COMPARISON OF SPECIES AND VARIETIES OF THE GENUS BACILLUS STRUCTURE AND NUCLEIC ACID CONTENT OF SPORES' PHILIP C. FITZ-JAMES2 AND I. ELIZABETH YOUNG3 Department of Bacteriology and Immunology, and Department of Biochemistry, University of Western Ontario, London, Canada Received for publication May 10, 1959 A preliminary study of the nucleic acid content of the spores of Bacillus cereus and of crystal- bearing organisms related to B. cereus revealed that although the medium could affect the spore composition, certain fractions, particularly the deoxyribonucleic acid, were remarkably constant when expressed on a per spore rather than a dry weight basis (Fitz-James, 1957). The character- istic content of deoxyribonucleic acid in spores of certain species prompted further analyses of this group of organisms and also of spores of other species in the genus Bacillus. This comparison is the subject of the present communication; the results of some comparisons of the structure of spores which arose from this work are presented separately. MATERIALS AND METHODS Details of the organisms studied are given in table 1. To facilitate this comparison they were grouped as outlined. Those assigned to the first group form spores which possess an exosporium. This structure, recognized by de Bary in 1884 and so named and further described by Lewis in 1934, can be readily seen on spores in water mounts and air-mounted nigrosin smears when observed with the oil immersion objective. Observations with the electron microscope, how- ever, have greatly facilitated the recognition and characterization of this saclike appendage (Robinow, 1951) (figures 10 and 11). It covers the entire spore but is usually closely applied only around the sides of the spore. Thus, it remains attached to the outer spore coat when the latter is separated from the spore body by disruption 1 Supported by a grant-in-aid from the National Research Council of Canada. 2 Medical Research Associate, National Re- search Council of Canada. 3Present address: Department of Bacteriology, University of Alberta, Edmonton, Canada. or is cast off following germination (figure 11). Its formation during spore development as a new and distinct layer within the sporangial cytoplasm can be seen in ultrathin sections of sporulating cells (Hannay, 1956). The second and third groups are comprised of organisms whose spores possess no exosporium. Those in the second were received as strains of Bacillus megaterium, whereas those in the third are two rapidly motile, sporeforming rods. The classification and nomenclature of Smith et al. (1952) have been followed where possible. Methods of spore production. The agar medium of Howie and Cruickshank (1940) supplemented with 0.5 per cent casamino acids (H and C) and a beef papain digest nutrient agar (Asheshov, 1941) were the two solid media used in most of the experiments to maintain cultures and to grow spore crops. Sporulated cultures were harvested and washed as described previously (Fitz-James, 1955a). When the spores or spore- crystal mixtures were freed of detectable vege- tative debris, sufficient 0.1 N NaOH was added at room temperature to raise the pH to 10.8 to 11.0. The spores could then be separated under any swollen parasporal material by centrifugation. The spores were resuspended and rewashed, twice with 0.5 N NaOH, once with 0.1 N HCl, and 4 or 5 times with water or saline. Any debris on the surface of the pellet was removed after each centrifugation. The final pellet of spores was made into a thick suspension (1010 per ml) in water and dispensed for dry weight determinations, counting, and analysis. The purity of the spore suspension was further checked in the electron microscope during the measurements of spore volume. For reasons of safety, B. cereus var. anthracis, and for comparison B. cereus strain N, were grown in an aerated liquid medium developed for studies of sporulation (Young, 1958). Spores 743 on February 17, 2021 by guest http://jb.asm.org/ Downloaded from
Transcript
COMPARISON OF SPECIES AND VARIETIES OF THE GENUSBACILLUS
STRUCTURE AND NUCLEIC ACID CONTENT OF SPORES'
PHILIP C. FITZ-JAMES2 AND I. ELIZABETH YOUNG3
Department of Bacteriology and Immunology, and Department of Biochemistry, University ofWestern Ontario, London, Canada
Received for publication May 10, 1959
A preliminary study of the nucleic acid contentof the spores of Bacillus cereus and of crystal-bearing organisms related to B. cereus revealedthat although the medium could affect the sporecomposition, certain fractions, particularly thedeoxyribonucleic acid, were remarkably constantwhen expressed on a per spore rather than a dryweight basis (Fitz-James, 1957). The character-istic content of deoxyribonucleic acid in spores ofcertain species prompted further analyses of thisgroup of organisms and also of spores of otherspecies in the genus Bacillus. This comparison isthe subject of the present communication; theresults of some comparisons of the structure ofspores which arose from this work are presentedseparately.
