic acid from a number of different strains of E . coli, Salmonella typhimurium and Aerobacter cloacae although a number of variations in the non-carbohydrate components of colanic acid were later shown (Garegg et al., 197 la, b). De-acetylated polysaccharide prepared by mild alkali treatment was also a substrate, but no activity was obtained using carboxyl- reduced polysaccharide in which all the glucuronic acid residues were converted to glucose.

A phagr! which induced a capsule dcpolymcw~o for K . pneumoniae type 2 strains was studied by Watson (1!)66). rl’hc! cnzymo doetroyed the immunochemical activity of thc polysaccharidas. I‘urthcr examination of this enzyme (Sutherland, 19724 rt~veulcd that t h c IJOIY- succharidcs from t t largo number of type 2 Kleb.uiellf6 Htr;iinn wcro all hydrolyscd to thcir component tet,rilHucotiaridcs, As thv lirikatgc cleaved is : munnosyl-glucose, the crizyiiie is a mannosidaso. lhzyme activity was unaffected by the prescnce or absence of a numbcr of non-carbohydrate residues on the polysaccharides. Activity WUH not detectable against carboxyl-reduced type 2 Klebsiella polyssccharide or against a, number of other polysaccharides tested, including some such as Klebsiella serotypes 30 and 69 which show serological cross-reaction with type 2 material. (I. Orskov, personal communication). As with the E. coli system described above, there was no correlation between phage- sensitivity of the bacteria and susceptibility of the capsular polysaccharide to the phage-induced enzymc.

BACTERIAL EXOPOLYSACOHARIDES 163 Probably the phage-induced enzyme systems which proved most

useful in providing inforyation about their substrates were those active against K . aerogenes type 54 exopolysaccharide (Sutherland, 1967 ; Sutherland and Wilkinson, 1968). Two distinct enzyme types were found, exemplified by those induced by phages F31 and F39 respectively. The F39-induced enzymes hydrolysed the substrate into octasaccharide repeating units, each of which contained one acetyl group arid two other groups subsequently identified as formyl (Sutherland, 1970a). In

/J - a GLC 1 --+ 4 GLWA -II; 3 FUC I- I; i . PGLC

(i) Klebaiella Typo 54 polysaccharide

1 13 a-GLCUA

(ii) Klebaiella Type 2 polysaccoharide

U 1 B GLC 1 3GLCUA 1 - 3 F U C 1-

cAr, _I ( i i i ) Eacherichiu coli K27 poly~accharido

FIG. 6. Structures of suhstratos for Klebxiellu phagct F34-iriclucocl unzymo.

contrast, the F31-induced cnzymc hydrolysed the polymccharitlc to equal quantities of two tetrasacoharitles, onc of which was ucctylatcd. The enzyme was also active against the octassccharidc rcpcating unit. Both enzymes hydrolysed do-acetylated polysaccharide and polymcr from which both acetyl and formyl groups had been removed by acid treatment. As with the other phage-induced polysaccheride depolymer- ases, activity was not found using carboxyl-reduced polysaccharide. The discovery of an E . coEi exopolysaccharide with a structure showing some similarity to K . aerogenes type 54 slime provided a possible substrate for the F31- and F39-induced enzymes (Jann et d., 1968). Although oge of the glucose residues in the natural substrate had been replaced in

'


E . coli K27 material by gdactose, both enzymes were active in hydrolysing it (Suthcrland et al., 1970). The products were an acetylated tetrasaccharide and mi octasaccharidc, respectivcly. An extension of these studies (Sutherlilnd, 19728) showed that type 2 Klebsiella polysaccharides also acted as substrates. The two type 64 hydrolases thus

TABLE 6. Some Enzymes of Value in Determining Oligosaccharide Structure

EC No. Name Rpccificity

3.2.1.67 Liictnso 3.2.1.4. ~ 0 1 1 ~ 1 ~ 4

3.2.1.30 a-(:lucositlaRo' ActH o i i rnariy a-I) -gIycopyr(LriOXiC1OH. 3.2.1.21 p-(:lllcoHid~~c~~ Acts on inariy p-r,-gliicopyl'urioHidoR, but

activity iw lower when the tormind rcducing sugar is not a hexose. Commercial proparations are normally impure, containing a-giucosidaae and /l-galactowidrtse activity as well aa other glycosidaaes.

3.2.1.92 a-Galactosidaso H ydroly ses several a -D -galactop yranosides . 3.2.1.23 p-Galactosidaae" H y d r o l y ~ c ~ numerous /l-D-galaotopyrano-

3.2.1.24 a-Mannosidase Hydrolyses a-D-mannopyranosides. 3.2.1.25 8-Mannosidme Hydrolyses 33-u-mannopyranosides. 3.2.1.27 o?-l,3-Glucosidaae Acts on several a- 1,3-~-ghcosides. 3.2.1.31 p-Glucuronidase" Although this enzyme hydrolyses many 8-D-

glucuronides, commercial preparations froquently contain a-glucuronidwe and other glycosidases.

sides.

3.2.1.38 p-L-I"ucosidnHo Acts on P-t-fucosides. -- a-L-Fucosiclam bfydl OlYHOH Ct-r..fllMJHidf'H 1Jllt, dOpOrldin~ 011

t h Hourw, may uoritiiiri fJ-r,-fiiccJHiduHo tin t i hoxoHcrrm i rifdaxox.

- a-N-acetylglucoxnminitlu~~~ The prepartitions from limphi coritrriri othor

- 8-N-acctyl- ' I ' h l H onzyrrio, propnrc:d from vnrioun noiirccw, glucosaminida~es hydr01yHf:H iriuriy ~ - ~ - u C o t y ~ - D - ~ ~ l ~ c o H -

g1yCOHitlfLHCH.

aminidex.

a Preparations of these enzymes can be obtained commercially.

appear to have the lowest specificity of any of the enzymes of thiR type studied so far. Their activities are unaffected by the presence of acetyl, formyl or pyruvyl substituents, and they hydrolyse three distinct polysaocharide types (Fig. a), all apparently to complction. Although these enzymes can be classified as fucosidases, they can also hydrolyae mannoside bonds provided certain criteria are fulfilled. The L-fucoae

IIACTEI1IAL RSOl'OI,Y8AC(YIIAI11 I)RH 165

or D-mNIlI1ORC i w i d i i i l must, form piwt, of i b t i ILlclobioul'oiii(* nc:itl witJi 1)-glticuron ic wid :tnd thc! trrniiiinl vrdwiiig r(v$ltit* of tlw f'ii(*wido or mannoside shodti bc ~-glucosr.

The hydrolase enzymes, whether from bacterial sources or induced in bacteriophage-infected bacteria, provide a very useful tool in studies on bacterial exopolysaccharides. They enable isolation of fragments from the native polysaecharides by mild methods. Acetyl and other labile groups are not detached as they are by virtually all of the classical chemical techniques. The enzymes are thus extremely valuable in structural studies, and may yield fragments which can in turn be degraded by other hydrolytic enzymes. The polysaccharide hydrolases also provide a highly sensitive and specific assay method for use in biosynthetic studies on the polysaccharides. From the results outlined above, it seems that different types of enzyme exist, including hydrolases and eliminascs ; cndoenzymos and oxoenzymea. J t is 1)o"siblc that all types can bc found among cithcr tho buctwial or t h o baoteriophage- induced enzymca.

The products of tho polysaccharide hydrolescs and of partial wid hydrolysis can yield furthrr information on treatment with other glycosidases. A numbcr of terminal non-reducing sugars can he removed i n this way. It should bc noted that such glycosidascs show variations in their activity due to aglyconc specificity and they may also be inhibited by the presence of acyl or ketal substituents. Several commercial glycosidases are available and others can be isolated end purified in the laboratory (Table 6). Many of these enzymes remain impure and the inclusion of adequate control substrates is necessary if accurate results are to be obtained.

F. STRUCTURES OF SOME EXOPOLYSACCHARIDES Very many bacterial exopolysaccharides have been examined to

determine their qualitative and qnantitativc sugar composition. In many preparations the presencc of monort~Ilcchrtridc: in II simplc niolar ratio has ennhlrd authors to predict t h i t t th rb polymc-rrr c:ontA t i 11, rc!pnt,- ing unit whose Rim and corn])~~itiori (:;LII bc postulated. Ihwj)it,c: thirr, relatively few polysaccharidcx have bccri rigorously invc!xtigattxl with II view to completc elucidation of their structure. It i H seldorn pOHHihle to predict in advance what tho cnomposition, still ~ C R R thc structurc, of thc polysaccharide isolated from n particu1;tr bacterial species or Ntrain will be. But, i t is clear from structural dotcbrminations on a number of exopolysaccharides that they u c formed from repcating unitx of varying complexity. Recent results elucidating the mode of biosynthcsis of these polymers would seem to indicate that all heteropolysaccharides and


possibly even some homopolysaccharide which are found in bacterial slime and capsules may be constructed in this way. In the following pages, sonie examples are talten from those bacterial genera in which the complete or partial structures of a number of exopolysaccharide types have been elucidated.

All polysaccharides, including bacterial exopolysaccharides, may be divided into homopolysaccharides and heteropolysaccharides, i.e. into polymers composed of a single type of monomer unit, and those formed from several types of monomcr, respectively. Homopolysaccharides most widely studied have h e n cellnlosc cxcretcd by Acetobacter species and the lovruis and dcxtrans Hynthcsized from sucrosc arid similar substrates by Leuconostoc rnesenteroides and othcr bacterial species. Glucans from Agrobacterium species and other soil bacteria have also been studird (Putman et al., 1960; Gorin et al., 1961). Other homopolysaccharide dimes and capsules undoubtedly exist and further knowlcdge of their structure and synthcsis must await improved methods of analysis. By thoir very simplicity, being composed of a ningle uniform type of monomer, studies on homopolysaccharides present problems which are either absent when studying heteropolysaccharides or are more readily attacked with these polymers.

Most bacterial exopolysaccharides studied, whether in detail or merely by detection of the component sugars, are heteropolysaccharides. These polymers can vary greatly in their complexity. Some, such as the capsular polysaccharide of D. pneunioniae type 111, contain only two types of monosaccharide, D-glucose and D-glucuronic acid (Reeves and Goebel, 1941). The presence of five or six different sugars has been reported in some bacterial exopolysaccharides but it is possible that all of these may not have becn truly derived from capsulc or slime. Poly- saccharides containing tlirco or foiir diffi:rc*nt rnorio.q~~n~:liarid~!~ tLrc commonly found. 1 t in cchrt;iin that c:xopoly.qaoc:haridoR i n g:cwcw,l iirv much less complex than are the polyswchuridc c!ntitios of thc lipopolysaccharide or somatic antigen found in the ccll wall^ of most Uram- negative bacteria. In these, even the so-called “basal Ntructure” in tho genus Salmonella contains five different sugars, while a further thrcc or four different monosaccharides may form the 0-antigenic determining “side-chains” (Luderitz et al., 1968).

1. Structure of Homopolysnccharides

Determination of the structure of homopolysaccharidcs may often be more difficult that that of heteropolysaccharides due to thc presence of a single monomer unit. Bacterial cellulose, produccd by Acetobucter species, has received much study arid was identified as a linear polymer


of 8-glucosyl 1+4 glucose residues (Barclay et al., 1962). The polysaccharide chains have an average length of 600 glucose residues and closely resemble the product from green plants. The difference between the plant and bacterial polymers lies in their location in the cells in which they are synthesized. In bacteria, cellulose is an extracellular polymer and does not form part of the cell structure as in plant cells. Loss of the ability to synthesize cellulose does not adversely affect the bacteria.

Other extracellular homopolysaccharides which have been the subject of much study arc the levans and dextrans. Levans are produced by many plant-pathogenic bacteria of the genera Pseudomonas and Xantho- monm as wcll ihs the Grim-pouitive genus Bacillus and Streptomus mlivarius. I,OViLIIA iw(' pOlyfl'UCtOHtt8, oftrn with vcry high molecular wuights of onc niillion or greatcr, in which tho predominant linkage is @-D-fructosyl S+8 n-fructosc (Coopcr and Preston, 1935). The products from Bacillus spccies have chain lengths of 10-1 2 fructose residues and we extremely labile to acid hydrolysis being completely hydrolysed by 0.03 AT HCl at 60" in only 30 min. (Lyne et al., 1940). Dextrans are essentially linear chains of a-D-glucosyl 1 +6 D-glucose with branch points at positions 2, 3 or 4. Their formation as extracellular polymers is confined to a small number of Gram-positive bacteria including, Leuco- nostoc mesenterioides, L. dextranicum and Streptococcus viridans. The chain length shows considerable variation, some preparations having average chain length of 6-40 monosaccharides while others contain up to 660 glucose residues (Peat et al., 1939).

Examination of a polysaccharide excreted by Agrobacterium tumefaciens indicated that it consists mainly of 8-D-glUCOpYranOSyl 1+2 D-glucopyranose linkages (Putman et al., 1960). Further work on polysaccharides from a number of Agrobacterium species confirmed that they too are similar linear glucans (Gorin et al., 1961), but this type of 1+2 linked structure has not hrm widely found o u t k l a Ctw gonux Agro- bacterium.

2. 8tructurc of Heteropolysmcharides

a. Gram-negative bacteria '

i. Escherichia coli. The large number of fitrains which are clarJwified aa E. coliare differentiated on the basisof the lipopolysaccharide (somatic) antigens, found in the cell walls. Within these many serotypes, two forms of eftopolysaccharide production were recognized. A large number of strains, together with strains of most Salmonella species and Aerobacter cloacae, are capable, under snitable conditions, of excreting an extracellular slime to which the name colanic acid was given (Uoebel, 1963; Anderson and Rogers, 1963; Grant et al., 1969). This polymer will be


considercd separtbtely. i n addition, a minority of E’. coli strains arc capsulate and may be recognized as siich by negative staining techniques. For diagnostic purposes, the capsulate strains of E. coli are subdivided into three groups termed A, B and L (Kauffmann and Vahlne, 1946). Of these the B and L types form very small capsules which could not be discerned using the light microscope except after growth under special cultural condition8 (Orskov, 1956) but, could bo idcntificd by swologicvd mcthods. ‘I’hc A t,ypr forrncd hrgw (VL~AIIIOR vir;liblc by liglit microscoy ty .

Jhc:scherich,in, coli typc 27. Tho ctaitlic: ItolyHacctiaridc from thiR strain contains four sugars, namely u-gluauronic acid, n-gluco~c, n-galactoso and L-fucose in oquimolar proportions (Jann et al., 1968). O-Acetyl groups were also detected. Acid hydrolysis yielded a number of charged oligosaccharides but no neutral oligosaccharides. On the basis of methylation, periodate oxidation and other data, a tetrasaccharide repeating unit was suggested but the site of the O-acetyl groups was not determined. The native polyeaccheride has the repeating unit :

-+(GIc 1 + 3 GlcA 1 3 3 Fuo)-

It has a high molecular weight which is decreased to about 10,000 by alkali treatment. This led to the suggestion that chains of about 140 repeating units are linkcd through ester bonds between the hydroxyl groups of monosaccharides and thc rarboxyl groups of glucuronio acid residues.

Escheriohia coli typc 30. Anothcr E. coli c:npHultv polymcohiuklc W ~ L H also an mid macromolcculc whicbli oontnincxl i)-glucuronio acid, 1)-

galactose and a-mannoso in tho molrir rtitio I : I : 1 (Hicngwwr PI al., 1967). Although acetate was present, thc molt~r ratio W ~ L H ICHH thitri oiiv, indicating either that not cvery r c p t i n g iinit in ncc4yhtc:d or thii t H o r n ( ! of the labile O-acetyl groups wcrc lofit during iHolrLtion r ~ r i t l piiriHc:ation of the polysaccharidc. The repeating un i t provcd to be a tri~acchariclc :

B [+ 3 Man 1 -P 2 GlcA 1 -+ 3 Gal 1-1-

It was suggested that the polysaccharide is formed from lincar subunits and has a molecular weight of about 150,000. Becausc of tho lability of the mannosidic bonds in the polymer and the relative stability of the aldobiouronic acid 3-O-~-D-gl~lcuronosy~-D-g&~actosc, partial acid hydrolysates of type 30 material were unusual in yielding a singlc product, the aldobiouronic acid. No other oligosaccharides were isolated.

E . coli type 42. Another simple trisaccharide repeating unit was found in type 42 E . coliexopolysaccharide (I?rskov et aE., 1963 ; Jann eta,!., 1965).

BACTERIAL EXOPOLYSACCHARIDES 1“

This polymer contains galacturonic arid, galactose and fucosca. 011 the basin of chemical studies on the polymer, a repeating unit structure was deduced :

As well as sugars, 0-acetyl groups (6.4% of the dry weight) are present in this polysaccharidc. Probably because of the structure of the aldo- biouroiiic acid, serological cross-rcnction with D. pneumonine type I capsular polysnccharidc occurred (Heidelberger et ul., 1968).

These three E . coli capsular polysaccharides, all of the A-type, thus have fairly simple repeating units composed of 3-4 monosaccharides and all are acetylated. Much more complicated polymers were found when two of the B-type polysaccharides were examined. E . coli type 85 was found to be an acid macromolecule containing glucuronic acid, mannose, N-acctylglucosamine and rhamnose in a molar ratio of 1 : 2 : 1 : 1 (Junn et al., 1966). A number of oligosaccharides were obtained by partial acid hydrolysis and, on the basis of their structure and other data, a pentanncoharide repeating unit was proposod :

4 3 Gel 1 -+ 3 GlcA 1 -+ 2 FUO 1-1-

--+ 2 or 4 [GlcA 1 -+ 2 or 0 Man 1 -+ 3 Man 1 --* 3 GIcNAc]- I

Rhe,

Later work (Heidelbcrger et al., 1968) indicated that the structure might be even more complcx and thus account for a very strong cross-reaction with antisera to type 11 and type V D . pneumoniae polysaccharides.

Another E . coli exopolysaccharide of unknown type contains rhamnose, galactose, glucosamine, glycerol and phosphate, but these components are also thought to be present in the cell-wall lipopolysaccharide of the same strain (Jann et al., 1970). It was suggested that a similar structure exists in both the lipopolysaccharide and theexopolysaccharide. Whether this is indeed so, or whether the “exopolysaccharide” represents an extracellular secretion of incomplete lipopolysaccharide, remains to be established. “Colanic acid”. It was long thought that a polysaccharide is produced

which is common to many species in the bacterial family Entero- bactcriaceae. Thc initial serological observations of Kauffmann (1936) were confirmcd and cxtcndrd by Hcnrikscn (1949, 1950). Further work (Anderson, 1901 ; Anderson w d ICogvrH, l!jCI) indiaatotl thtt t many &rains of ”almonelh or Escherichia could form a dime polyHaccharidc when cultured under suitablc conditions. It was aho known that mucoid mutants could be obtained from E’. coli Htrain K12 (Beiwr and lhviH, 1967). A similar polysaccharide was isolated from another E . coli strain along with a type-specific capsular antigen (Idrskov et a,?., 1063) which waa serologically and chemically distinct. Ooebcl (1963) applied the name “colanic acid” to the slime and observed that it contains 30-32% fucose,


33-34% galactose, 16-17% glucose and 17-20% glucuronic acid. It was also confirmed that antigenically similar material could be isolated from mucoid mutants of E . coli K12 (Sapelli and Goebel, 1964). Material of the same chemotype was obtained from Aerobacter cloacae strains and, along with other colanic acid preparations, scrved as substrates for a group of phagsinduced enzymes (Sutherland and Wilkinson, 1965). The control of colanic acid synthesis has been the subject of a number of studies (e.g. Kang and Markovitz, 1967; Grant et aZ., 1969) which showed that, with the exception of strains which have a defect in a gene coding for an enzyme responsible for synthesis of a portion of the

Pyruvntr Gul

' I f 1 4

7: C k A

Gal Acetyl I I I , t 4 a

j. B

__.+ 3 CIC 1 ~ F w 1 - ~ F U C 1- or 3

FIG. 0. Structure of colanic acid.

molecule, all h!. coli K12 strains j ) o w w t h o grmhtic ability to synthcsizt: colanic acid. Although strains wort normcilly non-mucoitl, undor Nuitahlc conditions polysaecharitlc synthrds was int1uc:cd in 11 umcrous Salmonella "species" und E . coli strairiA. In 1111 ('BXOH the po1yrnt.r proved to contuin the same monosaccharidca and aotcd ns u substriLt(? for phagc-induccrl enzymcs. Despite the large number of studics (JII colunic acid, its structuro was for long neglected. Partid acid hydrolysis of tllo Id, coli caI)sulnr polymer yielded two charged oligosaccharides, onc of which was idcntified as the aldobiouronic acid, /3-n-glucuronosyll--dl n-galactose (lloden and Markovitz, 1966). Re-examination xhowcd that three preparations from E . coli, A . cloacae and S. typhimurium each contain the four sugar8 already mentioned, together with acctate and pyruvate (Sutherland, 1969). The presence of the polysaccharjde constituents in un approximato molar ratio of fucose :gnlactosc : glucuronic acid : acetate : pyruvnte, 2 : 2 : 1 : 1 : 1 : 1, indicated the possibility of hexasaccharide repeating

BACTERIAL EKOPOLYSACCNAHIDES 171 unit which ww both acetylnted niid pgnivylated. MetJ~ylation ; ~ i i d htisr- catalysed fragmentation indicatod that pynivcitc! W ~ R prcsciit as t i

ketal forming 4,6-0( 1’-carboxycthylidciie)-~-galactoae (Lawsoti et al., 1969). The overall results confirmed that the basic structure was a hexasaccharide (Fig. 6) which was constructed as a trisaccharide branch on a trisaccharide portion of a linear chain composed of residues of glucose and fucose. The position of tho acetyl group remained am- biguous despite the recent isolation of the repeating unit by enzymic hydrolysis (Sutherland, 1972b). Further studies showed that, although the carbohydrate composition of colanic acid from a number of E . coli and S. typhimurium strains is constant, variations occurred among the non-carbohydrate constituents (Garegg et al., 1971a, b). Thus, one strain of Salmonella produced a polysaccharide lacking acetate, while other preparations were acetylated on the non-branching fucose residue. The greatest variations were in the substituents on the terminal galactose residue. In two mucoid mutants of S. typhimurium of common parentage, tho polysaccharidcs contain a 3,4-O-c?thylidene group and a 3,4-0-carboxycthylidcne group respectively. In all, six out of nine proparation8 tcsted contain a 4,0-0- carboxyethylidene group, while one polysaccliaride is unusual in containing a methylene group linked to positions 4 and 6. The various structures detected are shown in Fig. 7.

From these results, it is clear that, at least in the strains examined, it is possible for a number of dissimilar bacterial strains to synthesize an exopolysaccharide of similar if not identical carbohydrate structure. The polymer does, however, show considerable variation in the non- carbohydrate residues present, both in regard to the nature of these residues and to their position on the carbohydrate cntitiss. It is unfortunate that, so far, extensive studies have not been made on the modifications to polysaccharides from strains of common ancestry.

ii. Klebeiella aerogenes and K . pneumoniae. All strains of Klebsiella are mucoid, and in many cases large amounts of capsular polysaccharides are formed. Like the polymers from the closely related E. coli several of the structures have been determined, and it has been shown that, unlike most other bacterial general, the type of extracellular polymcr is remarkably uniform. Few contain morc than three sugars (Table 7) and those in which the structure has been completely determined ~ O H H C B H tetrasaccharide repeating units.

Type 1. The monosaccharides in this cxpopolysaccharidc were identified as fucose, glucose and glucuronic acid (Erikmn arid Hcnrikson, 1959) but more recent work has shown that pyruvate is also present (W. F. Dudman, unpublished work). The polymer is unusually susceptible to alkali and is unaffected by periodate (Barker et al., 1903a, b).


This was interpreted 11s boing due to t lw I'rcdoriiins1lc:c~ of ( 1 t9) linlcngchs bctwccn thr. 8 U p ' H . 0 1 1 0 O h I ' V t L t i O l i Which appear8 U!lIl?iUid Wt1s that some glucuronic acid was n h s e d on mild acid hydrolysis (0.01 N H,SO, at 80" for 30 min.). This would seem to indicate that a t least some of the glucuronic acid in the polymer occurs linkcd in a manner different from the normally acid-resistun t uldobiouronic acids. Altcvxiativcly , if

I I1

H

I11 IV

FIG. 7. Ketals attached to the terminal reducing galactow rusiducs of colanic acids from different Atrains of Eacherichia coli and A'almnnella typhimurdum. From Oaregg et al. (1071a, b).

electrophoretic separation was used, a pyruvylated fragment might have been mistaken for glucuronic acid. A number of oligosaccharides have been isolated from the polymer produced by strain A 1, by partial acid hydrolysis and by autohydrolysis (I. W. Sutherland, unpublished results). Several of these were found to contain pyruvate but their complete structures have not yet been determined.

Type 2. The monomer constitucnts of this serotype were shown to be mannose, glucose and glucuronic acid (Barker et aZ., 1958). On the basis of methylation, periodate oxidation and oligosaccharide sequcnccH, it

BACTERIAL EXOPOLYSAOCHARIDES 173

WHR suggrated that tho exoI’olysacchiirido c:ontiLins IL Ittrgr “rqwciting i ini t” aontdning tibout 40 monoscirrlitiridcs. I 3 y c!oiitrt~st~, oxnminutioii

Type Monosaccharides References

1 * 2 3 4 5 8 9

11

Fucose, glucose, glucuronic acid Mannose, glucose, glucuronic acid Mannose, galactose, galaoturonic acid Mannose, galactose, glucose, uronic acid Mannose, galactose, glucose, uronic acid Galactose, glucose, glucuronic acid Rhamnose, galactose, glucuronic acid Mannose, galactose, glucose, glucuronic acid

21 Mannose, galactose, uronic acid 23 Rhamnose, glucose, glucuronic acid 26 29 Mannose, galactose, uronic acid 30

Mannose, galactose. glucose, uronic acid

Mannose, galactose, glucoso, wonk acid

32 46 47 52 54 57 61 64 69

Rhamnose, galactose Rhamnose, glucose, glucuronic acid Rhamnose, galactose, glucuronic acid Rhsmnose, galactose, gliicitronic acid Fucose, glucose, glucuronic acid Mannose, galactose, uronic acid Galactose, glucose, glucuronic acid Rhamnose, mannose, glucow, glucuronic acid Mannose, glucose, galactose, glucuronic acid

71 Rhamnose, glucose, glucuronic acid 72 Rhamnose, glucose, glucuronic acid

~

Barker et al. (19634 Gahan et d. (1967) Henriksen and Eriksen (1962) Eriksen (1962) Henriksen et al. (1961) Dudman and Wilkinson (1956) Heidelbergor et al. (1970) Nimmich (1969) ; Eriksen and

Eriksen and Henriksen (1063) Nimmich (1960) Dudman and Wilkinson (1056) Dudman and Wilkinson (1956) I. W. Sutherland (unpublished

Heidelberger el al. (1970) Nimmich ( 1960) Heidelberger et al. (1970) Hsidolberger et al. (1970) Dudman and Wilkinson (1956) Dudman and Wilkinson (1966) Nimmich (1960) Barker et al. (1 068) I. W. Sutherland (unpubliahed

Nimmich (1069) Nimmich (1069)

Henriksen (1963)

data)

data)

of material from four different strains showed that each contained a relatively simple tetrasacharide repeating unit (Gahan et al., 1967) :

B a B [+ 4 Glc + 3 Clc I +4 Man 1-1

t 3

The availability of a phage-iriduccd onzymc ( WatRon, 1966) dcpolymer- izing type 2 Klebsiella polysaccharides permitted a further study of their structures. A preliminary examination (Sutherland, 197 lb) Rhowcd that different polysaccharide preparations contained acetate, formatc and, in one case, pyruvate. All acted as substrates for the phage-induced


enzyines as well as for similar enzymes from type 54 Klebsiella strains. The hydrolysis products differed, depending on the polysaccharide composition but, in each cam, a tetrasaccharide with the samc carbohydrate strucfurc as the repeating unit of the polysaccharide was isolated These oligosaccharides were either formylated, acetylated or pyruvylated and their properties varied with the different substituents. All the type 2 polysaccharides contttincd formato, but three distinct typos wcrc distinguished, namcly (i) containing formatc only ; (ii) containing formate and acetate, and (iii) containing firmate and pyruvate. The prcparations in the first group appeared to posscss formate uniformly distributed on the polymer, as a single formylated tetrasaccharide was found in enzymic digests. The second group of type 2 exopolysaccharides were also uniformly acetylated and formylated, as enzymic hydrolysis yielded only one tetrasaccharidc! composed of the same monomers in the same molar ratio as the polysaccharide. The single polymer which contained formete and pyruvate was unusual in that enzymic hydrolysis produced equal amounts of two tetrasaccharides, one of which was formylated and the other pyruvylated. The formylated oligosaccharide was indistinguishable from that isolated from the other type 2 polysaccharides. It is therefore probable that the polymer is composed of tetrasaccharide repeating units which are alternately formylated and pyruvylated. There is some similurit'y to a K . aerogenes type 64 strain in which alternate tetrasaccharides are acetylated (Sutherland and Wilkinson, 1968).

Type 3. The polysaccharides from six type 3 Klebsiella strains all contain mannose, galactose and galacturonic acid (Eriksen, 1965a). Although the complctc structures wcw not elucidated, the re~ults indicated that the polymtw probably contain a relativcly Himple rcpeating unit in which thcrc is an aldobiouronic acid containing galacturonic acid and mannose. The polymers from type 3 strainrJ classified as K . ozaenae, K . pneumoniae, K . rhinoscleromcllis and lr'. aerogenes proved to be serologically indistinguishable (Eriksen, 1 O65b).

Type 8. The component sugars of a type 8 exopolysaccharidc wcrc identified as glucose, galactose and glucuronic acid in tho molar ratio 1 :2 : 1 (Dudman and Wilkinson, 1056). On the basis of pcriodate oxidation, carboxyl reduction and partial acid hydrolysis, a tetrasaccharide repeating unit was proposed (Sutherland, 1970b) :

-[D-Gal 1 -+ 3 D-Gel 1 --c 3 Glc 1-1- t

D-GlcA

Acetyl, formyl or pyruvyl residues were not found in the preparation examined. Recent studies (I. W. Sutherland, unpublished work) on ~1

number of other Klebsialla type 8 strains showed that the exopoly-

BAOTBRIAL EXOPOLYSACOHARIDES 175 saccharide from one contained acetate and pyruvate; the others were devoid of acyl groups. The pyruvylated sugar was galactose and, as it could be obtained by mild acid hydrolysis, it was concluded that it did not form part of the aldobiouronic acid. From the chromstographic mobility of the carbowethylidene galactose in a number of solvents (Sutherland, 1971a) it appeared to be identical with 3,4( 1’-carboxy- ctliylidciiegalnctose). If tliis obscrvtbtion is c:orrc!ct, it irnpliw that thc carbohydrate structure of tho polyst~collrwitlc must diffcr slightly from that previously reported for K . aerogenes type 8 (strain A4) (Sutherland, 1970b) in at least some of thc glycosidic linkages.

Klebsiella aerogenes type 64. Studies using the A3 strain of this serotype showed that the slime and capsular polysaccharides have the same chemical composition, which is unaffected by the carbon source in the growth medium (Wilkinson et al., 1955; Aspinall et al., 1956). It was thought to be a highly branched polymer containing 10% fucose, 27% glucuronic acid and 46% glucose. A re-examination of the polymer by Sandford and Conrad (1966) using improved methylation and other procedures indicated that fucose, glucose and glucuronic acid were present in the simple molar ratio 1 : 2 : 1. The presence of a tetrasaccharide

repeating unit was later confirmed (Conrad ~t al., 1966). This structurc was complicated by the prosence of acetate first indicated from serological properties of the polysaccharidos (Davies et al., 1958) and later confirmed by analysis (Sutherland, 1967 ; Sutherland and Wilkinson, 1968), and of formate (Sutherland, 1970b). Enzymic hydrolysis produced equal quantities of two tetrasaccharides, both of which contained formate and one of which was acetylated. The isolation and characterization of an octasaccharide which could be further hydrolyscd to the t w o tetrnsaccharides indicated that every alternate tetramccharidc rcpeating unit is acetylated and that formibte is present uniformly on c t d i

tetrasaccharide. Although the positions of the acyl group8 were not identified, the isolation of trisaccharides (Fig. 8) showed that they are not attached to the terminal non-reducing glucose residues. Examination of the products of enzymic hydrolysis of several other type 54 exopolysaccharides (I. W. Sutherland, unpublishcd results) showed that all of the polysaccharides are formylated. Acetate is absent from some of the polysaccharides and is present on all the tctrasaccharides of other preparations. Pyruvate does not appear to bc present in this uerotype.


Another K . nerogenes cxopolysaccharides of nnknown serotype was recently shown to contain a tetrasaccharide repeating unit of the same general form as those found in other capsular preparations from this genus (Yurewicz et al., 1971 ; Troy el al., 1971). Thc polymer contitin8 D-galactose, D-maniios(? cud n-glu(?ironic acid in tho niolnr ru th 2 : I : 1 . As with other prepartttion~ ~tudic~tl, tho polyHnrrtiu,ridc c o n ~ i s t ~ ( ~ ~ ( v i -

P3H Enzyme I Qlo-QlcUA-Fuc-(~lc-GIrlJA-For *T' )

I / /

Qlo Glc

F38 Enzymc

NnOH QIc-GIcUA-FUC +--- / /

ale Glc

Cellulase J Cellulnso I Acetyl

I

NnO1[ I Ulc-QlcUA-Fuc - Clc-ClcUA--Fuc

FIG. 8. Courso of hydrolysis of acotylatod fractions from Klebsiella A3 poly- sancherids (typo 64). From Suthurlniicl nntl WdkinHon ( 1 968). All oligosaocharitlcm later provod to bo formylatetl.

tially of a linear chain to which siiiglc monomer rcsiduw arc uttachc*rJ. Tho structure W ~ R apparently free from ucyl substitucnts :

-[-Gal 1 3 3 Mart 1 -+ 3 Gal]-

I T' fi-Gl~A

A number of Klebsiella polysaccharidcs were found to contain galsc- tose, mannose, glucose and a uronic acid (Table 7). I n some preparations, the uronic acid was identified as D-glucuronic acid. Types 30 and 69 are known to cross-react with serotype 2 (1. Orskov, personal communication) but the chemical structures on which the serological cross-reactions arc dependent are not known. Neither of those two exopolysaccharides is hydrolysed by the type 2 polysaccharide depolymcruse (1. W. Sutlrc*r- land, unpublished results).

BAUTBRIAL EXOPOLYSAOOHARIDES 177

It is clear that from the results obtained with serotypes 2, 8 and 54, as with colanic acid, numerous strains can synthesize a polysaccharide with the same carbohydrate structure. The non-carbohydrate substituents, such as acetate, formate and pyruvate, are however dependent on the strain examined, and show a considerable degree of variation. The exopolysaccharides of the genus Klebsiella which have received detailed study to datc are remarkable since they a11 conform to a uniform general pattern, usually a tetrasaccharide repeating unit from which a trisaccharide forms a linear molecule with a side chain consisting of a single carbohydrate residue. The number and nature of the monomer constituents are also much more limited than in other bacterial genera which have received comparable study.

iii. Miscellaneous bacterial exopolysacchuridee from Gram-negative ape- cim. Despite the increasing knowledge of the structure of bacterial exopolysaccharides, it is still not possible to predict their physical properties. Very few exopolysaccharides, other than the dextran group, have been examined with aview to determining their actual or potential commercial use. A few polymers from diversc bacterial genera are now being investigated to determine their suitability as replacements or substitutes for the traditional industrial polysaccharides suoh &B starch and alginates. As yet, insufficient is known about their structure and physical chemistry to enable the production of tailor-made polymers for a, particular purpose as can be done with dextrans and starches, but some knowledge of their structures is now available.

Arthrobacter v iscww. One of the few bacterial exopolysaccharides known to possess gelling, as opposed to paeudo-gelling, properties, is the polymer from A . viscosus strain NRRL B-1973. Solutions were shown to form a gel after autoclaving and cooling (Cadmus et al., 1963). A t certain concentrations, various salts promoted gel formation and the viscosity of the solutions was unaltered by heating in the presence of these salts. Despite these unusual properties, the chemical composition of the polysaccharide shows no unusual features. The components found were D-glucose, D-galactose and D-mannuronic acid in equimolar amounts (Jeanes et al., 1965). As well tw sugars, the polymer contains 25% acetate. Thus, almost two-thirds of the available free hydroxyl groups possess O-acetyl groups, a higher proportion than in any similar polymer so far examined. The only monomer in common with other gel- forming polysaccharides is D-mannuronic acid which is a major constituent of alginates, but the possibility of other common propertieu such as specific glycosidic linkages has not yet been reported.

Xanthomonas campestris. A second polysaccharide with gel characteristics resembling those of sodium alginate or other commercial plant gums was isolated from cultures of X . campestris strain NRRL B-1469

178 I. W. SUTIIEIELAND

by Jeanes et al. (1961). It resembles that of A . viscosus in the atypical resistance of its viscosity in solutions to heat and to salt effects. The viscosity of solutions is enhanced by monovalent cations at neutral pH values. Although 0-acetyl groups are present, their removal does not affect these properties. The polysaccharide contains n-glucose, D-man- nom, n-glucnronic acid, ncrtcito iind pyriivats in t h o iq)proxirnntm molor ratio 3.H :3*0 : 2.0 : 1.7 : O * O (Slorrcblwr t i n t 1 , J C ~ I I ( I H , llfH2). (:rsdrd acid hydrolysis rrleased one molc of moniiosc and eventually several charged oligosaccharides, the major one being an aldobiouronic acid 2-0-8-D- glucuronosyl-D-mannose. Smaller amounts of a trisaccharide and a tetrasaccharide were also isolated. These both contain the aldobiouronic acid together with one and two moles of glucose, respectively. Furthcr studies (Sloneker et al., 1964) using periodate oxidation and other techniques led to the suggestion that the polysaccharide consists of a structure with a repeating unit of 16 monomer residues. Most of the polymer was thought to be a linear molecule of glucuronic acid, mannose and glucose residues to which were attachcd other mannose residues, 0-acetyl group and the pyruvate ketal. If this structure is correct, i t indicates the possible involvement of a highly complex biosynthetic system.

Azotobacter species. Azotobacter vinelandii normally produces exopolysaccharides in laboratory culture, but shows some variability as to whether discrete capsules or slime are produced. The polymers were thought to contain various sugars depending on the strain studied. Cohcn and Johnstone (1964) rcportcrl tho presonac of glucose, rhttmnose iind gulacturoriic acid us well a8 u laatonc rwembling mannuronolactonc in somc of its properties. The galucturoriic acid was the major component of the polymer, a8 identified by colorimetric away. Acetate was also found in several preparations. An unconfirmed report (&us, 1985) suggests that polysaccharides froni several A . vinelandii strains contain rhamnose and a keto acid, which was reactive in the thiobarbituratc assay. It was identified as 2-keto-3-dcoxy-galactonic acid on the basis of its chromatographic behaviour, oxidation and reduction produrhs. It was preferentially releascd from the polyfmchtbride on mild acid hydrolysis.

Several exopolysaccharides from strains of A . agile, A . vinelarulii and A . chroococcum have also been examined. It was suggcstcd that, unlike most other bacterial exopolysaccharides, their composition varics with the culture medium, in particular with regard to the prcscnce of fructose in the polymers (Zaitpeva et al., 1959). Glucose is present in all preparations as a major component, while rhamnose has also been detected, Although traces of other sugars were identified on the basis of their chromatographic mobilities, the possible prcsencc of uronic acid8 W~LR

BACTERIAL EXOPOLYYACCIIARIDPB 179

apparently not considered. It i R , howcvcr, probablo that thcsc polymers twc similar to those describcd by Cohcn and Johnstonc (1964). An extracellular polysaccharide from A . i d i c a (Beijerinckia indica) was examined by Quinnell et al. (1957) and was initially thought to contain n-glucose, D-glucuronic acid and a hcptose in tho molar ratio 3 : 2 : 1. Ltrter the polymer was subjcotcd to mcthylirtion m d periodatc oxidation, and it apparcntly contains a tristiccharidc rcpcating unit composed of glucuronic acid, glucose and D-glycero-D-mannoheptose (Parikh and Jones, 1963) :

-[-0-~-QlaA (1 + 3) O-D-QlC (1 -+ 2) O-D-glyoero-D-m&nnoheptose-l]-

A further preparation studied by Haug and Larsen (1970) resembled this A . indim polysaccharide in containing the same neutral sugars, but lacked D-glucuronic acid, which was replaced by L-guluronic acid, identified by its optical rotation and other properties. Acetate was also present.

The exopolysaccharide from another A . vinelandii strain differs from those described earlier from this bacterial species by its close similarity to alginic acid (Gorin and Spcnccr, 1966). It is a polyuronide in which D- mannuronic acid is the major component with lesser amounts of L- guluronic acid and acetate. The kinetics of periodate uptake were very similar to those for alginic acid from Laminaria species. The mannurono- syl residues are linked togcther through the 4-position and the only appreciable diffcrcnce from tho a lp1 polymer is the higher mannuronic acid: guluronic acid ratio and thc presence of acetate. These particular results werc cxtcndcd by the report from Larsen and Haug (1971) on alginate-like polysaccharides from thrce furthcr strains of A . vinelandii. A fractionation procedure was adopted to separate the uronic acid- containing polysaccharide from polymeric material which contains glucose and protein. The ratio of mannuronic acid to guluronic acid in all three preparations was very similar (about 0.56), while the acetyl content was calculated as one per five uronic acid residues. When partial acid hydrolysis was applied in a manner analagous to that wed for seawecd alginate, it appeared that each polymer contains sequencee of each uronic acid, i.e. sequences of contiguous D-mannuronic acid residues separated from similar L-guluronic acid sequences by portions of the molccule in which the two types of monomer alternated. Thcse structures are vcry similar to those described for othcr alginatc preparations, although minor differences between the three Azotobacter exopolysaccharides wcrc observed. In view of these results, some of the earlier reports of othcr monomer constituents in the cxopolymccharides from Azotobacter, species may have been due to dcgradntion products or to other artctfacts and should be re-evaluatcd. Anothcr important feibturc of the Azo- tobucter alginate is thc charactcrimtion of an exopolysaccherido which

180 I, W. SUTliEHLAND

apparently is not fornwd from idriitird blocks of rcy)~wtiiig units but which is a liiirar molrculc with differing stxuat,urr in diflrrcwt portioiw of the polymcr. Associatrd with this fcitturc wihs tho isolatjion of an extracellular enzyme capable of modifying the polysacchwide structurc (Haug and Larsen, 1971). The iniplications of this are considcred with other work on exopolysaccharide biosynthcsis (p. 198).

Pseudomonas species. The production of polymers resembling alginic acid is not confined $0 a single bactcrial genus. Linker and Jonrs (1964, 1966) isolated a mucoid Gram-negative bacterium from a patient with aystic fibrosis and identified it as a Pseudomonas species. The polysaccharide hydrolysate rcscmbled hydrolysatw of alginic acid, containing marinuronic acid as the major component, together with guluronic acid and the two corresponding lactones. Infrared spectra of the polysaccharide closely resembled those obtained from sodium alginate and both polymers are susceptible to an inducible “alginase” prepared from a yellow-pigmented bacterial strain. Cornpariaon with similar preparations from other mucoid Pseudomonas isolates revealed one polymer containing mannuronic acid only, and others in which the molar ratio of mannuronic acid to guluronic acid ranged from 4 : 1 to 20 : 1, respectively. In a commercial sodium alginate, tho ratio was 5 : 2. The initial preparation mcthod used alkaline extraction with consequent 10~s of 0-acrtyl groups. Omission of this preparativc Rtsge gavo a product containing 9-11 YO acetate, which was much morc resistant to digestion with the bacterial alginase.