MATERIALS AND METHODS
Details of the organisms studied are given intable 1. To facilitate this comparison they weregrouped as outlined. Those assigned to the firstgroup form spores which possess an exosporium.This structure, recognized by de Bary in 1884and so named and further described by Lewis in1934, can be readily seen on spores in watermounts and air-mounted nigrosin smears whenobserved with the oil immersion objective.Observations with the electron microscope, how-ever, have greatly facilitated the recognition andcharacterization of this saclike appendage(Robinow, 1951) (figures 10 and 11). It covers theentire spore but is usually closely applied onlyaround the sides of the spore. Thus, it remainsattached to the outer spore coat when the latteris separated from the spore body by disruption
1 Supported by a grant-in-aid from the NationalResearch Council of Canada.
2 Medical Research Associate, National Re-search Council of Canada.
3Present address: Department of Bacteriology,University of Alberta, Edmonton, Canada.
or is cast off following germination (figure 11). Itsformation during spore development as a newand distinct layer within the sporangial cytoplasmcan be seen in ultrathin sections of sporulatingcells (Hannay, 1956).The second and third groups are comprised of
organisms whose spores possess no exosporium.Those in the second were received as strains ofBacillus megaterium, whereas those in the thirdare two rapidly motile, sporeforming rods.The classification and nomenclature of Smith
et al. (1952) have been followed where possible.Methods of spore production. The agar medium
of Howie and Cruickshank (1940) supplementedwith 0.5 per cent casamino acids (H and C) anda beef papain digest nutrient agar (Asheshov,1941) were the two solid media used in most ofthe experiments to maintain cultures and togrow spore crops. Sporulated cultures wereharvested and washed as described previously(Fitz-James, 1955a). When the spores or spore-crystal mixtures were freed of detectable vege-tative debris, sufficient 0.1 N NaOH was addedat room temperature to raise the pH to 10.8 to11.0. The spores could then be separated underany swollen parasporal material by centrifugation.The spores were resuspended and rewashed, twicewith 0.5 N NaOH, once with 0.1 N HCl, and 4 or5 times with water or saline. Any debris on thesurface of the pellet was removed after eachcentrifugation. The final pellet of spores was madeinto a thick suspension (1010 per ml) in water anddispensed for dry weight determinations,counting, and analysis. The purity of the sporesuspension was further checked in the electronmicroscope during the measurements of sporevolume.
For reasons of safety, B. cereus var. anthracis,and for comparison B. cereus strain N, weregrown in an aerated liquid medium developedfor studies of sporulation (Young, 1958). Spores
Dr. C. F. Robinow. This organism showed atypical smooth colony type of growth onagar.
Dr. T. Angus, Sault Ste. Marie, Canada.This insect pathogen forms a spore and aparasporal crystal in each cell (figure 3).
Isolated from platings of heat-activatedspores which had been diluted in formal-dehyde-saline. These colonies appearedidentical to the T+ strain but only onehalf of the cells produced a crystal (figure4). Six separate isolates have been made.The spores did not lie obliquely in thosecells which did not contain a crystal.
Isolated in the same way as the T± strain.Cells of this isolate formed only spores(figure 5). The spores did not lie obliquelywithin the sporangium.
Dr. T. Angus, Sault Ste. Marie, Canada.This insect pathogen forms both a sporeand parasporal crystal in each cell(figure 1).
Isolated from platings of heat-activatedspores of strain S+ which had been dilutedin formaldehyde-saline. Cells in theseisolates produce only spores (figure 2).
Dr. C. Toumanoff, Pasteur Institute. Iso-lated from diseased silkworm larvae, thisorganism forms both a spore andparasporal crystal in each cell.
Isolated from a plating of the A+ strain, thisorganism resembled the parent strain inregard to size of spores and colonial formbut the cells did not form crystals.
Dr. C. Toumanoff, Pasteur Institute. Thisorganism formed uniformly large crystals.
Dr. C. Toumanoff, Pasteur Institute. Afterculturing strain A for 31 passages on al-kaline medium, pH 9.0, this strain whichhad lost its crystal-forming ability wasisolated.
Dr. C. Toumanoff, Pasteur Institute. Thisorganism is classified at the Pasteur In-stitute as a typical B. cereus.
Dr. C. Toumanoff, Pasteur Institute. Iso-lated following 8 passages through thebody cavity of the larvae of Galleria mel-lonella of strain A30. Each cell forms aspore, a small crystal and a larger, tri-angular-shaped inclusion (figure 6).
Name of Organism Strain Source and Characteristics Reference
-13. B. cereus
14. B. cereus
-15. Bacillus cereus var.anthracis
16. Bacillus cereus var.mycoides
17. B. cereus var. mycoides
18. Bacillus medusa
Group 21. Bacillus megaterium
2. B. megaterium
3. B. megaterium
4. Bacillus 350
Group 31. Bacillus subtilis
2. Bacillus apiarius
Dr. C. Toumanoff, Pasteur Institute. Iso-lated following 2 passages of strain B30-1per os through larvae of Galleria mellonella.This isolate resembles B. thuringiensis.
Dr. C. Toumanoff, Pasteur Institute. Iso-lated from larvae of Galleria mellonellainto which an asporogenous culture of a
Bacillus species had been previously in-jected. Morphologically this isolate re-
sembles B. thuringiensis but has a
mesenteric type of growth on agar.
Isolated in 1954 from a fatal case of bovineanthrax.
Dr. C. F. Robinow. Typical whorls of whitegrowth on agar.
Dr. C. F. Robinow. Typical whorls of growthon agar; produces a yellow pigment duringvegetative growth.
Dr. C. F. Robinow. Isolated from cow dung,this organism produces a large spore andan ellipsoidal or spherical parasporal bodyin each cell (figure 7).
Dr. C. F. Robinow. Isolated from boiledsliced carrots by the method of Koch(1888).
Isolated following repeated platings on
papain-digest agar of the asporogenous
strain received from Dr. S. Spiegelman,University of Illinois.
Dr. E. D. DeLamater, University of Penn-sylvania
Dr. John Risbeth, Botany School, Cam-bridge, England. Isolated from a sampleof African soil and classified by Dr.Risbeth as a B. megaterium (figure 8).Vegetative cells are sensitive to lysozyme(figure 9) but do not grow on glucose-nitrate agar.
Dr. Ruth Gordon, New Jersey ExperimentalStation, New Brunswick, New Jersey.
Dr. H. Katznelson, Science Service Labora-tory, Ottawa, Canada.
Hannay (1956)
Katznelson(1955)
were harvested and washed free of debris bycycles of centrifugation and resuspension.
Another fluid medium developed for the study
of spore and crystal formation in B. cereus var.
alesti (Young, 1958) was also used for the pro-duction of spores of this and closely relatedstrains.
Analytical methods. (1) Dry weights:-Dryweights to the nearest 0.1 mg were determined induplicate on 0.5 or 1.0 ml of the final sporesuspension. After 10 to 20 hr at 60 C, they were
dried to a constant weight over phosphoruspentoxide. Duplicates were within 1 per cent ofthe mean weight.
Figures 1 to 6 Water mounts of sporulated cells from 40- to 48hr cultures on agar.Figure 1. Bactillus sotto Ishiwata in final stages of sporulation forming a spore and small crystal in-
clusion in each cell.Figure 2. Two chains of a nonerystal-forming strain isolated from Bacillus sotto. Except for the ab-
sence of the crystals the morphology of growth and sporulation was very similar to the parent strain.Figure 3. Bacillus thur-ingiensis Berliner.Figure 4. A varient isolated from the parent culture of B. thuringiensis, which, during sporulation,
forms slightly smaller crystals in some cells and none in others.Figure 6. Another variant isolated from B. thuringiensis which forms only spores.Figure 6. Bacillus sp. B30-1 which forms a triangular (t) and diamond shaped (c) inclusion during
sporulation.Figure 7. Bacillus medusa showing the refractile parasporal bodies in a 22-hr fluid culture.Figure 8. A water mount of Bacillus 350 treated for 5 mi in 0.15 per cent KMnO4and stained 4 mmn
in 0.25 per cent thionine. The lightly stained spores focused below the plane of refractility show thecoat wrinkles characteristic of this large spore former. Photograph courtesy of Dr. C. IF'. Robinow.