A further examination of mucoid Ps. aeruginosa strains (Carlson and Matthews, 1966) confirmed that the exopolysaccharidcs secreted by these bacteria are polyuronic acids in which the principal monomer component is D-mannuronic acid. Varying amounts of 1,-guluronicacid weredetectcd. The identities of the uronic ctcids wcre further confirmed by caterification, reduction and characterization of the methyl esters. Unlike tho production of “colanic acid” by various species of Enterobacteriaceae most of which are sufficiently closely related to permit genetic compatibility, “alginic acid” production apprars to be a f(?nturc? of bacterial genera which are not a t present considcred to be closely related. ThuH, the dissimilarity of the genera Azotobacter and Psuedomonas irJ indicatcd by their respective DNA G-C proportions of 56and 64%. By cornparifion, the bacterial strains which excrete colanic acid all b c h g to t t group with 50-66% G + C in thcir DNA.

6. Gram- Positive Uncleria

Diplococcus pneunaoniae (I’neumococc~i,r). Although a numbcr of goncrct of Gram-positive bacteria produce cxopolysaccharides, few have been


extensively studied. Due mainly to their medical importance, and to the pioneering studies of Griffith (1928) on bacterial transformation, a considerable amount of information about pneumococcal polysaccharides has been obtained. The polysaccharides contain a much wider range of monosaccharides and other components, and are more varied in structure than the Klebsiella group, although so far the structures of only a few serotypes have been completely established.

Type I. The capsular polysnccharide of this strain was one of the first such polymers to receive a considerable degree of chemical and immunochemical investigation. Both 0-acetyl and D-galacturonic acid residues are present and immunologically important (Avery and Goebel, 1933; Hoidelbergcr, 1902). Further Rtudiw ((1 uy et al., 1 067) idcntified both gluoosttminc uiid gttluc:tommiii(h ILH ~)oly~t~oaht~ridt! compori(wt,r. The preHciiw of i ~ - g I i ~ ~ : o ~ c , c!cirlic~ tctiitt~tivc!ly id(bntificd, w t t ~ confirmod, but ~ L ~ I ~ L O ~ O ~ C wtts not found. Although tho c:ornplote structure of 11. pneumonine type I exopolysaccharido h t t ~ not yet bcen elucidated, part of the structure is considered to be a trisaccharide sequence :

-[-D-galacturonosyl-( 1 -+ 3)-glucosaminyi-( 1 -+ 3) galtacturonosyl-]-

Type 11. This exopolyseccharide contains L-rhamnose (48%), D-

glucose (35%) and D-glucuronic acid (16%) (Barker et aZ., 1965). From methylation studies, it was cclncluded that the L-rhamnose is linked through positions 1 and 3, while the n-glucuronic acid is linked at positions 1 and 4 as well as occurring as the non-reducing terminal residue. The D-glucose is involved in 1, 4, 6 branch points. A 8-linked trisaccharide, isolated by enzymic hydrolysis, contained only L-rhamnose. The application of sequential enzyme induction revealed further facets of the structure not detected by earlier studies (Barker et al., 1967). The trisaccharide sequence : P-L-Rha (1+3) B-L-Rha (1 +3) a-L-Rha, was attached to the remainder of the exopolysaccharide molecule by an a (1+4) linkage. On the basis of these and other rosults, a complex repeating unit structure wax PONtultttCd for the polymer (h’ig. !)).

Type IZI. This polymer possc~nse~ the simplest known rcpcating unit found in a bactcrial extracallular hcteropolysttccharide. In (L HoricH of studies, Goebel and his colleagues (Hotchkisg and Chobel, 1937 ; Adams et al., 1941; Reeves and Uoebel, 1941) found that the polymer is composed of D-glucose and D-glucuronic acid, forming a disaccharide repeating unit which is a polymer of cellobiuronic acid and is, as already mentioned, the substrate for a hydrolase produced by a Bacillus species :

Type V. The capsulgr mpterial of this serotypc proved to contain a more complex pattern of sugars. Acid hydrolysis of the purified polymer released D-glucose, p-glqcuropic scid and two amino sugars, narnely

t[-3-8-D- QlcA (1 -+ 4) fl-D-GlC-]I-

I


L-fucosamine (2-amino-2,6 dideoxy-L-galactose) and L-pneuniosamine (2-amino 2,6 dideoxy-L-talose) (Barker et al., 1960). Although acetate was present, this formed part of iV-acetylamino sugars and not 0- acetyl groups. By the use of methylntion studies and periodate oxidation, some structural details were determined by Barker et al. (1966b). A disaccharide repeating unit of 3-O-~-glucuronosyl-N-acetyl-~-fucosamine was found, to which were attached residiies of D-glucose and N-acetyl-L-pneumosamine.

FIG. Q. Structure of the repeating unit of the polynaccharitlo from Diplococcw, pneurnoniae type 2. From Barker et nl. (1967).

Diplococcuspneumoniae type VI polysaccharide is onc of a group which contains ribitol phosphate. Unlike many other such polymers, thc only other sugars present were tho nciitrd ~ C X O H ( : H J~-gillconc tmrl r)- galactoRe and the methylpcntoso r,-rhiimti(~~c (Jtchcrs r d Hctirlr!lh~:rgc~r, 1969, 1961). On the basis of chornioibl atliLIYH(!H n r i d othcr tctc:hnicj i i~, IL simple tetrasaccharide repeating unit WLLH ~~ropo~(’:d :

-42-0-a-~-&d (l+ J) .o-a-~-Glc (1 -P 3) O.a-T,-Ilha (1 -+8)-1Cibitol 1 or 2 4 ) . I 3 . ( J 1-

0 ONa / \

Types X I A and X V I I I . Exopolymccharidcs of them serotypcs are among several which contain glycerol phofiphatc residucs (Shabarova

I ~ ~ ~ ~ ' I ' K I t I A 1 ~ K \ ~ l l ' O l , Y S l ( V ' I l A l t l IlIfS I N3

el nl., IN;?) i ~ i i d tliiis h i ~ r LL suprrlicitd rcweniblsiiw tm thr tcic*lioic* twitls which are of widcspreitd occurrence in the cell walls of Gram-positive bacteria. Type X IA exopolysaecharide cwiitniirx in addition. n-glucose. D-galtIrtOSe and iwatate (Kentidy ~t nl. . I !)(in). 'rht~ ruolar proportions of

A partial strncture for the polymer, based on the results of periodate oxidation and alkaline hydrolysis. is

D-glUCOSC, D-g.dW!tOst', gly WrOl, 1)hORPhiLtC iLlld iL ate are 2 : 2 : 1 : 1 : 2.

-[-:W-u-Gal (1 4) n-Glc ( I -b 6) D-Glc ( I -> 4) n-Gal-]-

P 4

3

glycrrril

J n it aerie8 of studies 011 wpsular material from U . pneumoniae type XVIII (Markowitz and Heidelberger, 1954; Estrada-Parra rt nl., 1962; Estrada-Parra mid Heidelberger, 19f13), a sugar composition showing some similarities to that of type XIA was noted. D-Ghirose, u-galartose, L-rhaninose and glycerol phosphate wcre detected along with 0-awtyl groups. Treatment with alkali removtld the glycerol phosphate but left the sugars intact. As a result, it was concluded that the polymer contains a linear chain of neutral sugars to one of which is attached the glycerol phosphate. On the basis of the chc>mic+iil i d immunochemical studies, the main chain of the polysacdiaricle fforn 1,. pnmmoniue type XVIIJ may cwnsist> of one of two possible* xtriicturw :

-[.3-1)-Gtd ( 1 - > 4) u-D-UIC ( I -> O ) . ~ - c l l c ( 1 -+ J)-r.-lthu. ( I -+ 4)-n-(>lo 1.1- or

- [ - ~ - D - G I I I ( 1 -+ 4)-U-Ok ( I +3 ) -~ -Hha ( 1 - > 4) Or-D-Uk ( I -+ 6 ) - ~ - 0 1 ~ I-]--

Although the sugars and othw romponmts present in u number of other I). pwunionine exopolysac~charidrs have hwn dc4ermincd (Table 8). in most other serotypes no attempt has beeii mad(* to dc+mninc what repeating units, if any, are present in the polymers. I t in dear that thcwc eapsular polysarcharides are exceedingly heterogenoun with rcsgard to t,heir nionomer components. containing a number of sugars arid other substances not previously found in exopolysaccharides. Care Hhould probably be taken in assessing some of the results, an contamination of capsular pol~~saccharide preparation8 with other polymers has bcen observed and a cell-wall polysaccharide from 1). pneurnoniae (Pneumo- coccal C polysaccharide) is known to contain among other subntanccH S-acetylgalectosRmin~ phosphatcl ((:ottsc.hlich and I i u , I !)A7). large volu r r i ~ of tlatiJ, on t h o irnrriiirioc~ltc~rnic.ul rchtionHllips of j)rl('tJrno-

(w*mI I)ol,vsiLc'c'ti:LritltIs h i ~ x h ( ~ t i ;tinuswtl. This W ~ L H wvicbwcvl t)y tic+]pl- l)cbrg(~ ( I !)fro) wlio sltowetl t l i d 1 1 i c ~ r . c ~ w(w* wrologiwd c+rosx-r'cwt ioti1.I betweeii tlifferont U . pneumoniw p01yiiit~rs uncl witti a r i u rri hcr of' ottitar

8

TABLE 8. Cuiiiponeiits of Some Expolywcharides from Diplococeua ptiercmon iae

Serotype _ _ ~

Reference

Heidelberger (1960) Barker et al. ( 1966b) Adaina et al. (1941) Barker et al. (1966~1, b) Rebers aiid Heidelberger (1961)

Heidelberger and Tyler (1961)

Rao and Heidelberger (1966) Heidelberger (1960) Estrada-Parra et al. (1962) Miigazaki aiid Tadomae (1971) Roy et al. (1970) Mills and Smith (1962) Robcrts et al. (1966)

- 4

- - Tyler and Heidelberger (1968) - Heidelberger (1960) 1

T: f: - z - -

IV. Biosynthesis

A. ENZYMES A N D I ’ R E C ~ I ~ I ~ S O ~ ~ S

The importjanrc of sugar nuc~leotides as glycwidc donors for the syn- thcXis of riiost polysuccliarides uiid glycan-containing molecules has bcwi known for nitany years. ‘I’hc first of these compounds tlo tw isolated and (.tiartr(.ti.i,izt.tl by (hpiitto rt (11. ( I!)%)) w i ~ s U1)I’-gluoosc~. Along witfli niutiy othw su(h cwniporinds i t h;rs since becn shown to function in , imong other systems, exoi)olys;toclittridt~ syntht4s. As ~ ~ 1 1 us their. direct rolr in providing sugrrrs for trtrnsft~ to form polymers, the sugar nuoleotitlcs provicle a mechanism for synthesis of particular types of monosaccharides. This is seen in the eonversion of UDP-glucose to UDP-galartose by UDY-galactose-4-epimerase and in the formation of GDP-fucose from GDP-mannose by the two enzymes of the GDP- fucose synthetase complex. Many of thege reactions are involved in the provision of sugars for other polymers in the cell, but the action of UDP- glucose deliydrogenase i n the fornirktion of glucuronic acid is normally

IJ I ) l’-yliiconr tlvh ytlroytwuuv lII~P-~luroHc~ .-* UDP-glururonir wid

2 NAD 2 NADH2

speciticully nssoc~itrtrd wit 11 i t s prov isioi i for rxoI)olvsckcc.lruridc.H. A rompreherisive list, of wgar i iu(~lt~otid~s arid ttwir isolatiori froin hac.trrial (und other) cells (’an be fourid i n t,hc rcvicbw

I t is obvious that, in provision of thc: j)rwursors for ~~olysi ic~c~~iar id~~ formation, u considerable number of (wzymes arc involved. Sornc: of thcsc are concerned primarily with the catabolism of monosaccharide substrates but are equally important in the early reactions leading to sugar nucleotide synthesis. A scheme for the enzymes leading from a carbohydrate substrate to the synthesis of the exopolysaccharide of K. aerogenes type 8 (Fig. 10) shows that, out of a probable total of at least ten enzymes, six are specific t o the processes of capsule formation.

The enzymes responsible for synthesis of sugar nucleotidcs involved in capsule production were studied in several strains of 1). pncumoniac! (Smith el al., 1967, 1959). Even i n non-capsulutc strains, high levels of sugar nucleotides were detected, indicating that control was probably exerted a t an early stage in pwursor formation tirid not, at tho sugar- transferuse level. I)efccts in cwzyrrws rw~)orisith for sirgar-riuc:Ieotitl~~

(iinsl)iirg ( i ! ) fbt ) .

t - 1.11'11)

H 0 7 n

(;lr-fl-P

l1 (Ilurow

FIU. 10. Pathway for syiithesis of thc. capiilar polynacclirir.rclrt ~ l ' h'h?/mkl&i nero- gene8 AS. The enzymes listtd iricliarle swwttl ( I 4) which mi! irivolwtl 111 ~ir(:r;ur~rii* formation for snverd different polgrnrw. 'l'ht: rcrnaintlor arc9 irivolvcd ~ 0 1 t . l ~ in oxopolysmoharide synthesis.

BACTERIAL EXOPOLY BACCl CABID E8 187

namely hexokinase, phosphoglucomutase and UDP-glucose pyrophosphorylase, are required to produce UDP-glucose. On the other hand, fucose, a frequent component of bacterial exopolysaccharides, is transferred from GDP-fucose. ' This necessitates the formation of GDP- mannose, involving two enzymes of the GDY-fucose synthetase complex, as well as prior stages in the conversion of glucose to mannose. Most of the potential glycosyl donors and the systems by which they tire Hyn- thrsizcrl ILrv wrll rcwgliizcd m d fiirtJic*r dc4ttiIs (:a11 br foiincl in the rovivw I)y (iinxhiirg ( I l ) t i l ) . Somo gtipx i n our kriowletlgc still remain, as iiiiclcotidc s i i g ~ s contltbining w v w d of the amino ~ i i g i i r ~ recbently disc*ovc*rcd il l I ) . pnvu?r/o/tinfJ i)olyxucc~hrLritl(~s hiivcb not! yct him itionti- fiod.

?'lie sugar nurlrotidcs day u d u d rolc i i i c1xoi)olysacoharidc synthesis, as in the forniation of other glycan-containing polymers. They provide a mechanism for sugar interconversion. This is seen in the formation of galactose and glucuronic acid as their UDP derivatives from glucose as UDP-glucose. Similarly, L-fucose is formed from D-mannose through the mediation of the GDP-liiiked sugars (Ginsburg, 1960). The second role of the sugar nucleotides is that of activating the monosaccharides. The simple sugars cannot be transferred directly to acceptors during polymer synthesis. The nucleotide diphospho sugars with relatively high (nege- tive) free energy of hydrolysis provide suitably activated monosaccharides. The free energy of hydrolysis of UDP-glucose is -7600 calories, and other sugar nucleotides probably give similar values. The only exopolysaccharides which are apparently not formed from activated glyoosyl donors w e levans and dextrans. Their synthesis requires the utilization of siicrosc or r thtcd oli~oN&~~:cih~iridcN from which part of the rnolwulc (friictoscs fi)r I(W;I I IH i~,rid glirwsr~ for tloxt r iu iN) ix polyrncvid. 0thc.r c*xtrncdlul;ir Iiorrrol)olyn;ic.~~liilri~i~~~ (sera 1). I HH) oonfimi to t lw stan(lart1 psttwii. t i t ilizing ~ i i p l r iiiwlwticlw 11s t Iw F,I ,YC*OHY~ t loriorn.

In the same way that several of t h o cwzymw of carbotiytlratc rncbtaho- lism are essential for exopolysacchsride formation, the involvornctnt of isoprenoid lipids or glycosyl carrier lipid dcmonntratcd hy 'I'roy ~1 al. (1971) extends the range and number of enzymes which must ttc considered. The C, s isoprenoid alcohol bactoprcnol, which contains on(* saturated isoprene residue, was identified in iJactohncilluR mwi ('l'horric! and Kodicek, 1966) where it was the major biosynthetic product frorn mevalonic acid. Recently complete synthesis of metabolically aotivtt glycoqd carrier lipid from d '-isopentenyl pyrophosphate and fttrneHy1 pyrophosphate was achieved (Chrietenson P t al., 1969). Particulate enzyme preparations from Salmonella newington catalysed synthesis of a C, , isoprenoid alcohol phosphate containing 1 1 unsaturated isoprene residues. A soluble enzyme system yielded lipids of shorter chain length.

188 1. \V. SIITIIEItLANI)

Whether the bartoprrnol inolated from 1,. r m r i Hervrti tlw H t m w function in, vivo as glycosyl carrier lipid is not clear. Cytologic!al studies (Barker and Thorne, 1970) indirated that it was almost all associatcd with the sphaeroplast mrmbrime. ‘I‘hiB would br the cxperted site for synthesis of polgrntw found ou twi th t IN* c v l l m w n h n o . I riaiifWcntt is yr t known dboiit, t,lw synthwin ofglyc*o~.yl cwric~r lipid t w l I)trc~tq)rcvrol to htoriiiiii(~ wlrc~tlrvr cvizy t r i ic . dc+v*tx in t h i r nyiithrwis I d h l , n i i t l what, wwndary (+fwt,s o w u r on polymcr forniutiori. (hntlitional mutants Hhould provide information ofthe effect of altercd synthcsis of glycosyl carrier lipid on polysacwharide formation. We bclieve that a group of mutants (see p. 20.5) showing temperature-sensitive (low temperature restrictive) polysarcharide synthesis fall within thiR ratrgory (Norval arid Suther- I t d , 1 W)).

AR well an sugar nucleotideH ant1 glyc:osyl currier lipid, a third type of p r w m o r must be considered for exopolysaccharide Hynthesis. These are required for the acyl substituentn commonly present. It is not yet clear what these precursors are. Formate might be added from a tetrahydro- folate compounds or from formyl-Coil. Similarly, two possible donors for acetate are acetyl phosphate and acetyl CoA. The latter is more probably involved as it is known to be the precursor of 0-acetyl groups found in the lipopolysaccharide of 8. newington (Keller, 196.5). Transfer of acetate from acetyl-CoA to oligosaccharides from K. aerogenes type 64 exopolysaccharide was also noted (Siitherland and Wilkinson, 1968). The rnost, probable donor for pyruvthb is phosphoenolpyruvate. Although polytiicw w i t h t h c b HUI~( ! (~t~i’t)oliytlr~~tc~ t&ruotiw but different acyl group titLv(1 k i i Htiidicd, only on(* c~xrmplt! h r m t ~ t w rqJortod where the I)rwtwid stxairirr W(TV of (‘on1 nion tLnc*(axtr,y. I t i l h w o N t m i n H , chungrn i t r t tw wyl group prtwwt oii c+olrLnic+ wid from A’. t,t/phirnurium wort! observed, one polymer being twetylated and the other not (Garegg el al., 1971a). Thus B defect in the acctylafle may Atill permit synthesis of a non-acetylated polymer. Whethcr thig i H also true for other polysaccharides and for other acyl groups has still be be extlrninc!d.

B. CELL FREE S Y N T H E ~ ~ H

Several attempt8 have been made to study the synthesk of exoply- saccharides by cell fractions. The earlier work on these aspects provided some information, but a real stimulus was obtained from the systematic studies on heteropolysaccharide synthesis by Mills and Smith (1 962) and their colleagues using preparations from D. pneumoniae. The value of specific methods of assay, particularly immuiiochemical and related methods, wag recognized during thwe Htudies and also in work on lipopolysaccharide syntheni8, hut h w riot proved widdy applicable i n

BAOTEHIAL EXOPOLY RACCH A M 1 1 ES 189

exopolysaccharide studies. Only recently, following the recognition of the role of lipid intermediates as polysacrharide precursors in numerous systems, have further developments been made with exopolysaccharide systems. The methods used have the disadvantages of all work which necessitates the use of particulate enzyme preparatione which cannot, be separated into their active cwnpononta. It, should rdao hc nwognizcd t h t b t

in ritm rmwlts rnriy not, prowwl iir chxac*t,l,y tliv HILIIW 111wiwr its is iiormrd for wholc (dls. I+w of tlw ccll-frw syHt (ww ntdicd HO fw cwnvcrt appreciable quantities of the sul)Htratcs provided into polymeric material, when compared with the rates of polysaccharide synthesis by whole cells in culture or in washed suspension. I t is therefore open to question whether the glycosyl donors and other Rubstr&tes used in such experiments are necessarily the sole compounds involved in the intact cells. At the same time, it should be remembered that preparation of the particulate enzyme systems necessitates disruption of what, in the intact cell, iR a well ordered fiiiictiond ayxtem for the formation and supply of the polymer precursora and their transfer from aqueous to lipophilic environments.

1. Homopolysncchorides

Denpite tho large number of baclteriii which secrete cxopolysaccharides few cell-free systems have been studied and moHt of the work on homo- pOlyHUC(:htLridCR has becw conwrned with leven and dextran system8 whic4i will not tw ctonHitlc!rcd I i c w . Sornc~ of t h rwwlts from studiw on h , ( : t w i i i I Hyrlt,hc!HiH of' (:(:Iluloac 1)y A rrdobc~drr qlinum twc partiaulrtrly relevant in view of the recent t1iHc:ovrry of the involvemcnt of lipids in polysaccharide formatiori. Ulaser ( l ! )RH) prepared a n insoluble enzyme system which catalysed the transfer of glucose from UDP-gluco~c to polymeric material. The product was soluble in watcr but insoluble in alkali. It was sensitive to cellulase and, on hydrolysin, fornit:d t h v characteristic oligosaccharides ohtaincd from cdlulone. Whon the enzymc! system was pretreated to remove endogenous polysaccharidc, cello- dextrins acted as primers. It was of particular interest that the glucorjyl donor in the system was UDP-glucose in contrast to cellulose synthesis in green plants in which GDP-glucose functions (Barber et al., 1984). Another intermediate was also detected in the A . xylinum system (Khan and Colvin, 1961a, b). Ethanol extracts of the cells yielded a product which, on incubation with soluble cell material, formed fibrils. These fibril8 were insoluble in alkali and IiydrolyNates showed ttie presence of glucose. No further evidence for the involvcmtwt of lipid iiitcrm~~diates in bacterial cellulose synthesis has twwr rc.portc4.

190 I . \V. Sl~'l't lEltl .AN1)

Synthesis of a gluran by Hhizobiuiii japoniczwi was studied by Uedon- der and Hassid ( 1964). A particulate system was involved, and incubation with UDP-glucose led to the formation of tt product identical with the extracellular gluean. Opt1inid synt,liesis required the prrscwx of Mg' ' or &In2 I and a pH vdue of 7.5. The polymer c:losely rcsrniblrt1 thr material isolated from dgr.ohoc./r,ii/it/ species ( h i t mtui at nl . , I !Is()) i n its high cwnttrnt (SO%)) ot'p I .:! linkctl gliicoxv tvkliiiw. Sonic oI ' thc~ gliiww WRP nlso I >:I or /3 I ~ ( i I in lwl .

2. IlrtPropol!jeccc.ciInridr8

l'rom the frw ~.xof)o13'saU"(ItiiLridrH wtiidi huvr rrcvivod study using biosynt,hctic. Aystrms, most information has hern glrltned from stiltlies on 1,. pnpumonina and h'. a p r o g t w s . Using coll-frc.e prepsrations from I>. pnc.urnonina type 111, Smith ~t nl. (I!t60) obtained net synthesis of polysacdiaride from U i)l'-glucose and Ul)I'-glucuronic acid. Lncorpora- tion of the t8wo sugars in a 1 : 1 molar ratio was comparable to that found in the native polymer, while the product, identifiable through the use of 14C-sugar nucleotides, was precipitable wi th antisera to type I11 Pneumo- coccus polysaccharide. Attempts to replace the UDP-sugars with glucose or glucose 1 -phosphate were unsuccessful. Further examination (Smith et al., 1961) showed that tho enzyme complex had a pH optimum a t 8-35 arid an optimal temperature for polymer formation a t 32". For full activity, Mg2 were rssentiiil ; in thcir tthsrnrc, polysac.charide synthesis was negligiblc. 1)iffcwnti:bl cwtrifugation of Lhc pt~rtic~iilatc enzyme preparation showcd that it scditnc~ntrtl hctwcon :30,000 and 1 15,000 g, ulthough slight activity (lrss than lo%;,) was found in the supernatant solution. Perhaps becausc of the simple structurr of t h e polymer and its requirements for only two glycoxyl donors, in exactly equirnolar proportions, a very high proportion of the sugarH (W?4) was inc.orporatcr1 into polymer under optimal mnditions, Another fctitiirr. of' t hr systrrn was a riiquircment for preformrd polyrnw, whic41 wiis prrsiirnibtjI,y essential as an acccptor for thc sugarx. 'I'hu dcpen(lon(v wan highly specific, as addition of pure exopolyxucchuridc had n o cffevt. St im t ~ l ~ t i o n of polymer synthesis was noted following the addition of po1ysat:chitride which had been depolymerizecl by enzymic treatment (Smith and Mills, 196%). After c*hromatographiv separation of the products of enzymic hydrolysis and testing fragments of different size, it was concludod that oligosaccharides composed of' 8-1 2 disaccharide units werc active as acceptors. The nature arid role of such acccptors is still not r:lear, ax wo observed (Xutherlantl w t l Norvnl. I !)To) that IL K . / L p r o ! j p / b p x xtrain, known to be clefectivr in ~ ~ l i o s ~ ~ t i o p l u c ~ o t r ~ u t ~ s c ~ , w t ~ x I I I I ~ L I ~ I V to fitrtrl polymer when cell-free ~ ) r q ) t ~ r t ~ t i o l i ~ from glilcosr-gr{Jwtt cc*lln ~ ~ p r ( 3 I I H ~ ~ i b r l d

BACTERIAL EXOPOLYSAC('X1 ARIDES 191

the appropriate sugar nucleotides supplied. Cells grown in galactose- containing media resembled wild-type bacteria in their ability to form polymer. This may indicate that ftdurc to form gluc~oso I-phosphate results in the illability to form Romp cicccptor or pritiicr niolccwlc. On tho other hand, UI )l?-g111 c o ~ c py loop 11 OH 1) I I 0ryl &LAC- AH 111 I I t m t,R A ho w c d normal polymer synthesis. 111 similwr Htwlics (Troy rt nl., 197 I) , VD1'- galactose epimerane-less mutants nlno had normal l)olymcr-synthesil.ing caparity, which apparently indiciitcn tho 111*~senc~ of siiitnhlr acreptora.

111 other po~yH~ic!oharide-Rynthc.Niaili~ Hystemn it llan not IL~WILYR boon ctlosr whcthcrr tlw plymor protliic!cd by c*oll-frw nyntcmH wan of com- PHrlbhh sizv to that formd by intitot colk. The polysaccharide from D. pmu7n(rnim typo I 11 prepartitions behaved on ~rnrn~~noel~~otrophor~s~n in cbxaotly tho Rtmc way as tho native polysaccliuridc! and it wan thorefore thought to hc of idcntiaal molecular wcight.

Attcmpt,s to obtain polymer nynthoclin in othcr I ) . pnru,monina Htrainn did not givr i w good irrc!orlwration of Nilgarti into polymwiv mritoriul (Smith tind Mills, 1962b; Mills tmd Smith, l!W). In 1). pncumoniue type I, addition of UDP-galacturonic acid and UDP-N-acetylglucosrtmine to a particulate fraction obtained from a non-capsulate mutant yielded polymeric material precipitable by homologous antisera. Some synthesis occurred if UDP-galacturonic acid alone was present in the incubation mixture, but it was not clear whether this was due to its addition to a pre-existing molecule or to the presence of some membrane-bound UDP- N-acetyl-glucosamine. Diplmoccus pneumoniae type VII I preparations also showed polymer Rynthcsis when the riridine diphonphatc derivatives of glucose, galactose and glucuronio acid were added to particulate enzymo preparationH, but the inc:orporation of iflotopically labelled glucose only rc!itchod 5%) of tlw totd avidabk~ un 1f~)~ ' -g~ i l (?onc . Some of the ylucoab was ctpimerizocl to g'tluntonc!, ~ l ~ l invwtigatiori ofttic! futc oftlta labelled sugar showed the ))rencnce of 14(C-gluc!0s0 and 14C'-gr~laato~tr in hydrolystttes of enzymically forrriod polymer. One unexplained rwult from these studies was the report that, in typo VIII preparationn, H ~ O ~ W

of the glucose from UDP-gluconc was oonvcrtcd to u non-ntduoing compound thought to be a dic.;rtc:cheritlcb, which produced only glIJC*OHCb on hydrolyc.ris. This reaction W ~ I H nori-wpwifir:, h ing ot)nc!rvc*(l iri ticv(1rtiJ

D. pneumoniae Ntrainn of diflwcnt scrotypo. It w t ~ s no t char wtlchthcbr t t l i w

compound was in apy way associutcd with cxopolyswoharide nyn thwis, and it may have been rather associated with some illtracellulltr or cell- wall component. No further observations on its nature and function have been reported.

The identification of lipid-linked intermediateN in the hiosynthesis of a iiuliiber of polysaccliarides or i~l~nucc~lreritlu-liktt mo\ecc.uler, contttinillg repeating oligosaccharides has bccii wr4 documcntcd. 'I'heHc eomymoundn,

1!P2 1. N’. S I I T I I E l t l A N 1)

which h w c rrcwitly been twmod glycosyl carrier lipids in the review of the subject by Rothfield and Romeo (1971), were first identified as intermediates in tho synthesis of lipopolysaccharides (Wright et al., 1966; Weiner et d., 196.5) and of niucopeptide (Anderson et aZ., 1966). Ethanol-soluble corn pounds had iilreiidy been recognized in bacterial cellulostb synt,hcnix (Kliiin iiiid (‘olvin, I!)tiLrt, 1 ) ) . It, wtin to bn expected t,het a rolr inigtit ILIHO 1 ) ~ found for g l ~ ~ ~ ~ y l cwrricr lipids in exopolysaccharide syiit,htbsis, tw t ticsc p l y inrrs renemblc lipopolysaccharides and mucopeptidcs ill thrw respects, niknicly : (i) they are composed of oligo- xacicharidw poI,ynic*rizc~tl i i t t o i L liircvir mrkcromoloculc; ( i i ) they are found outjwittli th(% l)iL(:f(viiiI WII nic~ml~rr~iw ; und (ii i) t ho glycosyl donor8 iirc rnaiiily forinti in the ititrtic*dllili~r iL(1ticoi1s cnvironment. The first

I*’lli. 11. Sb~llCtlll?’ l l f f$,VCOXYl l ’ l l l ’ ~ l < b l ’ I l l B I l l X Ir I \ ’ l ) lV(Yl 111 I ) ” l y H l l c . C t l l ~ ~ i t l O HyllthOHi8

in Klebsiella arroprrx. X i r id i r ib t fw &I moiicinttcchrtritb I . t ~ H l d \ l ( ~ .

report of the involvement of glycosyl rurrier lipid in exopolysaccharide synthesis (Troy and Heath. 1!W) was followed by its subsequent identificat,ion on the bitsis of its mass Rpectrum as an isoprenoid alcohol (Fig. 11; Troy et nl . , 1!)71). In its active form, the terminal isoprenoid alcohol group is attached through a pyrophosphat,e bridge to a monosaccharide (Fig. 1 I ) . The compound is thus similar, if not identical, to that participating in thv fbrinution of the oligostlccharidc repeating units of both the mucopeptide and lipopolysitccheride constituents of bacterial cell walls (Rothfield tint1 Romeo, 1!)7l) , It, differs from the glycosyl carrier l i pitls req)oii~iihle for ni iin no1 i pid S,ytlth(!HiH i m l for certtti 11

modifications t o l i ~ ) o 1 ) ~ ~ l y ~ ~ ~ ( ~ ( ~ t i a r i ( l ( ~ ( i t 1 sonic lynogcmic: t)clc:tcrial HtrrtinH) as these are linkod to t h o rc:tluc*ing cwd of morlo~aoohtlridt:~ or oligosaccharides through a phosphate: t t i d not, a pyrophosphrttc! grot1 p.

Using a strain of K , nerogen~s which synthosizcs a polyNacchtlride with a tetrasaccharide repeating unit:

- ( ( ; t l l 1 r :I .\I1111 I * :I (:Id)

1 T‘ ( i l l . . \

Troy et al. (1!)71) found that seyucntial transfer of HugarH from nucleotide donors to glycosyl cwrier lipid ( C X ? L ) occurretl. The sequence of reactions is:

I‘DP.Ud I C i ( ” l A I’ (:(*I, I’ I’.(;III 1 1‘JIP UCL-P-PP-C:al k UDP-Man 4 ~ ~ ~ ~ ~ ~ - ~ . € ’ . ~ ~ ~ l ~ . \ l ~ l l + (UDP)


GCL-P.P.Gal.h.lan + UDP-GlcA + (:CL-P-Y-Gal-Man-GlcA + ( U D P ) GCL-P-P-Gal-Man-GloA + UDP-Gel r (:CL-P-P-Cai-Meii-GeI + (UDP)

t GlcA

The isolation and study of a series of‘ rnutunts from another K . aerogents strain that synthesizes a tetrmaccharide repeating unit (Sutherland and Norval, 1970) confirmed these results. The unit has the strrwtmc :

The first rctwtioii in thc scv~iictic+c~ l’or this struin is :

This was shown by isolation of UMl’ from the rewtion mixture and by its inhibitory effect on glucoxe l-~)honphtit(* trutinfrr. Subscquently two moles of galactose were transferred to thcb glyc*osyl cwricr lipid and their transfer was partially inhibited by UDP. Mutants blocked at various stages of transfer of sugur to lipid were isolated and characterized (Table 9). As in other forms of sequential transfer, as found in lipopolysaccharide formation, failure to transfer one sugar led to non-transfer of subsequent monosacc+harides. Thus, mutants unable to transfer glucose 1-phosphate to the lipid phosphate failed to transfer galactose, and all such transferase-less mutants were unable to form polymer. One noticeable difference in this respect from the liT)o1)olysacch&ride- synthesizing systems was in thc procwm for forming t h e monosrtccharide sidr-chainn. 1 t i the prcytiriLtions iisctl t)y ‘I’roy P I (11. ( l!l7 I ) t h e addition of‘thr nitlv h i t i (glucwrotric~ cwitl) W ~ L H ii Iwcwbciiiinitc- l iw t h c h tultlitioti of‘

‘rAi i ih ; 0. ‘ ~ r i ~ i t d i ~ of SIIKWH l o 1,ipiii I I I Mtitciiitx id ~ 1 f ‘ / i ~ & h

Type 8 (Straiii A 4 )

UDI’-(iIc 1 GCIA-1’ > ( ~ ~ ‘ l ~ - l ’ - l ’ - G l ~ ~ I LJMP

Strain

Sugar trarixfix (nmol./hr./mg. protth)

I:lucosc~ (Jalac*towc* liut io __ - -~ _ _

A4 034 036 027 03 7 029 038 05 2 03 1

0.124 0.053 0*053 0.022 0 0.013 0.020 0.061 0.061

. .

1.1 1.3

Y ?

(~liicosc t rarrsfwase Galactose transferam I

194 r. w. s u m ~ R C A N r)

the second galactose residue. By contrast, in the formation of the anti- geriic portion: of 8. typhiniurium lipopolysaccharide, the trisaccharide mannosyl-rhamnosyl-galactose is formed on the glycosyl carrier lipid and could be polymerized even in the absence of the side-chain sugar, which in this polymer was the 3,O dideoxyhexoso abequose attached to the mannosybresidues (Osborn tmd Weiner, 1968).

The lipid rbquirernent for the exopolysaccharide enzyme system was demonstrated by the use of solvent extraction and careful reconstruction of an active complex (Troy et al., 1971). The largest oligosaccharide identified as k ing attachcd to lipid was un octasaccharide (i.0. two units of the polymc~r) and it is still not olrtir how large a fragrncnt is eventually u,(:(:iinitilit,tod ot i thc! lipid prior to trundor to an uc:coptor. I n the only oomptirdh x!yntcw which hthrt h v t i Htudiod, t h o thntigori portion of l i i~ol~olyxiic~c~ti~i~i(~(!H (Koi i t uiicl ( h h r t i , l!NM), tho ctrit,irtr Hid(!-(!Iiaiti of about ($$it tcttrunswhuride iinitn uttuchud to glyco~yl carrier lipid wall iHoIated from *m iitant strains unable to transfer it to acoeptor portionx of the rnucrornoleculu. Unfortunatcly the accwptor molecule in capsule Rynthonix hw-not beon identified, und x(!lection of mutant8 similar to these is not possible. It is however certain from the ixolation of slime- forming mutants unable to attach the polysaccharide formed fo the cell surface (Wilkinson et al., 1964; Norval, 1969) that some specific site is involved. The possible disorganization of this site during particulate enzyme preparation may account for the relatively low levels of polymer formed by cell-free systems.

Of particular interest in the exopolysaccharide biosynthetic system is the role of the glycosyl carrier lipid vie Ci via its function in lipopolysaccharide and mucopeptidc synthesis. I t is possible that there are slight anomeric differences between the glycosyl carrier lipids which render them specific to the differelit MyRtamH which utilize them. Tho amount of glycosyl carriqr lipid in hactcrid cc*h i H c!xtrc!mcly low ; th viiluct of 042% of the ccll rntiwi was xtiggestcd for that involvetl in lii)~)~~(Jly~it,Cc!tittri(I(~ formation in 8. ne&nylon (Ihtikwt rl d., l!Ml6) ant1 of 40 nrriol./g. wet weight for the analogous compound ili 45'. typhiwmrium (Kont and Otjborn, 1968), while Wright (197 1) indicetcd 1 O5 moleculen/cell UH the value for glucosyl lipid in lysogenic SnlmonPlh. The glycoHyl carrier lipid from K. uerogenm membranes ('1'ro.y el al., 1!)71) functioned in vitrr, in tho bionyiithcxin of'a rnttntian i n Micrococolwr i!pfJdniklictu, whil(! irrtcrchungct bet w w n t hc mcico pept id(?- tLnd I i 1)o~)olynucoharide-form ing xyxt(!mx WUH

earlier reported. In the growing c*ell. m ucopoptide and lif~'r~)ly~a(:"haride syntheses are assumed to occur xiriiiilt~ti~o~~NIy, arid would therefore require glycosyl carrier lipid at thc xame time. Exopolysaccharide formation could occb when there i8 IJO direct requirement for glymsyl carrier lipid in the other system and certainly occurs in non-growing

BACTERIAL EXOPOLYSACCRARIDES 196

cells. If the lipid were used first for synthesis of the cell-wall components, then for extracellular polymers it might be expected that bacitracin, which prevents dephosphoryvltttion of the glycosyl carrier lipid pyrophosphate, would have an iiihibi tory cffeot on cxopolyswchtwide synthesis. In fact, 26% inhibition of tranefer of sugars involvcd in exopolysaccharide synthesis to lipid was observed (Sutherland and Norval, 1970). This may indicate that a portion of the glycosyl carrier lipid normally utilized for mucopeptide synthsRis can function in cxopoly- Necohurido formation. Othor ovidertot! comes from K. mrol~enn~v nctrotype 2 (Htrtbin 243) i t1 which gdR(:toe(! is clhH(!llt from the sxopolysc~c!c:tt,iriclc

,

but is the major component, of thc lijiopolyxaecharidc (Kc~leltzow el nl. , 1968). Addition of UDP-galactose to cell-free syetems depressed t tic incorporation of glucose and mannose into glycosyl carrier lipid end exopolysaccharide (Sutherland et al., 1971). Thus, formation of galacto- sylated glycosyl carrier lipid appears t,o decrease the amount available for exopolysaccharide synthesis. In this system, as in K. aerogenes type 8, glucose is the first sugar t’ransferred to lipid in exopolysaccharidc formation. Incorporation is again diminished in the presence of UMP.

Fig. 13(n)

IIA("I'I~:ItI A L I ~ ~ ~ O J ' O I , Y H , ~ ( ' ~ ' I I A l l 1 I) KH I 97

M i b n i m u * , froiii ( 1 I ) l ' - i n t i ~ i i i i o x ~ ~ , ix t,rcuixftw(d i d t c b r g I i i ~ ~ c * , iw1 IittJv is transferred iii t lit. t c b R t w ! c ~ of prior gliioosylation (14'ig. 12). lhrthor

, support for the sharing of the glycosyl carrier lipid between the systems is seen in some recent results from our laboratory (Norvul and Sutherlend, l!MQ). A new group of muteiitw wcrv iNol l t t t4 from t81ircc different A'. wrogenes stmius of' diflerrii t s(wt:)' ph. 'I'lic~sv in i i tm ts, iiisinly obt9taiticd after aminopuriiie niutagenesis, had curious culture morphology, resembling the wild type at8 36' but possessing characteristic colony type

FIG. 13. Colonies of Iilebsietla aerogenee CH. mutant grown on a nitrogen-deficient agar-containing medium for (a) 72 hi*. at 3S0, (b) for 72 hr. at 30°, (c) for 96 hr. 86 20".

at 30" and lower temperatures (Fig. 13). In liquid media autoagglutina- tion was Reen at temlmetiime hclow 30". A t 95" Hynthesix of both l i p - polyeaccharide trnd ~!xoI)Olye~t':':hllrid(! wtw iiormd but, at 20" on ~olicl media, the amount, of t!XOpolyeaat:I~atrid(! HyrithcNizt!d won dcon!uned by epproximafely 75% and in liquid nicttliti hy ahout, 60'%,. A t tho Ham(! time, the lipopolyxuac:hiLr.itlc! aontcwt wihH cl(!c:rc:uxc4 1b.y 60 t!O'%. I'rorri these results, it i R prot)oblo tht. t h i n type! of' r r i i r t l t i i t ((!It rnutant) itivolven a conditional fault ill glyoo~yl c:rhrric.r lipid N y l l t h O N i H . A t t h c ! lower temperature insufficicnt glyco~yl otirricrr lipid may I)(! uvailahlf! to enable

BAUTERIAL EXOPOLYSACORARIDES 199 Examination of acylation of exopolysaccharides should prove inter-

esting and fruitful in this respect, especially since the recent discovery of polysaccharides with various acyl modifications (Garrgg et al., 197 lb). It is possible that such polysaccharides nre formed with 11 bnsic cwyl group, probably pyruvato, which could then be modified by cnzyrrics found either extracellularly or in the pcriplasmic rrgion. A coinpnriblc qystem wa8 dcscribed by Kcller (19G5) iiivolvirig the acctylation of some Salnionalla lipopolysaccharides. In this xystcm, the donor was acctyl- CoA and various compounds acted as itcceptors. These included de- acetylated lipopolysaccharide, oligosaccharides, oligosaccharide- 1 - phenylflavozoles and a number of compounds in which L-rhamnosyl ( 1 -+3)-~-galactosyl sequences were present. Surprisingly, the intermediate involved in lipopolysaccharide synthesis, namely L-rhamnosyl- D-galactosyl-phospholipid, was not an effective acceptor. This result, combined with the use of various mutaiits defective in complete lipopolysaccharide synthesis, suggested that, normally, acetylation might occur either on the complete lipopolysaccharide or at the stage of polymerized trisaccharide attached to the carrier lipid. If the exopolysaccharide biosynthetic system is similar, acetyl-CoA from the cell membrane or the periplasmic spacc might again function as the acyl donor, while the enzymes involved might be part of the membrane or alternativrly, some of the small molecular-weight protcins discovered in the periplasm. Thc variety of acyl substitucnts so far discovcrcd indicatrs the posgibility of either a rarigc of suittbble donors or, more probably, a number of different enzymes capable of modifying a smaller number of added groupings such as acetate and pyruvate, i.e. :

PS t (X) +PS - x

PS t (A) -+ PS - A -+ PS - X or

modifying enzyme

Preliminary experiments with a serotype 8 K . aerogenes strain, in which the exopolysaccharide contains acetate and pyruvate in addition to the normal carbohydrate structure (p. 193), showed that addition of the potential acyl donors, acetyl CoA or acetyl phosphate and phosphoenolpyruvate did not stimulate transfer of sugars to lipid or polymer (I. W. Sutherland, unpublished results).

From all these results, one can propose a generalized model for PXO- polysaccharide synthesis in bacteria (Pig. 15).

In this model, ulrrio& all of the fir nutionn am' Ijrirncirily ux,roc.iutccl with thc bartcrid W I I triwnbraii(~. Otily t I i c * c w l y n t r h p x iri n~~ti-xj)wili(: carbolrydrate mc4ul)olinrn (ill t tw chxrwiplv c4tcvl , thaw Ic*idirig to IJ f j lJ- glucose formation) are intracellulw in location. All o t h w iiro loo~itcd lit

9

200 I. W

. SU

TH

ER

LA

ND

BACTERIAL EXOPOLYSACUHARIDES 201

thc cell membrane, and suggcst a high degree of localization and regulation within the membrane. It is also clear that, for the synthesis of any heteropolysaccharide capsule or slime, ti considerable number of highly specific enzyrries are involvcd. This in turn implies ttlitt, a significant portion of the bacterial genome is devoted to provision of thc necessary genetic information.

C. CONTROL One can maltc an arbitrary diviRion of tho enzymes leading to exo-

polysaccharide formation into several groups: (1) enzymes such as hexokinaw responsiblc for thc initial metabolism of a carbohydrate substrate ; (2) enzymes involved in sugar nucleotide synthesis and interconversion, e.g. UDP-glucose pyrophosphorylase and UDP-glucose dehydrogenase ; (3) transfernses forming the repeating unit attached to glycosyl carrier lipid ; and (4) the translocaseR or polymerases that form the polymer.

Possibilities exist for exerting control over polysaccharide synthesis a t any of these four levels, and mutants lacking enzymes of any group fail to synthesize exopolysacchrtride. The first group, being involved in so many other cell processes, cannot bo involvcd in cxerting any specific control and arc generally found intracellularly . Insufficient is known about the final group to consider thcm hcre. Tht:y are ulmost certainly piirticuluttb, iks ilrc thc glycoxyl trensfiww:H tirid sornc cnzy mcs in the sccond group. ‘I’hose cnzymcts which havc h e n Nhown to exert control arc the nuclaotida phosphorylases. T h y wwo shown (Ginsburg, 1964 ; Bernstcin and Robbins, 1965) to control the transfer of monosaccharides into polymer either through the base specificity of thc sugar nucleotide or through ncgative fccdbaok mcohaninms opcrating a t t h c b lovcl of enzyme action. The actual lcvels of these enzymes rcmain conatant cvcn in non-mucoid mutants of several of the exopolysaccharide-Hynthesizing strains examined. Exceptions are found in E . coli and will be considered separately. In a study of synthesis of GDP-mannose and GDP-fucosc (Kornfeld and Ginsburg, 1966) a feedback inhibition mechanism permitted independent control of the rate of formation of the two sugar nucleotides from the same precursors. Such fine-control mechanisms would obviously be extremely valuable to the cell in regulating exopolysaccharide precursor formation, especially in those bacterial strains, such as some Balmonella species, in which mannose isl present in the lipopolysaccharide of the cell wall and fucow is found in colanic acid.

An interesting example of the control of polyaaucharide synthesis was seen in the observation of binary capmlution in I). pneumoniuc! (Austrian and Uernheimcr, 1!359). Following trtmxformtition of B non-c:;t~)x~iliitcd strain (originally type 111) with DNA from u hoterologous c a p d a t e


( t y p I ) ~ t ~ t ~ i i 1 , ib Rmiill 1111 t n I w of bitirvy (ttL1iHllltltod (\olimic\s w ( w d ( b t j ( y ( : -

t d . ‘ ~ ’ I I o H ( ~ ~woducvd IL iioriiird ttttioititt oL‘t,ypo 111 etqiHiil:tr ~)~ily~itc,~.hririd~! together with a ninall amouiit of type I polymer. Use of the DNA from the binary capsulated recombinants for the transformntion of heterologous (typc 11) nonlmucoid strains produced three types of progeny, namely cnl)Huli1tc typo I , ~~oil-ciil)sL~ltit~~ type: 111, t~d biniiry type (I + 111) cells‘. Capsulate type I11 cells were not dotected. Prom these results, it was concluded that repair of the type I11 genome had not taken place, and consequently no recombination of the type I DNA with the imperfect type 111 genome. It was assumed that tho type I DNA co- existed with the non-capsulatc genome, permitting the formation of some type I material from several of tho precursors normally used for the type I11 polysaccharide. There are thus two parallel and interacting biosynthetic pathways in the binary cells (Fig. 16). Some of the binary capsulate recombinants were unstable and reverted to the non-mucoid

4 3 Type I polymcr

Fra. 16. Pathways of polysaccharido synthesis in binary capsulated DipZococcus pneuinoniae cells.

type at high frequency (Bernheimer and Wermundsen, 1969). The instability was thought to be caused by the type of mutation in the non- mucoid (typo 111) strains. The heterologous genetic material was probably integrated at at leust two loci, one of which was adjacent to the part of the recipient genome rcsponsible for capsule synthesis and the other some way from it.

Production of colanic acid in the Enterobacteriacouo seems to possess a unique regulation mechanism. A regulator gene, denignated cup K (Markovitz, 1064; Markovitz und Iloscmhuum, l!IfX), WRH identifictl on the chromosome of h’. CO& t h d j i h ( : ( ’ l l t to tho pro 1oc:urt (rt:nporiHiihlth for prolirie biosynthcHiR). It ooil trolhd n(vcru1 enzyrnc+~ thought to bc involved in oolanic acid HyntlwHis. ‘l’hw! inc:lud(:d UJ)?’-~ulaoton(!-4- epimcmsc and UDI’-fucwc* Hynt,ti(*ttLse. ‘I‘rawduc:tion WHH Iiwd t ( J forrn heterozygoten. Whc*n t h gorw was intogratotl on t h c b cttromosomc*, t ti(?

non-mucoid condition was dominant. Transfer of the cup& locus us part

BACTERIAL EXOPOLYSACORARIDES 203

of an episome produced the reverse effect, mucoidness, i.e. production of colanic acid, being dominant. By culture in medium containing p - fluorophenylalanine, the regulator gene produced an inactive product, resulting in colanic acid production in strains which were normally non-mucoid (Kang and Markovitz, 1007; Grant et al., 1069). Simul- taneously an incrcasc was notcd in tho lc!vol of sevcral of tho cnzymrs involved in cobnic acid nynthcsis, iiicluding phosplio tnat Inow itiomertw, UDP-glucose dehydrogonasc, UI)P-galucto~c-4-epimcrasc, GDP-mannoso pyropliosphorylasc and GDP-fucose synthetasc. The increase in enzyme levels was 2.5-3.3-fold for Home of thc enzymes, but as high a8 13-fold for UDP-glucose pyrophospllorylase (Lieberman et al., 1970). The capacity to form colanic acid secms to extend to most E . coli and Salmonella species as Grant et al. (1969, 1970) showed that, under appropriate conditions, colanic acid was produced unless a recognized defect was present in an enzyme essential for its synthesis. This was also reflected by high levels of GDP-fucose and UDP-glucuronic acid in the nucleotide pools of cells synthesizing the exopolysaccharide. The increase in the two sugar nucleotide levels occurred simultaneously when non-mucoid repressed cells were cultured in the presence of p- fluorophenylalanine. It thus seemed that control was normally through the reaction of a single repressor rendered inactive by the amino-acid analogue. These results were therefore in good agreement with those of Markovitz and his colleagues. The genera Escherichia and Salmonella togethrr with Aerobacter cloacae thus fall into tliree groups : (1) strains normally producing colanic wid, including A . cloacae and many E . coli K12 substrains; ( 2 ) strains producing the polymer when grown undcr nitrogen-dcficicnt, cnergy-rich conditions, including most E. coli and some Salmonella types; and (3) strains which givc rise to colonics which were only mucoid in media containing fluorophrnylalaninct. It still remains to be determined whether the repressor is part of an opcron controlling all of the genes involved in colanic acid synthcsk. If this is so, an appreciable part of'the bacterial genome may be involved, as a largc number of enzymes are thought to be involved in the formation of the complex oligosaccharide unit which is polymerized to colanic acid

Numerous mutations can affect the production of bacterial exopolysaccharides. The use of several such mutants in work on polymer production has already been mentioned. Non-mucoid colonies were frequently noticed during subculture of mucoid bacterial strains. Such cultures provided much information during studies on capsulc Rynthcsis by Diplococcus pnezomoniae, a systcm which had the advantage that transformation provided a means of genetic intorchange bctwocn non- mucoid strains.

(Fig. 17) . 1\

Gal : :Pyr

GlcA I

I Gal +c

11 I I UDP UDP-Glc & VDP-GlcA

Gel

G-1-P

4 G-6-P GDP-Jfann

4 4

Yam- I-P

MM~-6-P

ENZYadES

1 HexokinsSe

2 Phoaphoglucomutase

3 LDP-glucose BrophosphoryLeSe

4 UDP-glucose Dehydrogenase

5 Phoaphoglucose Iaomeraae

6 Phoaphommnoae Isomer-

7 Phosphomannomutsse

8 GDP-Mannose Pyrophosphorylase

9’98 GDP-Fucose S j m t h e b

10 Fucoae Transferaae 1

1 1 Glucuronic acid Tranaferesa

12 Glucoae Transferase

13 Fucose Transferase 2

14 Acetylase

15 Acylase

16 UDP Galactoae-4epimenuw

17 Galactose Tranaferase 1

18 Galaetose Tranafenrse 2

19 Polymerase

FIG. 17. Pathway for the biosynthcsis of colanie acid from its precursors. Soverd enzymes (1-3) and (5-7) are involved in general rarbohydmte metabolism. Others are involved in the synthesis of precursors for esopolysaccharide atid lipopolysaccheride (16) or exopolysaccharide done (4,8,15. 17-19). GDP-4R6DMm indicates panosine diphosphate-4-keto-6-deoxym~nnose.

BAOTERIAL EXOPOLYSACCIIARIDEY 305

Most mutations affecting enzymes known to participcitr in cxopolysaccharide synthesis lead to non-mucoid (0) drrivutivcs. Srwh mutnnts resemble the wild-type bacteria with regard to most of tlic oinymcs leading to exopolysaccharide formation. Thus, in I). pneumoniae. wild- type and non-capsulate cells both contain tho enzymes involved in metabolism of UDP-sugar nucleotides, and the actual sugar nucleotides were isolated and identified (Smith et nl., 1957). Thc loss of exopolysaccharide synthesis may be due to deletions in precursor synthesis, in transferases, or in polymerase. Because of interrelated systems coming under specific control mechanisms, it might be expected that loss of one enzyme in a sequence would lead to altered levels of others. Few attempts to measure the levels of enzymes involved in different stages of polysaccharide synthesis have been reported. Changes in levels of several enzymes leading to colanic acid formation in E. coli were observed (Markovitz et al., 1967) in mutants. Non-capsulate mutants of E. coli K27 varied greatly in the levels of UIIP-glucosc! ~)yroph(~~~’lioryla~sc,

WILI) TYPE (Capsiilcs uiid Slitric.)

Baci tr:ieiii I resistance 0 Mutants (No exopolysacoharide) t--- CR-0 mutants (No exopolysaccharide)

FIU. 18. Mutations affecting exopolysaccharide synthesis in Klebsielb aerogenea.

phosphoglucomutase, UDY-galactose-4-epimerase and GDP-mannose pyrophosphorylase (Olson et al., 1969) indicating a lack of correlated control in this strain at least. Similar results were obtained using non- mucoid derivatives from two different serotypes of K. aerogenes (Norval, 1970).

The mutants already mentioned, which havc lost thc ability to form capsule but retain the ability to mcrc!te slime:, can iindorgo fur thr mutation involving ~ O R H of polyrnor-h.yrithcaiziri~ triipttcity . ‘ h w iiro thus two pathwiiy,q of mutation loading to prodiiotion of 0 ( ~ I ~ ~ ~ I - ~ ~ I I I ~ ~ ~ J ~ ~ I ) mutants (Fig. 18). ‘l’lrc conditional mirtwtx ((:IC-rriiitantn) : h o trri’ti-

tioned can also undergo IL ~ecorrd rnuLatiori 1,o product r d l x c:ornlh:tc:ty unable to form polymcr. Thcsc Clt-0 miltarits rc:wnihb: the Olt typ: in auto-agglutinability at low incubation tcmpcraturc arid in their altr:rc:d phage-sensitivity (Norval and Suthclrland, 1 !tfl!j). ‘I’hr:y HIHO rrwmblo 0 mutants in their stability. Fcw revertantH to rnuoc~idnc~~ httvc hccm obtained (e.g. Norval, 1970; I. W. Sutherland, unpuhlishccl remlts).

‘

206 I. W. SUTRERLAND

The CR mutants differ in that they are less stable, and revertants to cupsulation at low temperature occur. Despite the low levels of reversion of CR-0 mutants to mucoidness, it is possible to isolate revertants to the 0-characteristic by selecting for bacitracin resistance (I. W. Suther- land, unpublished results; Fig. 18). This tends to confirm the postulated involvemctnt of a glycosyl carrier lipid dofcct in tho CIt mutuiits. Rnci. trncin-msiL;ltant mutant8 probably hava c!lcvcitc!d lovc~11.l of glycoxyl crvriw liIk1 to niwurnvcvit tho firiluro to t l ~ ~ ~ ) l i c i ~ i ~ l ~ o i ~ ~ y l i i t o g l y ~ ~ ~ y l ccirriw lii)itl-~)y~oplio~pPhut(~ wtiioli l i r ~ btwi idoiitiliotl tm tho motlo of action of this antibiotic (Sicwcrt iknd Stromingor, 1967).

V. Function of Exopolysaccharides Various hypothctical functions hava been suggested for bacterial

exopolysaccharides. Most of these have implied a protective function, such as against desiccation, against phngocytosis or against bacteriophages. While there may certainly be some possibility of the first two roles being correct, the occurrence of a number of phages capable of inducing capsule-destroying enzymes suggests that capsules frequently present no real barrier to phage infection. They may even, in fact, act as a receptor for certain phages (M. L. Wilson, unpublished results) as a Klebsiella phage absorbed with much greater efficiency to capsulate than to non-capsulate strains. Thus the capsulc might function as a primary viral receptor while the cell wall is a secondary receptor.

Protection against phagocytosis may also be associated with R function for tho cxopolysaccharides iLA tigressins in puthogonin bactorial stmins. ‘I’hoy inight inhibit lysoxymc., tho mid l)(JlyrtrLc!c~lirLri(l(! cotn1)ininK with ttho biuk protoin, nn(l o t h b r iLIil,i~)~L(~t,~!rii~l whitmimi. ‘ I ’ t i c b ( : I L I ) N I J ~ ( L H

may dno inhibit bactorial cngulfmont by : L i r i o e h o . l’rotoction rhgeitirtb desiccation in most likely to bc of importunce t o soil Rpcdcrt, and it, i~ certainly true that many of thcse produce exopolynacchuridon. A fnrthcr role could be in the adsorption and provision of nutrients and ions. The numerous free carboxylic acid groiips of the uronic acid8 and ketih available in many of theso polymers could permit this. Also, the amount of water bound to the capsule (which is 99% water) might allow some of it to be utilized by the cell. Further elucidation of these possible roles must await adequate data, on the binding and exchange of molecules to exopolysaccharide material,

VI. Unanswered Questions Despitc the volume of work on bacterial exopolysaccharidcs which has

been amassed, there arc still a largc number of unanswcwd qucstionn. These include :

BACTERIAL EXOPOLYSACOHARIDES 207

(1) The real function of exopolysaccharides is still obscure and i t rcmains perplexing that microbial cells should expend so much energy and exert such complex regulatory and genetic mechanisms on their synthesis.

(2) The difference bctween slimc and capsulc is thought to exist in the loss of a spccific receptor or binding site from mutant cells which, although originally capsulate, become slime-fortning. This remains unproven and other possibilities such as loss of a binding enzyme, may be considered.

(3) When a capsulate cell divides, does the capsule undergo some parallel form of division? It would be intorcsting to know whcther, and to what rxtcvit, the capsule 011 cithor tleughtcr ccll in dcrivod from cixisting i ~ n d IIOW pc)lYHILC(:hilrid(!, ilnd to wllibt cxtcsnt the ])rOC(!HH roscrnbLw doposition of muao~)c.ptidc in th(: 1matc~ri;d ooll wall.

(4) The mrclranism of polyssccharidc acylation and modification is still obscure. In particular, i t remains to bc seen whethcr acylation normally occurs through a limitcd range of donors and subsequent modification of the acylated polymer. Tho stage a t which polymerization occurs is also unknown.

( 5 ) It is clear that, during exopolysaccharide synthesis, carrier lipids are involved and that these polyisoprenoid phosphates are inter- changeable in the synthesis of other repeating unit polymers, including lipopolysaccharide and mucopcptide, in vitro. What is not clear is whether there is any specificity in the use of these lipids, either through slight modifications to the lipid or through spatial separation of polymer-synthesizing sitw on the cell membrane. Alternatively, is there discontinuous synthesis of lipid-rcquiring polymers?

(6) The Rize of the rcpcating unit formod o n tho lipid is iiot c:l(.ar, rior i n the nature of thc acceptor to which it is tr:innfi:rrcA. ‘this nnlwct rnlty be rclatcd to (2). Thc loss, through mllt,iLti(m, of a n m:c.ptor rnoh:aulrt might lead to rdease of ~olublc d i r n c from thc: g l y ~ ~ ~ y l orwriur lipid instead of normal capsulc form iL t’ 1011.

\ VII. Conclusions Bacterial exopolysaccharides arc a complex group of polymers con-

taining a variety of monosaccharides and acyl and other substituents. They are essentially linear strands, many of which posseas side-chains of one or more nioiiosaccharides attached a t regular intcrval~ to tho chain. Almost all such exopolysaccharides are probably formcd from


repeating units of 2-6 monosaccharide residues which are assembled from glycosyl donors on to a polyisoprenoid phosphate carrier by membrane-bound enzymes. After polymerization, the molecules are extruded from the cell surface to form either a slime unattached in any way to the cell or a discrete capsule which has nn attachment mechanism UR yet undotonninod, binding it, to tlw o i i f ~ 1riyc.r of‘th(r wII wdl . AH i b rtwiIfd of tlicir rogiiltw da.iwbiin~, t w w r t d cisoli(’l,yHtLc!olirlri(l~~~ ibrv iho nubntmt1(iN of cnzy tnon from hcttorologoiirJ ttiioro-orb”ininmN or from plitlgc-infoctcd btbctcriti, which degrade thorn to tlioir component oligorJaccharide8. One group of bacterial exopolymccheride8, the bacterial alginateu, are oxceptional in that their components, and consoquently their properties, can be modified by extracellular enzymes after their excretion into the medium.


I am grateful to numerous colleagues for helpful discussions and particularly to Dr. W. D. Grant and Dr. Mary Norval for their suggestions during preparation of this manuscript.

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28, (52.


Physiology of the Bdellovibrios

I . htrodrlction . . 215

A. IaoltLtion . . 218 13. Cultiiro Media . . 218 C. Maintenance of Bdellovibrio Cultures . . 219 D. Distribution and Importance in Nature . . 220 E. A Taxonomic and Terminological Note , . 221

111. Structure and Chemical Composition of Rdellovibrio . . 222 A. Morphology and U1traRtriictut-e . . 224 T3. (”hcwiicul Composition of Bdellovibrio Cells . . 226

1 1. THOlabiOn, (h~tlVcttl(Jll, ~ ~ l % ~ l * l ~ J l l ~ l O l i , t%ll(l ‘~’tlX(J1l~JIny of IjdellOVibri<J . 218

1 V. Ryinbrosia 13etweon Host -Depentl(wt Bdellouibrios a i d Host C ~ I N , 228 A. Life Cycle of Host-Thpendcwt Bdellovibrios, , . 229 W. Effects of Bdellovibvio on the Host Cell . . 239 C. Merhankms of LYHM of the Host ('ell by Hdellovibrio . . 242 1). Kinoties of Host-IhpendPnt C:IYJWth . . 246 14:. NIltl.IbloIl Of ~f/e~/O?Ji/JT’ifJ . . 246 14’. HlJtitj ~p(’clfk*ity , . 249

v. Mf~ttLbo~rHrn of’ Bdellovibrio . . 262 VI. Host-liidepondent Derivtrtivc% of‘ / ~ r l ~ l k n i b r k . . 264

VII. Bdellovibrio Bacteriophages . . 256 VIII Acknou.leclgpinriitn . . 257

I{faft.Ti3 I W H . . 267

I. Introduction

Heinz Stolp was’honored in 1968 with the prestigious Robert Koch Prize for his discovery (Stolp and Petzold, 1962) in German soils of a small vibrioid bacterium which is parasitic upon other bacteria. Addi- tional cultures of this unusual organism were isolated in California, i t wa8 further characterized, and i t was named Bdellovihrio hucteriovorw by Stolp and Starr (1963b). “Bdello-” is a combining form which meam “leech”; “-vibrio” represents the comma shape of the parmito; “bmtori- ovoru~” means bacteria-eater.

1 0 ‘71;

living host bacteria in miid media (more rarely I L I ~ only iirttlor H p w i t i I conditions, as related below, with heat-killed hoHt bttctoria). lldcllowibrio could be distinguished from bacteriopliagen hy tho kinetics of devctlo 11-

merit of the plaques on host IawriH (Ntolp and h t , d d , l!#fi2; Stolp und Starr, l963b). Phage P~UCIUCH are usually fiilly rlctvt*lopc!rl within 12-24 hr., while Hdellovdbrio pluciuon IJocorno viHi t)lc on h w n n only tdtw 2 4 dayH ttiid thcn cnlargo progrowivcly with oontinuc!tl i i i c : i i h t r t , i o n u p to Nix days. Bdellovihrio W(LH ~ 1 1 ~ ; churaoturizocl by its huirrg ar1 twtively motile, rather Hmall, vibroid buatorium which attaohw to the bacterial

1'11 Y XlOJA)( 1 Y ( ) 11' TI I 15 I1 I ) ICI~IJ I V I Bit 1OS 21 7

host cell (Stolp tirid l'etzold, 1W2: Moll, and Btnrr, l!Wb). AR will be made clear presently, tohe entrance of Hdellovibrio into the bactorictl host cell and itla development therein are necessary slemente in the life of this micro-orpailiain. It haR been pos~ihlr to obt,uin host-independent ("Ry)roi'h~tic") l~dcllovibrio polbiiln t i o n H (dcfinncl hy tfhc shility to grow i l l t Iw tilwwiiw oi' lioNt I)cic*tcviri) I'roiii I i o H 1 , - t l ( ~ l ) ( ' i i ( l ( . l l l I d d l o v i l ) r i o

FIG. 2. Lytio action of Brlellrmibrio baclm*owwe Wain Rd. 100) on cobnim of hoherickia coli B. A mixture of bdellovibrias and hoRt bacteria W&R n t m k e d 011

nutrient agar and the plate was photographed after fotir &ye. Pig. 7 of Stolp and Starr (1963b).

populations ; host-independent popillations mn revert to host-clependent populations.

Additionctl insights about, microbial interreltltionships in general would be gained if host-parasite interactions were better understood. Pertinent to this better understanding are the studies of host-parasite relationships in host-dependent protista, including mdaria protozoa (Trager, 1960; Moulder, 1962); riist (Shaw, 1983) and other fungi (Barnett, 1963); Mycohmtariumr (Hank#, lnfttl); rickattsiw and pnit- tacine bacteria (Monltler, 1W2, 18f14, I !NM) ; Mp-qnh,rmn (Hrtyfiick,

218 ill. 1'. STAltll AN1) J. C' . - ( ' IlIJAh(4

1969) ; D i c / ! p h r t w , ('yclobort~r, and other predatory t)ii&*rirc (l'crtil'uv anci Gabc, 1969) ; parasitcv antl commensals of green algao (Chomov and Mamketew, 1966, 1071 ) ; and bactcriophagr (Adams. l!LW). Also pertinent, is the on-going conc*eptrial analysis of synrbiosi.q, iiic~lircliirg parasitisin ( H . li. Hthixe a i i t l M . 1'. Strcrr, ~inpuhlinhrd c l r \ t c i L ) .

Udcllotiihrio t hus rncrits r x t 8 r i i n i v r study I )wiius(* i t l piriisitimw 1)cwt~cri t i . Hrlrllloiiihrio li r Ls I )(Y' I I t t 1 c b K I I t) j ( of' other rcvivws rwtl sum- msrics (Stdp and Starr, l!NX3b; Starr arid Skarman, 1965; Shilo, l96f1, 1909; Stolp, 1968, 196h, t); Ucmatsii anci Wakimoto, 1O69a; Wakimoto, 1970 ; 8trm rtncl Seicllcr, 197 1) . The prc!sr!nt treatment, which emphasizes the physiology and biochemistry of tho bdellovibrios, is complementary to anothcr essay from our group (Starr and Seidler, 11371) which stresses the morphological, ecological, and taxonomic aspects of the burgeoning literature on thc bdellovibrios.

11. Isolation, Cultivation, Distribution, and Taxonomy of Bdellovibrio

A. ISOLATION

The proccdurc first used for isolating Bdellovibrio (Stolp and Petzold, 1962; Stolp and Starr, 11)63a, b ; Stolp, 1!)65) involved centrifugation and differential filtrations of suspensions of soil, sewage, or other materials through a set of Millipore menibrane filters with pore sizes ranging through 3, 1.2, 0.8, 0.66, and 0.48 pm. The filtrate from the 0.46 p m . filter was plated with prospective host cells in scmi-solid agar using the double-layer technique HR for bnrt,eriophage (Adams, 1969). Enrichment of the Uddlovihrio popitlation with susc!eptiblc bacteria was considertd undcsirablr by S t d p ~ i n d Stiirr ( I !tfi:3b) hcwausc it inarcwud intorferc~c* from t)uc.trriophiqq~s, whilo t i iimg ( I!M!b) fbunrl strati rnrichrnc~rit beneficial when host cwlt iirrs w ( w iiscrl which woro renisttint, to hwtcrio- phagcs. The cult ures thus ohtiiiiicvl wwr sut)scqu(~nt~y pur'ifietl IJy thrw successive single-plaqur isolations and wcro further c.hrokcnl rnivro- scopically for the presencir of Bdrllocihrio, hascvl on t,hn Hmall siw, active motility, antl pHrltsiti('lpret1utor.y hehaviortr (Stolp and Sturr, 1963b). Whcr isolation antl en umcration proccclurss have hocn us(vl (see Section II.D, p. 220).

B. CULTURE MEDIA

Stolp and Pctzold ( 1962) isolated Hdollovibrio in riutrierrt ttrotti- agar using the double-layer twhniyue. The top arid hottom layers contained, in addition to nutrient broth, 0.9% and 1.8% (w/v) Bacto agar, respectively. A somewhat richer peptone-yeast extract agar was used by Stolp and Starr (1963b) to grow host-independent bdellovibrios;

1'11 Y sic )I,( i Y c ) I(* 'r I I IC I I I 1 14: I , I ,I ) v I II I( I I )s 219

i t w t i H i s t s I ) I ' 1.0% (w/v) Iwl)tonc~. tb:i'%~ ( w / v ) y ~ i ~ s t , c b x l r i i d , ( I ' t l 0 . H ) . Hiill ivull i i t i t l ( ' ; w i t h ( I !W) i i s c ~ l 1 l i t 1 ( ' ~ i n i 1 ~ 1 ~ c 4 1 - I l o h itw(Iiuit~ or 11

in prospective host, biac riii M liich l i d 1 ) c ~ t ~ n ( d t ~ i v t ~ t ~ ~ l in ti vc tlifferont culture media.

Yeast peptoiie broth (0.3%, w/v, yeast extract, o.O6%, w/v, peptone, pH 7.2; Stolp and Starr, 1963b) is widely used (Dias and Bhat, 1966; Klein and Casida, 1967; Parker and Grove, 1970) because its relatively low nutrient content gives host lawns of proper density and physiological conditions to favour Bdellovibrio development. A 10-fold dilution of nutrient broth (Stolp and Petzold, 1962) or a four-fold dilution of yeast peptone (Shilo and Bruff, 1965) are also used for the Rame reason.

Starr and Baigont (1966) incorporated tris-HC1 buffer (0.06 M; pH 7.5) into the yeast peptme broth to obtain a constant pH value. Schcrff ~t nl. (1966) cdtivated host bacteria Reparately in a complex mediiim iiritl ttirn grew Bddkovihrio in c r l l suspensions of host bacteria in distilled wtitchr, hoping thereby to itvoid unknown effects of the complex c.ulturc medium. (JudiJi PI nl. (1967) employed the same voiicspt8, but suspendctl thc host t):iateria in diddled water containing s d t s (KCI, C h ( : I 2 , M g H 0 4 , r ~ t i t l NILOI). Wator agar has bcun usod sucoess- fiil1,y b y (:illis r ~ n t l Ntikninrrrti (1!)70).

'I'he S I I ~ V C ~ of cwlt,urc rnctlirb Icudn to the! conclusion that the complex media have some limitations in connection with the undefined composition of the medium, the metabolism of the host bacterium, and the accumulation of metabolic products possibly inhibitory for the bdellovibrio. Some of these problems might be avoided by suspending washed cells of t h host t t n d Hdcdovihrio in tris-HCl (StolI) and Starr, 1963h) or H E PES (N-2 - l r~droxyeth~ l -~~ i~~er~~ int~ -N' -2 -r thnne srrlphonic acid)- NuOH buffer (042.5 41 ; pH 7.5) in the presence of rcquiretl cations, CaZ ' and/or Mg'+ (Sinipson and Robinson, 19611; Huang, 1968; Huang and Starr, 1971a). In any case, rational design of culture mctdia m u s t await exploration of the exad nutritional rocpircmonts of JidPllovibrio (HW: Section IV.E, p. 246).

IllOdifiCVl J<rON'lI'S l l l ~ Y ~ ~ l l l l l . l ~ l l l ' ~ ( ' l ' i'/ i t / . ( I !)tix) glV'U /k?f ' / / fJdhrifJ \v

c. RfAINTEKASC'E OF Bdrllovibrio C'L'LTUREH

In the present state of kiiowleclgc, maintaining cultnrcs of M P l l o - vibrio is a rather exiisperating art. ~-loRt-(lar)t!n'lt~iit IidPllovibrio iHolitt(:s

were maintainrtl (Stolp and Starr, l!+li:jt)) hy c lcpoxi t ing IL drop of f'rcdl BdpZlovibrio lys thb on the tol) li~yor of yotLst pq)t,ono ngrrr twnttiitiing host hacteria in n flask ; subairlt urc wiis incltlt: monthly. HoHt-inticpc!ndont cultures were maintained a t room temperature on peptono-yeast extract agar plate8 and transferred monthlv (Stoln and Starr. 1963131.

220 h1. 1'. W A I t I I \ N I ) , I . (I. I I I IANl i

l~ l l~g(qu ( ' I / I / . ( I!)(#) (i)llll(l t I l i l I j 1 INb V i i l l h * ( '01JlI~ 01' IIOIJl - l h l ~ M ~ l l ~ h b l l ~ ~

]i(/V//fJf*jh/'iO \\' ( Y 1 l l H ~ ~ ~ V ' l W l M Y ~ ~'I'OlIl ~ ~ ~ " / l l l ~ . 1 0 I ( ) / l l l l . k \ ' l l V l l l<l' l)b 111.1 (I

lyncitc. for :{ti ( I I I ~ N . A h i n i i i i t l Ih iv iH (I!) ' / ( ) ) rq)oi*l 1 Iiril iiiorv tJiriii !MV!4 of t h HtielloviBrb ccl ln lost tlicir ii~fcrt~ivity iii tho Iysliton Hftm t,lirrr dtlyfi at room temperature. On tlw other haiid, Much (ll)W), Seidler (lQU8), and Hiiang (1960) indirate that they were riblo to maintain Rdol/onibrio Iynates fit, 4" for four to oiglit, months.

I,yo~)hilizcit,ioii of skim-inilk HuspcnHionH of fleZZfJvibrio obtained from coiiHiientlly lynorl plates has gonerelly been satisfactory. However, the results are sometimes erratic. The milk suspension of BdeZZovibrio in this case contains also the semi-solid agar from the lawn, and the viability seems to be influenced by the composition of the medium employed for propagation of the host and bdellovibrio. Media which support limited growth of the hoRt bacteria usually give maximal recovery of the Bdel- Zovihrio after lyophilization. A horse serum-nutrient broth plus 7.6% glucose (tinal concent'ration) has been employed successfully a8 a suspending mt*dium in lyophilizcition (Huang, 1969). Sullivan and Casida (1968) reported that the slime produced by Azotobactw helped to pre- serve Bdellovihrio in the frozen state. Freezing Bdellovihrio lysates with 10% dirncth,yl~iilphoxide or glyctcrol added, oithor alonct or p l u ~ glucose tin in tjho prwticc witch bactcriophtigc! (Ychlrt ant1 Doi, 1966), ha8 been suggostctl by H . Stolp (perRonal rommiinic!ation). BdeZlovibrio may bo preserved frozen at -20' and -78" for a t least 40 days with excellent rccovery (K. Nakamura, personal communication). However, Abram and Davis (1970) found that five strains of BdeZZovibrio were highly susceptible to a frecze-thaw cycle.

1). I)lSlvItl13UTl(JN A N D IM1WRTANC:E IN NATlJItE

Bdellovibrios are widely distributed in nature. They have boon found in sewage, river and lake water, seawater, soil, and in rice paddy soil and irrigation water. The population has been estimatccl at about 40-200 bdellavibrios/g. of Californian soils (Stolp and Starr, 1963h) ; about 1,C)00-70,000 hdcllovibrios/g. in 29 soils from I3 states in oa~tarn and central United Stkites (Klein el al., 1966; Klein and Casida, 1967); Western Australian soils scored 2-1,200 bdcllovitJrion/g. (Parker and Grove, 1970) ; Newage in l n r h cmtained lip to 864 hrl~!llovihrios/ml. ( I h and Hhat, 1965) ttnd powage from I+itrrcxt HH rni idi tts I f ~ ) , f ~ f ~ f ) hd(!hJ- vibrion/ml. ((At1c;lin P/ d., l ! ) ~ ) ; ihoiit 40 MI ~~( l ( ! l l (~vi t~r i (~H/ml , won3 found in s w or pard wiitc-r i r i I x r r ~ ~ l (Shilo, I !tnfi).

Tho xovc!ral mothoclx which httvv hwi i i i ~ a d rmult in r r luoh vurietion in rccovcry of tith!~hvi tJrioH. 'lltro or#&riinrn h r b H h n c!tirrtnc~retc:d t)y thr! douhle-1agc.r pIa(411e terhnic~ir~ directly wi thout filtration (Klein untl

PHY8IOLOOY OF THE BDELLOVIBRIOS 221

Casida, 1967), by the most-probable-number method after centrifugation of a sewage suspcn,qion ( I h e ixtld RhtLt, l!)M), or on double-la.ver lawns after centrifupatiou followed hy diflerential liltrutioii (Stolp and Starr, l963a, b ; Parker and Grokt, 1970). Vuron utitl Shilo (l!NA) and Varon (1968) have applied differential centrifugation in linear Picoll gradientt3, and have suggested that two successive different id filtrations (first 1-2 prn, tmd then 0.22 pm. pore i z e ) of the upper leyer of the lqicoll griidietit could I)c used for quantitative ussay of Ildallovibrio i n nature (Shilo, 1 96!1).

The possi bk importance of’ Bdallovihrio in the biological control of pathogenic and suprophytic microfloru in nature was suggested by Stolp atid Petzold ( l!f(i2) and Stolp a i i d Sturr ( 1963b). This ha# been estab- lishvd to nortw vxtcwt in rivc8r wiLtw ( ( i t i d i n I)/ i l l , , l!Mi’, 196Hu, I ) , c‘,

l!)6!);iq v ; (iti6liii i i i ~ l I m t i k ) l i i i , I!)fM, i!)(i7, i!)W), i i i nc:iLwafm (Mitdrc4 I,/ d., i!)fG’; M i t v t i c ~ l l t m l hlorris, I!)(;!); ( j i idit i ~ i i i t l (hhiooh, I!b70), i i i soil (Kloi t i t in t i (heiclu, l!)ti7), t L i d prohubly i~ l so in ncwagt ( D i m and l h t , 1‘365). Furthcr rcmarlw on this su1)jcc.t will be found in Section 1V.F (p. 24!)).

H. A ‘i’AXONOMI( AND ~’RRMINC)LOOI(’AT, NOTE

The poesibhlt~ relationship of the gon us Hdellovibrio to the genera Spirillunr, ant1 i’ibrio has been unalysed by Sturr and Seidler (1971), who also have attempted to develop a concept t ~ i i c l u definition of Bdellovihrio.

A plurality of species has now been discerned within the genus Bdellovibrio (Seidler and Starr, 1968b; Seidler et al., 1969, 1972; Starr and Seidler, 1971). The use of the epithet “Hdellovibrio bacteriovorus” should be limited to the nomenclatural type specimen of that species- strain Bd. 100, as designated by Stolp and Starr (1969b)-and to closely related strains (e.g. Bd. lo!)). On the basis of rnolecular arid other evidence, Seidlcr et nl . (1!)72) have reccwtly clolinc1;ltod two u&litional species; namely fidPlbuibrir> s/olpii (with st riiiri /id. IJKi2 (le4giidx:tl I)y t hc:rn ;is t h (1 no rn ( * n c l i L t 11 ril I typck sj)wi it I ( ’ t i ) i i I i f 1 /idP//f)77ibPio durrii ( w i t h strriitr lkl A3.12 tl(.nigtlilt(.tl t)y 1 Iic.tti ;is t l i v I i o f t r c ~ l l c : i ; L ~ ~ l J r r l ~ tylicu sptvirnc.n). ‘1’0 avoitl norncbiic+l;d iiriil i i if i*lic*it I W i l l t t i t ! prcwtvit (:HxIL,~. w(’ will simply tine straiii tlesigniitioitn: c . g . lidPllovibrio st ruiir A3. Id (or Nd.

11-1 (for host-independent,), tl-I) (for host -dcpen(leiit), end b’-P (for facultatively-parasitic). Sections lV.A.5 (1). 23%) and JV.E (p. 849) contain remarks about the differencc~s w l i i d i vxist twtwcwt thv Ihvin anti +Jerusalem strain# of fid. 109. Iprirt hcr cwrfiision woii ld h c a iivoidc.d if they were separatd! clesigrtatetl i i t t h futurt. an /id. I O H ( Ihv in ) and hd. 109 (Jeruzlalrrn ) , rest wc t i vet y .

A3.12). In addition, where ntwssary for chrity, iw wil l use thc. p r d 1xes

233 M. P. STARR AND .T. C . 4 . IIUANO

111. Structure and Chomicsl Composition of Bdellovibrio

A. MORPHOI,OOY A N D ULTRASTR~ICTUI~B:

Bdellovib14os are usual1,y sniull, curved, Cram-ticgativc rods, 0-26-0.4 pm. wide and 04-1.2 pm. long (Stolp and Petzold, 1962). Vibrios of slightly different proportions have also been described (Stolp and

Sturr, 1963b; Murray, 1964; 131trnliurn ct d., 1970; Abram und Ihvis, 1970). The variation depends oti #train and specicN differences, on the stage in the life cycle at which t h u ct!lln are meaautstl (Starr and Baigent, 1966), on the medium used (rich media, e.g. trypticaHe-yeast extract or nutrient broth, give 6-20% long form8 which sre straight or coiled, according to Abram and Davis, 1970), and on incubation oonditiom (Mishustin and Nikitina, 1970).

BdeZlovibm'b has an extremely thick flagellurn (Fig. 3 ; 28 nm. according to Seidler aiid Starr, 1967, 1W8a; 21-2.5 nm. according to Ahram and

.) .> ‘{

Davis, 1970), which consists of a sheath (7.5 nm. thick) surrounding a core (13 nm. diameter) (Seidler and Starr, 1967, 196th). Slightly different proportions have also been reported (Shilo, 1966; Burger P I nl., 1968; Burnhani et ul . , 1970; Abrnin wid Davis, 1!)70: Uematsu and M’akimoto, 1970). The shruth, which is c+ontinuoux wi th the c ~ l l ~ 1 1 1 (Miirrwy, l!)(i.t.; Yvidlw ; i t id S t w . I!)( iHti; l~iiiwliitin F/ t t l . , l ! ~ N i ) ; Ahrtbiri t r 1 1 1 1

Ihvis, 1!)70), I I I ~ I ~ ~ ~ t b l l , brcbak, ~ L I I ~ S C ’ ~ ) ~ L I X ~ C ! fro111 tl~v ( 1 0 1 ~ 1 i i i t hci presence of (i ill-urea (Seidler and Sttirr, 1908~) . Even though the wall tbppears to be morphologically continuous with the sheath, i t is not known why urea affects only the sheath but not the cell wall. How- evrr, A l m m and Davis (1970) believe that the flagellar sheath material might h a morc susrcptible to disruption than the rell wall.

Appcndagcs or extrusions have frequcntly been observed in preparationn stained with phosphotungstir acid in the electron microscope (Huti i ig rt al., 1966; Shilo, 1966). Shilo (1966) and Abram and Shilo (1967) found spike-like fil~mcwts, 4-5- 5.5 nni. in diameter and 0.8 pm. in Irngth, located a t the pole distal to thc flagellum, and suggested that the filaments ma.v play a role in the attachment and penetration of the host cell since this pole arts in attachment to the host cell. These structures are believed by some writers to be artifacts formed during fixation or staining. Murray (1968) noted that zinc ionx were required in the fixiitivr for preserving the cell wtdi r~nd rell membrane of Bdellovibrio ; ill thr ttbs(w(*r of ziiic, thc wll wall rn;Ly twcomc? loonencxl. Ilddlovibrio hw a cvll-wall profilr typicitl of ( : I W ~ I - I I ~ & ~ : ; L ~ ~ V C ha(-trriu, but it tius hem said t,o hick the usual pc~~~titloglvc~tii~ la,ycr commonly illustratcd in ot>her cram-negative bacteria (Murray and Maier, lBM), although chemical analysis (Tinelli at nl., 1970) of the cell wall of Bd. 109 has proven the presence of peptidoglycan components.

Abram and Davis ( 1 970) observed intric~~tcb surface projevtions from the cell of negatively stained hdollovi hiox. Thcsr projet-tionH werc varyingly induced (depending upon the type of stains applied), and they were easily detached from the cells and fused to form vcsiclesand tubulen which surrounded the cell. Uranyl acetate, which stains Bddlovihrio cells positively and negativcly, was found not to have these effects and preparations stained with it show smooth surfaces with many folds. The stains which cause the formation of projections from Hdellovibrio cells also caused dispersion of the isolated cell wall. An undulate outermost surface was seen in freeze-etched preparations. The isolated cell wall has two amooth surface layers. The inner-wall layer is separate from the outer layer. Scattered particles (6-1 0 nm. i n tliamt.%t,er) and ptctchcs of inner layer frorn the wall COV(T tlw o i i t n i f l ( * o f i h cytopli~nmic. rrwmhrtLIipn (Ahram untl Davis, 1!470).

A “hold fast” has hcelr rcportcd at thv tlntc~ior (dliLgdIaic!d) c . 1 ~ 1 of’

PtIYRIOCO(: Y OY THE BLIELLO\’IRRIOS --.

“‘‘4 11 I>. S T ~ I ~ I L + \ I ) . I (‘ -(*. i i i r n i w

some Hdelloi*ibrio strains (Sliilo. l!)(i(i: Abriitn and Shilo, l!Ni7 : H itiit~g, 1!369: “infcbotion cushion”, Srhwff P t nl., 1I)BG, Srlierff, l!MM: “lar,ye convoliition“. Btanhtun ct n l . , 1UtiHb). Scherff el n l . ( 1 9Ni) arid others have siiggested that this “holdftixt” structure might nitvliate itttach- nierit and penetration of HdPZlovihrio. However, Starr and Raigent (1$)66) tmd Abrnni and 1)nvis (1!170) were unithle> to find sudi t~ tlrviw i l l thcir prq)itribtions, i t t l t l tho c~rtrrcwt writings fi-onl t w o groiips (Hitrn- I ~ t ~ t n / I / . , 1!)70; AI)rtiin i ~ t t d I h v i x , 1!)70) siigp4, t h t t his “ l i o l d f t t ~ t ”

1niL.y I)v t i t i wtifiwt,, cvtusc4 t)y disorgiinization o f t h r cell sitrfitce. A t t h e anterior c w i of Hddovibrio oclls, two distinct features have

t r c w obscrved (Abrant and Dtivis, 1970). Fibres whirh vary in length ( u p to 1.6 pm.) and i n diameter (8-10 nm.) emerge from the anterior end. ‘I’hcy may be straight or erirved with ttngular bends. A cell generally bears two to three fibres and occ*asionttlly as mtitiy as six. These structures tire not won in the round form of BdPZZovihrio or in aged cells. Six to 13 (+otron-tlcnsc circles (ring striietures) with nn outer diameter of !J to 12 t ir i i . , whicih arc h i l t into thc cell wtill i tntl nssocic~t~cd with the proto- I)Itwt, i ~ r ~ tilso visihlo l i t ttw iint,cbrior t*n(ls; two to ttircbc f i t m n cwierg!t. from it . ‘I’ho ring strrwtiiro rntt*y tw H(*iLttCr(l(l or in clttsters. Attram and Ihvis (1!)70) twlicvc thtLt t l i t w ring stxirctumn and thc fibres rnay he related to tho partisit ic itctivity of HdPlZovihrio.