Figure 9. Bacillus 350 protoplasts formed 10 min after the addition of lysozyme to vegetative cellsgrown in 2 per cent peptone and stabilized in sucrose (0.3 m).
All, with the exception of figure 8, which is bright field, are dark phase contrast photomicrographs.Magnification is approximately 4000X as indicated by the 5 jA marker.
Figure 10. An electron micrograph of an unshadowed spore of Bacillus cereus var. alesti illustratingthe characteristic exosporium found with minor variations of the spores of B. cereus and varietiesthereof (X39,000).
Figure 11. Carbon replica of spores and spore coat of B. cereus var. alesti, after the method of Bradleyand Williams (1957).
a + Normal crystal-former; +, partial crystal-former; -, noncrystal-former; RNA, ribonucleicacid; DNA, deoxyribonucleic acid; SEM, standard error of mean.
'Values expressed as 10-16 g P per spore.c By the orcinol reaction on trichloroacetic acid extracts (Schneider, 1945).d By the diphenylamine reaction on trichloroacetic acid extracts of theDNA separated by the Schmidt
and Thannhauser (1945) procedure as in Fitz-James (1955a).e The spore residue remaining after N KOH extraction (Schmidt and Thannhauser, 1945) plus hot
trichloracetic acid extraction; composed of spore coat.
TABLE 3Comparison of the size, weight, and composition ofthe spores of Bacillus cereus and those of tworelated organisms harvested from nutrient agar
a Strain designation symbols, abbreviations,and description of phosphorus fractions as infootnotes of table 2.
(2) Spore counts:-Counts were made in thePetroff-Hausser counting chamber using thephase-contrast condenser and 40 X phase-contrastobjective (Zeiss). Spore suspensions were dilutedin either saline or formaldehyde-saline (0.9 percent NaCl containing 1 per cent formaldehyde)until the count fell between 20 and 80 spores perlarge square of the chamber. Six or more largesquares were counted for each of at least fourapplications or until the average number ofspores was within 5 per cent of the mean of allapplications.
(3) Phosphorus-containing compounds:-Thecompounds were fractionated and analyzed ac-cording to methods already described (Fitz-James, 1955a). To begin the fractionation, sporeswere disrupted in methanol. Cold trichloroaceticacid extraction followed removal of the lipidfraction.
Total nitrogen (N) was estimated by micro-Kjeldahl procedure using a microadaptation ofthe ashing procedure of Beet (1955).When possible, the chemical data were calcu-
TABLE 4Comparison of the weight, volume, and phosphorus fractions of the spores of Bacillus cereus and closely
related Bacillus species (varieties) harvested from aerated fluid cultures"B. cereus and Variants in B. cereus var. alesti and Related Strains in Medium BMedium A
Comparison of SporesB cereus B. cereus B.cereus B. cereus B. cereus B. cereus B. cereus
(N) var. var. var. alesti var. alesti var. alesti var. alesti B.cereusanthrax mycoides (A+) (A-) (B C')) (31-9()) (-i)
Wt per spore (1012 g). 1.03 0.86 1.61 1.35 2.35 0.80Volume per spore (M3 X 102 :1:
SEM).. 35.2 i 24.8 :i 40.6 27.5 i 25.8 33.9 :1 36.3 46.7 2.5 1.4 1.2 0.98 1.7 1.4
a Strain designation symbols, abbreviations, and description of phosphorus fractions as in footnotesof table 2.
lated both as a function of dry weight and ofspore count (g X 10-16 per spore). Only thelatter values have been included in the tables;those for the per cent dry weight can be calculatedby dividing the amount of the fraction per spore(10-16 g) by the average weight per spore (10-12g) and adjusting the decimal.