Coinpact hdies ( I 50-300 nm. long, 70-1 20 nm. wide) are frequently observed in t~ certain negativrly st ;lined preparation ; these structures wpr‘c not, s ~ c n in freeze-etched cells (Abram and Davis, 1970) . Vibrioid (~11s huvc! un average of two, and long form8 have ~ ~ v e r r t l , such structures. Theso hodirs show ib wJsiiIar ltmiriiLtetl struc:tjurc\ which upI)earH as “fingw-print” pi riin when stiiinetl with lit,hiurn tungstate and potrts- sium ~)hospIiotungstat(~ ttt IJH 7.6. wiicwus h o t h Iamintited ~ t n d vesicu- lated structures were seen in preparations sttiitwtl with pntarisiiim phosphotunpstate at p H 8.1. ‘I’hesc h l i c s arid intracc!lliilar laminittc*rl structures tire believed to cxtrude from thr prntoplast as a rcsult of an osmotic taffect exerted 011 the cchlls by t h c strain (Ahram and Ihvis, 1 !) 7 ( )) .

Densely sttiined reginiis are frec~ucntly seen emheddod in thc! nuulr:ur areas which are surroii ndctl hy rihosomw. I~lccbtrori-rlonHc in(-lusion bodies aw often swn ( H uatig, I {Jfi!); Atjram unfl Ihvis, I!j70). Ilpitic.r a11d Shilo ( I WfJ) fOlt l l ( l thtit, fiJrnItitiot1 O f thi!H(! i t i ( * l t i s i ~ t i t J O f j i ( w wiis fuvourrd k1.y growing /iduZlotiihrio cdls i t i o i ic~-tc~nttr st rwgt t i ntitjricattt, both containing hcttt-killd vt4ls (65 ’, mirl.) of / ’ . ~ ~ ~ ~ ~ / ~ / , ~ n ~ ~ ~ , / ~ ~ /Lpr/L-

ginosa Mesnsoni(+i httvc~ t w t i otwrvtvl at tJtw t~titc.rior (q1(1 of thp clr.11 (Starr nnrl Haig:(wt, IUW: K i i r i i l i t i r n ut d. , I I I f J H h : tiitarig, i!jf;II). ‘l’ttf. I I I C ~ O S O I I ~ C S t h u g h t to tw t i w i ( - i i i t o d wit,ti i i t t t w j i r l l ( q l t (IjIIr11IIatII et d., 19681,) &lid c d l division (Biirilltain 4 4 nl. , 1970).

PHYRIOLOGY OF THE l3UELtOVIl3RIOS

2211 M . 1’. ST4Itlt AN11 ,I - ( I . IlITANC:

Burger ef al . ( 1 968) observed that some cells of Bd. I!‘ were “encysted” withi i i thv tlisrupttd host cdls of’ Rhonospi,i//uni rrrbr?it//; t l w fin(. striicttirc of t twsc ( w i ~ j . s t c d c~*lls (iilso c*allrd ‘ ‘ r ( ~ 4 ing I ) o t l i c w ” ) is riit I i c ~ diKereiit from t litit of 1lor11ii11 intri~ci4111tw I)ctdlovibrio ( + P I I S . tlotwigcr P I r r l . (1!)72), who studit4 i n (hatiti1 the dt.\~rlol)tii(~i’t ikiid strnrturo of‘

tlicse rtwting hoditis or cysts i t i NrJ. \V grown in h’. rubrum (Fig. 4 ) - report that t l i e redrig body or cyst is 1.2 pni. long by O*t i pm. widc, i ~ n d that i t starts forming Home three Iioars after the establishment of host- Hdellovihrio cwltiire by deposition of amorphorus niaterirtls around the pcriphery of’ the Hdellovihrio cell. Thtm two layers, an outermost c e l l wid1 t ~ n d ~ L I I innermost plit,qmu rnembrarw, itre formed ant1 they encloxc t I N & grrwulur tytoplibsm c ~ n t l ti t)rillar nurleoplasni rcgions. LJpn niatura- t i o i i , t h ri~rrting h l y (cyst) witsirrts of i L t,iiic*k outw luyor (30-40 nni . ) L L I I ~ t i (VII wall tliffi.rentiut~c~i into u tripurtite inner layer. The resting botly (cyst) hus been otxwrved only in Hd. W grown in R. ruhrum, but not in Hd. M’ grown in F I . coli nor in Ha. I o!) grown in F I . coli l3 (Hocriiger rl /I/., l!172),

13. CHEMICAL COMPOSITION OF l~ddlov i lwio CELLS

The DNA content of host-inde~,t~ndent Bd. AX12 was found by Seidler et nl. (1969) to be 5.01 -1 0.04% DNA oil it dry weight basis. Chromatography of t h e uric1 hydrolyrrute of the ni~clci(: acids (Pig. 5).

r i i ~ ~ ~ o ~ , o i : v 010 '1'11 E ~ i ) ~ r , ~ o v i i t i t i i i* 327

isolated from host-independent, Ikl. A3.12 and host-independent Bd 109 by Mnrmur's (1961) mrtliocl, rcvritled the prosonce of d-TMP, UMP, ribose, d-CMP, adenine and guanine (Seidler d nl., 1969). Based on the GC content of DNA its determined by the buoyantJ density in caesium chloride gradients (Mandel et al., 1 W i X ) and by an optical melting method

0

(Marmur and h t y , l9W), Scitllcr ~1 al. ( I !jfi!j, 1!)72) w ( w i th l t : to divitltr the strains of host-dependent ~ i t l host - i i i ( I ( ~ ~ ~ w h i L hdc~llovi hriox in to two distinctive groups, namely a Iiigh4iC groiip (most Htrrtins) with 50-61% U C and i t low-GO group (Hd. A3.12, / Z d . UKi2, ant1 /Id. 321) with 42-43% in their UNAs. These sttitlies did not, howcver, show any unusual structure or composition in the DNAs of t h e bdellovi brios.

Rittenberg and Shilo (1970) found that Bd. 109 contains 0.42 & 0.05

248 M. P. NTAPl~ AN I ) .I. V.4'. IlLrAN(I

mg. protein per loLo cells 110 min. digestjioii in NaOH (by the method of Lowry et at?., 195l)l. Host-independent strainA of BdelZovibrio have protein contents of 60-(16% dry weight (Seidler and Starr, 1908b). All of the usual amino acids are present (Pig. 6).

The major phospholipids of Bd. UKi2 are phosphatidylethanolamine and phosphatidylglycrrol ; there i w a lesser uinoiint of phosphatidic acid (Stchcr ttiid Coiiti, 11170). Arat,tLto- or sc?ritict-ltlbc.llcd lilkl W ~ L H prctparecl try inoorportAiig I I ,4-'4CJ iwt?ttLte, I : ) - I4 ( ! / nwiiio, or I j21' I orthophosphate into yemt extrat~t-p~~~)tonc mttdiuni. Alkalino hydrolysin of the lipid revealed the prwencft of f'utty twiclx on the ohmmutogram. The ulkali- Httible r(widuv of'the lipid coi i tu i t in Nphingolipid, whir+ in ram! in 1)acteriu (Stc!irior i m l Ooriti, 1970). 'l'lio j)olt~r portioti of' tho two itiujor nphiago- lipidn of' / I d . IJK i2, u p ) n tlrtgriulet,ioii, givv riw to thc H t L r r i c ~)honphorun- ooiittbitiilig prodwtN (~to i l ic r I./ d., 1!)7 t ). Aftor hytlro!,ynis with ~ N - H C I ( I W, 4 Iir,), t,ho wutcr-nolublc* portiorr of ttic procluctt aoiinintw of& Ningle m ujor uo t n pound w hie h con tui IIH phoe p t ior LIB und is I iiii h y d r i n -posi tive , Alkaliiio phosphatusu (frorn E. coli) or hydrolynis in ~ N - H C ~ (125", 100 hr.) does not release inorganic phosphorus from this compound. This characteristic of tolerance to hydrochloric acid and alkaline phosphatase suggests that the uncharaoterized compound contains a carbon-phosphorus bond, and that phosphonolipids are present in Bd. UKi2. A third phosphorus-containing sphingolipid was found, which is alkali-labile like other phospholipids and capable of releasing inorgunic phosphorus when hydrolysed with hydrochloric acid.

Tinelli at al. (1970) determined the chemical composition of the cell wall of Bd. 1011 harvmted from u 20 hr.-aulturc with B. coli B. The iNoIutctd cdl WUIIR, uftor hydrolJ%iH in f jN- H(11 ( IW, 20 hr.), contain two umino ~ugaiw (murumic* ocicl aiitl g1uc:oHurviino) in udrlition to I3 other amino acids, all of which ure Himilur to thc U R I J ~ L ~ (tell-wdl oompotiontn of pro karyoti (7 m icro-orga n i H rn H.

IV. Symbiosis Between Host-Dependent Bdellovibrios and HoNt Celh

In the earliest studies 011 the nyrrihiotk usnociation Iwtwcm ljdello- vibrio and it8 bacterial host, Stolp wid l't!txoltl ( I !jfi2) followt!d thc interaction by means of phusewiitrt~wt microscopy arid whudowed preparations in the electron microacope. They found that the bdellovibrio collided violently with, and attached to, the hoet cell surface, like a leech attaches to an animal's Rkiri, within a few minutes after establishment of the two-membered culture. The host cclln subNequently lyaed. Further observation# of this sort (Pig. 7) led Stolp und Starr (1963b) to describe Jidellovibrio a8 a predatory, octoparasitic, and bacteriolytic micro-orgttoim. The intermediate intritoellular n tep

A , 1,lp 15 :V( $1, E 0 Iz 11 f WI’ - I 15 I ’ E h 1) 14; “I’ I< 1) ICI,I,OV I lilt1 OH

Starr and Htiigent (l!j66), Huang rl d. ( I ! M j ) , ant1 Scharft rl ad. (1966) reported simulttlneously and intl(~pcndcntly that Reverd c h i t i n c t steps are involved in the host-Bddloaibrio interaction (Fig. 8). In the light of current knowledge and our present purpose, these s t e p might he designated as fol lon~~ : (1) “recognition of prey” and movement toward i t ; (2) attachment of Bdellovibrio to its host cell: (3) entrance into the host cell by the bdellovibrio ; (4) rounding up (“sphaeroplasting”) or other morphological chrmges in the liost cell: (5) developrnent and multiplication of the bdellovibrio within t h e hont cell; and (6) disruption of t h c hont c d l and rc4ciisc. of t tw /M&ivihrio progrny. ‘l’I~~:w reports xho N t h ii t / k / r l l n d w i o ( I vfi 11 i 1 (4). is i I I I ‘ ‘ c * 1) c I ( I uinisi t (1. ’ , I I I c I r~ c~orrwtly ISttwr u i i t l Sc~i(llc~, l ! )71 , d i i i g 11w o ] l i i i i o t t 01’ 11. It. t I ( i n c . : t i t e l A l . 1’. Stair (uinpiitJlishcd obscrvat ions) 1, ui i iut ri~iiiiirt~l or i t ~ t r i ~ - i i i t ( 1 ~ ~ i r n ( ~ i i t ~ . I

33 I

232 M . P. HI’Altlt A N I ) J. (’.-(‘. HIIANO

ptLriwitcc, iiot, i u i “C’Cto~)(iiiUNit,(”’ I ~ H cltuc!r’il)cd iii thc first py)(m (Stolp wid Yctzold, lOti2; 8tolp and Starr, 1963a, b; Shilo and Bruff, 1966; Klein aiid Casida, 1967) and, rather puzzlingly, in a recent article (Uematsu aiid Wakimoto, 1970).

I . Altaihmnt

Chciriotuxie (presently under study by M. P. Starr and hie colleaguefi) haa been Huggcst!ed as playing e role in attachment, bewu8e Bdcllovibrio bc?tltbvee ~ L R though it cttn “recogtiizc” the proqmotive host cell in a mixcd c:ult,urc! of non-~u~cept ibl~ iiiid Huficcptibla bactoria (Starr and Baigent, I(l(i(i; Stolp, 1068; set’, U ~ R O , the remarks about ho8t specificity in Hetrtion I V. I”, p. 249).

(~encrally the non-flagellated end of the bdellovibrio attaches to the host cell (Scherff et al., 1966; Starr and Baigent, 1066; Stolp and Petzold, 1962; Stolp and Starr, 1963b; Burnham et al., 19fl8b). Murray (1964), however, seems to say that the flagellated end attaches, but this is probably a lap8u8 calami. Attachment of Bdellovibrio to the host cell occuri in seconds. An actively motile bdellovibrio, moving at an unusually high velocity (Stolp, IH67a, b; Simpson and Robinson, 1968), hits a host cell with such force that it may push it for several cell lengths (Stolp and Starr, 1963b). Stolp and Starr (1963b) suggested that the actual collision between bdellovibrio and host, and the drilling action (or an arm-in-socket type of motion, according to Starr and Baigent, 1966) of the motile bdellovibrio against the host cell, are necessary factorH in eRtablishing the attachment. The excellent time-lapse films of Stolp (Il)67a, 1)) Reem to r up pod thiH hypotheeis.

In the earliest fitagcfi, attachmerit is reverdhle (Stolp end Starr, 1963b; Starr and Baigent, 1 U M l ; I3urnharn el d., 1HtlHb). Infoatioii of a single host cell by wvcrtil Hdt?Zlovihrio ceiln ti&^ been rioted (Stolp and Starr, 1963b; Stolp, 1 9 6 7 ~ ; Sehurff e6 d., 1RH6; 8hilo arid Hrufl’, 1005) ; this depends in part upon the relative number of host and parasite aells in the two-membered culturw (Rtolp and Starr, l963b; Stolp, 1964). Sometimes, such multiple attachmenh to a single host cell lead to rapid disintegration of the host cell-“IysiR-from-without”-with no apparent intracellular multiplication (Abram and Shilo, 1967). Bdellowibrio Itttaohes, penetrates, and multiplies in all growth phasex of the host culture (Shilo, 1969).

More Bdellovibrio celh attach to host cells when the relative concentra- tioiis are one bdellovihrio re11 to I 0 hoHt cells (multiplicity of infection = 0. I ) tJhrtn wider. itllJ’ otlirr cwnrlitiorin t(!st(!il ( V w m and Shilo, 1 W8). Unctttctchotl t ~ i i t l tittuchc!tl fiddltrvil~rirr c: tr l l~ , i i i t h i n ( t t b ~ p , wor(h enurnrrcttcd after dif€urctitial filtration of‘ tho two-rn(jrnhcr(.<J (:uiturc.

PIIYSIOLO(’IY O F THE: UI~EIAA~VIUltIOS 233

The multiplicity of infection, however, is not crit ival for overdl growth since an initially few Bdellovibrio cells are able to wusc the cvinplete lysis of a concentrated bacterial cell suspension (Stolp and Starr, 1963b). The multiplicity of infection affects the growth rate, but not the yield, of Bdrllovibrio.

Many other factors arc known to i i f fk t t I i c tittwhment of Utlrllovihrio to the host cell in addition to multiplicity of infection; these include composition and pH value of the niedium, oxygen tension, and incubation tetnpc~raturc~ (Varon and Shilo, 196%). Motility of F2deNovibrio is canc !n t id for attwlitnont (Stolp and Starr, I!Nit); Stollb, I !M; Varon m t l Sliilo, l!Mki: I)icvlrid\ o/ ~ r l , I !M!J, I !J70; A h r n i i i i c l I h v i s , l!J70). I l ( k i ~ t - I c i l l c v l Iiosf, c*dls ~ I , W h*ss si i i f i h h l Ihr iittiwtriiic~tit, I)y / idvIhi /&o t , I~ i i i i iwc living wlls. /Ud /o i ) ib r io is rej)orttvl ,gc~tic~itll,y r io t to iLt twli to (:runi-positivc& t)acntctria (scv Scctioti 1V.F, 1). 261 ) ; tiowever, there have been ohswvations in our labortitory which show t,httt i2drZlovibrio can attac4i to f he cells of Bacillus .vutitiliy which are unc.ongenia1 for Bdplloiiibrio growt 11, arid even to cover glasses arid slides.

Starr and Baigent (1966) postulated that host and parasite were held together by “a strong surface bonding”, since a cytologictll device which might mediate the attachment seemed to be lacking. However, as detailed in Section III.A (p. 223), other workers have variously reported (or denied the existence of) such “hold fast” devices.

Varon and Shilo (1969a) measured the ability of Bd. 109 and Bd. GB to attach to mutant cells of E. coli €3 and J‘almonella typhimurium genetically different in cell-wall chemical composition (rhemotypes). l‘hcy found that chernotypc Ra (containing a conipletr “rough” core hut Iwking 0-specific8 side rhibins) Wits a 1)cW.r r( Ibtor for Hrlollovibtio thaii thv wilcl-tyl)c (stnootli) host strthitis. Olwtiiot,ypc~ I{ 1) ( i i l )sc~i ic*c~ of gluc.osanriticx) stiowed a Icss firm i L t h d i m v t i t Aclditiotirrl (l(4i(di(vi(-iw in the 11 antigen (chernotypc. It(.) f u r t h d ~ ~ ( w i i w e 1 rcwl)tor iwt i v i t y . Varon ; ~ n d Shilo ( I !JB!la) illtc~rprvtcrl t hPir (ItItiL ILH slcfqpf i i r ~ I h f the. location of rrrcptors for /2ddlovilirio i~ itr t h e . I ( ;ititigc*tr hiyw Aclclit i o t t of rough host cells to a systcam cotitaiiiitig smooth strsins c ~ f , S ( i / r ~ / o ~ / , ~ l / ~ i typhirnurium and H d . lo!) iiirreusccl thc attiic-hrnc~t. ‘ I ’ t i c h high Ic.vc4 of attachment, howwer, seems not to tx! esscbritial for growth of l i d u l l o - uibrio in thr host (*ell, beraitsc Bdellovibrio t l t * v c 4 o p c(~lliill.~ w d l i r i cc4ls defirient am1 atlec~trate in attachment sites J. ( ‘ -0. Hirang (unpiit)lishetl data), based on ii similar working hypotht~sis, fount1 thzit Nd. fj-5-S parasitized equally well the wilt1-tyj)ci and 1 1 -dic~motypc~ rri ictartt.s of Snlmonelln typhimurium (Kessel ~t a / . , 1 Wi), ii~rng re11 suspctisiions in tris-HC1 buffer containing (;a2 ‘ and Mg2 ’ . Klein and Casidu ( 1967) found that Bd. 0 x 9 - 1 tilid B d . 167-1 parasitized members of all 25 E . coli snbproaps which they tested as well its the S and 1% f o r m of E. c d i

234 M. P. RTAltlt A N D J. C.-C!. IilTANI;

MGH6. Encapsulation, which occurred in a mutant strain of Aerobmter c2mcne in the presence of' 1 %I Inctosc, did not nffonl ~)t~(ittwtion tigainHt tLt8t,twl; t)y Hd. Ar* IIR dt~t~oriniiictl

MWI tilid MWll, sirnilw tro the oiitmor;t lrsyt!r oti t h wall of". ~ ~ r p r n ~ u VHA (Murray, 1965; Miirrty. rt ccl.. 1!1(17; 1311~!I~iniro ~ t i t l Mwriiy, l970), ( i p p ~ t i i ~ tzo protoc.t. t h I i o H t , ( * ( + l l ~ I'roiii ~)}irti~it.ixnt,ioii by IkchJloviOrio (Huang, 1 NllH ; I3iioktniro, 1 I)7 1). h n i o v d of tho H~~rUVtilr~d layer (protein in nature; Buckmire and Murray, 1970) by treatment of host cells with EDTA and sodium lauryl sulphate, by heating, or by spontane- ous mutation, rendered them susceptible to parasitization. This factor might account for our being able to show the susceptibility of& 8erpen.9 VHL (defective structured layer) to Bd. W, whoreas Burger et a2. (1968) reported it would not parasitize their Ntrains of A!. 8erpenu (which may havc had u complete structured layer).

Surviving horJt clones reshtant to LldeZZovihrio havc iiot yet been found in two-mumbered cu1trrrc:rJ (Stolp c ~ r i c l Htarr, 1Rff:Jh ; Shilo, 1968). 8uch it, I~rlelbvihrio-roxiRtunt, c 4 o i i c woiild nquiro tho (:iiIwity to block tho approach, attachmctiit, pciwtrutiori, and wnirrrilatiori of ha& materials for growth and rcl)r(id uc!t,iott tjy Udellowibrio, and/or the ability to rolewe i tself from t h o hoHt crivclopcn. 8 1 . I$. Sriellun and M. P. Sturr (unpublished dattL) oxitmilied mwiy preparations of the survivors of Bdellovibrio attack; in every case, the propagated clones of the sur- vivolw were &ill seemingly iss susceptible to attack by BdeZZovibrio as the origiiid host strainx.

2. Penetralion info Hod Cell Monk obwrvcrs agree t,hut, following attachment to u congenial

host cell, the Bddlovibrio breuclies thc hont (!ell wall within a few minutes and completes the intramural (intra-iiitegumentsl) penetration of the host cell several minutes therellfter (Fig. 8). Stolp (1907a, b), using cinematography, observed that penetration waa completed within seconds in the system he studied but, more typically, the penetration is reported to begin within 1-20 min. after attachment (3-20 min.; Burgeret at., 1968) and to be complete within 5-60 min. after attachment (Starr and Bai@nt, 1UUfI; l~)-80 miir. : I3urgw a! J., J M X ) . 'I'hc paraHitc! losos its flagellum diiriiig atfachnieiit or itivwioti.

Vuron i ir ic l Shilo ( I WiH) rncrw~iirctl the ~iontb~.tioit of l~drlloviBrio into

1)Itqi i t t i i )r i irc \ t8 io i i ( M i i d i . I !)(in). Tlw o\rt,c.nnost! Htrlrct~llrl~tl luyw 011 t'llo 1 d I W%ll of Spi r i l l t! ) t ) nPrpP?L"

Pra. I).

230 M. P. BTARR AND J. C.-U. IIUANG

the host cell by preparing * 4C-labelled Hdellovitkio and determining radioactivity in the host cell after scvcre niechanical agitation. Since penetration iu completely blocked by inhibitors of protein synthesis, such ay streptomycin, puroniycin and chloramphenicol, they suggest that iiiducible enzymes have to be formed by the parasite before it is able to penetrate the host cell. ThiR induction is believed by them to require a dirert contact between Bdellovibrio and its host cell or between BdelZo- vibrio and the inducer; the postulated inducer is masked by the host cell wall and is exposed to the Bdellovibrio onlyafterattachment. It would be of intorcst to uee whether Ruth poutulatcd inducible enzymes are formed i 11 u N y n t o m cot1 tuin ilia t 1 isr 11 1 )t,c!tl l i o n t uolls awl in tuct ildallovibrio ocllx.

‘ l ’ h nit!c:huiiinrri fbr tho forrrinthw of 8 pore ( !piga I)) in tho hoHt c t d l wid1 i N not iinniotlistctly (:lour. Htolp i ~ i i d Htilrr ( I WBb) pontulutotl that thr violent bullistic cdlisioii of the purwite with ttre hod cull and/or thc niibr;lcyiiont, rotibtioii (up to 100 revolutions per sec. according to Stolp, I ~ 6 7 a , b) of the purasite are renpoiisiblc! for pore formation. The work of Upinu et ul. (1907) and Burnham st al. (1968b) seeme to support this concept, since the peptidoglycan, which is responsible for rigidity of the host cell wall, was observed unchanged in electron micrographs during the earliest stages of the host-parasite interaction; not all of the host cells attacked by Bdellovibrio turned into spherical bodies at this stage. An abstract by Abram and Chou (1971) indicates that the Bdellovibrio must be able to approach and attach to the host protoplast in order for penetration to be completed.

Burnham el aE. (1988a,b) postulated that the Bdellovibrio penetrated through a bulge preformed on the host cell wall. At the point of attachment, the Nddlovibrio flattens uguinst i ts host aell at the outermout mcmbrunc in the holdfust region. The /jfk!sf!~ovibrio then ~ U R ~ C H into thr! centro of the bulge and breakH through the hoHt (!ell wuII. l d p i i i c 01 (11.

(1987) reportcd thu t hulgo forrncbt,ion on the hnnt, cell dirritig t h c t oirrly Htagen of nttwhmerit wus induced hy t h u i i i tmiul prcwurc t h ! timi, Cell. Huutlg (1060) UlNO OhHWVd U t)Ulgc! formc!d in H O r n f ! ttont ct!lh infected by Bdellovibrio. A oorri hintLtion of fiatom, including clurniLg:c! of the host cell and turgor preHstire, likely aontributo to the developrriont, of the bulge on the cell wall of thc hod,.

Constriction of the Bdellovibrio cell is usually How a~ the bdellovibrio advances through its restricted point of entry into thc ho8t cell. An intimate “arm+-socket? arrangement or “strong bon(]ing” between the Bdellovibrio and its hoclt cell has been poRtulated for thiH stage of t h o interaction. Thus, after primary penetration, tho Bdellovibrio ie not readily separated from its host cell by violent shaking or mixing (Burnham el a!., 1968b). Scherff el al. (1966) reported that the “infwtion cushion”, which they believe is a11 integral p r t of the parasite, becomes enlarged;

P I l Y sIoLol$ Y 0 Y ‘PH E 1% I ) E IJI 40v I I~ILIOS 237 i t is this part of the parasite which penetrates the host cell wall first. Thereafter, the Bdellovibrio cell pushes into the host cell; the host cell wall becomes distorted and is separated from the plasma membrane, forming vacuolated areas (“bubbles” : “balloons”) between the cytoplasmic membrane and the cell wall wlicre t he hdcllovibrios develop. Schcrff ct nl . (1966) observed tliitt i is niaiiy (IS sis ZMdlovihrio cells entered and reproduced wit’hin a single p a o t i d o ~ ~ i o i u ~ t l host cell.

3. (/r.o/rlh of 13tlcllovibrio lVi/hin Ifon/ C ~ l l n

I ti thv (lirys t)oforc> thc intnirniiral or iritrit-iiitcgiitnc?iital locus of Zjrldlovibrio WILH known, Stolp nncl Stwr ( 19A3h) suggestctl that Bdello- vibrio multijdicd c~xtracellularly by binary fission. Starr t m t l Waigcnt (1966) suhsrc~iic~ntly hhowed the intermediate formation within the host cell of a Itirgc. helical Bdellorihrio cell, which dividcd by multiple constriction to form several vibrioitl progeny. Although there arc varia- tioriti in the dct)ails, ScherfT ct t r l . (196fi), L@pine et al. (l967), Burnham et al. (1969, I970), and Huang (1969) also report that the bdellovibrios penetrate the host cell wall (but not the membrane) and develop intracellularly by thickening and elongation to form a helical filament. The filament, upon maturation, segments into individual vibrioid units, presumably by constriction (Abram and Shilo, 1967) since cross-wall formation has not been observed. The division starts with an asym- metric. constriction of the cytoplasmic membrane of the filamcntti, according to I3urnham 41 nl. (1069, 1970). ‘I‘hcii, thc outer lttyer of t h c s mothcr c d I wall breakti a t the dividing rcgionti. ‘I’hfl flagcllum and thc flagellar shcrbt,h form at, on(’ cmtl of the (lalighter c*rl l before it wparatw coniplctc1,y from the filamcwtoiis nrotlic~r c * c 4 (Nurnhr~m ot al., 1969, 1970). ‘rho btlrllovibrio prog(’ny withi t i t h g h o ~ t , ~ of t l i ~ host, cdls Inovcb actively (Starr and Baigent, lWi6; h r g ( * r a/ ul. , I W H ; rind oth(:rs). Whether or not the daughter cells arc inftvtivc bcfore thcir flngclla are formed is still obscure. Scherff ul 111. (19RR) claim that growth was obsorved to occur from both ends of the intracellular ljdellovihrio; the end which entered the host cell was allegedly the first to grow. Tho number of Bdellovibrio progeny produced within a cell of Pseudomnnm fiirorescens varies between 6 and 30 although a more usual range is 8 to IS (Srherff et nl., 1966), 14 (Abram and Shilo, 1967) or 5.7 (Seidler and Starr, 19698) in E. coli.

13. EFFECTS OF Bdellovibrio ON THE HOST C m A

Stolp and Starr (1963b) observed that Rhodospirillum rubrum lost its motility five seconds after attachment by Bd. 100. The motility of Spirillum serpens strain VHL, similarly, was reported to cease 12 min. after attachment by Bdellovibrio 6-543 (Huang, 1969). How Bdellovibrio infection in its initial stages could affect the motility of host cells is still unknown.

Most students of Bdellovibrio have observed that the host cells form into sphericul t)oditlR (“sphaeropliists”) within a few minutes after iitt,twhrncwt by MolIovi/)rio. I II thc. (*iLs(l of long host c:chllx, II l o d i z ( 4 , soinctirncw rnirlt8ipl(~ “t)it.iiooiiirig” or ‘ ‘ 1 ) ~ t)l)lv formitt ion’’ hiis h n obxrrvcrl, riithcbr t l i t i i i 11 rountlirig ul) of‘thcb cbtitirc! host wll. Most? writers on the suf)jwt, hitvc stiggcvkcvl that, thin rnorl)liologiu~tI oliiingc* iri t hc host cdl is (*iLitscbtl inainly 1j.y tlanit~gr t o t h c b rigid i)(Il)tidogl,yc,iiii cr,rnporwiit of‘ the host cdl w d l by Hddovibrio (illtttotigll, UH notcd i n Section I V.A.2, p. 236, the clectroii miurosc*opr evidence is still cquivord). These spherical bodies may be rchtcd morpliologiod1y to sphwroj)ltists produced from bacterial celln by ciththr lysozyma or pwicilliii in r n c d i a containing sucrose ant1 cations as supporting iig(t1its. ‘I’hc. torin “sphwro- plast” is conventionally used to desigriutr the sl)hericd, osmotically sensitive form of the bacterinl cell in which t h e cell wall has hecn modifiotl (McQuillen, 1960; Martin, 1963). There is still some question (see Section IV.C.1, p. 242, and Starr and Seidler, 1971) as to whether the spherical host form resulting from attack by Bdullovihrio is act iiully a (*on- ventionalsphaeroplast. since it does not seem to he osmotically sensitive.

Shilo and his coworkers investigated the early effects of Bdellooihrio on cells of E. coli. Shilo (1966) and Abram and Shilo (l967), using phosphotungstic acid-stained and shadow-ca~t preparations in an electron rnicromope, showed that holes or pits upprw on t , h hoHt, w I I surface shortly lifter attack hy l~dPllotiibrio is i r i i t i r i t d . L‘iiroii P / d . (1969) found that fidpllovibrio incorporittcd iLmirio i i ( : i (h i l l ) ( l irriwil poorly, arid produced inRigriificarit nmoirntn of‘P-tF;Llji(.loqi(lUIs(:, w h w c m

240 M. P. RTARR AND J. C -C. ITUANQ

the host cell (either R. coli B or E. coli K-12) was competent with respect to these chtrracters. They enhanced the early effects of Hdellovibrio by infecting tht> Iiost cwlturr. with a high miiltiplirity of itikction (X-17), so that 50% of t h r hont d l s hr(*iitii(> non-viablv within 2 -4 rnin. aftm

ion, t i t w h ic ti ti iiit’ no 1) iosy I i t Ii(& of bdcllov i h i o n h i d vet 1)cyyi I I .

~)r(www id itti iti(Iiiwr, ($1 l i ~ ~ l - f l - i ~ - t i t i o g i t i i i ~ ~ t o n i ~ l ~ ~ , R . cooli XJ‘II-

t l i c w i m v l ~ - ~ i t l i t ~ ~ f o n i ~ l i i n i ~ \ v l i i ( - l i is iloti*(~fiilil(~ Iby f Iii. i i i ( w v i N ( * 01’ wtl ~)igiii(lti( ILN t h i * ( h i riiifit i o t i 01. o - i i i t r i)~~h(~ii ,yl-/! l t i ~ t i l i i c ~ t o ~ ~ y r i r r t o n i t l ~ ~

l)row(vlN. S , y i i f h i H of’ fl-gtiItrc,tonitltix(’ Iirtltcrl whc~ii t t i cs coultiire of E . coli wtis iiitihc.tctl wit I i /Id. 1 o!) ( J i w i s i i k r n ) , ~ ( v i it1 t t i(& j)rwcriw: of iiicl U(YT. ‘I’licsr tiitthorn iiitcq)r(~tctl t htbir datu t ~ s indic.titing that Hddlo- vibrio i i i f ivt ioti intrrf(ws with thc synt ticisis of p-gulitc~tosi(lttHe-Hr,cc’;tic. tn-ICNA of the ho& ‘l’o prov(! the point that sytithesix of IINA arid protein i n t ho host werr af l i~ted, they further exuminetl incorporation of ’H-urwil into an acid-precipitublc portion of E. coli B, and incorporation of ‘‘C-leucine as specific markers for RNA and protein syntheses, respectively. They found that RNA synthesis in the host was inhibited 3 min. after Bdellovibrio infcction ; protein synthesis was inhibited a t 8-9 min. Varon and Shilo (1968) found that streptomycin specifically prevents the penetration of Bdellovibrio into, but riot attachment to, the host cell. Since the rate of jH-uracil incorporation was decreased in the presence of streptomycin, Vuron ~f al. (1969) concluded that the host ItNA synthesis httltcd before thcb ptirasitc’ penetrates the hoHt ccll w d l , i t . the at,twhrnc*rit of Hdidloaihrio o t i the sitrfaw of tho host, W I I wiix sutiicic~nt to c t ~ i i s ( ~ t h e (wwtioii of t 1 1 t h hont’s ICNA qwttiwis.

arid Sliilo ( 1 !)70) ~ ~ x i i m i t i c v ~ the e f h ~ t ~ of‘ /id. 109 ( J c m - ion 011 thc riLti1 of rc.nl)irut ion of Iti( : to~(l , Huwiiiutc, o r coli M1,35 or E . roli I < r ~ t i ( 1 o t i the pormcthilit,v to lwtow,

actetutr, sucschate, or O- t i i t ro~~t i t~ t i .~ lg~ i luc tos i~ l~ . ‘l’tic host Ntraiti, 4, coli rUlL36, is lactosc.-c,r,yr)tic: (lm i -z y ), i . ( b . c*apahle of s.ytithesizing fl-galuctosidase (z ‘) but luvking g ~ I i i ~ t ( ~ s i ( 1 ~ ~ perrncaso ( y -1 Itnd u rcgu- latory gene ( i - ) which woidcl othcrwisc produce r e [ ~ r c ~ s o r ~ to control thc /3-galuc.tosidtLsc operutor 1t i their hystems, a high multiplicity of infbc- tion (two or more) was usctl. A l o ~ v c*onwntrution (5 prriolrs/ml.) o f tho substrate (lactose, succitiate, or uretate) was used to avoid its possible effect on the growth or respiration of the host cell, but it did serve as an indicator to det the chcingw which occurred in cultures of the host Bdellovibrio.

Thc respiration rate rose steadily in the control culture (no itdded substrate) of E . coli-Hdellovibrio, and reached a maximum at 3 hr. at which time the lysis of host cells 1 J e g t i t i . Lysis was completod in 4 hr, and the respiration rate dropped whcrt t h r Hdrllovihrio progctiy werp releriscd from the gtiotit(v1 h t ~ ~ t c * c l l . .l(hlit,ion of laclosf* to t h hoNt-

1'1 1 YSI 0 1 AM I Y 0 I ' 7'1 I b; II I I E I , I , I ) v I II It 11 )S 24 I

/ h k l l o / ~ i l w i i ~ cwlt I I W riipitily ~ t i i i i i ~ l i i t ( ~ I rcwl~iriition o v c ~ i i 24 i n i n . -

pcriotl aftcar t h c t wo-incnil)cwt1 cwlt III'(~ M'ILS o~t t i l~ l i s l icd : t l i ix i n ( w w ( ~ was then followed by a rapid dcrrcasc. Addition of succinatc, lactate, glucose, or tnalate to the host-Bdellovihrio system gave no increase in respiration: instead, starting at 10 min. after infection, the rate of respiration dropped rapidly (Rittenberg and Shilo, 1970).

Close scrutiny of the changes in respiratory patterns showctl that the increase in the lactose respiration rate was tl(4,ectetl between 5 and 10 min. and reached a tnaximum 20 min. after the establishment of the two-membered culture The decline in succinatc respiration started a t the time when an increase in thc rate of lactose respiration was noticed. The cvhanges in respiration were influenced by several factors including tigc of the host, wlls, nirmbcr o f rrlls at i~ fixcd multiplicity of infection, or t hc mirltip1icit;y of infiec~tion iLt IL fixccl host-cvll popiiltLtion (Itittenbwg entl Stiihi, l!)70).

'I't i~ rat v of 0- I I it roll tic~iiylgt~I~~c~to~i( I ( * Iiy(1 rol,ysis, w h i d i iq i I r ( I iwt ivcl of irnmiLxkitrg of'8-giilac~t,osirltisc~ t ~ ( ~ t i v i t y i i i 1 t i ( - vryptia host, cwritiiiircd to rise for 4.5 rnin., although the riktc of rrspirtLtion of lactose incrcascd rapidly and started dropping at 25 min. Beta-galactosidase activity was detected only in trace amounts in the supernatant fluids of the hoet- Bddlovibrio system. Addition of sodium chloride or phosphate, which prevent attachment of the Rd~llovihrio to host cells, did not damage permeability control and the respiration of the host cell. The earlier the addition of streptomycin (250 pp./ml.) to the system, the slower the unmasking of j3-galactosidase activity. Puromycin and chloramphenicol had similar effects. Rittenberg and Shilo (1970) state that the increase in permeability and disruption of respiratory activity of thc host cell might suggest that one early cffwt of Bdpllovihrio attack i N damagc to the host cell membrane. Manifestation of these effects did not require the complete penetration of the BdPlZovihrio into the host cell. I,eakag!t. of ultraviolet-absorbing materials, ''C-labclled host rcll w m ponrrits, amino acids, and host 8-gahbctosidasc was low, hiit thwv H r i h t i t n w s

could be dete~tctl 20 rnin. aftw in f i iorr im1 twfiwr* vrirr ip lvt ion o f t t i c first growth cychl of HdPllovibrio (I)riic.kw, I !MV). Alkrt l i rw f'ti"sfiIIILi,Ilnrl,

an enzyme locttlixed in thc! p(.ripltisrn of' h' rnli, rlirl not I w k orrt i rn- mediately after penetration of t h e hoxt wII ant1 its cwnvcmiori into II

spherical body. These data suggcst to Shilo ( I 969) that tho mzyrnc~ i H effectively bound within the pcriplasni and/or that immotliatc: r rpir ing of the damaged wall takes place.

During growth of Bd. 6-5-8 in a living c*ulturr of E . coli ML35, compounds absorbing a t 260 nm. and reacting with Folin-Ciocalteau reagent were released, and total cell protein (presumably host-plus- Rd~llovihrio) decreaNed (Crothers nnd Robinson, 1970. 1971). The rate

242 h1. 1'. Y'L'AItR ANI) ,I. C.-L', lIIJAN(;

of O-iiitroplieiiylgalartoside hydrolysis iricreamd, but tioltiblo /?-galactosidase was not detected in the supernatant. The Bdellovibrio-infeuted cell of E. coli becsmc brightly fluorescent when a fluorescent dye (8- anilino-l-naphthalene sulphonic acid) was added. These data lead to the euggestion that, upon damage of the cell membrane by the attack of Bdellovibrio (which would allow passage of O-nitropheaylgalactoside and the fluorescent cIj .o) , a lcwge amoiint, of Iwt cellirliir components is still hold intr~wollularly for nouriwhing thcl hont-tiaponcicnt, BdPZZovibrio C d 1H.

(j. M I N ~ I I A N I N M H 014. I , Y H I H OIC IWC Iiowr CM~I, ISY Ih?ellovibrio

I . IV od1p .J' lidwioiy1ic Action

l,*ysiH of t)eu:i,cbrieL I)y thlogic:cll or noti-biologiosl agetitti, arid the possible tneohairimw involved, hiivn been revicwed extensively by McQuilleii (19fiO), Work (19Ul), Martin (1903, 1966), Perkins (1963), Brown (l964), Saltoii (1964), Weidel and Yelzer (1964), MacLeod (lees), Rogers (1965), Ghuysen et al. (1966, lees), Stolp and Starr (lees), Shockman (1965), Strominger and Chuysen (1907), Ghuysen (19ti8), and Gum (19BH). Lysis of filamentow fungi by Streptomyces sp. (Aguirre et al., 1963; Hsu and Lockwood, lQ6Q; Jones et al., 1908), by other soil micro-organisms (Carter and Lockwood, 1957; KO and Lock- wood, 1970), by Bacillus circulans (Horikoshi and Iida, 1Q5Q), and by other bacteria (Mitchell and Alexander, 1963) has been well documented.

Actually, little j, yet lrnown about the mechanism of bacteriolysis byl3dellooihrio. Wc mighttirxt rcvicw t,hcfac:twahoirt tho conversionofthc hoHi, ( v I I ititgo IL Hl)h(3r i (* i i l botly (HOC Swthil IV. 13, 1). 23I4, ~uid Sttirr und N ( + l l w , I!)7l). ICxistc:iic*c! of' t h Hl)t I (* l ' i ( ' id I.)o(ly NllgK(!HtH thtbt tho rigid ~iej~ticlt~~l,vc:ciii hiyw of' thc: will1 hrin t)cr!n tiof'ttm!d rmi that tho porct formed in the host c d l mill (1 uring penetration hy J-jdelloiArio may haw been sealed by some cLgglomerciting rritLtcrilt1 (Ciudin d d. , 1067) or by a wall-repairing mechanism (Shilo, 1 Sfig), The mochanim of formation of the wpherical body, a~ well ~ L H thc rolc of thiN Htructure in the hoHt- parasite relationship, are &ill unknown. The nature of these Hpherical bodies and particularly their existence in non-osmotically supported media, if they are indeed sphaeroplasts, require8 further investigation. The identity of these spherical hodies to the sphaeroplasts formed by lysozyme-EDTA is questionable, although Shilo (1969) has mggested that the spherical bodies formed during Bdellovibrio infection re8emblo the penicillin- or cycloserinc-induced NphaeroplaatR which retain the alkaline phosphatase.

The continued interaction with Bdellovibrio eventually results in considerable disorganization of' the host cell8 (8tarr and Rltigent, 1966;

Yil YSlOLO(i Y Oh' ' V I I b; I ~ l ~ I C L I ~ O V i l I l t i O ~ 2.13

Sclicrff rt d., 1066). Nuolear materids disperse and disappwr. Hibo- somes and cytoplasmic niateritils become granular m t l unevenly distributed, and the peptidoglycan It~yrr of the host cell wid1 is partially digested. Although extensive disintegration of the host8 cell wti11 is evident in later stages, this disintegration might be caused by ciizymes produced by Bdellovibrio (see Section IV.C.2, p. 243), by autolytic enzymes produced by the damagcd host c d l (Stolp arid Stan., 1!)65), or by both.

' l ' t i t b I.yt i c e c w a y m w ~ ) r o d r i t * c v l by lldrllonibrio rnrby irlso alli~rt i t x ow11 vitd)ilii8,y, t w I might ( * I L I I S ( ' t Iiv riq)icl c l c d i r i c b in i i i i r r i l w r ~ of vitd)lca W I I H obxcwcvl i r r l i c ~ i i i t l (wlturtw 'l'his Ii.vlwtCirnis ix I~is(d on thr ftwt thiit (1 il I I t i o i i 01' Ndrllonibrio ( ' I I 1 t 11 rtw 1 )rolorigs t Ii ix via t)i l i ty . A 1 tcarn ti ti ve or culdit ioiiitl ('xl)libii;itiotis o w u r to us; ttiwr i i i d i h a~(-(*~irniilrition o f

iliid other toxicbxu b+ttitri(*CH, ii~id cIxhtLustion ofiiutricnts. I'iirthw work oii this sub jwt is c*lcarly rrcluircd.

3. Muraw idnsr

Early uttcniptrJ to demonstrate muramidase and other lytic. enzymes in Bdellovihrio and to relate these enzymes to the disintegration of the host, cell were hampered by tho use of two-membered systems containing living host cells, with the consequent unccrtairity as to whether the enzymes originatcd in the hdellovibrio or host cells. Several Nrlellovihrio strains were shown (Huang aiid Starr, 1971a , b) to multiply readily in heat-killed (70" or 100"; 1 0 min. ; 131", 5 or 1A min.) cell suspensions of t,he host in tris-HCl huffrr (0.025 V ; p H 7 .5 , cwitaining 2 mM each of MqS04 t in t i CaC1,) iind to l y s c b thr host c*rlls. (irowth of lldrllovibrio i t l i t l S ~ I L ~ I I ~ ~ ~ ~ I I C W U S diw~lirtioii of t t l ( b l l c ~ i ~ t - k i l l ( ~ ~ l h 0 H t C * O I I S W ~ - I Y * t i ( # -

c*omI)aiiirtl t)y t h v ii~)~~cvwatic*(~ iii t l i r c+iiltiirt* of solublv tt~ilri~l~ii(* ; ic*it l

and of siit)mic~rosc.e)~)io ptirticlw w l i i d i c~)~i t i i incr l t m i r i o SiiUiLrx. AH enzyme or a mixturc of enzynic~s whit-ti is rc~ltwrtl t)y ttw growiiig Bdellovihrio antl whivh dcgrades t,ht* host ( d i n is t)c4icvcd to I i t)t.r;it(. t / I ( ,

particulate matter containing amino sugars ( H iiang timl I i o h i i i s o i i ,

1969; Huang antl Starr, 197 I h). 'I'hc crude ena,ymc! propttr~ition prv- cipitated from filtrates of a culture of Bd. 6-,543 grown on heat-killed host cells by ammonium sulphate or cold acetone, solubilizes the pepti- doglvcaii isolated from the cell of Spirillum serpens VHI, and releases reducing sugars into the supernatant solution. The crude tmzyme preparation also lyses 14Cl-lttbelled peptidoglycan and releasex so111 hle I4C-labelled materials into the filtrate (Huang and Starr, 197 1 b). Using this radiochemicul analysis as indicator, lysozyme-like enzymes have been isolated and characterized (Huang and Strtrr, 1971 h) from the crude enzyme preparation by chrotnatography on a DEAE-cellulose column

2 I 4 hl I' S T A I l K 4 Y I l J ( ' ( ' III1Ah(l

o r S ~ l i I i ; i ( k ~ (( 100) #PI filtratioti. 'I'hc molccdar weight of'tliis lysozyme- lilw ctizynic is 12,600, its cletcrmin~d by Sephrtdex pel filtriitioli. There is 110 cluwtioii, i is thrrc may have becn with preliminary studies on this sribjcvt thtit these tire Rdellovihrio enzymes ; any such enzymes from the host cells would have been inactivatcd by the heat t,reatment, and the heat-killed host cclls could, of coiirse, not produce uriy such enzymes.

An abstrurt, by l~atckrrll (J/ nl. ( 1970) also rcports the purification (60 70 fold). from Bncllovihrio-l~ost ciilture lysates, of a bacteriolytir enzymc which tlegraclc,s isolated c e l l wt~lls of Spirillum, stvywns VH L. The cwzymr, which is siiid to IiiLve t~ molcc~ular weightj of approxirnat,cly .tf),of)f), nlso dt*grtdtv 1)iirititvl ~ i ~ p t i d o g l y ( ~ i ~ n isoliktd from A'. .smpmx.

PHYRIO1,OOY OF THE BDELLOVTBRTOS 243

Lpsis of the host culture usually precedes the detectable formation of protease in the two-membered culture (Huung and Stitrr, 1971b). At a low multiplicity of infertion ( O . ? ) , a mild lysis was observed over a period of rb5 t)o 8 hr. : however, significant amounts of the protense were not produced until 9 hr. after mixing Bdellovibrio with the host cells. When a high multiplicity of infection (50 ) was used, immediutc lysis of the host cultmc occurred (within 0.5 hr.) itiid yet the protease ciould not be detected until 3.5 hr. later. These results indicate that the host cells lyse extensively before protease product ioii reaches a maximum. Whrn t tic. pro(Iiic*t ion of t~ c.asc~iii-liy(lrolysiiig zone b y i k normally 1 )ri it (v~s(1- )xi t i v(1 Ikldloribrio is i I i t i i t ) i 1 (11 1 1 by tLi Id it ioii o f ;mi t i i i )I i i ii rri

w l p l i t b i ( ~ , IiIw1iii.s itri’ st i l l h r t i i ( v 1 oti t tie Iiost Iiiwtt (tliirmg ;uid Stwr. 1!)7 I I)). “ I ’ t ~ o t c ~ i ~ s c ~ - i i c ~ ~ t ~ t ~ i v ~ ~ ’ ’ mtrtiitit,s, i t 1 1 his (*;LSP I M f ~ l l o d ~ r i o strtLitis wliic4i (lo t ioi Iiydrol~ysc Azoc*oll or (*tis(iri (ooll~gt!nasc~- rtntl i*aseinase- negative), w c ~ e isolated from protease-positive Hd. 6-5s using nitro- xoguaniditie treutniont or I)y the selection of sporitaric~ous mutants. “I-’roteuse-ticgutivt.” nii~tarit~s grtw slowly and formed sniall plaques (less than 1 mrri. i n diameter in fivca days), while the protease-positive strains formed plaques 6 mm. in diameter in two days in a moist environ- merit. Although ‘ ‘protease-negative” bdellovibrios do not hydrolyse collagen or casein, they do, like the protease-positive strains, produce carboxypeptiduse which hydrolyses N,N-dimethylpolypeptides obtained from trypin-treated casein (Huttng and Starr, 1971b).

The protease produced by host-dependent Hcl. 6-5-S has been purified 65-fold using acetone fractionation and DEAE cellulose separation (Hueng and Starr, 1971b). One niujor peak, which retains u strong activity in hydrolysing Azoroll, W;LS obscwetl. The partially purified enzyine releases groups from hacwiogiobiti3 geitdiIi, and a1t)umili wtiicoh reart with tlitiitrofluoroht~iiz~nt~. ‘l’hv I)rot(vw chtuiwd frorri t h v fractionatioti on 1)15Ali; wIIi ihe I o s w its Axoc.oll-(iigl*sf iiig rbkility (about 5O‘Xt) aftcv tlitLlysis for 24 h r . i i i t ris-l lOl o r c l in t i l l v i l wiLt(1r. Cul(*iurn, but r i o t niagnwiiini, ioiis tm’ rvvjiiirc.tl to st i i tJ i l ixv i t iv I)rotiv~sc+ activity during dialysis. Atltlitioii of‘ (‘aL ’ ; ~ r i ( l Mg2 ’ to ttw reac+tion mixture of tris-HCl buffer contuinirig Ijrotwsc and Aaoi*oll nc.i,iviitc.s enzyme activity. The optimcll pH valiic~ is 8.5 t i t i i t t tie optirrd vottwtitrti-

tion of tris-HCI buffer is 0.25 M . ‘I’tre i\lic.hac~lis ~oristerit (~ f ‘ 1 h v I)rotcvisc~ for N,N-diniathylcasein is S.1 / lW5 ill. The molwuiar weight, S ~ H

measured by Sephwdex gel filtration, is ahorit 11,000 (liuang tmtl Sturr, 107 1 b).

4. Lipusv

A lipase, which is capable of hydrolysing tributyriti incorporated into agar, was dt4ected in the mld-acchtone prcvipitibte of t,ho

I). KINETICS OF HOST-DEPENDENT GROWTH

The kinetics of growth of Bdellovibrio and of death of the host cell were first illustrated by Stolp and Starr (1963b) before the intracellular nature of the Bdellovihrio parasitism was known. Bruff (1964) found that plaque formation by Bdellovibrio followed the formula KP = CNvX (where NY = number of plaque; sNv -- concentration of the bdellovibrio; x = the number of bdellovibrios necessary to form a plaque; and C = u oonstant) with x equal to 1.2. This meaiiH that one bdellovibrio WUR siitfic*ic!iit to initiate fortnation of a single pltquo in the syRtorn

Bcitllor and Btarr ( 1f)tiBu) fo l lowd tho noti-synchr~oiious growth of l i d , 109 in E . coli H in terms of pliquo-forming unih. 'I'hero was a lag period of about 4 hr., arid a rneun goneration time of 1.3 hr. (the calcula- tion assumes tha t bdellovibrio divideR by binary finsion, which it does not). On average, eltch cell of E. coli B gives rim to 5.7 BdelZovibrio celh. The lag period depends on the physiological Htate of the parasite, the pH value, and the temperature of incubation. In the temperature range of 25-38", the average bur&, size and lag period are inversely proportional to the temperature; at 42", there is no burst. The hurst coefficient (a quotient of the average buret Hize and the latent period in hours) is highest in the range 30-35" (an optimum rango of growth temperature).

An optimum temperature ranging from 30" to 32" and optimum pH value range of 7-0 -8.6 were notcd for lid. N-6801 (Uometsu el J., 1971). The thermal death point of onft drain of Udellovibrio i H reported to be approximately 14, min. a t 51" (K. Nukttmiirtl, prwonttl communica- t.ion).

E. NUTRITION oc Ii~.ellovibrio Early attampte to grow ho,qt-cl~cI)r!ndorrt J M & d w i o or1 tiori-livi rig

hoHt celk or on extracts of ccll~ did not ,wcc:nc!cxl (Stol p et id Stthrr, 1 M1:h). In these tl'irth, &kllOVibrio W t ~ n u I l t ~ h l ~ * ( I ) l,ynC* r t d grow i l l hOHf, c:t!lln

NliC IlHt!(l.

PIIYSIOLOQY OF TIIE BDELLOVIBRIOS 247

killed by heat ( loo'), chloroform, toluene, or ltoccal disinfectant. This inability to grow was not related to differences of pH value or redox potential in the cultures of host bacteria. I n only one case did Stolp and Starr (1963b) find that plaques developed on heat-killed cells, and this case may have depended on the carry-over of nutrients from tho Bd. A3.12 lysate (host-independent growth has been reported with this strain; Shilo and Bruff, 1965). The nature of the material supposedly carried over has not yet been determined.

The results of the single extant study of the nutritional requirements of a host-independent Bdellovibrio might profitably be related here. Yeast extract was shown to be an excellent substrate for growth of host-independent Bd. A3.12 (Bruff, 1964; Shilo and Bruff, 1965), but casein hydrolysate could not be utilized as a sole sourco of carbon and encrgy. Small quantities of yeast extract were needed to obtain good growth yields when peptone was used as the major carbon and energy source; this suggests that yeast extract contains one or more growth factors for Bdellovibrio. A mixturo of B-group vitamins (riboflavin, calcium pantothenate, B, 2, biotin, p-aminobenzoic acid, pyridoxal phosphate, and pyridoxamine hydrochloride) could replace yeast extract in this system.

A number of workers (Huang, 1969; Varon and Shilo, 1969b ; Crothers et al., 1970; Huang and Starr, 1971a) have observed that washed cells of host-dependent bdellovibrios develop in the presence of, and lyse, washed host cells which have been heat-killed (70' or loo', 10 rnin., or autoclaved, Hnang and Starr, 19718; 70', 15 rnin,, Varon and Shilo, 1969b). However, extensive autoclaving (e.g. 40 rnin.) renders such cells unsuitable as Bdellovibrio nutrient (Huang and Starr, 1971a). Lysis of such preparations by Bdellovibrio in broth or tris-HC1 buffer suspension is extensive, but generally with very low plaquing efficiencies on lawns. Growth of Bd. W on heat-killed hoRt ccl l~ (go', 20 min., Rhodospirillum rubrum) was observed by Burger et al. (1968) ; again tho plaquing efficiency was very low. Although Bdellovibrio attaches very poorly to heat-killed cells (70', 15 min., Varon and Shilo, 1968), some development of Bdellovibrio was observed in such preparations (Varon and Shilo, 1969b). Gillis and Nakamura (1970) reported that Bdello- vibrio grew on lawns of heat-killed (66O, 20 min. ; 121', 15 min.) cells of Shigella boydii in non-nutrient agar. These observations eliminate the likelihood that bdellovibrios require nutrients from the culture medium in addition to those they get from the bacterial host cells, and they support the view that development of Bdellovihrio depends primarily on some host-cell material or metabolic product@, as suggested originally by Stolp and Starr (1!363b). This concept is further supportotl by tho growth of Bdellovibrio in host bacteria at all PhaHes of the host growth

11

248 M. P. STARR ANI) J . P.-('. HUANG

cycle (mitl-log phnse growth of' tjlic host , ILS with bnctcriophugcs, is unntrtlssary ), by Bdellovibrio dcvelopment in host l)wt,cria t l i i b t had beeii inactivtited by streptomycin or by ultraviolet irradiation (Huang, 1969 ; Varoii and Shilo, 19G9b), a i d by niultiplication of Bdellovibrio in cell-free extracts of susceptiblc or non-susceptiblc host organisms (see Section ZV.A.4, p. 238, and the next paragraph).

Bdellovibrio N-6801 multiplies in suspensions of autoclaved cells or in cell-free extracts of bacteria which wore either susceptible or non- susceptible as hosts for the parasitic phase ; these bacteria include Corynebacterium, michiganense, Bacillus subtilis, Bacillus megaterium, Bacillus cereus, Agrobacteriurn tumefaciens, and Pseudomonas,fluorescens. Bddlovibrio N-680 I also grows and forms colonies on agar containing autoclaved host cells, a kind of host-dependent but not parasitic growth (Uematsu and Wakimoto, 1969a, b, 1971). The colonies produced in these trials werc light yellow in colour and produced lytic zones, probably due to proteolytic activity on the dead cells. The lytic zone was not seen when cells of autoclavcd bacteria, which were non-susceptible to parasitic attack by R d . N-0801, wrre incorporated into the ngar. Sincc thc numbrr of ph~~ue-forn~ing iinits on the lawn of living host cells corresponded with thc nurnbcr of colony-forming units on the lawn of autoclavcd host cells (the ratios being unity), Bd. N-6801 is considered to be a facultatively parasitic strain of Bdellovibrio. It is, however, still dependent on nutrients from the host cell or other non-susceptible bacteria. Thus B d . N-6801 behaves differently from the host-independent bdellovibrio strains or facultativcly parasitic Bd. UKi2 (see Section VI, p. 256).

Growth and lytio activity in liqnid culture are influenced considerably by the composition of the medium used for cultivating the host bacteria (Stolp and Stnrr, 1963b). Somewhat retarded growth of the host bacteria generally favours development of the parasite; t i more rapid host growth rate might somehow suppress the more slowly growing Bdellovibrio, and host nietabolitcs might inhibit the Bdellovibrio. One-tenth strength nutrient broth or quarter strength yeast peptone medium generally are best for this purposc.

There have been several studies regarding thc role of tlivalcnt cation# in Bddlovibrio devclopmt>nt. Rokiinson and Huang ( 1007) reportccJ that Ruspcnsiom of wrmhctl It. coli 1J/r hnrvt:Htotl from conrplox ar1d from synthetic media and suspcndetl in 0.026 M triH-f1CI tjtifT(8r (pi1 7-51 supported growth of 13delZovibrio only when Ca2 + and/or Mg2 ' wor(9 added (similarly with E. coli MLM; Crothers and ItotJinRon, 1970). Huang and Strtrr (1971a) noted that Ud. 0-5-8, U d . 100, Hd. 109 (IJnviH), and B d . A3.12 all multipliccl in the presence of living or heat-killed (70'' or 100") 10 min. ; 120°, 5 or 15 niin.) Spiril lum sewens VHL in HEYE8-

PHY$IOtOCIY OF THE BDELLOVIBRIOS 249 NaOH (for B d . 6 - 8 4 , see p. 243) or tris-HC1 buffers or distilled water, when supplemented with Ca2 + and/or MgZ +,

Multiplication of Bdellovibrio in the presence of Ca2+ and Mgzt is associated with the release of ultraviolet-absorbing materials, presumably from the host cell, and the release into the culture supernatant solution of amino sugars; soluble amino sugars were not released in the absence of added cations in this system. Even in the presence of added cations, the growth rate of Bdellovibrio in heat-killed host cells is lower than in viable but non-prolifera,ting host cells. Late addition of the cations shortens the growth lag time of Bdellovibrio. Calcium or magnesium ions (chloride or sulphute) each support growth independently. Bdellovibrio 100, Bd. 109 ( lhv i s and Jerusalem strains), Bd. A3.12, and Bd. 6-5-8 require Cat+ for growth in either thcir homologous or heterologous host, bacteria. Ildellovibrio 109 (Jerusalem), but not always Bd. 109 (Davis), Bd. 100, B d . A3.12, or Bd. 6-54, is capable of growing in the presence of the low concentration of Cat bound in situ on its host E. coli B (when grown in complex medium). After repeated washings of such host cells or upon growing the host cells in a Cat+-depleted minimal medium, B d . 109 (Jerusalem) requires added cations for development (Huang and Starr, 1971a). This distinction in cation requirements between B d . 109 (Davis) and B d . 109 (Jerusalem) supports the view expressed in Sections 1.E and IV.A.5 (pp. 221 and 239) to the effect that these twostrains are markedly different. Addition of EDTA (0.01 M ; pH 7.5) prevents growth of B d . 6-5-5; growth is restored by addition of CaZt and Mgz+. A requirement for Mgz+ and/or Cat+ in the system B d . 109 (Davis)-E. coli B was also observed by Seidler and Starr (1969a), who suggest that the possible effects of cations in this system might be associated with attachment to the host cells, diminution of the latent period, support of the host “sphaeroplasts”, and increase in t h o average burst size. The fact that Bd. 6-6-S multiplies in dead host cells only in the presence of added cations suggests to Huang arid Starr (1971a) that these cations are needed, also, for other aspects of Bdellovibrio development and in addition, since release of soluble amino sugarn into the culture supernatant occurs only in the presence of added cations, also for activating lytic enzymes.

One other fact on Bdellovibrio mineral nutrition might hc rccorded. Marine bdellovibrios isolated from the Mediterranean 8ea (Shilo, 1966) or the Atlantic Ocean (Mitchell el al., 1967; Mitchell and Morrin, 1969) form plaques only on lawns containing 3% (w/v) NaCI.

F. HOST SPECIFICITY Several procedures are used for determining the host range of bdello-

vibrios, namely (1) attachment of Bdellovibrio to the host cell as observed

250 nf P. STARR AND J , c.-c. IIUANQ

inn phase-contrt& microscope (Stolp wid Starr, 1963,) or by the differential iiltratioii itiethod (Vuron iuid Shilo, 1968); ( 2 ) clctwing of a host culture in broth (Stolp and Starr, 196313) or t i host, cwll suspcnxion in buffer (Hnaiig, I!)69) ; (3) plnque lbrtriiiticw (Klciii t in t i C'twiidtL, 1987 ; using Thornton's medium) or cwnllurnt lyHis (Stolp aiid Strcrr, l!)Kjl) ; using yeast-pcptone nicdiuni) ; aiid (4) total count by Coultcr countcr (Uruff, 1964; Shilo and BrufY, 1965). Shilo and Uruff (1965) found that the host range was broader in liquid media (turbidity change) than on agar media (plaques) ; the broader host range might be attributed to the highcr niiiltiplicity of the parasite. The use of washed host cells suspended in bufl'cr together with washed cells of Bdellovibrio generally gives a consistently wider host spectrum than the other procedures (Huang, 1969). G. l h w s (personal comrnuiiication), however, observed that the growth rate of Bd. W is very low when the host cells (Rhodospirillum rubrum) wcre washed, suspended in buffer, arid infected with the Bdello- vibrio; thc rates of growth and infection are increased somewhat by addition to the buffered cell suspcnsion of a carbon source, but much more so by ammonium chloride.

Many factors affect the development of BdelEovibrio and, consequently, lysis of tho host cell. These would have a bcaring, too, on host specificity. These factors include: number (Stolp and Starr, 1963b), age (Wood and Hirsch, 196G), and metabolic activity of the hoat cell (Stolp and Starr, 1965); uccumulation of metabolic products of the host; composition of the medium including the prescncc of Ca2 ' and/or Mg*+ (Robinson and I-Iuang, 1967; Seidler and Starr, 1969a; Crothers et al., 1970; Crothers and Robinson, 1971; IIuang and Starr, 1971a; see also Section IV.E, p. 249). The efficiency of plaque formation is affected also by the host density on the lawn, water content and thickness of the semi-aolid agar top layer, incubation ternpcrature, and hiirnidity of the incubator (Ueniatsu et nl., 1971). Burger et al. (1968) expressed host specificity in terms of plaquing efficiency. The plrtquing efficiency of their B d . W varied from 0.00001 with Serraliu n w c e s c e n s to 10 with Yroteus vulgaris ; the playuing efficiency with Rhodospirillum rubrum was arbitrarily designated as unity.

(w/v), 2,3,6-triphenyltetrazolium chloride has been incorporated into media (Seidler and Stam, 1969a; Jackson, 1967). J. C.-C. Huang and M. P. Starr (unpublifihed data) found that addition of Hkim milk (2%) to the xcmi-solid agar also increased the contrast for plaque counting.

Despite the undcniable relcvancc of host fipecificity to an undorrJtand- ing of tho Bdellovibrio-host symbioHiH, very littlct syfitemltic work hm been donc on this subjcct. Twelve strailis of Ldellovik~rios coultl be divided into five groups on the busis of host specificity (Stolp and Stan ,

To facilitate plaque counting, 0.1

19G3b). Of 120 strains of psciidoiiiolllLtle)i~~~(ls tC\sLod, 23% werc ututl’fectc!d by Bd. 321 (Stolp and Petzold, 1902). Bdellovibrio N-0801, Bd. N-6804, and Bd. N-6805 could attack about 50%, while Bd. N-6802 and Bd. N-6806 could attack only about 5%, of the 43 strains of Pseudomonas tested (Uematsu and Waliirnoto, 1970). Xanthomonas campestris and X. cucurbitae are resistant to all strains of Bdellovibrio isolated by these workers, nilti Bd. N-6802 und ]Id. N-0800 could not attack Erwinia sp., E . coli, or Aerobacter NP.

Bdellovibrios which parasitize Azotobacter and Rhizobium were sought but at first not found (Stolp and Starr, 1963b; Postgab, 1967). However, a Bdellovibrio isolated by using E. coli as the host can parasitize Azoto- bacter chroococcurn, but not Azotobacter vinelandii or Rhizobium species (Sullivan and Casida, 1968). Recently, Parker and Grove (1970) isolated, from Western Australian soils, strains of Bdellovibrio, which are capable of parasitizing Rhizobiurn meliloti, R. trifolii, Agrobacterium tumefaciens, and A . radiobacter .

Using an agar-block transfer method, Gillis and Nakatnura (1970) studied the susceptibility of Shigella boydii, S. Jlexneri, and ”3. sonnei to parasitizution by bdellovibrios isolated from sewage. An agar block (one em. square) is cut from the centre of the Bdellovibrio plaque on one host lawn and transferred to another. The agar block, according to these authors, provided little or no carry-over of the f i s t host, and formed a clear zone on lawns of the latter. All Shigella species tested were susceptible to these bdellovibrios. J. R. Gillis and K. Nakamura (personal communication) injected Bd. 109 into a rabbit eye infected with Shigella jlezneri and found that the Bdellovibrio partially protected the eye from Shigella keratocon junctivitis.

The presently known bdellovibrios usually attack only Gram-negative bacteria. The existence of bdellovibrios capable of attacking Gram- positive bacteria was expected by Stolp and Starr (1963b), even though these workers did not find any such strain. GuBlin etal. (1968c, 1969b, c) found that bacterial parasite Xpfr (which they at first claimed was a Bdellovibrio species) could parasitize the Gram-positive bacteria, Clostridium perfringens and C. histolyticum. There is no evidence in any of their papers that this bacterial parasite (Xpfr) actually enters the clostridial cells and there is some question a8 to its being a Bdello- vibrio species (A. GuBlin, personal communication ; Rtarr and Rcidler, 1971). Burger et nl. (1968) isolated Bd. W which they claimed could parasitize, in addition to some Uram-negative bactcria (including Agrobacterium tumefaciens), the Oram-positive Streptococcus faecalis and Lactobacillus plantarum. Bdellovibrio W was reported not able to parasitize Pseudomonas aeruginosa or Spirillum serpens. We have found that Bd. W multiplies in Rhodospirillum rubrum and Spirillum serpem

252 M. P. STARR AND J. C.-C. IIUANQ

VHL, but that i t does not parasitize or lyse available strains of one of the alleged Gram-positive hosts, Stwptococcus faeculis (we have not tricd L. plantarum). The reasons for the discrepant findings are presently unknown (see Section lV.A.1, p. 234 for comments about Bd. W and Spirillum serpens wall structure).

I t is intc~resting to notc that lk/1~11~~?7ihrio (or OrgiLiiiHiw which I i r ~ v c x

soirio posd)ly H I I porlic-ird rrHcvnbln i i w tto Iklcllonilwio) Iinvv b01)ii rc!portcttl to priisit,ixct, bcnitlo t,lio l):Lctc!riiL rofiwocl to above, Ilyphomicrobium (Wood and Hirsch, l966), Cylophuga (Much, 1965), and Chlorellu vulgaris (Mamkaeva, 1968; Gromov and Mamkaeva, 1966, 1971). Commcnts on those findings will bc found in Stttrr and Seidler (1971).

The ability of Bd. 6-543 to infect micc, fertilized cggs, and a variety of tissue cultures has been tested (F. J. Simpon, personal communication), The results show that the Bdellovibrio does not attack any of the mammalian cells tested with the exception of one egg which was believed to be infected with a Gram-negative bacterium.

V. Metabolism of Bdellovibrio

Since host-dependent bdellovibrios do not grow on any as yet defined medium, studies of their metabolism and nutrition have been possiblc only in the presence of the living or dead host cells. Somewhat more has been achieved in studying the metabolism of host-independent strains, although their precise nutritional requirements are also not yet known.

'J'hiLt Ildellovibrio grows only cwrohically h i ~ s 1)ocn rioted by Stolp and Starr (k!)K$b), Simpson arid 1Cobinson (L96H), and others. Burger P t al. (1968) observed that Bd. W grew ant1 infkctcd the host cell only under oxygen partial prcssures of 4-6 mm. Hg and higher.

Prcshly harvested Rd. 6-5-S has an endogenous respiration rate of 20 x pl. O,/cell/hr. a t 35' (Simpson and Robinson, 1968). Thirty- five per cent of this respiration rate was lost when Bd. 6-543 was stored in tris-HC1 buffer (ZO, 4 days). Potassium cyanide and sodium azide did, but carbon monoxide did not, inhibit the endogenous respiration of

Under their experimental conditions, Simpson and RobinRon ( 1 968) found that Spirillum serpens started lysing at 120 min. after Bd. 6-5-S and host cells were mixed. The lysis, which was completed at 200 min., was accompanied by a large increase in total respiration, and this increase continued after the host was completely disintegratcd. They believe that the material released from the host likely supported respiration, since the consumption of oxygen was too grcat to be accounted for by endogenous rcspiration of the Bdellovihrio alone. A~pe~rr~gir~e cind

Bd. 6-543.

PI1YSIOI,OllY O F 'PI1 E HI)ILCOVIllRIO?l 253

gluttamate, and cwpwiidly glii t i m iiw, R t i in 1 1 I t i t (r roq)irtLtio I I , I t i t.ttcwl)crly twd Shilo ( 1!)70) roporkvl t h t , lwptoiiv, y w t 4 cdrric-1, or crinoiii Iiytlro- lysltte increased the respirtitioii rste of anothcr struin.

According to Simpson and Robinson (1968), the particulate fraction of extracts of Bd. 6-6-S contains cytochromes a (absorption peak at 606 mm.), a3 (445 nm.), b (659, 628, and 426 nm.), and c (566-652, 626-622, and 424-422 nm.). Scidler and Starr (1969b) showcd that 16 strains of host-independent Bdcllovibrio contain cytochrome c (522-524 nm.). Two patterns wero found among the host-independent bdellovibrios at the absorption peaks corresponding to cytochrome c and the Soret region; host-independent B d . A3.12 has peaks a t 607 nm. and 417 nm., and the rest of these bdellovibrios have peaks a t 596-600 nm. and 421-423 nm. (Seidler and Starr, 1969b). Bdellovibrio 6-6-5 possesses cytochrome oxidase, as determined with N,N'-dimethyl-p- phenylene diamine. A cell-free extract of B d . 6-5-5 contains NADH, and NADPH, oxidases; the rate of oxidation was twice as high with NADH, than with NADPH,; the oxidation of NADH, by B d . 6-5-S extracts was markedly inhibited by potassium cyanide and sodium azide, but rotenone or a 4 : 1 carbon monoxide-oxygcn gas mixture did not inhibit it; the oxidation of NADPH, was not inhibited by any of these reagents; all of the NADPH, oxidase and 10% of the NADH, oxidase were found in the soluble fraction (Simpson and Robinson, 1968).

Polarographic studies showed that glutamate, a-ketoglutarate, succinate, fumarate, malate, pyruvate, acetate, a-glycerophosphate, 8-hydroxybutyrate, NAD, or NADP could not support consumption of oxygen by the crude B d . 6-5-5 cell extracts; NADH, and NADPH,, however, did support it (Simpson and Robinson, 1968). Cell extracts of Bd. 6-6-S contained ATPase which mostly resided in the particulate fraction. The extracts were also capable of catalysing an ATP fs lzP, exchange. Since arsenate, azide, and 2,4-dinitrophenol did not inhibit this exchange, the process is probably catalysed by reactions other than those associated with oxidative phosphorylation. Tho cell extractu possibly contain two systems for oxidizing NADH,; one is stimulated by flavins (FAD or FMN), is insensitive to potassium cyanide and leads to the production of hydrogen peroxide; the other is sensitive to potau- sium cyanide, and likely leads to the production of hydrogcn pcroxide via the cytochromes (Simpson and Robinson, 1968). Somc Ntrainn of Bdellovibrio are rich in catalase (SimpRon and Robinson, 1968 ; Seidler and Starr, 1969b; Diedrich et al., 1969, 1970), while othcru lack it (Stolp and Starr, 1963b; Seidler and Starr, 1969h).

Bdellovibrio 6-5-8 apparently contains tho ctnzymcx of the tricrtrhoxylic acid cycle in soluble from and succinatt! dehydrogenaee in bound

254 M. P. STARR AND J. U.-U. RUANQ

form. Six enzymes of the cycle (isocitrate dehydrogenase, a-kotoglutar- ate dehydrogenase, succinyl-CoA synthetase, succinatede hydrogenase, fumarate hydratase, and malate dehydrogenase) were found to be present. The absence of a glyoxylate cycle and the inability to utilize carbohydrates, alcohols, and similar sources of carbon and energy suggests that Bdellovibrio might be dependent on proteins, peptides, amino acids, and nucleic acids (Simpson and Robinson, 1968). Mitchell et al. (1967), on the other hand, report that a marine Bdellovibrio was capable of utilizing cell walls of E . coli as & sole source of organic carbon.

Seidler et al. (1972) identified, from seven strains of host-independent bdellovibrios, four dehydrogenases (alanine, glutamate, malate, isocitrate), fumarate hydratase, adenylate kinase, quinono reductaso, and tetrazolium oxidam. By comparison of thin-layor elcctrophorctic migration rutas through starch gels, theso authors were ablo to classify thesc seven strains of Bdellovibrio into five distinctive zymogen groups with important taxonomic utility (Seidler et al., 1972; Starr and Seidler, 1971).

Wehr and Klein (1971) used the disc-assay technique to test the activity of commercial herbicides against a Bdellovibrio strain (not specified) and its pseudomonad host. The sensitivity of the Bdellovibrio to the herbicides was measured: (1) by growth inhibition of the Bdello- wibrio plaque obtained by placing tangentially to a plaque edge (six days old) a herbicide test disc and a control disc and incubating further for 4-14 days at 27’; and (2) by enumerating the decrease of plaque- forming units in the host- Bdellovibrio cultures containing a graded concentration of herbicide. Of the 17 herbicides tested, 11 including urea, carbamate bromacil, and three phenoxyacetic acid herbicides inhibited plaque formation of the Bdellovibrio on lawns of P.uewbnona8 sp., while only 4,6-dinitro-O-sac-butylphenol in hibitod tho p s ( ~ ( l ( ~ - monad host. Linuron [3-(3,4-dichlorophenyl)-l-methoxy-l-mc~thyli~rc:a~ decreased the net multiplication of Bdellovibrio. Wohr and Kloin (1871) showed that free Bdellovibrio cells, in tho ahsencc of host c o l l ~ , wcro sensitive to linuron thus indicating to thorn “that linuron j)rirnttrily increased the death rate rather than influencing only tho sccontlary characteristics of motility, attachment and penetration”.

VI. Host-Independent Derivatives of Bdellovibrio

Stolp and Petzold (1962) and Stolp and Rtarr (1963b) sclectccl ho&- independent derivatives from cultures of host-dependent bdellovibrioa. These host-independent derivatives were unable to lyse living bacteria (Stolp and Starr, 1Q63b). They were generally less homogeneous in

PHYSIOLOGY OF THE BDELLOVIBRIOS 255 appearance than the parental type. Although they usually have a single sheathed flagellum, one strain has been reported to have as many as three flagella at either one or both ends (Stolp and Petzold, 1962; Stolp, 1968).

The frequency of occurrence of host-independent bdellovibrios in host-dependent populations was at first reported to vary from strain to strain. Values of 1:106 to 1:109 have been quoted (Stolp and Starr, l963b; Stolp, 1967c, 1968) but most workers now agree that it is about 1:106 in many strains (Seidler and Starr, 1969b). The reversion of host-independent populations to host-depcndent populations occurs at about the same frequency when host-independent bdellovibrios are massively inoculated into congenial host cultures. Whether the shift is genetic or eiivironmental or both is not yct conclusively known, nor is it clear whethor individual cells (as opposcd to populations) are both independent and dependent on a host. These and related questions are considered in more detail by Starr and Seidlcr (1971).

Three mrthods have been used for isolating host-independent derivatives. Thc first method (Stolp and Starr, l(t6Sh) involves the inoculation of a coiicentriited suspension of host-dependent cells (over lo9 cells/ml.) on to a yeast-peptone agar plate or into nutrient broth. The second method (Shilo and Bruff, 1965) uses an enrichment technique. Host-independent derivatives arc concentrated by the successive addition of host cells and host-dependent Bdellovibrio cells, and the host cells plus the Bdellovibrio cells (those attached to the host cell) are separated from the independent derivatives by differential filtration. The host- independent cells in the filtrate are transferred to full strength or one- tenth strength nutrient both containing heat-killed ( 120°, 15 min.) host cells (109/ml.) and shaken at 28". The third method (Seidler and Starr, 1969b) consists first in selecting strcptomycin-resistant (Sm') host- dependent Bdellovibrio clones on Sm' host cells. Streptomycin-resistant host-independent bdellovibrios develop spontaneously in the two- membered culture. Then, thc: 8m' host-dependent Bdellovibrio culture is grown on streptomycin-sensitive (SmS) host cells. A lysate containing large numbers of Sm'host-dependent Bdellovibrio cells and somc remaining Sms host cells, as well as Sm' host-independent Bdellovibrio cella, is tranaferrod to a nutrient selection medium which contain8 Rtrcptomycin. Thc Sm' host cells in the lysate are killed, the Sm' host-depcndcnt Bdellovibrio cells cannot grow under these conditions in tho absenco of viable host cells, and the Sm* host-independent Bdellovibrio clones develop.

Whether the host-independent strains of Bdellovibrio kolated by them several methods are genetically and physiologically idcntical remains to be investigated. A still somewhat sketchy assemblagc of the hacterio- logical traits of the host-independent bdellovibrios will bo found in the

266 M . P. STARR AND J . C.-C. HUANQ

review by Starr and Seidler (1971) ; much remains to be done in depicting these organisms completely.

A facultatively parasitic strain of Bdellovibrio (strain UKi2) has been selected from obligately parasitic Bd. UK, using E . coli B/r as a host bacterium (Burnham et al., 1969, 1970; Diedrich et al., 1969, 1970). The cells are 0.8-1.2 pm. in length and 0.3-0.4 pm. i n width; they are motile, curvcd r ~ d ~ cvich with IL sing10 polttr hgd lu rn , 1mig Npirtdn (up to GO p m ,) t ~ r ( b rcgulrtrly obsc~r.vc!tl. I%(: growth curve in ymst pcptonc broth has a lag period of ti hr. Ihring this period of time, the comma- shaped cell develops into spirals, then 7-10 vibrioid cells are produced from each spiral cell. The host-independent phase in this exceptional strain involves thickening, coiling, and fragmenting into new progeny, quite similar to t h c parasitic intracellular pham which han hcen obmved in most Bddovibrio strtiiiis. Facultativaly partwitio Bd. UKi2 produces a WhitiAh-groy colony surrountlctl by t i circulw plaquc-liko clearing zone, a trait wliicth (:nabled IXcdrich P t al. ( 1 869, 1970) to differcntiatc it from the obligate parasito which forms u olcar plaque. ‘I’his facultatively parasitic strain has physiological characteristics very similar to those of host-independent strains. Proteases are produced in the supernatant of yeast-peptone broth culture which lyse autoclaved E . coli B/r cells and degrade Azocoll. Further remarks about this very interesting Bd. UKi2 strain and about facultative parasitism in Bdellovibrio will bo found in Starr and Seidler (1971).

VII. Bdellovibrio Bacteriophages

Hashimoto et al. (1970) isolated from sewage a hexagonal, tail-less phage (HDC-1) which is infective for facultatively parasitic Bd. UKi2. The phage has a size of between 60 and 70 nm. and appears in preparations st,ained with 0.5% uranyl acetate to be composed of two distinct coats (capsomer layers). Acridinr, ortLngc staining followctl 1)y molybdic acid treatment revealed that, thc: ptiiLgc contains ~iri~lc-~tr.nritlc,cl DNA. Bdellouibrio bacteriophage H I N - 1 infccts and lyso~ I M . UK i2 spwifically and yields 5 r: lolo phage particle8 per mi. Strilin 1-1 I X - 1 iu relatively stable to heat (99% inactivation at GO”, 2 hr.), osmotic shock, storugc, and over a wide range of pH 2 to 10.5 (Diedric h et al., 197 1 ).

Twelve additional phages for host-independent bdellovibrios were isolated from raw sewage in Kentucky (Althauser and Conti, 1971). Negative stains of these phage8 show three distinctivc morphological types, designated R, C, ant1 E, according to Bradley’s nomenclature. A positive correlation between the three morphological types and the host range was observed when the 13 phages were tested against 19 strains of host-independent bdellovibrios.

PHYSIOLOGY OF THE BDELLOVIBRIOS 267 The 13 strains of bdellovibrio phages were further classified into

five groups according to thcir host specificity: (1) MAC-9 and MAC-6 lyse only host-independent Bd. A3.12 and Bd. 0x9-2; (2) HDC-1 and HDC-2 attack only Bd. UKi2; (3) MAC-7 attacks host-independent Bd. B and H-I Bd. E; (4) the remaining seven strains have a broad host range, encompassing most available Bdellovibrio strains ; and ( 6 ) MAC-3, the only phage that attacks host-dependent Bd. 120, has the same wide host range &B (4) except that it does not lyse host-independent Bd. Xty. Some of the bdellovibrio phages attack parasitic strains of bdellovibrios in three-membered (bdellovibrio, host, and phage) systems, a very interesting example of hyperparasitism.

The analysed correlations among phage typing (as studied by Conti and his collaborators), base composition of Bdellovibrio DNA (Seidler et al., 1969), zymogen analysis (Seidler et al., 1972), molecular heterogeneity in terms of DNA-DNA or RNA-DNA hybridization (Seidler and Mandel, 1971; Seidler et al., 1972), host specificity, and other traits of Bdellovibrio are eagerly awaited, particularly for the clarification of important issues in the taxonomy and parasitism of Bdellovibrio which these should provide.


The research from this laboratory referred to in the review was supported, in part, by research grant AI-08426 from the National Institute of Allergy and Infectious Diseases, United States Public Health Service, and by a Fellowship (to J. C.-C. H.) from the Medical Research Council (Canada). We appreciate very much the editorial and bibliographic assistance of P. B. Starr. We are greatly obliged for critical reading of the manuscript by A. K. Chatterjee, H. R. Heise, J. E. Snellen, and J. W. Nunley.

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Mitchell, R. and Alexander, M. (1963). Can. J. Microbiol. 9, 109.

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biology, University of Maryland, College Park, Maryland.

Giittingon.

st.OlI), Ha ( 1969tl). /?nngP, kl t?f /?kUl ]’/LO10 I~a~JfJrh / l f l C / l f ! 80, 2.

pross.

518.


AUTHOR INDEX

Numbers in italic0 rejet to the pagea on which reference8 are lieted at the end of each article.

A A~WIMII, I ) . , 220, 292, 223, 224, 232, 233,

Ailerrin, (1. A., 12, 14, 27, 54 AdernH, M. H., 159, 181, 184, 808, 218,

Adelberg, E. A., 49, 57 Afenas'eve, T. P., 178, 213 Aguirre, M. J. R., 242, 257 Ailheud, Q., 29, 30, 57 Ailhaud, 0. P., 30, 51, 52 Akagi, J. M., 81, 99 Akamatsu, Y., 28, 40, 54 Akashi, S., 18, 51 Alberts, A. W., 19, 22, 51, 55, 56 Albertsson, P. A., 108, 109, 137, 140,

Albro, P. W., 6, 7, 8, 10, 11, 18, 37. 38, 39,

Aldelmen, 8. L., 131, 137 Aleem, M. I. H.. 7, 37, 57 Alexander,M., 113,130,135,238,139,242,

Alkam~tsu, Y., 51 Allen, A. D., 93, 98 Allen, C. M., 34, 51 Allison, M. J., 3, 4, 21, 51 Alper, R., 55 Althauser, M., 266, 257 Alworth, W., 34, 51 Amaral, D., 161, 208 Amemiye, K., 238, 257 Ames, Q. F., 38, 51 Anderson, E. S., 167, 169, 192, 208 Anderson, J. S., 208 Anderson, R. F., 176, 209 Amon, D. I., 65, 73, 74, 76, 77, 79, 80, 81,

82, 83, 84, 86. 86. 98, 99, 103, 104 Arf~enio, C., 161, 212 Artom, C.. 7, 31, 51 Ashcroft, J., 129, 137 Aepinell, 0. O., 146, 176, 208, 213 Asselineeu, J., 1, 14, 16, 17, 27, 36, 42, 47,

2311, 237, 258, 239, 252

257

141

40, 41, 43, 46, 51, 53, 56

259

48, 49. 51, 52, 55. 67

AUHtriLbn, It., 160, 186, 190, 201, 208,

Avogiio, P., 116, f 39 Avory, 0. 'l'., 158, 181, 208, N!) Avi-lhr, Y., 132, 199 Asoracl, It., 28, 53

212

6 Bechofen, R., 82, 98 Bacon, J. 9. D., 242, 259 Baddiley, J., 13, 14, 16, 43, 46, 51, 57, 58,

Beigent, N. L., 222,224,229,231,232,233.