Morphological methods. Spore volumes werecalculated from measurements taken directlyfrom the electron microscope screen and fromelectron micrographs of shadowed and un-shadowed material. The screen measurementswere made at a magnification of 20,000X withreference to the 1 ,u marker of the microscope(Philips E.M. 100A) and the measurements fromthe micrographs were similarly made after pro-jection from an enlarger. As the majority of thespores were elliptical in outline, the formula7rab2/6 (where a is the length and b the width ofa prolate spheroid) was applied to 10 or morespores in each group. The standard error for themean volume was calculated. However, as thevariation due to the magnification on the screenof the microscope was found to vary at differenttimes of measurement by about 10 per cent, onlythe major differences in spore volume can beconsidered significant.Methods of fixation, hydrolysis, and staining
smears for light microscopy have been describedin detail elsewhere (Robinow, 1951; Fitz-James,1955a).
RESULTS
Analysis of spores in group 1 (table 1). (1)Ribonucleic acid content and spore volume.-Theanalysis of the spores in group 1 are summarizedin tables 2 to 5. Of the three crystal-formingorganisms, B. cereus var. alesti forms the largestand Bacillus sotto the smallest spores. The sporesof Bacillus medusa, like those of B. cereus var.alesti are approximately twice the size of B.cereus strain N whereas those of B. cereus var.mycoides are approximately three times larger.The content of ribonucleic acid (RNA-P)
varied with the spore volume, larger sporescontaining up to twice as much RNA as thesmaller ones (table 2). However, a change inmedium could affect both the size and RNA-Pcontent of the spores; for instance, those of thecrystal-forming organisms were smaller andpossessed a considerably reduced content of RNAwhen grown on nutrient agar (table 3) or inaerated fluid medium (table 4). While spores ofB. cereus strain N appeared unaffected by suchchanges in media (tables 2 to 4) those of B.cereus var. mycoides were also decreased inRNA-P content and spore volume (table 2 and4).
It has also been a consistent observation thatspores of the crystal-forming organisms formedon nutrient agar are prone to spontaneousgermination and lysis when kept in aqueoussuspension. These spores, although possessingthe decreased amount of RNA are enriched in
TABLE 5Comparison of the size, weight, and amounts of thephosphorus fractions of the spores of Bacilluscereus ASO and of the spores of two differentcrystal-forming bacilli isolated from Galleriamellonella following injection and feeding ofculture ASO
a Spores for this comparison were grown onpapain digest agar. Abbreviations and descriptionof phosphorus fractions as in footnotes of table 2.
b Of the same width as the other two organismsbut slightly longer.
nitrogen (tables 2 and 3). It should be empha-sized, however, that the crystals formed on eithermedium were indistinguishable.
(2) Deoxyribonucleic acid content:-It becameapparent from these various analyses that thequantity of one component, deoxyribonucleicacid (DNA-P) remained remarkably constantfor each species regardless of the growth mediumor attendant changes in volume of RNA-Pcontent of the spores. For example, spores of B.cereus strain N contain a similar DNA-P contenton all media so far employed for their production.Separate studies have further revealed that therough strain of this organism has this sameamount of DNA-P which is also possessed byB. cereus var. anthracis. However, this amount ofDNA-P per spore is not common to all membersof group 1 (table 1) for B. cereus var. alesti andB. medusa contain approximately twice thisamount, but Bacillus thuringiensis and B. cereusvar. mycoides contain an amount slightly greaterthan B. cereus strain N. It should be noted,furthermore, that the non- and partial-crystal-forming organisms isolated from crystal-formerspossess the same DNA-P content as the parentorganisms (tables 2, 4, and 5).
(3) Residue phosphorus:-The amount ofphosphorus in the residue fraction was alsouseful for distinguishing certain members of thisgroup. Associated with the spore coat remnants,this residue-P was similar in amount in B. cereusstrain N, B. sotto and B. cereus var. alesti. How-ever, spores of B. thuringiensis possessed almost10 times this quantity. Similarly, those strains ofB. thuringiensis which had lost partially orcompletely the crystal-forming ability still con-tained this same high content of phosphorus inthe residue fraction (table 2).
It is also of interest to note the level of residue-P in the spores of the two crystal-forming organ-isms isolated from Galleria mellonella followinginjection and feeding of B. cereus strain A30(Toumanoff, 1956). The organism B30-1, whichformed two parasporal inclusions formed sporeswhich contained four times the amount ofresidue-P as did B. cereus strain A30, whereas thesecond isolate (B. cereus strain B30-2) producedspores which contained a further 10-fold increasein this fraction (table 5).