Bekal, M., 67, 101 Balekjien, A. Y., 131, 138 Bellou, c. E., 43, 49, 51, 56 Bamburg, J. R., 176, 209 Beptiet, J. N., 267, 260 Barber, C. A,, 189, 208 Berber, a. A,, 208 Berclay, K. 8.. 167, 208 Barer, It., 132, 137 Berkor, I). U., 188, 20X Ijarkor, H. A., 146. 167, 159, 172, 173, 181,

Ijurkor, T., 101, 203 Bernott, E. V., IlG, 137 Bsrnott, H. L., 217, 257 Barney, J. A., 116, 140 Barnick, J. A., 108, 139 Baronoweky, P., 22, 51 Beronowsky, P. E., 23, 24, 26, 57 Barridge, J. K., 7, 36, 37, 51 Bertell, P. F., 160, 209 Baumenn, N. A., 7, 11. 12, 28, 33, 44, 46,

Booking, J. H., 00, 61, 64, 98 Beijerinck, M. W., 60, 61, 98 Beinert. H., 81, 84, 103 Becker, Q. E., 169, 209 Beieer, 8. M., 169, 209 Belozorskii, A. N., 178, 213

182, 183, 184, 211, 212

234, 237, 238, 242, 261

1x2. 184, OUd, PUB, 2fP

48, 51

12 %03

2G4 AUTlIOR INDEX

Bone~ntinn, J. I t , 60, 65, 68, 73, 74, 75, 77, 79, 80, 81, 82 , 84, 85, 86, 94, 96, 98, 102, 1/14

h n n o t t , It., 77, 08 Dcwson, A 11.. 6, H, 37, 40, 41. .il, 57

13orgorsim. 1'. J., 110, 01, 6(i, 67. (is. (;!I, 97,

Burgordon, I?. , I , 0 2 , O(i, !/S BorgoIwn, I d . I ) . , ! I , 51; Bernharcl, K., 23, 51 Berliner, JC. , 114, 1Xi

Hornlicriniur, H , l'., 160, 185, 100, 201, 202,

13orrrst,oin, 1%. 1, , 201, 209 Uoutntrr, iZ. H., 115, 137 13hat, J . V., 220, 221, 258 Bick, R. M., 182, 1x4, 208, 209 Rior, M., 108, 137 Biggin#, I ) . It, M, 67, 91, 92, 9s Hiuhop, I). CX , 25, 27, 44, 51

Uergcw, L. It., 7, 16, 37, 51;

101

&ll . tH( 'h , I,. JJ. , 41, 44, 48, 51

208, 209, 212

Ulntor, v., 160, 213 ~~JoI '~ldld, H., 157, 209 UIC~IIIC, PI. L., 5, 33, 53, 88 1310(~}1, JC., 2, 17, 18, 20, 22, 23, 24, 25,

27, 2X, 3 0 , 34, 42, 5 1 , 52, 5 4 , 55, 5U, 57

Ulorh, W., 25, ,;!2 Ulorrr~trorn, I ) . (2. . 83, 98

I%owi, V . , 128,

Uond, C., 60, 98 13o1iner, H., J r . , 29, 56 BonFcon, P. 1'. M., 9, 32, 41, 44, 48, 5 1 , 51,

Uounio, E. J., 107, 20,Y Bowman, It. L. , 112, 1:7 Bloornfiold, I). I<., 22, , ; I Bottir,, If., 78, 82, 84, 98, 100, 10.7

I M C I U ~ I ~ , o. E. n., 100, 121, 122, 140

55, 56

13mdfitdd, J. It. G., 132, 141 l3radEo1d, L. 1, , 1 15, l J 8 Brndy, It. O., 19, 51 Brashc~nr, D. A,, 108, 1.39 Braun, W., 233, 2.59 Fhrod, It. s., 0, 3 5 , hf Brogoff, )I. RI., (~8, 100 Brornc>r, J., 7, 31, 'i/ Brcxnnim, 1'. J., 43, $0, 'j/

Brtximik, 1'. I,., 96, 98 Brion, IC. T., 130, f.:/ 13rioiIey. C: , 8, .;2 Brighton, W. 1) . 115, 1.7; DrilJ, W. J., Of;, 100, 10.1

I ~ ~ o \ v I : I , A. K., / l 7

Brimncombo, J. B., 182, 184, 208, 209 Hrindloy, I). N., 20, 5 1 Brintzingor, H., 92, 98 Brown, A. D., 242, 257

Ilrown, 11., 16(1, P I / /

Ijrtiff, U. , 244. 247, 250, 255, 260 I h f f , 11. R., 232, 246, 247, 250, 257 Ihwntliah, 1,. LC., 14, 43, 4G, 51 Uryant, M. P., 3, 4, 21, $1 Buchanan, R., 80 , I01 13uchanan, 13. I L , 77, 80, 81, 82, 83, 98, 99,

Buchanan, J. G., 182, 183, 184, 211, 212 lhekmiro, F. L. A, 234, 258 Bui, P. T., 92, 99 Rull~ii, W. A,, 64, 65, 06, 07, 68, 69, 76,

13unn, C. R., 39, 51 Bunt, J. R., 96, 09 Durgor, A., 219, 223, 226, 234, 237, 238,

Burnham, J. C., 30, 57, 222, 223, 224, 232,

13roWl1, J . l'., 27, 55

H Y U C I ~ I I O I ~ , (i-. c., 108, / ; i i

100

84, 88, 89, 90, 92, 93, 96, 99, 100, 102

247, 250, 251, 252, 258

23fi, 237, 25fJ, 288 ~%lll'nH, It. C., (10, (14, 66, 06, 67, 68, 70,

X8, 90, !)2, 93, 94, !15, 96, 99, 100, I01

I h r r i a , It. €I., 60, 60, 67, 68, 69, 71, 76, 88, 8'3, 90, 91, 02, 95, $9, 100, IOI, 102, 10.7, I04

I~urto11, l'., 82, 99 I k t o n , A. J., 3, 16, 17, 51

J ~ U S S O , D., 7, 38, 42, 67 httcrworth, 1'. 11. w., 22, 52

AUTHOR INDEX

Carter, 0. B., 132, 133, 137 Carter, H. E., 16, 18, 51, 52 Carter, H. P., 242, 258 Carter, J. R., 3, 17, 30, 52 Carter, J. R., Jr., 30, 52 Casida, L. E., Jr., 210, 220, 221, 232, 233,

260, 261, 259, 261 CRstb, J. E., 64, 68. 70. 71, 88, 99 CRtO, E. P., 113, 130, 139 Cevenaugh, L. A., 122, 141 Chekraberty, A. M., 160, 209 Chang, Y.-Y., 8, 30, 31, 52, 5Y Chett, J., 93, 99 Cholodny, N., 123, 138 Chou, D., 236, 257 Christenson, J. O., 187, 209 Chung, A. E., 26, 52, 74, 99 Churoher, a., 116, 140 Clark, 8. P., 116, 139 Cleus, D., 178. 209 Clivcr, D. O., 108, 109, 138 Clo~s, O., 120, 127, 1.48 Coffey, E. M., 115, 138 Cohon, O. H., 178, 179, 20.9 Cohon-%uh, G., 08, 102 CollinR, V. G., 122, ZSX Colvin, J. It., 189, 102, 211 Conklin, C. J., 118, 119, 140 Conrad,H. E., 148,167,173,176,195,209,

Constentopoulos, G., 42, 52 Conti, S. F., 30, 57, 222, 224, 228, 232, 236,

237, 238, 244, 253, 256, 257, 258, 259, 261

210,211,212

Cook, T., 15, 53 Cooke, K. O., 117, 1,?8 Cookmy, K. E., 96, 99 Coons, A. H., 114,138 Cooper, E. A., 167, 209 Cooper, F. C., 108, 137 Corbin, J. L., 93, 102 Corpe, W. A., 147, 148, 209 Cote-ltobles, E. H., 76, 99 Cottral, Q. E., 118, 139 Coty, V. F., 61, 99 Courtney, R. J., 116, 133, 140 Cox, R. M., 68, 77, 78, 99 Crecch, H. J., 114, 138 Cronan, J. E., Jr., 8, 27, 20, $2, tti Croson, M., 19. 55 Crother8, R. F., 241, 247, 248, 260,

Cruickshank, R., 146, 209 Crumpton, J. E., 131, 132, 140 Crumpton, M. J., 176, 209 Currie, J. F., 126, 120, 139

Cymbslkta, 8.. 108, 140

258

CUtts, J. H., 44, 52

265

D I)UOSCh, G., 68, 69, 99 Dahlen, J. V., 91, 99 Dalton, H., 60, 67, 69, 04, 99, 104 Deniol, J. J., 7, 31, 55 l)triiiol, It. M., 76, 104 I)eniolHxon, I)., 115, 120, 127, 1J8 I)anlcort, M., lB, 34, 58, 192, 194, 209,

Dark, I?. A., 110, 121, 140 Dauchy, S., 27, 52 Davidson, J. B., 31, 52 Devies, D. A. L., 175. 209 Davis, B. D., 169, 209 Davis, B. K., 220, 223, 224, 233, 237,

Davis, J. B., 61, 99 Dawes, E. A., 19, ri2 Dawson, M. D., 97, 103 Dewson, R. C., 97, 99 Ilawson, R. M. C., 36, 68

Dockor, K. , 74, 101, 103 Dodoncicr, It. A., 190, 200 13)oiI)ol, H., 192, 138 I)uland, F. H., 126 Dolaney, R., 19, 57 Delwiche, C. C., 97, 99 Donisov, N., 94, 103 Dannis, S. C., 220, 259 Denny, C. F., 233,244,263,260,258 Depinto, J. A., 16, 52 Der Vartanien, D. V., 81, 10.3 Detroy, R. W., 89, 91, 95, 99 D’Eustachio, A. J., 64, 66, 72, 75, 80, 83,

Devanathen, T., 66, 81, 90, 99, 101 Do VRY, J. E., 224,229, 232, 236, 237,243,

Dies, F. F., 220, 221, 258 Dicks, J. W., 147, 209 Diedrich, D. L., 233, 244, 253, 256, 258,

Diena, B. B., 132,138 Dilworth, J. R., 93, 99 IXlworth, M. J., 60, 06, 71, 99, 100, 102 Dinglingcr, F., 43, 49, 57 Didor , J., 34, 54 I)ittrrior, J . C., 6, 7, A, 10, 11, 18, 35. 37, 38,

213

257

Thy, J. I. E., 27, 28, 33, 52

93, 99,100, 101

260

259

30, 40, 41, 4f1, 61, 66, 68 IkJOrIl~Jrly, l’., g:j, I03 Uoi, It. H., 220, 281 lhllhopo, E’. I,., 116, 139 Doinagk, G I . F., 161, 209 Dorfman, A., 168, 211 Doty, P., 227, 259 Doudoroff, M., 46, 49, 51, 5Y, 161, 211

266 AUTHOR INDEX

Drow8, U., 223, 220, 234, 237, 238, 247,

Ikozd, J.. 06, 07, 100 Druckor, E., 239, 258 Drucker, 1.. 240, 241, 281 Uruott,, H. A,, 108, 139 Dua, R. D., 88, 100 Dubos, R., 168, 209 Dubourdiou, 0.. 79, 84, 100 I>ucltworCh, I). H., 170, 213 Dudnun, W. B., 140, 147, 148, 158, 173,

174, 175, 209, 210, 213

250, 261, 362, 258

Dugan, 1’. it., 9% I02 Duguid, J . L’., 145, 147, 194, 20!/, 213 I)urn, J. 11.. 67, 100

E Eckor, H. I<,, 123, 1 3 8 Edorihaidor, Q., 86, 101 Edrnundn, P. N., 147, 194, 213 Egpobraton, L., 45, 53 Efirnov, O., 94, 105 Eggloston, L. V., 73, 104 EiduR, L., 132, 138 Eisenstoin, K. I<., 82, 100 Eklund, C., 169, 160, 161, 209 Elbein, A. D., 189, 208 Elgart, R. I,., 11 R, I40 Elkan, G. H., 30, 51 Ellis, M. E., 7, 32, 39, 47, 48, 52 Kl Nakhal, H., 124, 141 Kploy, J. D., 148, 176, 195. $09, 211 Kriksoni, J., 20-9 Errksnri, J., 171, 173, 209, PI0 ErikHcn, J. I , . , POX Erlich, 1C., 123, 1.48 Est,rada-Pnrrci, S., 160, 183, 184, PO!),

Estroumza, J., 30, 57 Evans, E. E., 116, 138 Evans, H. C., 82. 99 Evens, H. J., 64, 76, 89, 90, 95, 97, 101,

Evans, M. C. W., 64, 08, 77, 80, 82, 91, 96,

Extorkate, F. A., 9, 43, 47, 52

210

103

98 ,100 ,103

F Fattal, B., 108, 140 Fackrell, H. B., 244, 247, 260, 258 Fermor, V. C., 242, 259 Farr, A. L., 228, 959 Fay, P., 07, 08, 70, 77, 78, 99, 100 Fedorov, M . V., 62 , 100

Folton, F. G., 116, 140 Fennossoy, P., 16, 34, 58 Ferrari, A., 112, 132, 138 Fey, H., 127, 159 Fikhmsn, B. A,, 132, 138 Finnerty, W. R., 30, 31, 32, 43, 48, 52, 55,

Fishor, R. J., 07, 102 Fischer, R. J., 72, 90, 100 Vischnr, W., 45, 52 FiizgoraItI, a. I?., 07, 08, 103

56

b’loirrcher, s., 8. 52 Pogg, (3. E., 67, 70, 77, 90, 100, 101 I l ’ O l H ; y t h , w. (3. c., 10, 953 14‘(JHtor, A. n., 172, 208 I’orrtor, E. M. , 3, 4, 12, 21, 58

l honke l , A. W., 70, 102 Francis, M . J. O., 9, 56 T’runzon, J. S . , 74, 99 Frcw, J. I., 90, 101 Freudnian, H. H., 233, 259 Frorman, F. E., 16, 34, 57, 176, 187, 191,

192, 193, 194. 213 Friok, G., 108, 109, 140 Fuchsmnn, W. H., 94, 99 Fulco, A. J., 23, 26, 52 Fuller, C. E., 130, 138 Fuller, R. C . , 77, 98 Furukawa, Y., 44, 58

FOUHt,, G . P., 82, 84, 100, 102

G Gaho, I). R., 218, 260

Cuhrm, I,. C., 173, 210

(~riric.c:tlo. C., 161, 812 ( ~ c L r c i n - ~ o h i b , I., 242, 267 C:circ.is-ltivwa, J . , 99 Garagg. 1’. J., IG6, 162, 171, 172, IHH, 199,

Carnor, W., 110, 131, 138 Glirtnor, H., 108,138 Gasclorf, H., 170, 209 Gonnaro, R. M . , 110, 131, 138 Georgi, C. I<., 44, 52 Gorke, J. H., 112, 138 Gest, H., 36, 57, 01, 08, 73, 76, 100, 101,

Ghslamhor, M . A., 176, 213 Ghuysen, J. M., 223, 228, 242, &fig, 261 Gibbons, N. E., 4, 12. 14, 19, 54, 57 Gibson, K. D., 7, 31, 52 Gilliland, R. B., 131, 138 Gillis, J. R., 247, 261, 258 Ginsburg, V., 186, 187, 201, 210, 211 Glasor, D. A., 112, 138

(:@by, w. L., 30, 67

~ > l l l f l I l f l H , (‘., 162, 168, 210, 21~1

210

102

AUTHOR INDEX

Gleser, L., 189, 210 Glaudemens. C. P. J., 184, 212 Ooebel, W. F., 166, 167, 169, 170,181,184,

208,210,212 Goldblum, N., 108, 140 Goldenson, J., 118, 138 Goldfine, H., 3, 4, 7, 11, 12, 17, 18, 22, 23,

24, 26, 26, 27, 28, 29, 30, 32, 33, 34,

58 36, 37, 39, 44, 47, 48, 51, 63, 53, 57,

Go~dmen, M., 114, 117, 138 Oomos, N. F., 48, 52 Gorchein, A.. 7, 14, 32, 36, 62 Gordon, I., 132,140 Gorin, P. A. J., 167, 179, 210 Oottschlich, E. C.. 183, 210 Gough, D. P., 34, 53 Gould, J. R., 113, 130, 138 Gould, R. M., 33, 43, 46, 53 Grant, W. D., 167, 170, 203, 210 Grey, 0. M., 27, 53

Green, M., 76, 100 Greenberg, D. M., 7, 31, 51 Greenburg, L., 132, 138 Greenwood, F. C., 128, 139 Grimth, F., 181, 210 Grindey, c?. U., 43, 53 Gromov, B. V., 21R, 262, 258

Grovo, P. L., 220, 221, 261, 260 UuBlin, A., 220, 221, 236, 237, 242, 261.

Gunselus, I. C., 160, 209 Gurney, E., 96, 97, 103 Gurr, M. I., 9, 53 Quthrie, R. K., 116, 127, 138 Guze, L. B., 242, 2.59 Guy, R. 0. E., 160, 181, 210

Grey, 0. w., 11, 53

GPORH, 8. K., 187, 209

258, 259

H Hedfield, K. L., 92, 100 Hagen, P. O., 4, 6, 7, 11. 12, 27. 28, 32, 33,

Hahon, N., 117, 133,138 Heinee, R. F., 116, 138

Hakamori, 8., 167, 210

Hall, C. W., 34, 56

34, 36, 37, 39, 44, 48, 52, 53

Hejrs, A. K., 33, 53

H ~ I ~ S . c. N., 115,139 ~811, D. 0.. 80, 82, 83, zeo ~811, M. o., 7, 31, 53 Hernmond, R. K., 43,40, 64 Hancock, I. C., 3, 39, ,5j Henkine, W. A., 117, 138 Hanks. J. J., 217, 259

\

267

Hensen, R. E., 84,103 Here, I., 16, 55 Hareda, T., 165, 210, 211, 212 Hardy, R. W. F., 60, 64,615, 66,68, 72, 73,

76,79,80,83,84,00,92,93,94,96,96, 99, 100, 101

Hare, A., 116, 139 Harper, C. L., 96, 98 r h p o r , 12. M., 206, 212 Hart, P., 30, 56 Hanhimoto, T., 222, 224, 232, 233, 236,

237, 238, 244, 253, 266, 258, 259 HaHkin, M., 182, 208 He~xid, w. z., 167, 189, 190, 208, 209,

Haug, A., 179, 180, 198, 210 Hewtrey, E., 46, 62 Hayflick, L., 217,218, 259 Heystead, N., 68,100, 103 Heyward, A. C., 19, 53 Heely, K., 33, 68 Heath,E.C., 16,34,57, 177,187,191,192,

193, 194, 213 Hebert, R. R., 101 Hechemy, K., 29, 53 Heos, M. A., 96, 99 Hoidolborger, M., 160, 166, 168, 169, 173,

181, 182, 183, 184, 209, 210, 211, 212, 215

212

Hoinor, G. V., 116, 139 HoniN, Y., 113, 130, 158 Henrikscn, R. D., 169, 171, 173, 209,

Hersh, R. T., 81, 99 Hornrning, F. W., 34, s53 Higeshi, Y., 15, 34, 53 Higuchi, T., 192, 213 Hildebrand, J. 0.. 3, 53 Hill, J. H. M., 118, 141 Hill, R. E. E., 94, 100 Hill, R. L., 19, 57 Hill, S., 62, 94, 100 Hirnee, R. H., 81, 99 Himmelspech, K., 168, 210 Hinkeon, J. W., 84, 86, 100 Hipsely, E. H., 61, 62, 97, 98 Hirase, S., 153, 210

Hirsch, P., 250, 262, 261 Hirst, 0. K., 116, I38 Hoch, Q. W., 66, 101 Hodgwon, It., 167, 190, 218 Hoeniger, J. 11’. M., 220, 226, 259 Hoorrnan, K. U., 131, 167 Hoorrman, K. ( J , , 131, 138 Hofmarin, K., 22, 23, 24, 26, 27, $3, b G Hoffmenn, K., 7, 38, 42, 57 Hoffman, P., 160, 211

210

Hir8pm8, O., 36, 53

268 AUTHOR INDEX

Holborow, E. J., 115, 138 Holdernan, 1,. V., 113, 130, 130 Holni-Hanaon, O., 62, I01 Holmo, T., 156, 162, 171, 172, 188, 19!1,

Holnten, H. I ) . , 68, 68, 90, 95, $)(I, !I$, 100,

Hong, J., 81, 101 Hoopor, N. K., 27, 53 Hopnor, Th., 85. 101 Hopton, J . W., 159, 181, 208 Horoolcc~r, 13. Id., 151, 168, 108, 20.8, 211,

I I~ t~ iko~J i i . I<.. 242, 25!/ ~ l o r t l l l l d , (I., 17. 57 H I I ~ I I I ~ , A. *J., ! t f i , 101 tlorning, hl. (:., 21, 5.1 Ilotchin, J. 16., 132, 138 Hotchkiss, It. I ) , , 1H1, 210

Hout~tnull(ir, I T . M . T., 8, 9, 41, 43, 44, 4(i,

How, M. J., 150, 181, 184, 208, 210 Howard, I). L., 96, 101 Howard, Is., 82, 96, 99 HBU, S. O. , 242, 250 Huang, J . C.-C., 218, 220, 223, 224, 22%

210

101

2I.Y

Hotta, K., Ifi, ri5

5.1, 56

234, 236, 237, 239, 243, 244, 245, 240, 247, 248, 249, 250, 259, 260

Hudak, C. E., 122, 140 Huennekons, F. M., 83, 104 Hugget, A. S. G., 161, 210 Hull, J . I?., OH, 104 Humptimy, 13. A., 152, 211) Huniphlnys, Q. O., 3, 53 Hutnphric~x, , I . (!,, 160, 160, 210

Huntor, J. I t . , 131, 132. / / 1 )

Huntiw, W. M., 128, I.1!1 Hussoy, I [ . , 10, 58 Iluston, C. I<., !), 14, 43, 4U, 5.1 Hutchison, A. R l . , 175, PO!/ Hyndman, I A . il., 16, 101

ti l ln~ltrar. , I ) . , 104, 210

I Tanotti, b;., 15, 5.3 Iiria, S., 212, 2.59 Ikawa, &I., 2, 7, 0, 16 , 14, 36, 40, 47,

Ikokawa, N., 17, 57 Imai, Y., 23, 53 Illsolera, R. V., 120, 140 Ionocia, T., 48, 52 Tshizuka, I., 46, 59 Ito, M., 117, 139 Ito, T., 166, 211

53

J Jackson, E. I<., 66, 68, 100, 101 Jackson, H. R., 260, 259 Jackson, R. W., 177, 209 Jnmos, A. T., 7, 14, 39, 42, 58 Jarnionon, R. S. P., 176, 208 Junn, B., 163, 164, 168, 169, 205, 210, 211,

Jtinn, K., 145, 163, 164, 100, 108, 169, 210, 213

211. 213

2 1; 0 JIirlrltLsCh, I f . W., 123, 159. 221, 24‘3, 254,

J u I I I I ~ ~ ! ~ ~ , S. I’., 122, 140 .JILVIIHH, 1,. I$ . , l:$z, /&/ Jtrur.6~uihor~y, G . , 2H, 53 Jiwifw, A., 177, 178, 210, 211, 212 Jong, 1). Y . , 66, ‘30, !)2, 101 Jonkin, H. M., 46, 50, 53, 54 Jarmyn, M. A., 150, 210 Jorno, N-K., 116, 139 Johnson, U. D., 115, 139 Johnson, R. C., 45, 50, 53, 54 Johnstone, D. B., 178, 179, 209 Jonos, D., 242, 259 Jones, J. K. N., 179, 211 Jones, K., 81, 101 Jones, R. N., 114, 138 Jonos, It. S., 180, 211 Joo, C. N., 4, 5, 12, 14, 54 Jost, R., 127, 139 Jukcs, T. H., 83 , 102 Jiingorrnann, I<., 74, 101, 103 Jiirtflhuk, P., 38, 54 .Ji,yco, G. IT., 43, 46, 54

K I<tr~i, I I . II., 118, 1 1 1 KIbjiy&fiiih, S., ! iO, 101 ICitlo, S., (i7, 101 Kaliriinkayt~, ‘1’. A., 02, 100 Kallio, It. E., 40, 52 Kamon, M. I)., 61, 68, 94, 98, 100, I01 Kamio, Y . , 3, 4, 6, 11, 12, 22, 48, 54 Kanclutxoh, A. A, 34, 54 Kanccltt, T., 21, 54 Kanoganaki, S., 3, 4, 11, 22, 48, 54 Kariornnsa, Y., 40, 54 Konoshiro, T., 7, 27, 32, 54 Kanfer, J., 30, 40, 54 Kana, 8.. 170, 203, 211 Karmon, A,, 21, 5.Y Krtrninkin, G . S., 122, 139 Katos, M., 1, 4, 5, 12, 14, 17, 18, 23, 27, 35,

Katz, I., 3, 4, 10, 21, 51,!54 42, 54

AUTHOR INDEX

Kauffmann, F., 168, 169, 211 Keufmann, 34, 54 Kowioy, M., 3, 4, 10, 15, 21, 51, 53, 54 Kointor, D. L., 86, 101 Koller, J. M., 188, 199, 211 Kolloy, W. 8., 194, 209 Ktillo~g, 11. S., 115, 141 Kvlly, M., 06, 67, 8!), 00, 91. 02, !KI, BS,

101 Kdly- VaIecyq I(’., I 5 1, 208 Ktiriiiotly, I) . A., 1x3, 2 1 / I(rrlll1otly. 14:. l’., x, 30, 31, 93, 40, $2, 54,

50, 57 Koiiriody, 1. It. , 65, 88, 69, 90, 101 Kent, J. L., 194, 911 Khan, A. W., 189, 192, 211 Ko~nol, 1%. W. I., 233, 259 Kim, J., 238, 257 Kim, K. C. , 5 , 10, 12, 54 Kimura, A,, 15, 54 Kitnura, T., 83, 10s Kiridt, T. J., 175, 209 Kipling, C., 122, 138 Kirby, A. L., 34, 53 Kito, M., 29, 54 Kiyaau, J. Y., 31, 54 Kloin, D. A., 220, 221, 232, 233, 250, 264,

259, 261 Klein, E., 117, 141 Klouwen, H., 8, c52 Kliictl~, R. V., 75, 8!), 9.5, 101 Knappe, J., 88, 101 Knight, 13. C. J. U., 130, 140 Knight, E., 83, 98 Knight, I<., Jr., (i0, 06, (10, 72, 73, 7!), 83,

H4, 93, IOU, 101 Knight, 8. U., 179, 212 Knoche, H. W., 26, 54 Knutron, C . A,, 177, 211 KO, W.-H., 242, 259 Kooh, A. L., 132, 139 Koch, B., 64, 90, 101 Koch, R., 89, 95, 101 Kocun, F. J., 8, 54 Kodicek, E., 16, 18, 34, 67, 187, 21: Koeltzow, D. E., 148, 195, 211 Koepfli, J. B., 16, 53 Kok, H., 82, 98 Kolin, A,, 109, 139 Koostra, W. L., 8, 33, 54 Kornborg, A., 30, 41, 44, 48, 51, 54 Kornfold, R. H., 201, 211 Krabok, W. B., 123, 140 Krobs, H. A., 73, 104 Kubitwhek, H. E., 108, 112, 124, ]XI Kuchynka, K., 93, 101 KuczynBki, M., 132, 139 Kuhn, N. J., 21, 30, 54

Kundig, F. D., 34, 54 Kunisowa, R., 68, 102 Kunnos, R. S., 29, 56 Kurkdjian, A., 39, 54 Kurokawct, M., 16, 55 Kurz, C., 151, 213

l ( I i H l l O t R , ,I . , 115, 13!/ ~ ~ ~ l H ~ l l i O ~ , I). J., 4, 12, 14, 54

~ ~ i r n i u i o n o . M., 44, 58 Kur,ontnov, 8., 122, JJ!) KwiLpiwki, 6. l 3 . , 116, I;]!)

269

L hbach , J. P., 14, 55 I,tlbZOfff&y, N. A., 109, 140 Laaavo, C., 16, 26, 27, 40, 56, 57 Ladwiy, R., 223, 226, 226, 234, 237, 238,

247, 260, 251, 262, 258, 258 Lagoda, A. A., 176, 209 Lam, G. K. H., 160, 209 Lamblin, D., 220, 221, 236, 237, 242, 251,

Lands, W. E. M.. 30.55 Laneele, 0.. 16, 49, 55 Lane&, M. A., 16, 26, 40, 55, 56 L a g , D. R., 8.44, 55 Laraen, B., 179, 180, 198, 210, 211 Lascelles, J., 7, 55 Laurell, Q., 116, 126, 127, 138 Laurent, M., 223, 228, 261 Law, J. H., 2,Y, 7,26,27, 28,31,32, 51,52,

53, 54, 55, 57, 58

258, 259

hWHo11, c. J., 182, 171, 211 IAI ( .hJlf l t ( l , J . I t . , (14, 06, 67, tix, fir), 7(1, 88,

MI, $10, !J2, 05, $9 IAodoror, I<., 2M, 411, 5.3, $5

Lao, L. H., 116, 140 Leo, c. c., 96, 99

LOC, S. Is., 76, 101 LOOS, H., 39, 52 Le Gall, J., 79, 84, 100 Lohano, D. P., 40, 81 Leiberman, M. M., 202, 205, 211 Leloir, L. F., 185, PO9 Lomoigno, M., 19, 55 Lsnfant, M., 28, 53 Lonnarz, W. J., 1, 2,3,9, 18,21, 22, 23, 24,

25, 27,28,30, 31,32,33, 34,43,46,48, 51, 53, 55, 56, 57

LBpine, P., 220, 221, 236, 237, 242, 261,

L c H ~ o ~ . S. M., 169, 211 2.58, 259

h H t l X ’ , 1%. Id., 31, 32, 35, 36, $7, $8, 22M, 261

Lettern, R., 7, 31, 6.5 Jlwin, I<., 34, 54

270 AUTHOR INDEX

Levin, E. Y., 18, 55 Levy, R., 23, 25. 52 Lowis, E. E., 122, 1JO Lewis, V. J., 130, 139 ]AX, M., 68, 109 Liaaon-Jonson, 8.. 18, 55 Lidwoll, 0. M., 116, 139 Lie, It., 94, 98 Liebermann, M. M.. 203, 211 Liepkalns, V., 33, 53 Light, M., 126, 126. 139 Light, R., 22, 23, 24, 51 Light, R. J., 23, 215, 55 Lindborg, R., 16G, 167, 162, 171, 172, 188,

199, 209, 210 Linker, A., 169, 180, 211 Lindstrairi, E. S., 62, 101 Liu, 0. U., 108, 139 Liu, T. Y., 23, 24, 26, 53, 55, 183,

Livermoro, €3. I?., 46, 60, 53, 54 Lockhart, W. R., 123, 138 Lockwood, J. L., 242, 258, 259 Lofland, H. B., Jr., 7, 31, 51 Losada, M., 76, 98 Lovenberg, W., 73, 80, 83, 101, 103 Lowry, 0. H., 228, 259 Liideritz, O., 16, 17, 34, 55, 58, 146, 160,

Ludoweig, J., 169, 211 Lundgrun, D. G. , 8, 19, 55 , 56

Lyne, R. I%., 187, 211 Lynen, F., $0, 21, 30, 54, 55

21 0

1151, 152, 158, 166, 210,211, 21.3

I r l l N t , O., 7, 31, 56

M McCarnen, R . E., 30, 55 MoCarthy. K., 83, I01 McClelland, 116, 139 MoCormick, D. B., 84, 102 MacDougall, J. C., 9, 55, 56 McDowell, R. H., 166, 212 McElroy, W. D., 120, 199 MecFerlane, M. G., 1, 8, 18,43,44,46, 48,

MoKenna, C. E., 94, 98 McKenna, E. C., 86, 94, 102 McKinnoy, J. A., 49, 56 McLoary, C. W., 162, 171, 211 MacLeod, P., 27, 55 MacLeod, R. A., 126. 126, 139, 242,

McNeill, J. J., 39, 51 MeQuillen, K., 239, 242, 259 MacPherson, I. A., 176, 209 Macree, A., 34, 51

55

259

Mager, J., 132, 139 Mague, T., 96, 103 Mahl, M. C., 64, 102 Maier, S. M., 223, 234, 260 Majerus, P. W., 19, 20, 22, $1, 55, 66 Malkin, R., 80, 83, I02 Makita, M., 16, 56 Makula, R., 30, 31, 32, 43, 46, 56 Mamkaeve, K. A,, 218, 262, 258, 259 Mandel, M., 226, 227, 264, 267, 259,

Manigault, P., 39, 54 Mansberg, H. P., 116, 139 Marchessault, R. H., 19, 55 MarcuR, L.. 62, 67, I02 Mare, I. J., 160, 211 Marinetti, G . V., 8, 43, 58 Markovitz, A., 202, 203, 206, 211, 212 Markowitz, H., 170, 183, 211 Marmur, J., 227, 259 Marr, A. G., 7, 27, 54, 76, 99 Marshall, R. D., 162, 211 Martin, D. B., 21, 53 Martin, H. H., 239, 242, 259 Martin, 9. M., 27, 54 Massey, V., 73, 82, 84, 102 Massey, W., 84, 100 Matsubera, H., 82, 83, 98, 102 Matnuhashi, M., 192, 208 Matsumera, S., 20, 26, 61, 56 Matsuki, T., 90, 101 Matthews, L. W., 180, 209 Mavis, R. U., 29. 56 May, K. R., 108, 139 Mayhow, S. C., 73, 82, 84, 100, 102 Meadow, P. M., 3, 39, 53 Merek, E. L., 112, 139 Murriok, J. M., 18, 56 Metcalf, T. G., 108, lJ9 Moyer, F., 11, 46, 40, 56 Meyer, H., 11, 45, 49, 50 Meyur, K., 169, 211 Miles, A. A,, 162, 211 Miles, L. E. M., 116, 139 Milhaud, Q., 19, 55 Militzer, W. A., 44, 52 Millbank, J. W., 60, 62, 102 Miller, E. J., 126, 139 Mills, C. T., 160, 184, 186, 188, 190, 191,

206, 211, 212 Misaki, A., 166, 211. 212 Miahustin, E. N., 61, 102. 222, 259 Mitchell, R., 221, 242, 249, 264, 269, 260 Mitruko, 8. M., 113, 130, 136, 139 Mitz, M. A., 106, 110, 112, 113, 114, 121,

Miyezaki, T., 184, 211 Mdckul, W., 101, IOf

260

122, 125, 131, 133, 139

AUTHOR INDEX 27 1

Mohanty, G. C., 118, 139 Molotkovsky, J. O., 9, 56 Moor, H., 226, 226, 259 Moore, W. E. C.. 113, 130, 139 Morgan, D. M., 132, 141 Morgan, W. T. J., 168, 211 Morman, M. R., 44, 56 Morris, J. A., 66, 69, 90, 92, 96, 101,

Morris, J. C., 221, 249, 260 Mortenson, L. E., 64, 66,66, 68, 69, 70, 71,

72, 74, 76, 80, 88. 90, 92, 94, 96, 96, 99, 101, 102, 104

Mortlock, R. P., 71, 102 Moss, C. W., 130, 131) Moultlor, J. W., 217, 260 Moustafa, E., 69, 90, 102 Movaca, It. Y.. 122, 141 Mowor, H. F., 64, OH, 70, 71, 88. 9.9 Much, A. M., 220, 234, 262, 260 Mudd, S. G., 16, 53 Mulvany, J. G., 108, 132, 139 Munson, T. O., 68, 71, 93, 99, 102 Murray, E. G. D.. 6, 36, 44, 51, 52 Murray, R. G. E., 222, 223, 229, 232, 234,

102

238, 259, 260

N Nagai, J., 23, 56 Nairn, R. C., 114, 116, 117, 139 Nakada, H. I., 162, 171, 211 Nakamura, K., 247, 261 Nakarnura, R. M., 117, 133, 138 Nakos, K., 90, 96, 102 Naritomi, L. S., 116, 138 Neilsen, A., 68, 102 Nelson, N., 86, 102 Nelson, R. A., 116, 121, 122, 139, 140 Nelson, 8. S., 106, 121, 140 Nosbitt, J. A. 111, 3, 32. 55, 56 Neubergsr, A., 32, 52,162, 211 Neufeld. E. F., 34, rj6 Neufeld, H. A., 118, 119, 140 Neurnan, J., 86. 102 Nowton, W. E., 93, 102 Niblack, J. F., 160, 209 Nichols, B. W., 7, 14, 39, 42, 58 Nielson, A., 68, 102 Niernann, C., 16, 53 Nikitina, E. S., 222, 259 Nirnich, W., 168, 173, 210 Nimmioh, W., 173, 211 Nishioka, K., 117, 140 Nison, E. H., 76. 99 Nixon, D. A., 161, 210 Nojirna, S., 40, 52, 54 Norris, A. T., 22, 23, 24, 26, 51, 56

Norton, W. T., 4, 56 Norval, M., 148, 149, 186, 188, 190, 193,

Nosoh, Y., 90, 101 Nowotny, A. H., 17, 56 Nozaki, M., 76, 98 Nurna, S., 22, 54 Nutrnan, P. 8.. 96, 102 Nyo, J. F., 7, 31, 32, 53, 67

194, 196, 197, 206, 211, 212

0 Ohno, K., 18, 52 Ohrloff, C., 74, 201, 203 O’Loary, W. M., 1, 17, 22, 23, 24, 26, 20,

O’Kanrj, D. J., 70, 104 Okunuki, K., 82, 204 Oloniacz, W. R., 106, 118, 119, 120, 140 Olson, A. C., 206, 211 Onn. T.. 166. 162. 171. 172. 188. 199. 210

27, 36, 42, 63, 56

op den Karnp, J. A. F:, 2,S, 9,43,44, 53, 56, 57

Oppenheim, J., 62, 67, 102 Orentas, D. G., 163, 164, 178, 212 Ormorod, J. G., 68,100 Ormerod, K. S., 68, 100 Orr, T. E., 160, 209 Orskov, F., 163, 168, 169, 210, 211 Orskov, I., 163, 168, 169, 210, 211 Osborn, M. J., 168, 192, 194, 211, 213 Otsuke, H., 16, 54 Otten, B. J., 47, 52 Overath, P., 30, 56 Oyama, V. I., 110, 140 Oyarne, Y. I., 112, 139

P I’eintur, T. J., 168, 211 l’alatlini, A. C., 18G, 209 Palarnota, B., 4, 12, 14, 84 r’alloroni, N. J., 151, 211 Palmor, G., 84, 102 Pangborn, M. C.. 49, 56 Panos, C., 4, 27, 28, 62 Pappenheimer, A. M., 168, 169, 209, 213 Pardoe, G. I., 169, 208 Parejko, R. A., 62, 89, 91, 96, 99, 102 Parikh, V. M., 179, 211 Park, B. H., 169, 160, 208, 211 Park, C. E., 7, 16, 37, 56 Parker, C. A., 60, 66, 100, 102, 220, 221,

Parshall, C. W., 66, 101 Patterson, P. H., 30, 31, 5G Pauli, G., 30, 56

261, 260

272 AUTHOR INDEX

Paulus, H., 31, 33, 34, 54, 5 6 Poarco,T. W., 106, 115, 128, 129, 133, 140 Pcarsn, A. C. E.. 116, 140 Pearson, H. W., 66, 68, 1 U 3 Poet, S., 167, 211, 212 Pock, H. D., Jr., 76, 102 Pelzer, H., 242, 261 Pongre, R . M., 68, 104 l’orcivnl, la:., l b 0 . 2 / 2 l’orfil’iw, I i . V., 218. 1’00 l’orkitis, 11. It., 281.2, 2M/ l’orry, M. P., 12, 14. 54 Potoring, I)., 84, 101‘ Potitproa, A., 251, 258 Poteold, H.. 216, 216, 215, 218, 221, 222,

PAster, R. M., 19, 56, 96, 101 Philipson, L., 108, 100, 140 I’hillip~, K. O., 61. 104 PhillipH, W. D., 83, 98 Phizsckorltty, 1’. *J. It., 9, 55, 56 Pioringer, R. A., 29, 31, 34, 54, 56 Pike, L., 161, 212 Pwie, N. W., 162, 211 Pisano,M. A., 106, 118, 119, 120, 140 Pital, A., 122, 140 Pittsley, J. E., 177, 178, 210, 211 Pieor, L., 29, 54 Ploem, J. S . , 115, 140 Porath, J., 109, 141 Postgate, J. R., 62, 66, 67, 69, 91, 98, 99,

Pottor, A . I,., 167, 190, 212

228, 232, 251, 254, 255, 261

100, 131, 132, 140, 261, 260

I’owflll, E. o., 100, 116, 128, 12!), 133.

i’oxtorl, 1. It., l!)5, 21:i I’rcttt, I ) . C., 70, / 0 2 Preston, d. V., 167, 2U!/ Pricor, W. E., Jr., 30, 54 Pringeheim, E. CJ., 67, 102 Promo, J. C., 15, 26, 40, 56 Proom, H., 130, 140 Prottey, C., 43, 49, 56 Pudlos, J., 48, 52

Putman, E. W., 166, 167, 190, 212

140

Pugh, E. L., 19, 857

Q Quesnel, L. B., 132, I40 Quirnhy, F. A., 112, 140 Quinnell, C. M., 179, 212

R Rabinowitz, J . C., 80, 81, 83, 101, 102 Radunz, A., 14, 42, 56

Randall, R. J., 228, 25.9 Itsndle, C. L., 5, 7, 8, 10, 11, 37, 38, 39,

40, 41, 56 Itno, C. V. N., 184, 212 Iteo, K. K., 83, 100 Reo, N. V., 109, 140 Rapp, F., 132, 140 I < i ~ p l i i ” t , M. M.. 4, 5f; I l I I H I I I I I ~ I V . A . ri., 123, 1 1 0 I h y , ‘1’. I(.. 21,. 6/i ltiwiri, S., 17, 57 Itihcrrn, 1’. A., 182, 183, 184, Z0!/, 21% ltixltbi, I., 8, M Hoodor, I). J . , 115, 127, /Xi RR(JR, D. A., 162, 171, 211 ROOVOS, R. I<. , 166, 181, 184, 208, 212 itctinor, A. M., 224, 238, 260 ltoinor, E., 110, 131, 140 It&, J., 32, 55

Richards, J. 13., 34, 53 Richards, 0. W., 123, 140 Richards, R. L., 93, 94, 99, 100 Riedoror-Hondcrson, ill. A., 61, 102 ltigopoulos, N., 77, 98 Ripko, R., 68, I02 ltippka, R., 68, 102 Itittenborg, 11.. 22, 57 Rittenhorg, R. C., 227, 240, 241, 2153, 260 Rizza, V., 14, 57 Robbinx, P. W., 187, 192, 194, 201, 209,

1lot)ortn. .J. R., 19, .i.l ItoltorLq, W. K., 184, PI2 RtjI)iiinoii, , I . , 223, 220, 232. 241, 243, 244,

Itiht)OtlH, 1). w., 19, 62

213

247, 248, 250, 252. 253, 254, 258, &.i!/, 260

l tot l i l lnrln, I t . , (i8, 100 K ~ ) t h i n H , 1’. W., 15, 34, $8 Ilodcm, L., 170, 212

Rogers, 13. J., 242, 260 Romeo, D., 192, 212 Roso, O., 17, 5 7

Itosoman, S., 34, 54 Roecn, S. M., 158, 211 Itosonfeld, M. H., 106, 118, 119, 120,

ItCJStXIhlLUm, N., 202, 206, 211 I ~ O H H , K. 1’. A,, 132, 137

Itothfioltl, L.. 158, 102, 211, 2 / 2 , Z/:j ltoy, H. E., 108, 137 Jtoy, N., 184, 212 ltupprccht, E., 74, 101, 1U.l HUHHO~, H., 82, 99 h ~ s e l , 8. A,. 82, 101

It~JfJ‘OrH, A. H., 167, 16fj, 208

Itosohrough, N. J., 228, 2.i!/

I40

ItlJqt, F. w. rj., 115, 140

AUTHOR INDEX 273

Russell, S., 64, 90, 95, 101 Russoll, S. A., 89, I04 Rutborg. L., 44, 5 1 Ryter, A., 39, 54

S Saito, H., 155, 211, 212 Saito, K., 18, 51 Salter, D. N., 128, 138 Saltmarnh-Androw, M., 192, 21.7 Selton, M. H. J., 242. 260 Samuel, D.. 30, 57 Samiiolwon, B., 44. 5 1 Sandborn, W., 131, 1.17 Sunclorn, J. IC., !#3, !/!I Suiidford. 1’. A., 157, 173, 175, 210,

212 SWl 1’10t 1’0, A.. 7(1, H(1, 101, /02, 10.Y Sunton MoLu, *J. M. I I I I H , 43, 57 Sapolli, 1%. V., 170, 218 Saetry, I’. S., 12, 54 Sattar, 9. A., 133, 140 Sauor, F., 19, 57 Scarborough, G . A., 32, 57 Schairor, H. U., 30, 56 Schacht, J., 101, 101 Schatzberg, G., 132, 139 Scher, M., 16, 34, 57 Scherff, R. H., 224, 229, 232, 236, 237,

Scheuorbrandt, G., 2, 23, 24, 25, 27, 28,

Schick, H. J., 68, 77, 7A, 102 Schildkraut, C. L., 227, 259 Schleoh, B. A., 38, 54 Schlesinger, G., 93, 103 Schluchterer, E., 167, 212 Schmidt, G., 169, 205, 210, 211 Schmidt, W. A. K., 108, l(J9, 140 Sohnaitman, C . , 19, 5.5 Sohnoider, K. C., 66, 101 Schneider, K. F., 163, 168, 210 Schneidor, P. W., 93, 1132 Schoonhoimor, R., 22, 87 Schollhorn, R., 6G, 10% Schrauzer, G. N., 93, 103 Schubert, K., 17, 57 Shieh, €1. S., 32, 57 Schwartz, G . , 81, 10.3 Schwinghamer, E. A., 97, 103 Scott, J. E., 150, 212 Scott, L. V., 116, 140 Scott, P. B., 66, 102 Segro, D., 116, 140 Seidter, R. J., 218, 220, 221, 222, 223, 226,

227, 228, 229, 237, 239, 242, 244, 246,

243, 260

51, 55, 57

248, 250, 251, 252, 253, 254, 255, 256, 257, 260, 261

Seligman, 8. J., 132, 140 Solinger, H. H., 120, 139 Somporo, J. M., 151, 812 Senti, F. R., 178, 210 Sepulveda, M. R., 115, 137 Sersichekes, H. R., 108, 139 Sevag, M. G., 149, 212 Shebarova, Z. A., 182, 183, 212 Shuparis, A., 203, 211 Shaw, M., 158, 212, 217, 260 Shaw, N., 13, 14, 43, 44, 46, 46, 49, 51, 57 Sheffiold, F. W., 116, 140 Ghonberg, E., 45, 67 Shophord, B. P., 128, 138 sll l lrr, s. I,, 32, 57 Shnt~hnr~, Y. I., 81. 82, 84, 10.3 shrill, c. w., 9B, ! / X Sliilo, M., 21X, 220. 221, 223, 224, 227,

241, 242, 244, 247, 24N, 240, 250, 259, 857, 260, 2Gl

22n, zu, 233, 234, 237, u n , 230, 240,

Shilov, A., 94, 103 Shilove, A., 94, 103 Shin, M., 86, 103 Shiomi, T., 246, 250, 201 Shivoly, J. M., 6, 7, 8, 26, 36, 37, 40, 41,

Shockman, G. D., 242, 260 Short, S. A., 7, 37, 57 Shrauzer, G. N.. 93,103 Shurcliff, W. A., 106, 121, 122,140 Shuval, H. I., 108, 109, 140 Shuvalov, N., 94, 103 Shuvalova, N., 94, 103 Gicklos, G . M., 158, 212 Siddiqui, I. R., 172, 208 Sirrrra, C. , 19, 57 Siuwort, G., 200, 212 Silhart, 1). b’., 25, 57 Silwr, W. S . , 00, 81, 62, !)(I, I 0 3 Silvr:ry, J. K. (j., 62, 88, I04 Silvi!Htcr, W. I $ . , 62, 103 Sirnrrwis , 1). A. H . , 151, 2 / 1 Sitrim, M. A., !M, 10.3 Simon, R. I)., 77, 104 Sirnpson, I?. J., 232, 252, 253, 254, 260 Sinha, D. B., 39, 67 Bider, F. D., 61, 103 Sisman, J. 236, 237, 261, 2Q8, 259 Skorman, V. B. D., 218, 201 Slonckcr, J. H., 153, 154, 178, 212 Smillic?, R. M., 79, H2, 84, 10.3 Smit, J. A., 160, 211 Smith, D. R., 62, 103 Smith, E. E. B., 150, 184, 185, 188, 190,

51, 54, 57

191, 206, 211, 212

274 AUTHOR INDEX

Smith, J. D., 7, 31, 57 Smith, J. E., 116, 140 Smith, N. R., 6, 51 Smith, P. F., 8, 14, 18, 33, 45, 54, 57 Smith, R. V., 04, 08, 77, 91, 96, 100, 103 Smith, W., 110, 140 Snyder, F., 33, 58 Sobel, B. E., 73, 103 Sojke, G. A., 30, 57 Somors, P. J., 181, 182, 184, 208, 209 Borgor, Q. J.. 90, 103 Bpoartt, U., 32, 57 Spencer, J. F. T., 166, 179, 210 Spendlove, R. S . , 116, 140 Btacoy, M., 146, 160, 169, 167, 172, 181,

182, 184, 208, 209, 210, 211, 212 SLanaaov, N. Z., 8, 31, 52, 57 Stanior, It. Y., 40, 49, 51, 57 Stenloy, J. P., 61, 99 Stark, 0. It., 148, 213 Btarr, M. P., 216, 216, 217, 218, 220, 221,

222, 223, 224, 226, 227, 228, 229, 231, 232, 233, 234, 230, 237, 238, 239, 242, 243, 244, 246, 240, 247, 248, 249. 260, 261, 262, 263, 264, 256, 266, 267, 259, 260, 261

Stead, D., 44, 57 Steed, P. D., 234, 260 Steiner, M. R., 31, 32, 57, 58 Steiner, S., 30, 67, 228, 261 Sterling, R., 82, 99 Stern, N., 46, 57 Stewart, W. D. P., 00, 61, 02, 06, 67, 08,

90, 100, 103 Still, J. L., 25, 27, 51 Stock, C. C., 117, 140 Stolp, H., 216, 216, 217, 218, 220, 221,

222, 228, 229, 232, 233, 234, 236, 237, 239, 242, 243, 244, 240, 247, 248, 260, 261, 262, 263, 264, 265, 261

Stolzenbach, F. E., 86, 101 Strandherg, C. W., 08, 103 Strange, R. E., 100, 110, 116, 121, 128,

Strehler, 13. L., 120, 140 Streichor, S., 90, 97, 103 Strominger, J. L., 16, 34, 53, 161, 192,

200, 208, 211, 212, 242, 258, 261 Struggcr, S., 132, 141 Sullivan, C. W., 219, 220, 251, 261 Sutherland, I. W., 163, 156, 157, 180, 101,

18% 163, 164, 107, 170, 171, 172, 173,

203, 206, 2I0, 211, 212, 213

129, 133, 140

174, 175, 176, 188, 190, 193, 196, 197,

Suzuki, I<., 83, 103 Svesson, S., 167, 209 Sweeley, C. C., 14, 16, 10, 34, 53, 57, $8 Szilagy, J. F., 7, 56

T Tachibene, T., 117, 140, 141 Tagewe, K., 73, 70, 82, 86, 98, 103, 104 Tageya, I., 117, 139 Tait, 0. H., 32, 52 Takeheshi, H., 3, 4, 6, 10, 11, 12, 22, 48,

Takenami, S . , 82, 104 Talamo, B., 19, 22, 34, 46, 51, 65 Talpaseyi, E. It. S., (17, 101 Tamrn, I., 132, 141 Taylor, 13. F., 90, 99 Taylor, C. E., 116, 1.39 Taylor, K. B., 90, 103 Telfor, A., 96, 103

Than-Tun, 76, 100

l‘hielo, 0. W., 7, 27, 38, 42, 67 Thomas, D. W., 49, 51 Thomas, P. J., 26, 27, 49, 57 Thompson, 0. A., Jr., 33, 57 Thorne, K. J. I., 16, 18, 34, 47, 57, 187,

188, 208, 213 Thornton, M. P., 33, 53 Tietz, A., 46, 57 Tietz, C. J., 123, 141 Tinelli, R., 223, 228, 261 Tipper, D. J., 242, 258 Tipton, C. L., 18, 52 Tiaelius, A., 109, I41 Tkeczyk, S., 132, 137 Tomasi, V., 2, 56 Tomlinson, A. H., 116, 141 Torrieni, A,, 168, 213 Totter, J. R., 120, 140 Touhiena, It., 28, 53 Tove, S. R., 02, 101 Towner, R. I)., 118, 119, 140 Trager, W., 217, 261 Trakatellifl, A. C., 81, 10.3 Traylor., T. O., 94, 98, I02

54

~ r o l n P ~ s t , I). w., 147, 209

Thever, R. K., 74, 101, 103

‘hjhHt, A., 84, 103 ‘L’rofirnnrrkofl, u., 08, 10J ‘hJy, E’. A,, 16, 34, 57, 170, 187, 191, 102,

193, 194, 21.3 Tuckar, A. N., 14, 15, 62, 68 Tully, J. a., 17, 67 Turner, 0. L., 06, 104 Tyler, J. M., 184. 210, 21.3

U Udenfriond, S., 7, 31, 62 Uometsu, T., 218, 223, 232, 238, 246, 248,

260, 261, 261

AUTHOB INDEX 276

Ulfeldt, M. V., 115, 138 Umbreit, W. W., 112, 138 Umetani, K., 8, 9, 44, 57 Urakami, C., 8, 9, 44, 57

v Vagdos, P. R., 19, 20, 21, 22, 28, 29, 30,

Vahlno, G., 168, 211 Valontine, R. C., 60, 66, 71, 73, 74, 77.

78, 79, 80, 81, 82, 84, 85, 86, 96, 97, 98,102, 103, 104, 132, 141

Van Deenen, L. L. M., 2, 8, 9, 32, 41, 43, 44, 46, 53, 55, 56, 57

Vandecmteele, J. P., 89, 90, 104” Van den Bosch, H., 29, 58 Van Tamelen, E. E., 93, 104 Varon, M., 221, 232, 233, 234, 238, 239,

Veech, R. L., 73, 104 Voerkamp, J. H., 9, 43, 47, 52 Vennesland, B., 159, 211 Verheij, H. M., 43, 45, 57 Vettor, H., Jr., 86, 101 Villanuova. J. lt., 242, 257 Volk, W. A., 162, 213 Von Bulow-KoMtor, J., 23, 51 Vorbock, M. L., 8, 43, 58 Vurok, G. G., 112, 137

51, 52, 53, 55, 56, 57, 58

240, 247, 248, 250, 261

W Wachtol, H., 17, 57 Wada, K., 82, 104 Wade, H. E., 110, 121, 132, 140, 141 Waechter, C. J., 31, 32, 58 Wagnor, H., 11, 23, 51 Wagner, H. N., 125,138 Wakil, S. J., 19, 22, 57, 58 Wakiruoto, S., 218, 223, 232, 238, 240, 248,

Wetby, J. K., 45, 60, 54 Wallenfols, K., 151, 213 Walsby, A .E., 67, 77, 100 Wang, J., 82, 100 Ward, M. A., 94, 104 Washington, T. A., 126, 141 Watanabo, A., 77, 104 Wetkinson, It. J . , 10, 58 Watson, P. H., 160, 162, 173, 177, 211 Watson, R. W., 112, 138 Wettenburg, W. H.. 112, 138 Webb, M. A,, 167, 208 Webley, D. M., 242, 259 Wegner, G. H., 3, 4, 12, 21, 58 Wehr. N. B., 254, 261

250, 261, 261

Woidd, W., 242, 261 Weinor, I., 192, 194, 213 Weiner, I. M., 194, 211 Weiss, S. B., 31, 54 Wells, M. A., 36, 58 Wermundsen, I. E., 202, 209 Westlake, D. W. S., 166, 210 Westphal, O., 16, 58, 150, 161, 168, 169,

210, 211, 213 Wostwood, J. C. N., 133, 140 Whatley, F. R.. 80, 100 Whatloy, R. F., 82, 104 Wheat, R., 146, 166, 211 Wheolock, E. F., 132, 141 Wheiner, J. R., 83, 98 White, D. C., 7, 11, 14, 16, 37, 38, 43, 44,

Whito, E. H., 118, 120, 126, 141 White, L. A., 115, 126, 139, 141 Whittenbury, R., 61, 104 Whittick, J. J., 122, 141 Wilkinson, J. F., 61, 104, 145, 146, 147,

148, 163, 160, 161, 162, 163, 167, 170, 171, 173, 174, 176, 188, 194, 203, 209,

Wilkititmn, S. G., 11, 13, 14, 40, 42, 53, 58 Williams, J. M., 182, 208 Williams, P. J. lo B., 7, 32, 36, 39, 53 Williamson, I. P., 22, 58 Willinson, J. F., 176, 208 Wilson, J. D., 7, 31, 52 Wilson, P. W., 61, 62, 64, 66, 07, 68, 72,

76, 81, 84, 89, 91, 95, 98, 99, 100, 101, 102, 103, 104, 179, 212

46, 54, 55, 56, 57, 58

210, 211, 212, 213

Winogradski, S., 61, 104 Wintor, F. H., 124, 141 Wintor, H. C., 69, 92, 93, 99, 104 Witz, D. F., 89, 96, 99 Wolfo, R. S., 70, 71, 102, 104 Wolk, C. P., 67, 77, 100, 104 Wong, P., 75, 82, 99 Wood, B. J. B., 7, 14, 39, 42, 58 Wood, 0. L., 260, 252, 261

Wood, R. M., 115, 138 Woodworth, H. C., 116, 240 Work, E., 242, 281 Wright, A., 16, 34, 58, 192, 194, 209, 213 Wyatt, J. T., (12, 68, 104

WYHH, O., 160, 100, 101, 20!/, 212

Wood, R., 33, ri8

Wyklo, I t . l.., 33, 58

Y Yadomae, T., 184, 211 Yamakawa, T., 46, 53 Yamanaka, T., 82, 104

276 AUTHOR INDEX

Yamonioto, Y., 77, 104 Yang, C. S., 83, 104 Yankofsky, S., 221, 249, 254, 260 Yano, I., 44, 58 Yatos, M. G., 67, 76, 81, 104 Yehle, C. O., 220, 261 Yeng, D. Y., 66, 90, 96 ,102 Yengoy'en, L. S., 12, 54 Yoch, 13. C., 06, 68, 73, 74, 77, 70, 81, 82,

84, 85, 98, 104 Yoho, C. W., 23, 24, 6.3 York, U. K., 124, 141

Yoshimure, T., 10, 55 Yu, P. I<. W., 125, 141 Yurowicx, 13. C., 170, 213

Z Zaforiou, O., 118, 141 Zsitsova, 0. N., 178, 213 Zalkin, H. , 7, 20, 32, 58 Z O C i l , H., 161, PO9 Zolozniok, L. l)., 168, 211 Zoboll, C. E., 61, 103

A At!ottrLo in I~iwhwiid iixiiI)i’IyHiti!i’Iiibriili~~,

A cetobaeter ucetigenum, extracellular poly-

A. zylknum, cellulose synthesis by cell-free

Acetobacters, cellulose excretion by, 166 Acetyl residues in bacterial exopoly-

Acetylated dehydrogenase in nitrogen

Acetylation of bacterial oxopolyHacehur-

Acetylene reduction, nitrogenaso assay by,

Acetylene, reduction of, by nitrogenase, 66 Acidic proteins, forredoxins and flavo-

Acidity of medium and bdellovibrio

Actinomycetes, nitrogen fixation by, 61 Action spectra of nitrogen fixation in

Activation of luminol by microbes in

Activo sites on tho nitrogenaso onxyme, 93 Acyl carrior protoin in filtty Wid Hynt,haHiH,

Acyl thio-esters in bacterial fatty-acid

Acylation of bacterial exopolysaccharides,

Adenosine diphosphate as an inhibitor of

Adenosine triphosphate, and nitrogen

153

saccharido production by, 147

extracts of, 189

saccharides, 163

fixation, 71

ides, 188

68

doxins as, 78

attachment, 233

Anabaeim cylindricu, 77

relation to detection, 118

10

synthesis, 26

188

nitrogen fixation, 69

fixation, 66 content of Aerobucler ckrogenes, 110 contents of microbes, 121

hyclrolysis by nitrogenase, 91 requirement of, in nitrpgen fjxat ion, 92 role of, in nitrogen fixation, 69

Adenosylmethionine as a methyl klonor in

Aeration, effect of, on bacterial exopoly-

Berobacter aerogenea, weight per cell

determination of, 120 1

phospholipid biosynthesis, 32

saccharide production, 148

relationship with, 110

SUBJECT INDEX

277

A. clonrae, nttuchnient of hdellovibrios to, 234

iroli i i i ic! wiil ~ w o i l u c c i i l I,y, 170 Atrrol~ic: iiilmgiin Hxors, 67 Ago, efloct of, on lipid composition of

of colls, offoct of, on bdellovibrio growth,

Agglutination of erythrocytes, me of, to

Agricultural importance of nitrogen Axa-

Agrobactoria, phoqholipid composition of,

hactoria, 40

250

detect viruses, 116

tion, 69

7 A grobaclerium spp., extracellular glucan

Agrobaclerium tumefaciena, fatty acids in, production by, 166

3 growth of bdellovibrios on, 248 phosphatidylethanolamine in, 8 phospholipid composition of, 41

Alanylphosphatidylglycerol in bacteria, 9 Alanylphosphatidylglycerol synthetase in

Alcaligenes faecalia, exopolysctccharide of,

Aldehydes, long chain, in bacterial lipids, 4 A Idobiuronic ircitl in oxor’olyxaccharides of

bacteria. 32

168

tinctciria, 176 AlgUl HtWl)lH, 18 Aiyinasos of bacteria, I80 Alginic acid, 179 Alkalino phosphatase activity of host call-

bdelk~vibrio cultures, 241 Alk-1 -enyl cthcrs, in bacterin, 3, 28

Alk-1 -my1 glycerides in hc twia , 10 Alkyl ethw lipids in tmntctiin. I2 Alkyl ethars in bacturial lipiilw, 4 Amino-acyl I’hosphatidylglycorc~ in hao-

Aminophthalate, production of, from

Amino sugars, in hacterial exopolymchar-

in hactcrial lipids, 2

teria, 8

luminol, 119

ides, 152 in hdollovihrios, 228

Ammonia, &tl f t fiupprcHHor of nitrogonaso

aa the first product of nitrogen fixation, synthesis, 00

64

278 SUBJEUT INDEX

Ammonia-continued

populations, 112 detection of, in cstimating microbial

formation, sasay of nitrogenase by, 66 inhibition of nitrogen fixa.tion by, 64 inhibitory effect of, on nitrogen fixation,

68 Anabaena cylindricn, nicotinamido nucloo-

tides as electxon donors in nitrogon fixation by, 76

nitrogon fixation by, 64, 77 nitrogenase in, 68 pathway of nitrogm fixation in, 96 properties of electron carriers in, 79

Anaerobes, plasmalogens in, 10 Anaerobic bacteria, plmmalogens in, 3 Anaerobic nitrogen fixation, 6 1 Anaerobic pathway of fatty-acid synthesis

Analysis of bactorial growth products,

Anteiao fatty acids in bactorial lipids, 2 Antibacterial globulins, iodinated, use of,

in estimation of microbial populations, 128

Antibodies, immune-purifiod, use of, in est*imating microbes, 128

Antibody method for detecting bacterial capsules, 146

Antibody technique, radioactive, for estimating microbes, 127

Antigens, formylated, of bacteria, 162 use of, in detecting microbes, 114

Apolar components of hacerial lipids, 2 Apolar lipids, bacterial, biosynthssis of,

Appendagw on hdellovibrios, 223 Arthrobac$er RPP., glycolipids in, 13 Arthrobacter vd8coaus, exopolysaccharido

Asparagine, respiration of, by bdellovibrios,

Assessment of sparso microbial popula-

Athiorhodaceae, lipids of, 38 Atmosphere, monitoring of, for small con-

centration of microbes, 106 Atmospheric nitrogen, fixation of, 59 Attachment of bdellovibrios, 229,232 Automated detectors for stained microbes,

Azide, reduction of, by nitrogenme, 66 Azocoll, hydrolysis of, by bdellovibrio

Azomonaa spp., nitrogen fixation by, 62 Azotobacter agile, exopolysaccharido from,

A. ugdlia, fatty acids in, 3

in bacteria, 23

130

19

from, 177

262

tions, 106

121

protease, 244

178

A. chroococcum, oxopolyHaccharidc from, 178

nitrogen fixation in, 87 sterols in, 17

ferredoxin, properties of, 81 Azotobacter, coupling factors from, 86

Azotobarter indicn, oxopolysaccharido from,

Azotobncter ~ p p . , purt8ioulnto nitrogon 179

fixation syHt,om in, (17 po~y-~-hydroxyt~iityrati~ in, 19 production of enzymos by, that hydro-

lyse bactcrial exopolysaccharides, 161 UEO of, in preserving bdcllovibrios, 220

Azotobacter winelandid, axotoflavin from, 84 clectron carrier from, 73 oxopolysaccharido from, 178 nitrogen fixation by, 61, 64 pathway of nitrogen fixation in, 96 propertios of electron carriers in, 79 properties of nitrogonascs from, 88 role of nicotinamide nucleotidos in

separation of nitrogenase proteins from, nitrogen fixation by, 74

a9 Azotobacteriaceae, lipids of, 38 Azotobacters, parasitizetion of, by bdello-

Azotoflevin, nature of, 84 vibrios, 261

structure of, 87

B Beoillacem, lipid composition of, 44,47 Bacilli, endoRporo formation in, and lipid

production of oxtmoohlar polysacchar-

Buci l lu~ cereun, chromntopnpny of [ml-

11. circulana, production of cnzyrnos hy, that hydrolyso bacterial exopolysaccharidos, 168

composition, 42

idos by, 167

dUCtH Of, 130

B. maceruna, nitrogen fixation by, 62 U . meguterium, phompholipid composition

of, 41 B. pah.atYi8, production of onzymem by,

that hydrolyse bacterial exopolysaccharides, 168

B. polynayxu, nicotinarnide nucleotidos &H electron donom in nitrogen fixation by, 76

nitrogen fixation by, 62 pathway of nitrogen fixation in, 96 proporties of cloctron carriers in, 79

Bucdllua spp., nitroRon fixation hv, 61 poly-8-hydroxybutyrato in, I <

SUBJECT INDEX 279

Bacillua atearothermophdua, detection of,

B. subtdia, growth of bdellovibrios on, 248

Bacteria, immunological properties of, in

methylation of phosphatidylethanol-

nitrogen fixing, 80 terminal steps in synthesis of unsatu-

virulence and extracellular polysacchar-

by chemiluminescence, 118

lipids in, 11


amine in, 32

rated fatty acids in, 24

ides, 144 Bactorial acyl carrier proteins, 20 Bacteriul collulose, structure of, 168 Bactmial onxymes, wo of, in cstimating

microbial populations, 11 3 Barterinl exopolysacchrtridos, 143

hiosynthosis of, 186 coll-frw synthoRiR of, IHB protlriction of, 146 structures of, 186

Bactorial ferredoxin, proporties of, 80 Bacterial glycolipids, formulae of, 13 Bacterial growth products, analysis of, 130 Bacterial heteropolysaccharida, structures

Bacterial hosts of bdellovibrios, 228 Bacterial lipid compositions, 35 Bacterial lipids, biosynthesis of, 10

comparative aspects of, 1 Bacterial neutral lipids, 17 Bacterial phosphoglyceddes, formulaa of,

6 Bacterial spores, methods for rapid awss-

ment of sparse populations of, 109 Bacteriolytic attack, mechanism of, by

bdellovibrios, 242 Bacteriolytic nature of bdellovibrios, 228 Bacteriophage, w a y of, 133 Bacteriophages, for bdellovibrios, 256

of, 167

that induce synthesis of enzymes that hydrolyse bacterial exopolywcharides, 181

Bacteroid ferrcdoxin, 76 Bacteroidee melaninogend~ua, sphingo-

B . ruminiwla, plasmalogens in, 11 B . euccinogenea, plasmalogene in, 11 Bacteroids, leghasmoglobin in, 87

Bactoprenol. in exopolysacrharido bio-

lipids in, 14

nitrogen fixation by, 80

synthesis by bacteria, 187 nature of, 34

Ballooning of host cells in bdellovibrio

Base composition of bdellovibrio deoxy- invaaion, 239

ribonucleic acid, 228

Bdellovibrio bacteriophages, 268 Bdellovibrio b a c t e h w , lytic action of,

Bd. bacterwvorua, 215,221 Bdellovibrio cultures, maintenance of, 219 Bdellovibrio cyste, 226 Bdellouibrio atartii, 221

Ed. atolpii, 221 Bdellovibrios, chemical composition of, 226

217

plaque formation by, 216

composition of, 222 cultivation of, 218 distribution of, 218 encystment of, 228 faculttLtively parasitio growth of, 238 growth of, in host cells, 237 host specifloity of, 249 importance of, in nature, 220 inolation of, 218 lipase of, 246 long forms of, 237 marine, 249 mcchaniems of lysis of host cells by, 242 metabolism of, 262 muramidase of, 243 nutrition of, 248 physiology of, 216 protease-negative mutents of, 246 protease of, 244 rate of respiration of, 240 structure of, 222 taxonomy of, 221 terminology of, 22 1 vitamin requirements of, 247

Beijerinckio indica, exopolysaccharide

h’eijerinckia spp., nitrogen fixation by, 61 Bifiobacterium bi;fld.um, lipid composition

Binary capsulation in diplococci, 201 Biochemistry of nitrogen fixation, 64 Biological control of microflora in nature,

221 BiomaRR in mothocle for rapid w m m e n t of

sparso microbial popiilatiollcr, I 10 Diosonmr, me of. indobctircg microhen, 1 22 Biosynthosi8, of bactorial cxopolysacoher-

from, 179

of, 43

idee, 185 of bacterial lipids, 19 of capsular polysaccharide of Klebaieila

of colanic acid, I70 of cyclopropane fatty acids in bactaria,

of ferredoxins, 81 of plaemalogens in bacteria, 28

aerogenes, pathway for, 186

28

Biotin, involvement in fatty acid synthesis in bacteria, 24

280 SUBJEOT INDEX

Blue-groen algae, oloctron donors from, during nitrogen fixation, 70

nitrogen fixing, 60 I3ranched-chain acids as primors in bao-

torial fatty-acid biosynt,hoais, 21 Branrhed fatty acids in bacterial lipids, 2 Broad -spectrum methods for dot,octing

Broken cell prcparations, nit,rogon fixation

J3romac~il. o f h i 4 of. 1111 l ~ ~ l ~ l l ~ ~ v i l ~ r i ~ i ~ , 254 13rurelltc , i id i t ,c i i&, lipidn of, 26 Urucellrc spp., phosphatidylethanolamiiio

Brucellaceae, fatty acids in, 3

Bubble formation in bdellovibrio invasion,

microbes, 107

IJy, 60

in, 6

lipids of, 38

239

C Calcium, effect of, nn bacterial

sarrharitlo production, 148

C,'(iridida albicrwia, dotechtion of, hy

C h ~ ) i I I t ~ r v 1 ubc sranner, natitro of, 1

effect Of, 011 hthllIJVibI'iCJf3, 249

Iunrinoaroi~ce, 119 . ..

Capuular polysaccharido of Klebsiello aerogenes, pathway for biosynthesis of, 186

C a p d a r polysaccharidos, of bacteria, 143 of Escherichia coli, 169 of pneumococci, 160

Capsule w welling techniquo, 146 Carbamate, effoct of, nn bdottovibrios, 254 Carbohydrato coritont of Aerobacter aero-

Carbon dioxide, detection of, in estimating

Carbon monoxide and nitrogen fixation, 66 Carbon :nitrogen ratio in medium, effect

of, on bacterial oxopolysaccharido production, 147

Carboxyl reduction of bacterial oxopolysaccharides, 157

Cardiolipin, bioflynthesis of, 28 composition of bacteria, 46 formula of, 0 in hactoria, 8

genes, 110

microbial populations, 112

Carotenoids in bactoria, 17 Catalaso in bdellovibrios, 253 Cationfi, effect of, on hdellovibrioa, 248 Casoin, hydrolysia of, by hdollovihrio

Cell-free extracts, nitrogen fixation by, 64 Cell-free synthesis of bacterial exopoly-

proteaso, 244

saccharides, 188

Colt momhrunos, offovts o f bdollovibrios on,

Collulase, u80 of, in tlotormining oligo-

Crlliilose, excretion by aroLohiictor~, 106

24 1

saooharitlo structiuo, 104

synthesis by bacteria, 147 synthesis of bactorial, in cell-frou sys-

tems, 189 ('011 voluunea, est,imation of, 124 ("011 walls, and t ~ ~ l ~ ~ l l o v i l i r i ~ ~ ~ionotration,

230 host, lysis of, by bddlovibrios, 243 of bdellovibrios, chemical composition

Centrifugation in methods for rapid

Coramido phosphorylethanolamine in bac-

Cetyl alcohol, biosynthoais of, in bacteria,

Chain elongation in hactorial fatty acid

Chain length of fatty acids in hactoriul

Chain lengths of bachoriul fatty ucidu, 20 Chomical compoaition of kJtlollOVibril)H,

222,220 Chtrrnical composition of microbes in ro-

tation of rapid asscnsment methods, 107

Chomilumin~~sconco of lurninol, use of, in microbial detection, 118

C'hornotaxis of bdollovibrios, 232 Chomotypes of baoteria and hdellovibrio

of, 228

assessment of microbes, 108

teria, 14

33

biosynthosis, 22

lipids, 2

attachmont, 233

vibrio penotration, 23fl

262

Chloramphenicol, CffeCt of, IJn bdljtllJ-

Ohlorella wu!gnria, offoct of bdollovihrios 011,

Chromatium sp., lipids Of, 36 Chromatiurn spp., propertiea of oloctron

Chromatogribphic propc:rticin of pyruvy- carriers in, 79

lated HUgal'H, 154 CtlrlJlnctt(J~)h(JrOU, llitrCJgf>n fixatilt11 Iiy, 77 Chrotnobaclerium uiolrrceum, oxtrw*c:lli~ler

Clostridia, fatty acids in, 3 polysacoharide ~ ~ r ~ d u c t i i ~ t ~ tJy, 147

ph~t~pholipid composit,iori r t f ; 8 , 48 ~~ho~phoroclastic,trl~c~astio rwtcliori in, 7 I ~ J l U H I ~ t l l ~ J @ J t l H in, 11

(hstriclial forrricloxin, priqmrf,itiH Of, 81) (:loHtriclial fltt~ocl(~~iii, 8:i fjlO6lridiUm bulyricum, tJilJHyIlth!HiH Of

pla~malogons in, 33 fatty acids in, 3 pleamalogon in, 4 unsaturated fatty acids in, 28

SUBJECT INDEX 281

Cl. kluyveri, adenosine triphosphate pro-

CI. panteurianum, elect,ron donors in duction in, 74

nitrogen fixation by, 70 fiavodoxin from, 83 nitrogen Axation by, 61, 64, 08 nitrogenase, properties of, 88 pathway of nitrogen fixation in, 96 propertics of electron carriers in, 79 use of adonosine triphosphate in nitrogen

Coenayme A ostors of fatty acids aa acyl

Colanic acid, biosynthesis, 242

fixation by, 69

donors, 30

hydrolysis of, 162 in bacterial exopolysacoharidt:~, 166 nature of, 167 structure of, 162, 160

Collagen, hydrolysis o'f, by hdollovibrio

Collision of bdellovibrios and hont cells,

Commenaale of green algae, 218 Compact bodies and bdellovibrios, 224 Comparative aapects of bacterial lipids, 1 Complex lipids, bacterial, biosynthesis of,

proteaao, 246

228

28 of Gram-negative bacteria, 36 of Gram-positive bacteria, 42

Components of exopolyaaccharides of

Composition of bacterial exopolysacchar- pneumococci, 184

ides, 160 and medium composition, 148

Composition of bdellovibrios, 222 Composition of colanic acid, 171 Composition of medium, effect of, on lysie

of host cells by bdellovibrios, 248 Composition of microbes in relation to

rapid assessment of sparae populations, 109

Compositions, lipid, of bacteria, 36 Concentration. effect of, in bdellovibrio

attachment, 233 Conditional mutants in bacterial exo-

polysaccharide bioaynthesis, 206 Continuous culture of nitrogen-fixing

bacteria, 68 Control of exopolysaccharide biosynthesis

in bacteria, 201 Corynebacteria, lipid composition of, 48 Corynebacteriaceae, lipid composition of,

Cwynebaclerium diphtheriae, lipid compo- 44

sition of, 48 neutral lipi& in, 18

vibrios in, 248 Coynebact. michagenense, growth of bdello-

Coynebact. sepedonicum, lipids in, 11 Corynomycolic acid in bacteria 48 Coulter counter, use of, in estimation of

epclrse microbial populations, 124 Coupling ftbctorR cif r l zutohc./er, 85 Coverslip monolayura, uso of, in estimating

Cboss reacting of nitrogenaees, 91 C substance from pneurnococci, 160 Cultivation of bdellovibrios, 218 Culture media for bdellovibrios, 2 I 8 Cyanide, &B an electron acceptor with

viruses, 133

nitrogenase, 91 inhibition ofnitrogen fixation by, 64 roduction of. by nitrogenase, 88

Cyclopropane alcohols in bacteria, 27 Cyclopropane aldehydes in bacteria, 27 Cyclopropane fatty acids, in baateria, 20,

42 in bactorial lipids, 2 in plants, 2

Cyclopropane synthetaae in bacteria, 27 Cysteine residues of ferredoxins, 83 Cysts on bdellovibrios, 226 Cytidine diphosphate diglyceride in bac-

Cytochromes of bdellovibrios, 263 Cytophaga, effect, of bdellovibrios on, 262

teria, 10

D Davis strain of bdellovibrio, 239 Decarboxylation of phosphatidylserine in

Decay, phosphorescent, urn of, in detection

Dohydrogonases, in nitroyon fixation, 70

Deoxyribonucleic acid content, of Aero-

bacteria, 30

of bacteria, 131

of bdellovibrios, 254

bacter aerogenea, 110 of bdellovibrios, 228 of microbes in rapid arrsensment methods,

Depolymeraaes of bacterial exopolyaac-

Deproteinization of bacterial exopoly-

Derivatives, host-independent, of bdello.

Desaturation of fatty acida in hactoria, 23 I)osiccation, uxopolynaccharitl~tn antl pro-

Desulfovibrio yiyaa, proportioR of' oloctron

I)eedfovUrio RIJP., flavodoxinn from, 84

109

charides, 181

saccharides, 149

vibrios, 264

taction againet, 2fJO

aarriors in, 79

plaamalogene in, 11

282 SUBJECT KNDEX

Deevlfowibrio vulgaris, nitrogen fixation by,

Determination of adenosine triphosphate,

Dextrans, bacterial, 188 biosynthesis of, in cell-free ByBtOmB, 189

Diacyl phosphoglycerides of bacteria, 6 Diauxio growth in nitrogen-fixing organ-

Dictyobacter spp., host-parasite relation-

Dideoxy sugars in bacterial exopoly-

Diether analogue of phosphatidylglycerol

Diether ylycnlipid in Hdobucteriurn culi-

t)i~lyc!tmyl tliglyccrriclos in huutmirr, I 3

81

120

isms, 88

ships with, 218

saccharides, ,160, 182

phosphate, formula of, 6

ruhrurn, 14

J)ihytlI’fJ]Jhyt,yl UklJhlll il l t IUl l J ]~ t l i l i l ! filLl!-

turie, 6 Dihydrostorcitlic acid in baclcria, 3,27 Diimide, inability to dcmonstrate a8 an

Dimannophosphoinositides in bacteria, 49 Dimer nature of nitrogenaae, 88 Diphosphatidylglycerol, formula of, 8

Diplococcua pneumoniae, capsular poly-

cell-free synthcsis of polysaccharidcs of,

exopolywcharides of, 180 extracellular polysaccharides of, 160 polysaccharides of, 144

intermediate in nitrogen fixation, 93

in bacteria, 8

saccharides of, 188

190

Disruption of host cells by bdellovibrios,

Distribution of bdollovibrios, 2 18

Distribution of phosphatidylcholino in

Dithionite, and nitrogen fixation, 85 in cell-free nitrogen fixation, 88 reduction of, by forredoxins, 80

238

in nature, 220

bacteria, 40

Divalent cations, offoct of, on tJdullovitJricJH,

Division of bdollovibrios in hotit C ~ H , 237 Douglaa fir, nitrogen fixation by, 81 Dyes, UBB of, to stain and detect microbes,

248

121

E Ecological importance of nitrogen fixation,

Ectoparesitic naturc of bdellovibrios, 228 Effect of p H value on lipid composition of

69

bwteria, 48

Effects of bdellovibrios on host cells, 239

Efficiency of plaque formation by bdelio- vibrios, 260

Electron activation, adenosine triphospha- tase aa in nitrogen fixation, 92

Electron carriers, in nitrogen fixation, 78 Electron donors, in nitrogen fixation, 70

need for, in nitrogen fixation, 89 nicotinamide nuoleotides as in nitrogen

fixation, 78 Electron mioroscopy of bdellovibrios, 223 Electronic particle counters, me of, in

estimation of microbial populations, 112

I~lectrciphorcei8 in rapid asvcsemcirit of viriw ~iiq)iiIt~tit~ns, 108

1Ei i c~c i~ )~~ t l i r t i i ) i i itricl titlollovitwio uttaah- I I I ~ I I ~ , 234

I4~lI~!yHtUlWlt Of bdOkJVibl*ilJS, 226 1Sntl producls of metaholiHrn, detoction of,

in e8timating microbial populations, 113

Endophytes, nitrogon fixation by, 60 Endospore formation in bacilli, and lipid

Energy generation by bdellovibrios, 253 Enterobacteria, fatty aoids in, 3 Entcrobactericeae, cyclopropane fatty

composition, 42

acids in, 27 lipids of, 38 production of colanic acid by, 189

Entrance of bdellovibrio into host cells.

Environment and nitrogen fixation, 97 Enzymes, and bdellovibrio penetration,

238 involved in biosynthosis of bacterial

exopolysaccharidos, 185 lytic, of bdellovibrios, 243 that hydrolyeo bacterial oxopr~lysacchar-

Enzymic acttivitios 0 1 niicrohos iri nhtiorl

lhzymic tligimtion of protoin from biictorial

Enzymic hydrolysis of bacterial OXfJ[>OlY-

Epimeraaos in biosynthesis of bacterial

Envinia arnylwwo, and bdellovibrios, 229

h’ac?ke&hiu coli, attachment of bdollo-

217,229

ides, 158

h J PUjJid UHHlJHHIlll~llt III6tLtllJdH, 107

cxopolys~~charidos, 160

eaccharides, 168

exopolysencharides, 188

attachment of bdellovibrios to, 234

vibrios to, 233 bioBynthcHiH of phoHphdipicla in, 29 chromatography of prlJdUCt8 Of, 130 extracellular hetrtropolysaccharides Of,

187

SUBJEUT INDEX z m

Eechstdchia. coli-continued oxtracellular polyaaccharide produclion

fatty acid synthesis in, 24 fatty acids in, 3 lipids in, 11

by, 147

Esoheriohieae, lipids of, 38 Esterases, use of, in estimating microbial

populations, 114 Ester.containing lipids of halophilic bac-

teria, 4 Ethanolamine phospholipids in Backroidea

auccinigenea, 12 Etha~iolaminepla9melogen in bacteria, 3,12 Eubacteriales, lipid composition of, 43

lipids of, 38 phosphatidylethanolamine in, 0

Eukaryotic micro-organisms, inability of,

Eukaryotic organisms, lipids of, 1 Excretion of bacterial polyssccharides,

Exogenous fatty acids, use of, by bacteria,

Exopolysaccharides. bacterial, 143

to fix nitrogen, 60

146

30

biosynthesie of, 185 cell-free synthosis of, 188 structures of, 106 functions of, 206

reaction, 92 Exothermic nature of the nitrogen fixation

Extractable polar lipids, in bacteria, 12

Extraterrestrial life, detection of, 112 Extreme halophiies, lipids of, 4 Extrusions on bdellovibrios, 223

of baoteria, 6

F Facultatively parasitic growth of bdello-

Fatty mid deaatureges in bacteria, 23 Fatty acide, bacterial, biosynthesis of, 19

vibrios, 238

in bacterial lipids, 2 of Gram-negative bacteria, 42

Fatty aldehydes in bacterial lipids, 4 Feedback inhibition of nitrogen fixation in

Ferredoxin, in cell-free nitrogen fixation, 66 bacteria, 69

in nitrogen fixation, 66. 78 photoohemical roduction of, 76 reduction of, by nicotinamide nucleo-

tides, 14 Ferredoxins, properties of, 80

reaction of, with nitrogeneses, 87

Forrous ions, effoct of, on bactorial poly-

l'ortility, soil, and bacterial exopoly-

Fibres on bdellovibrios. 224 Filter-immunofluorescence methods for

Firefiy luminescent method for determin-

Fiacherelb spp., micrograph of, 63 Fixation of nitrogen, pathways of, 68 Flagella of bdellovibrios, 222 Flagellation in bdellovibrios, 232 Flavodoxin, and nitrogen fixation, 66

Flavodoxina, properties of, 83 Plavoproteins aa electron carriers in nitro-

Fluorescin isothiocynate method for de-

Formate, 88 a reducing agent with nitro-

dehydrogenaee activities in bacteria, 72 Formyl residues in bacterial exopoly-

Formylated antigens of bacteria, 162 Porrnylation of bmterial exopolymchar-

Froezirig of bdellovibrios, 220 Fucoaamine in bacterial exopolyaacchar-

Fucose epimeraae, use of, in sugar analysis,

Fucose in KlebeaeEla exopolysaccharides,

Fuoosidases, me of, in determining oligo-

Fucosidic linkages in bacterial exopoly-

Functiona of exopolysaccharidcs, 206 Fungal sterols, 18 Fungi, phosphatidylcholine in, 7

naccharide production, 148

aaccheridea, 144

estimating microbes, 126

ing adenosine triphosphate, 120

in nitrogen fixation, 78

gen fixation, 83

tecting microbes, 122

genaae, 12

saccharides, 162

ides, 188

ides, 182

161

173

aaccharide structure, 164

saccharidoe, 158

G Galactosamine in bacterial exopolyseccher.

Galactose dehydrogenase, use of, in sugar

Galactose, in bacterial oxopolysecchwidee,

in Klebeielh oxopolysaccharides, 173 oxidme, use of, in sugar analysis, 161

/3-Galactosih activity of host cell-

fl-Galactosidam synthesis by bdellovibrios,

ides, 162

analysis, 16 1

160

bdellovibrio cultures, 241

240

2 84 SUBJECT INDEX

Galart~osyldiglyrericles in troponcmos, 60 Galac.trironic acid in KleOsielln rxopdy-

CUR chromatfogrrtl)hy of bacLorial products,

Gas chromatography, llRC of, t o detect

Gelatin, hydrolysis of, by brlellovihrio

Qonotics of nitrogen fixation, 90 Glucan of Hhizobiurn jnponicum, wll-frno

Olucans proiluccd extrac!ellirlarly I)y hric-

(:lucos~rnino. in 1~itcI.crial c ~ x o i ~ o l y x i ~ c ~ c ~ l i i ~ ~ -

saccharides, 173

130

viruses, 136

proteaso, 244

Synt,haHiH 190

torick, 1 (It!

idos, 162 in I)tlollovibrios, 228

(: luc.ositniiiiidas(Is, IWO of, i i i tlotmmini ng

Qliicofluminyl derivatives of phosptlatidyl-

( l luco~e, in bact.orial exol~cJlysaco~iaritli.n,

in Klebsiella exoI)(Jlyflnachw.ides, 173 oxitlaso, use of, in Hugar analysis, I B I

GhcosidaHos, use of, in dotci*mining ohgo-

Glucuroriic acid in Klebsiella cxopoly-

Glutamine, respiration of, by bdellovibrios,

Glycerol in bacterial exopolysaccharides,

Glycerol 3-phosphate in bacterial phospho-

Glycolipids, biosynthosis of, in bacteria, 34

Glycosyl carrior lipids, riatmu 01; I92 Clyco~yl wrriirrs in tJactc!rial oxoliolg-

G I ~ c o H ~ I dorivativas ~ l ' r~~)lyis[)r)r'rri~iI~ i t 1

Glycosyl diglycoridas in hnctrv-ia, 12, 42 Clycosyl donors in bactcriul OXOiJCJl?.-

saccharide hiosynthcsis, 1x9 Glyoxylate cycle in brlellovihrios, 254 Gradient centrifugation in rapid asmss-

Gram-negativo hactoris,, cyclopropanc

l)lig(JHu~!Chnl'idO Htl.UCt~llI'OH, 1 84

glycerol in bacteria, 9, 4H

160

saccharide structuro, I04

saccharides, 173

263

I60

glyceride synthesis, 28

in bacteria, 12

HaC<!haI'itf(J hiOSyllLhoHiH, I 88

bacteria, 16

ment of niicrobial populthons, 109

fatty acids in, 27 fatty acids of, 42 lipid A in, 17 lipids of, 36 miscollanoous exopolgaaccharides from,

phosphatidylot hanolamine in, 6 struct,ures of c~xopolyHacchltritlcs p r ~ -

177

duoed by, 187

(>ram-positivo hcter ia , 1)dcrllovibrios and. 261

c:oinplex lipids of, 42 cyclopropanc fatty acids in, 27 exopolysaccharides of, 180

Green algae, commonsals of, 218 Growing cultures, production of bacterial

exopolysaccharides in, 146 Growth characteristics of microbes in

rolation to rapid asscwsment, niothotls, 107

Growth, kinotics of, in host-tltrpondont bdullovit~rir~~, 24t1

UHO of, to rwtiiniLtc rriiorobiul ~iopulations, 126

(if'li\Vtsh Vf I)ddl<JVihriOH i f 1 h(JHt (!fllh, 237 (2rowt.h phaw, offriot, of, i ) n tJmtorial

oxopolynucc:liuri~~~ product.ion, 1 47 oI'fwt of, on bactoriul lipid composition,

40 Growth products, bacterial, analysis of, I30 Ciillivcr, naturo of, 112, 126 Culuronic acid, biosynthosis of, 198

H Haernatin content of micro-organisms, 1 1 1 Hnernophilus ,in$wewzue, estimation of, 127 Haemophilua s p p , phosphatidylethanol-

Halobacterium cutirubrurn. lipids in, 12

Halophilic bactcria, lipids of, 4 Hont-killed cells, for growth of bdello-

amine in, 0

lipids of, 6

vibrios, 247 of host, growth of btfwllovibrios on, 247

ficrhicidos, aativit,y against htlollovihrios,

ifi:tfwO(!yHtH, ntul tiiim)Kiiii fixtrt i o r i i t 1 t d w -

rdi ; IJI ' , i i i riitri)prri fixittior1 i r i bliiii.

HotoroiJoly~ceharido Hylithf:HiH IJY cbc: l l -

~loteropolysaccharitlos, Lacterial, 1 G5 bacterial oxtracullular, 146 microbial, coll-frce synthosis (If, 190

264

Kl'f!t:fi Ul&Mr, fiz

grocti alyiu,, 07

fWC HYHt(rITI8, 188

Hoxokinase in biosyrlthoHiH Of hctoriltl

Hoxoscs in bmtr>rial oxo~~~~lys~rcc:h~rrirlo~,

Higher organisms, lipids Of, 2 Holdfast on bdellovibrios, 224 T3omoIogy of nitrogenascs, $1 1 Homopol ysaccharides, bactotial, 1 05

exopolysacchurides, IXIi

I GO

oxtracelliilnr, 147 structures of, 188

biosynt,hefiie of, in cell-freu HystemR, 189

SUBJEOT INDEX 285

Horse serum, use of, in preserving Bdello-

Host cell membrane, effect of bdellovibrios

Host cell wall, disintegration of, by

Host cells, disruption of, by bdellovibrios,

vibrio cultures, 220

on, 241

bdellovibrios, 243

238 offi,cts of l~dollovibrios tin, 230 growth of liclollovibrios in, 237 niochanisms of lysis by bdellovibrios, 242 rionetration of hdellovibrios into, 234

Host dopendont bdellovibrios, and sym - biosis, 228

life cyalo of, 229 Hont-depondent growth of bdcillovibrios,

kinotics of, 24(i ,HoHt,-indopontfent bdellovibrio popula-

tions, 217 Host-independent derivatives of bdello-

vibrios, 264 Host range of bacteriophages that induce

synthesis of enzymes that hydrolyse bacterial exopolysaccharides, 161

Host specificity groups of bdellovibrios, 260 Host specificity of bdellovibrios, 249 Hydrated electrons, adenosine triphos-

phate &B in nitrogen fixation, 02 Hydrazino, inability to demonstrate, as an

intermodieto in nitrogen fixntion, 93 Hyclrocarbon composition of bacbrwia, 46 Hydrocarbons in micro-organisrnn, 18 1-1 y tlrogon, nntl nitmigcin fixation, 76

concont,rat.ion of mudiiim, effect of, on lipid cornrionition of bactorio, 4ti

c:volution, nitrogonuno assay by. 66 inhibition of nitrogon fixation I J ~ , fi4 metabolism and nitrogen fixation, 64 sulphide, dotection of, in estimating

Hydrogenase, relation of, to nitrogenm,

Hydrolasee, use of, in studying oligo-

Hydrophilic nature of baqteriai capsules,

8-Hydroxydecanoic acid in bacterial fatty-

8-Hydroxydecanoic acids in bacteria, 3 8-Hydroxy fatty acids in bacterih, 3 8-Hydroxylauric acid in bacteria, 3 8-Hydroxymyristic aci4 in bactoria, 3 Hyphomicrobiales, lipids of, 36, 37 Hyphomicrobium, effect of bdellovibrios

Hyphomicrobium sp., phospholipids of, 32 Hyphomicrobium d g a r e , phospholipid


76

saccharide structure, 166

144

acid synthesis, 28

on, 262

composition of, 7

I Jriirnuiie adhorcnco madion, IlRtUlrI Of, I 18 Immuno-adhoronco, aasay, I33

iwo of, in estimating viruses, 138 Immunofluorescence methods for estima-

ting microbial growth, 126 Imrnunofluoroscont mothod for deter-

mining virusos, 132 Irnmiiiiological acbivition o f micrtihos in

rolation to rapid u8~ossmont, 107 Immuncilogical dotoction of viruses, 116 immunological proporties, of bacteria in

of microbes in relation to detection, 114 lmmuno-purified antibodies, w e of, in

Impcntanco of bdellovibrios in nature, 220 India ink muthod for visualizing bacterial

capsules, 146 Indirect methods for rapid determination

of microbial viability, 131 Induction of synthesis of enzymes t,hst

hydrolyse bacterial exopo~ysacchar-

relation to dotoction, 114

estimating microbes, 128

ides, 158 Infection cushion on bdellovibrios, 224 Inhibition of nitrogen fixation by hydro-

gen, 76 Inorganic phosphate, hydrolysis of, by

nitrogtmase, 65 Inorganic substituents in bect,erial exo-

polyaaccharicles. 168 Int,ra~!ytiipluRmic mombranen of hsctoria.

and phoephohpid composition, 39 Iritra-intcgiimental parmito, Bdollovi brio

as, 220 I ntramival ptjrasito, 13dollovitwio m, 228 lotlinetion of antibwtarial globulins, 128 lodino, radioective, WM of, ir i estimating

radio-imtopes of, we of, in ostirnating microhos, 127

microbial populations, 116 I Oris and bdellovibrio attachmentn, 233 Iron content of ferredoxins, 80 Iron-protein naturo of nitrogenem, 90 Iron, requirement for, in nitrogen fixation.

180 fatty acids in bacterial lipids. 2 Isolation, of bacterial exopolysclccharides,

64

149 of hdellovihrios, 218 of host-indopcndent derivetivos o f

bdellovibrios, 266 of nitrogenases, 88

synthesis, 21

lipids, 192

Isoleucine in bacterial fatty-acid bio-

Isoprenoid aloohols, &B glycosyl carrier

286 SUBJECT INDEX

Isoprenoid alcohols--continued in biosynthesis of bacterinl exopoly-

Tsovaleric acid in bacterial fatt,y-acid saccharidns, 187

biosynthesis, 21

J Jerusalcm strain of hdellovibrio, 239

K Kotals attached to torminal reducing

gcllucto8o residues of colania acids, 172 a-Kntohutyrato as a substrate wlth

nitrogenme, 72 Killed host cells, growth of bdellovibrios in,

247 Kinetics of extracollular polysaccharide

produot,ion by bacteria, 148 Kineties of host-dependent growth of

bdcllovibrios, 246 Klebaiella aerogenea, capsular anatomy of,

145 oell-free synthesis of heteropolysacchar-

ide of, 190 mutants deficient in exopolysaccharide

biosynthesis, 196 pathway of biosynthcsis of capsular

polyseccharido of, 186 production of enzymes by, that hydro-

l y ~ e bactcrial oxopolysaccharides, 169 time-course of polysaccharide produc-

tion by, 149 Klebsielb ozaenae, oxopolysaccharide~ of,

174 Klebaiella phages, substrates for, 163 Klebaiella pneumoniae, and formato &R an

electron donor in nitrogen fixation by, 72

fnrredoxin frorn, 82 iiitrogen fixation by, 64 pathway of nitrogen fixation in, 95

Klc baiella polysaccharides, monosacchai*-

Klebaiella rhinoacleromatia, exopoly.

Klebaiella spp., extracollular polysacchar-

ides in, 173

saccharides of, 174

ides of, 17 1 nitrogen fixation by, 61

L Labile sulphide in nitrogcnasc, 89 Lactam, use of, in determining oligo-

saccharide structure, 164 Lactic acid bacteria, complex lipids of, 42

Lmtobacillaceas, lipid composition of, 43 Lactobacilli, fatty acids in, 3

glycolipirle in, 14 polyisoprenoid alcohol in, 16

formula of, 2

by, 187

vibrio aultures, 240

Lactobacillic acid, biosynthesis of, 26

Lactobacillw c a d , bactoprenol production

Lactose respiration by host cell-bdello-

Leakage of purines by bacteria, 132 Locithin, formula of, 6 Loghaomogtobin, function of, in bacteroids,

Logumow, nitrogcn fixation by, 60 Leguminow plants, nitrogen fixing, 60 Leptoapira canicola, lipid composition of,

Leuconoaloc rneaenteroklea, polysaccharide

Levans, bacterial, 160

Life cycle, of bdellovibrioe, 222

Light scattering methods in eatimationa of microbial populations, 112

Limitations, nutrient, effect of, on bacterial exopolyaaccharide production, 147

Linkages in bacterial exopo~yeaccharides, 166

Linuran, effect of, on bdellovibrioe, 254 Lipme of bdellovibrios, 246 Lipid A in bacteria, 16 Lipid compositions of bacteria, 36 Lipid content of Aerobacter aerogenea, 110 Lipid intermediates in exopolysenoharide

Lipid-linked intermediates in exopoly-

Lipids, bacterial, comparativn cucpocts of, I complex, of Gram-positivo bacteria, 42 of bdcllovibrios, 227 of Gram-negative bacteria, 35

A7

46

production by, 166

biosynthesis of, in cell-free systems, 189

of host-dependcnt bdellovibrios, 229

biosynthesis, 189

sartcharidcw nynthcnis, 101

Lipo-amino acids in bacteria, 8 Lipopotyeaccharides, biosynthesis of, in

extracelluhr, of Gram-nogative badaria,

lipid intermediates in biosynthosie of, I02 of bacteria, 143

Lister& monocytogenea, lipid Composition

Long-chain bases in bacterial lipids, 14 Long forms of bdellovibrioe, 237 Low redox potential of ferredoxim and

Luciferase, me of, in determining d e n o -

bacteria, 15

I67

of, 44

Aavodoxina, 78

sine triphosphate, 120

SUBJECT INDEX 287

Luciferin, use of. in determining adenosine

Lucigenin, activation of, by micro-organ-

LuminoI chemiluminemnce in microbial

Lyophilization of Bdellovibrio cultures, 220 Lysine, in bacterial lipids, 9 Lysine-containing lipid in bacteria, 16 Lysis, from without by bdellovibrios, 238

mechanisms of, by bdellovibrios, 242 of bacteria, 242 of host cells by bdellovibrios, 240

triphosphate, 120

isms, 120

detection, 118

Lysophosphatides in bacteria, 10 Lyeophosphatidic acid in biosynthesis of

bacterial phosphoglycorides, 29 Lysylphosphatidylglycerol, biofiynthesis

of, in bacteria, 33 Lysyl-t-RNA ribonuclcic acid in bactorial

lipid biosynthesis, 32 Lytic: action of I I d d M i o bac~r iovor117~,

217 Lytic enzyrnosof bdollovibrios, 243

M Magnesium, effect of, on bdellovibrioe, 249 Maintenance of Bdellovibrio cultures, 2 19 Malaria protozoa, 217 Malonyl-coenzyme A in fatty-acid bio-

synthesis, 19 Mannan, biosynthesis of, in Micrococcua

lydeikticuu, 16 Mannolipid biosynthesis, 192 Mannoaamine in bacterial exopolysacchar-

Mannoae, in bacterial exopolysaccharides,

in Klebsiella exopolysaccharides, 173 ieomereee, UBB of, in sugar analysis, 161

Mannosidaeea, use of, in determining

Mannosidic linkages in bacterial exopoly-

Mannosyl diglycerides in bacteria, 13 Mannuronic acid, biosynthesis of, 188

Marine bdellovibrios, 249 Mess of microbes in relation to rapid

Mechanism of action of nitrogenaaes, 91 Mechanisms of lyeis of host cells by bdello-

Media for culturing bdellovibrioe, 218 Membrane Altere, use of, in staining

ides. 162

150

oligoaaccharide structures, 164

naccharicles, 168

in bacterial exopolynaccharidee, 180

eeaesament methods, 107

vibrios, 242

methods for detecting microbes, 122

Membrane filtration in methods for rapid

Membranes, cell, effect of bdellovibrios on,

Memsomes of bdellovibrios, 224 Metabolic activity. effect of, on bdello-

Metabolism, and microbial growth, 11 1

aaaeesment of miorobes. 108

241

vibrio growth, 260

microbial. UBB of, to estimate microbial populations, 126

of bdellovibrios, 262 Methane-oxidizing bacteria, nitrogen fixa-

Methane production in estimation of

Methylated sugars in bactorial exopoly-

Mnthylation, of bactorial exopolysacchar-

of phosphatidylethanolaniirio, by IJW-

tion by, 61


saccharides, 160

idos, 166

toria, 6 i n bacteria, 31

biosynthesis, 21 2-Methylbutyrab in bactorial fatty-acid

Methyloctadecaaphinganine in bacteria, 15 Methylsulphymyl carbanion aa a methyl-

Methyluronio acids in bacterial exopoly-

Methyl viologen, and nitrogen fixation, 66 reduction of, by nitrogen-fixing bacteria,

Microbacterium lacticum, glycolipida in, 13 M . thermoephactum, lipid composition of,

Microbes, immunological properties of, in

Microbial concentration, increase in awexi-

Microbial growth and metabolism, 11 1 Microbial microculture methods, 132 Microbial populations, sparse, rapid detec-

Microbial viability, rapid doteaticin (Jf,

Microcapsular polyll~~charides of bactoria,

Micrococcaceae, complex lipida of, 42

ating agent, 167

saccharides, 162

71

49


ments, 106

tion of, 105

131

143

fatty acids in, 3 lipid composition of, 43 phosphatidylinositol in, 9

Micrococcus cerificuns, anomaloua lipid

M . lyeadeikticua, biosynthesia of gtyco- composition of, 46

lipids in, 34 biosynthesis of mannan in, 16 detection of, by chemiluminwence, 118 glycolipida in, 13

288 SUBJECT INDEX

Microculture methods, URO of, in estimating

Micro-organisms, nitrogon fixing, 60 Minimum love1 of dotect.ion u i th niicrol)cs,

Minor phospholipids in bacteria, 9 Miscellaneous bacttrrinl oxo~~~r lyxnc t~ l i i~ r~

Node of bactmiolyt ic nthwlc by l i d i r l l i i .

Model for oxopolysiic!ctiari(i~~ biosynt h(wiH,

Model syst.onis of nitrogonase, 93 Molecular hydrogen, ILH an oloctreii t1i)noc

in nilrogori fixation, 76 (lotaction of, in oet,imat iriK microliial

pol)ulations, 11 2

mierobon, 132

106

i t l tss, 177

vibrioe, 242

200

Molnciilur nitrogon, fixation of, (i0 kloloc:ul~~r~ o x y p n , involvomeiit (if, in

bioayri thesis of bactminl unsaturat id fatt,y acids, 23

Moloculnr woight of nitrogonaso, 90 Rlolocular woights, of bacterial exopoly-

sawharitlos, 166 of ferredoxins and flavodoxins, 79

Molybdonum in nitrogenaso, 89 Molybdenum-iron nature of nitrogonase, 90 Molybdenum, requirement for, in nit r c g o n

fixation, 64 role of, in nitrogon fixation, 93

Mono-unsaturatod fatty acids, biosynthosis of, in hacteriu, 22

in bavtoria.1 lipitle, 2 Morphulogy of Iidollovihrios. 222 Motilit#y of bdollovihrioa, 232 Rlultiple attachrnonts of btlollovihrios,

Miiltiplicatiori of hdollovibrios, 229 Multiplicity of infection with bdollovibriiJs.

Murainic acid in hdollovihrioH, 228 Muinmidaso of btft~llovibrios, 243 Mutants, and nitrogeii fixation, 96

232

233

uf Klebsielln ueroyenes deficiorit in <!xu- polysaccharide hiosynthesin, 196

protease-nogativo, of hdollovibrioa, 245 !tIycotJncteria, host,-parasite rclationnhipn

with, 217 lipid8 of, 14, 49 ornithinc-contaiiiirrg lipid in, 15

2l.lycobucteriurn bovis, lipids of, 26 Ililycobacterium spj)., nitrogon fixation by,

Mycolic acids in bacteria, 48 hlyc!opltLsnins, ~ O H ~ - I ) U ~ U X I L O rclationshi[is

(i 2

with, 21 7 sttrrols in, 17

Myc:oeicli.n i r i tm(!twin, 14

N Nature, distribution of bdollovibrios in, 220 h'eisserin catarrhalis, clotaction of, by

Neurosporn sp., methylation of phospha-

Noiilml lipid r v 1 I iiiiwi 1, iot 1 of Strrriti (I hrl ( ' rr ,

Neutral IiFids o f biioturin, 17 Niootinamido nuclootitlc?s, and nitrogon

rhomiluminoscence, 119

t,idylothaiioluiiiitio in, 3 1

40

fixation, 65 i n nitrogen fixation, 73 (if' bdollovibrios, 263

Nit,robactorillcouo, lipids of, 36 Nitrocytitia oceanua, phospholipid ctornp~i-

Nitroyon fixation, I)ioohwnirrt,ry of, 64 nition of, 7

genetics of, 96 in Azotobmter, pathway of, 87 mechanism of, 91 mutants and, 96 pathways of, 59

Nitrogen-fixing micro-organisms, 60 Nitrogen incorporation, nitrogonase assay

Nitrogeneso, assay of, 06 inducible noture of, 68 nature of, 59 properties of, 66, 88 purification of, 88

Nitrogonasos, cross roucting, 91 rnochaniem of uction of, 1)1

Nitrogenous ccinipoiiritlrs, effect of do- ficioncy of, on bacturial exopoly- saccharitlo production, 147

Nitrous oxitlo. inhibition of nitrognn fixation hy, (14

by, 66

roduction fJf, by riitrrigma~o, AB

Noii-oxtractahlo IiIkls in bwtwiu, I fi Non-haern iron in iiitrogontmo, 89 Non-inuuoid nrut,arrt~~, ~f titr(:h!riu. 144

Non-sugar cornpanutits in tJactr!rinl vxo-

Son-synchronous growth of hdcllovitirioic.

Sucleic acid content of micro-organisms,

Nucloic acids, of bdollovibrios, 226

S O I l - ~ ! ~ ~ ) H U l t t t C rlllltiIJl~H of tJUCh!I'lU, I4(1

of Klebaiello ueroyenes, 206

polyaaccharidecl, 162

246

111

removal from hactorial exopolysaccharides, 149

NucleotidoH, in bdollovibrios, 226 sugar, in IJiOHynthosiH of hactcrinl exn.

Niiljriwil~H, oflijut of, on hni:t,oriitl c1x~q~oly- r)olysecctiaritlos, 186

HJLD(:hlLridlJ pr<J'luc:t,ion, 147

SUBJECT INDEX 289

Nutrition of bdellovibrios. 246 Nutritional requirements of host-indepen-

dent B d e l l d r i o , 247

0 0-Amino acyl phosphatidylglycerols, bio-

synthesis of, in bacteria, 32 Octadecaaphirrganino in bacteria, 15 Octnsacchtrritlo attachod to glycosyl carrior

Oligmacchuritlo structuro, determination

Organic acids in bacterial exopolyeacchar-

Ornithine amides in bacteria, 16 Ornithino in baotorial lipids, 9 Oxygen, effect of; on bacterial oxopoly-

lipid, 194

of, 184

ides, 162

saccharide production, 148 inhibition of nitrogen fixation by, 84 inhibitory effect of, on nitrogon fixation,

partial pressure, effect of, on bdello-

sensitivity of nitrogenaaes, 67 tension of medium and bdellovibrio

88

vibrios, 262

attachment, 233

P Palmitaldehyde, biosynthesis of, in bac-

Pulmitoleic acid, occurrence of, in bacteria,

l’uranitic hilollovihrios, 216 l’urtiul arid hydrolysis of bwtorial OXO-

~~olysaccharitlos, 157 l’artichrome inwtrumont, URB of, 121 Particle counter, Royce, 124 Paateurella huemolyticn, estimation of, 127 Pathways of nitrogen fixation, 69

Peas, nitrogon fixation by, 80 Penetration of bdellovihriow into host colls,

Pentoses in bacterial exopolysaccharidoe,

Peptidoglycan, and bdellovibrio penotra-

teria, 33

2

in micro-organiems, 96

234

160

tion, 238 bioaynthesis of, in bscterie, 18 lysis of, by bdellovibrioe, 243 softening of, in bacteriolysis, 242

Peptone aa a growth substrate for bdello-

Peptoatreptococcw eladenii, flavodoxin from vibrios, 247

84 plasmalogens in, 11

Periodate oxidation of bacterial exopoly-

Permeability of hoet cells to eolutee, effeat

Phage T7, composition of, 11 1 I’hagos, ostimetion of, 134

saccharides, 167

of bdellovibrios on, 240

production of enzymes by, that hydrolyse bacterial exopolysaacharides,

l’hugocytosis, protection again&, exo-

I’hir~n-contrast microscopy of bdellovibrio

I’honoxyacetic acid herbicides, effoct of, on

Phosphataee, u80 of, in estimating

Phosphatases of bdellovibrios, 228 Phosphate, in bacterial exopolynacchar-

labollod, estimation of, in assay of

169

polysaccharidos in, 206

invasion. 231

hdollovibrios, 264

microbial populations, 11 3

ides, 166

miorobial growth, 126 Phosphatidic acid, formula of, 6

in bdellovibrios, 227 in biosynthesis of bacterial phospho-

Phosphatidylcholine, biosyntheeis of, in glycerides, 29

bacteria, 31 formula of, 6 in bacteria, 7

Phosphatidylethanolamine, biosynthesie of, 28

formula of, 6 in bacilli, 47 in bacteria, 5 in hdollovihrion, 227

formula of, 8 i n hacteria, 7 in hclollovihrios, 227

I’ho~phatitlylalycurol, hiosyntheeis of, 28

1’hosphatidylglycorophoHphate in bacteria,

Phosphatidylinositol, hioflynthesh of, 33 10,30

formula of, 8 in bacteria, 9

Phosphatidylw,rino, hiosyrithowin of, in bacteria, 29

in bactcria, 10 Phosphoenolpyruvate BR a pyruvete group

donor in biosynthcsis of bacterial exopolysaccharides, 188

Phoephoglucomutam in biosynthesis of bacterial exopolysaccherides, 186

Phosphoglycerides, biosynthesis of, 28

Phospholipid compositions of bacterial

Phospholipids of bdellovibrios, 227

polar, of bacteria, 6

changes in, 41

290 SUBJECT INDEX

4-Phosphopantetheine &B a prosthetic

Phosphorescent decay, use of, in detection

Phosphoroclastio reaotion, as the driving

group, 19

of bacteria, 131

force in nitrogen fixation, 71 in bacteria. 70 in nitrogen fixation in clostridia, 71

Phosphorus compounds, offeot of doflcioncy of, on production of bacterial exopolysaccharides, 147

Photochemical reduction of forredoxin, 76 Photosynthotic bacteria, electron donors

during ni trogon fixation by, 76 glycolipids in, 14 nitrogen fixing, 6 1 phospholipid composition of, 7

Photosynthetic olectron donors in nitrogon fixation, 76

Physical attachment of exopolysaccharidos to bacteria, 149

Physical methods for determination of sparse microbial populations, 124

Physical properties of microbes in relation to rapid assessment methods, 108

Physiology of tho bdellovibrios, 216 Phytoflavin, nature of, 84 Plant ferredoxins, properties of, 82 Plaque formation, by Bdellovibrw atarrii,

216 by bdellovibrios, 260

Plasmalogens, biosynthesis of, in bacteria, 33

in bact,eria, 3, 10 Pnoumococcal capsule dopolymerasus, 162 Pneumococcal exopolysaccharides, hy.

I’nnumococci, components of cxapoly- drolysis of, 160

saccharides of, 184 phtrspholipid composition of, 8

~neumococcus spp., biosynthesis of glycn-

Pncumosamine in exopolysaccharides of

Polyfruotoses produced extracellularly h y

Poly-p-hydroxybutyric acid in bacteria,

Polyisoprenoids, biosynthesis of, in bac-

Polyisopronols, glycosyl derivatives of, in

Polymerase8 in biosynthesis of bacterial

Polysaccharide biosynthesis in diplococci,

Polysacoharides, extracellular, bacterial,

lipids in, 34

pneumococci, 182

bacteria, 167

18

teria, 34

bacteria, 16

exopolysaccharides, 186

202

143

Polyunsaturated fatty acids in bacterial

Ponds, bdellovibrios in, 220 Pores and bdellovibrio penetration, 236 Potassium, effect of, on bacterial exo-

polysaccharide production, 148 limitation, effect of, on produotion of

baoterial oxopolysaocharides, 147 Prooursors involved in biosynthesis of

bacterial oxopolysnccharides, 186 Predatory bactmia, 21 8 Principles of rapid microbial sssessment

methods, 107 Production of bactorial oxopolyeaccher-

ides, 146 Progeny, rolemo of bdollovibrio, 238 Prokaryotic micro-organisms, nitrogen

Prokaryotio organisms, lipids of, 1 Properties of bacterial exopolymcharides,

Properties of electron carriers in nitrogen

Properties of nitrogenmas, 88 Propionibacteria, lipid composition of, 48

Propionibaoteriaceae, lipid composition

Prosthetic groups of ferredoxins, 82 Protease, lipids of, 38 Protease-negative mutants of bdello.

vibrios, 246 Pratease of bdellovibrios, 244 Protection of respiratory systems in

Protein content of Aerobncter aerogenes, 110 I’rotoin content of bdoilovibrios, 227 Protoin, ruloaso of; fnlk~wing host, coll

I’rotoinH, immune adhorence roaction with,

Proteua rettgeri, estimation of, 127 P. vulyaria, chromatography of products

lipids, 2

fixing, 60

149

fixation, 79

plasmalogens in, 11

of, 43

nitrogen fixers, 67

cornhination with bdol~ovibrios, 241

116

from, 130 phospholipid cornposition of, 10, 4 I

l’roton source, adenosine triphospheto an,

Protozoa, methylation of phosphatidyl-

I’scudomonadacoae, lipids fJf, 37 PselLdomOIIndfib8, lipirh of, 36 Pseudomow aemginoaa, exopolysecchar-

in nitrogon fixation, 92

ethenolemine in, 31

ido from, 180 growth of bdellovibrios in, 238

p8. diminuta, glycolipids in, 14 Pa. fluoreacena, detection of, by chemi-

luminescence, 119 growth of bdellovibrios in, 248

SUBJEOT INDEX 291

Pa. ~ ~ - c & i n u d

Pa. p t i d a , atteck of, by Bdeuovibrio

P8. rubeacenu, glycolipids in, 14 PeeudOrnon&e spp., exopolyeeocharides

lipids in, 10

atarrii, 2 1 0

from, 180 glycolipids in, 13

Psi ttacosis virua, dotoction of, 1 17 Peyrhtriu bacteriophila, and nitrogen

PuriAcation, of bacterial exopolyrurochar- fixation, 68

idea, 148 of nitrogoneso, 88

Purinoe, leakago of, hy kactoria, 132 ' Piirornycin, uffcct of, on bdollovitwio

Purplo photosynthetic bacteria, ferre-

Pyrograms, uao of, in microbial dotection,

Pyrolysis methods for detecting bacteria,

Pyrolysis products of bacteria, and estima-

Pyruvate, as a substrate with nitrogensee,

aa an electron donor in nitrogen fixation.

in bacterial exopolyeeccharides, 153

ponetration, 280

doxins from, 82

131

131

tion 110

72

64, 89, 70

Pyruvyl residuca in bacterial exopoly-

Pyruvylated sugars, properties of, 164,166 Pyruvylfucom in bacterial oxopolyaacchar-

Pyruvylgalactoae rceiduos in bacterial exo-

mcharides, 163

idea, 164

polyeeccharides, 163

Q Quatornary ammonium salts, uao of, to

purify bacterial exopolymccharidoa, 160

Quellung reaction, 146 Quinone coenzymes in bacteria, 18

R Radioaotive antibody technique for esti-

Radioactive gases. estimation of, to essay

Radioaaaay of microbial populations, 129 Radioisotopes of iodine. use of. in eatima-

mating microbes, 127


Rapid broad-spectrum methods in mi-

Rapid deteotion, of sparse miorobiel crobial deteotion, 118

populations, 106 of virueee, 132

Rapid determination of microbial viebility,

Rapid speoiflc identification methods for

Rate of respiration of bdellovibrios, 240 Recognition of host by bdellovibrios, 232 Recognition of prey by bdellovibrios, 228 Recovery of bdellovibrios, 220 ltodox dyos, reduction of, by bacteria. 132 Rodox properties of flavodoxina, 84 ltedox protoina in nitrogen fixation, 88 ltaducing clectrona in nitrogen flxation,

Rcducing sito in blue-green algal hetero-

Reducing potential of f e d o x i n s , 82 Reductant, need for, in nitrogen fixation,

Reduction of atmospheric nitrogen, 60 Reduction of nitrogen- by nicotinemide

Reduction of redox dyes by bacteria, 132 Refractive index, changes of, due to

Regulation of nitrogen fixation in bacteria,

Releese of bdellovibrio progeny, 238 Release of compounds following bdello-

Repeating units in pneumococcal exo.

Ronpiration of bdellovibrios, 262 Jlospiration rate of host-k~dullovibrio BRRO-

hI5ting bodion in bdOllfJVitJri~JbJ, 226

131

microbos, 120

00

oysta, 68

69

nucleotides, 73

microbial activity, 132

09

vibrio invasion, 241

polysaccharidos, 182

ciatione, 240

Rhamnofm, in capsular polynacaharirhu of

in Klobniolla oxopolysaccharides, I73 isomorme, UBB of, in sugar analysin, 15 1

Rhizobia, parenitization of, by hdello-

Rhizobiaceaa, lipids of, 38 Rhizobium ferredoxin, propertiea of, 82 Rhizobium japonicum, cell-free synthesis of

nicotinamide nuoleotidm in nitrogen

nitrogen fixation by. 80, 84 pathway of nitrogen fixation in, 96 properties of eleptron carriers in, 79

Hacherichio mli, 169

vibrior, 261

glucan by, 190

fixation by, 75

R. meliloti, extracellular polyeecoharide nroduction bv. 147

ting mhobial populations; 115 Hhoiopaeudorno&ds, lipids of, 38

292 SUBJECT INDEX

Rhodopaeudomonaa epheroidea, ornithine- containing lipid in, 15

Rhodoepirillurn rubrum, effect of bdellovibrio invasion on motility of, 239

infection of, by bdellovibrios, 225 lipids in, 1 1 nitrogen fixation by, 61, 64 riitrogon fixation in, 68 pathway of nit,rognri fixation in, 95

Rhodonpirillum spp., lipids of, 3B Itibitd phoNpha1,o in pnoumococ:cal nxo-

I t i l ~ o n ~ i ~ ~ l i ~ i ~ ~ aoitl contont of Aero6arlr:r IJi)lyMni:c!hariil(iH, 1 82

trrrogrnrx, I 10 11 ItJoHltlll* IiI\ktigfjH 111 I)lLC~~lrial

Itibosomox of bdollovihrios, 224 ICickelhrciti, immuno adhoronco

Hflcl*ha~lt~Ofl, 1 68

W l l h , 1 I ( % RlVfWH, bddlCJVlbrlOH In, 220 Root nodules, nitrogen fixing, 60 Royce ~)srtirle counter, 124 Rumen barterla, branched fatty

21

oxrq~oly-

rcac tJ i on

acids in

Runiinococczsa flavefaciens, plasmalogens in, 1 1

S Sarcharom~ces cerevCaine, detection of, by

growth of I~tlallovil~rios in coll-fro0

Strlmonelln newington, polyisopronoicls of,

A'. lyphiniurzum. attachmorit of h d d h -

f;almonclleao, lipitlrj of, 38 Saprophytic bdellovihrio pnpulationH, 2 17 Surcina lulea, dotaction of, by chomi-

chem~lum~~~csrorico, I 19

OXbl'tM!t,H CJf. !?:I8

187

VihriOH to, 233

IumincHcenco, 1 1 f l neiitrcl.1 lipid compoeitiori iif, 46

h'fkrcina Hpp., phospholipid compi)Hition I J ~ .

Saturated fatty acids in bacteria, 3, 42 Seawater, bdellovibrios in, 220 Setemononas ruminantiurn, plasmalogens

Scmliki forest virun, composition of, 11 1 Separation of nitrogenase proteins from

Sequences in bact(eria1 exopol ysaccharides,

Serotypes, of Eacherirhia coli, 167

Serratia marceacene. biosvntliesiH of C V C ~ O -

8

in, 11

Azotobacter vinelnndii, 80

156

of pneumococci, 181

Serratin marcement-continued detection of, by chemiluminescence, 119 fatty acids in, 3 unsaturated fatty acids in, 25

Serratieae, lipids of, 38 Serum virus antibody, detection of, 11 7 Sewage, a~ asource of bdellovihrio bactorio-

phages, 266 brldtovibrios in, 220

Shoath on flapilla of hddlovihrioH, 223 A'/+plltr, l ~ ~ ? j d i i , grciwttl of bdollovihrioa 1 1 1 1 ,

Sidorr)c!aliHiL(:I)aO, lipiilti of, 37 Siltiiig indox, IiiLturo of , I12 Sitos, activn, on t,ho r r i l ~ r c ~ ~ ~ r i i ~ ~ o c ~ ~ ~ z y r ~ i t r , 03 Sizo of miortrt)cs in rdiitiori to rupid UHNOHH-

Skim milk, 1180 of, in prosnrving IjdeZZo-

Slimo polysaccharidcs of hactcria, 143 Sodium chlorido requirements of marine

Soil bacteria, extracellular glucan pro-

extracellular polysaccharides of, 144

fertility, importance of bacterial cxo-

247

mont mothnds, I07

VibriO C!Ult#UreH, 220

bdellovibrios, 249

duction by, 166

Soil, bdellovibrios in, 220

polysaccharides and, 144 Solute penetration by bdellovibrios, 240 Soybean root nodule, ferredoxins isolated

from, 82 Soybean root nodulnn, nicotinamide nucloo-

titles arid riitrogon fixatioii in, 74 Soybnl~ns, nibrogon fixrhon hy, 60 Sparso inicrohiid popiiIih)iiH, rapid clctc~c:.

Spi:ifin onzyrnic: rnnt.hr~tls for UHHIL.~ IJf tion of, 105

I)Uf'l.Orild ~ 1 X ~ J ~ J f J ~ ~ H l ~ f : l ~ ~ l l ~ f ~ ~ f ~ ~ ~ H I I ~ I W H ,

161

ing rnicrcihc:~, 107 Sliocific! idont.ific:atiori rn~:thr~tls for cli:tc:c:f,-

Specific: mr:thoiln, riqiiil, for rrii(:rol)i:H, 1 2fj Spof:ificiL,y in rapid rr~c:I~hiitln for tli~toc:l.irlK

Spooifici by of hdollovi twio r~tt,itnhmorit, 29:) rriicrohon. 106

~ ~ p h f k s r O p h O r W ridiCUkJaU6, pt%i~nU~~igorlH in, I 1

f+haoroptaHting of hdt3lIOVibriOH, 229 ~qphoerolilue nutona, c:xtrncclluler p l y -

Sphingolipids, in bacteria, 14

Spinach chloroplast ferredoxin reductam,

Spirillurnapp., relations of bdellovibrios to,

<~erpenn, attachinorit, 01' 1,rlollovi brio8 to.

saccharido pror1uc:tiiJn tiy, 148

of bdellovibrios, 228

82

221 . "

propane fatty acids in, 26 234

SUBJECT INDBlX 298

Sp+-ocheetales, lipid composition of, 43,45

Spore-forming bacilli, phosphatidyl-

Staining methods for detecting miri-obos,

Staining of bacterial capsules, 144 Staphylococci, glycolipids in, 14 Slaphgllowccw aureua, phospholipid com-

Starch granules, immunn adherence roar-

Sterol requirement of mycoplasmas, 17 Sterols, possible presence in bacteria, 17 Straight-chain fatty acids, biosynthesis of,

Streptococci, fatty acids in, 3

lipids in, 49

glycerol in, 8

121

position of, 41

tion with, 116

in bacteria, 19

production of extracellular polysacchar-

Slre~tocoocwfaeculi, biosynthesis of glyco- ides by, 167

lipids in, 34 chromatography of products of, 130

Rtreptomycer aureofaciena, lipids in, 1 I Strep. olivuceur, storoh in, 17 Rtmptomycctos, nitrogen fixation by, 01 Rtroptornycin, offect of, on bdellovibrio

Rtructural featuros of bacterial oxnpoly-

Rtructuro of bdohvibrios, 222 Rtructures, of bacterial exopolysnrohnr.

of bacterial heteropolysaccharidcs, 167 of bacterial homopolyaaccharides, 166

Substrate-binding site of nitrogenme, 93 Subunit nature of nitrogenme, 90 Succinate dehydrogenase in bdellovibrios,

263 Succinate in bacterial exopolysaccharides,

166 Sugar nucleotides in biosynthesis of hac-

terial exopolysaccharides, 186 Sugar residues in bacterial exopolyaacchar-

ides, 160 Sulphate ester lipids in halophilic bacteria,

12 Sulphur compounds, effect of deficiency

of, on production of bactcriul 0x0- polysaccharides, 147

penetration, 230

saccharidea, 160

ides, 166

Sulphur content of ferredoxins, 80 Surface bonding and bdollovibrio attuch-

ment, 233 Survival, microbial, and extracol1ult-w

polysacchuridos, 144 Symbiosis and bdellovibrios, 228 Symbiotic nitrogen fixation, 00 Syphilis, fiuorescunt antibody test for,

116

T Talose of bacterial exopolysRccharidns, 182 Taxonomic significaiiro of hnrtcriol lipid

Taxononiy of bdellovibrion. 22 I Teichoic acids of bacteria, 143 Temperature, and bdellovibrio attachment,

offoot of, on bdollovit)rio-hoHt wll

Tcrminal steps in synthesis of unsaturated

Terminally branched fatty acids, bio-

Terminology of bdellovibrios, 221 Thiobacillua novellua, phospholipid com-

T . thdo-oxidam, lipids in, 26

conipoaitions. 36

233

kinetic#, 246

fatty acids in bacteria, 24

synthesis of, in bacteria, 21

position of, 7

phosphatidylethmolamine in, 6 phospholipid composition of, 41

Thiobwteriaoese, lipids of, 36 Time-course of polysaccharide synthefiis by

Klebeiella aerogenea, 149 Traneaoylation in bacterial phospholipid

biosynthesis, 30 Transfer of sugars to lipids in polysacchar-

ide biosynthesis, 193 Transforesea in biosynthesie of bacterial

exopolysacoharidos, 193 Trepnemo pallidurn, lipide in, 49

Treponemataceae, lipid composition of, 46 Tricarboxylic acid cycle in bdellovibrios.

Triglycerides in micro-organisms, 18 Tubcrculostearic acid in bacteria, 27 Two-membered cultures of hdellnvikrios,

Typing of bdellovibrio hacteriophagoe, 267 Types of pneumococcal cxopolyclscchar-

plasmalogens in, 11

253

241

._

ides, 181 . .

U Ultracentrifugatirn in rncthoclw for rapid

Ultrastructuro Of bclrtllovihrios, 222 Ultrtrviolct microncopy in dotoctirig mi.

Untlocapronol pliotiphatctn i n bactwia, I 7 Urariyl ucutato and stairling ~ l ' IJdd to-

UNJU, effect of, on hdellovibrion, 254 Uridinediphospho Rugurn in lirJxynlhc:Hin

a8wsHmont of viral pophtiorin, 1 OH

crobes, 114

vihrion, 223

Of hactnrial HXor)lJ~yHtK:lc:C:hlbri~~l!~, 18fi

294 SUBJEOT INDEX

Uronic acid in Klebsiella exopolysacchar-

Uronic acids in bacterial exopolyeacchar-

Use of adenosine triphosphate in nitrogen

Utilization of extracellular polysacchar-

ides, 173

ides, 162

fixation, 69

ides by bacteria, 148

V ck-Vaccenic acid, formula of, 2 Vaccinia virus, tlotormination of, 139 Vanadium urid nitrogon fixation, 64 Vanadium -containing nitrogenasos, 94 Variation of lipid composition in a spocion

Variola virus, determination of, 133 Veillonella gazogenea, plasmalogens in, 11 Viability, microbial, rapid determination

Vibrio app., relation of bdellovibrios to, 221 Viral antigen, detection of, 117 Viruses, detection of, 116

of bacterium, 46

of, 131

immune adherence reaction with, 116 rapid deteotion of, 106,132

Viscous slimes produced by bacteria, 149 Vishniac, Wolf, contribution of, to estima-

tion of microbial populations, 126 Vital staining of bacteria, 131 Vitamin requirements of bdellovibrios, 247

w Walls of bdellovibrios, composition of, 228

Washed ccll suspensions, production of polymceharides by bacteria in, 148

Weights of microbes in relation to rapid assessment methods, 108

Wolf trap, nature of, 112, 126

X Xanthomonne campentria, compoei tion of

oxopolysacchariclo from, 1113 oxol)olyflaochciridu from, 177

X . plrrcueoli, productiori ol'onzymos by, that hydrolyeo t)a.oturiul oxopolysacchar-

X-ltay diffraction analyHis of furredoxins, idOE, 169

83

Y Yeaat extract as a substrate for bdello.

Yeast, fatty acid synthesis in, 20 vibrioa, 24

methylation of phosphatidylethanol-

Yeaets, immune adherence reaction with, amine in, 31

116

Z Zinc, and nitrogenaso, 91

and preservation of hdnllovihrios, 223

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