Analysis of spores in group 2 (table 1). Thethree strains of B. megaterium, L, KM, and Penn,all possessed similar amounts of DNA-P perspore (table 6). The larger and heavier spores of
TABLE 6Comparison of the size, weight, and amounts of thephosphorus fractions of the spores of varieties ofBacillus megaterium (grown on H and C agarexcept Bacillus 350, potato agar)
a Abbreviations and description of phosphorusfractions as in footnotes of table 2.
Bacillus 350, on the other hand, contained abouttwice as much DNA-P as the others and werericher in N and RNA-P. A comparison of thesespores with those of B. megaterium strain Lgrown on the same medium (potato agar) indi-cated that these differences were not due to themedium.
In a previous comparison of spores, the largequantity of residue-P associated with the sporecoat in B. megaterium compared with that in B.cereus strain N was emphasized (Fitz-James,1955a). From table 6 it is obvious that a largecontent of phosphorus in this residue is not acharacteristic of all strains of B. megaterium. Intwo strains, the residue phosphorus is over oneper cent of the dry weight of the spores, whereasin the other it is practically nil. This apparentdifference in spore coat composition prompted afurther structural comparison (Fitz-James andYoung, 1959).
Analysis of spores in group 3 (table 1). Spores ofBacillus subtilis as those of Bacillus laterosporus(Fitz-Jamesand Young, 1958) havea much smallervolume than those just described (table 7). Sporesof Bacillus apiarius, although they appear large,have a remarkably thick outer coat which gives anerroneously large estimate of the true volumeof the spore protoplast. Thus, in keeping withthe smaller volume of the actual spore proto-plast, the spores of both these organisms con-tain an average amount of DNA-P which ishalf that found in the smaller spores in group 1(figure 12) and an amount of RNA which is
20
15
I.*t
BOcllus cersus vaalestl crystal-former enon crystal-former o
Bgcillus 350 VBacillus medusoa
w ~~~~~~~~~whitestrain w.[,, Y |D Bacillus cereus vor mycoldes e strain
YI yellow strdnvo full crystol- forrmer e
*|C Bacilus thuringlensis portiol crystal-former 2non crystol-formor O
AV a Bacillus corsus Ntx B Bacillus megaterium strs L, K M and Penn. V
5I to xVA Bocillus solto crystol- former A'-a---' non crystol-former a
X. Bacillus cereus vor anthrocls +
z
Bacillus subtilis.2 6 Bacillus oplarius 2
I 4 A Bacillus lsntimorbus 35 . '---------' Bacillus popilliae 4
Bodllus laterosporus 5
Figure 12. A compilation of the average deoxy-ribonucleic acid (DNA-P) content of the sporesof some 22 species, subspecies, and variants of thegenus Bacillus taken from this and companionpublications. Except for Bacillus thuringiensis,the DNA-P/spore tends to be a multiple of theamount found in the smallest spores (group A).
also much lower. In this respect they are againsimilar to the spores of B. laterosporus (Fitz-James and Young, 1958) and those of Bacilluslentimorbus and Bacillus popilliae Dutky (Fitz-James, unpublished data); the DNA content ofthese organisms has been included in figure 12).As would be expected, the amount of DNA-P
in the spores was reflected in the size of thenuclear bodies seen after hydrolysis and stain-ing; those with the smallest DNA-P contentalso possessed the smallest nuclear bodies.
DISCUSSION
As a criterion for taxonomy, the presence of anexosporium about the spore can be of consider-able value. Such a covering is not present onspores of B. megaterium or B. subtilis but ispresent on those of B. cereus and other organismsbelieved to be varieties or close relatives of it.It must not, however, be confused with the cell-wall remnant which remains attached to theripe spores of some other species. Within thisB. cereus group, the organisms can now be furtheridentified according to the average content ofDNA within the spores. The average amount of
this compound is constant and characteristic ofeach organism and strain derived from it andremains unaltered during nutritionally inducedchanges in spore size and RNA content. Threesubgroups are thus formed each containing bothcrystal and noncrystal-forming organisms. Thatsubgroup of which B. thuringiensis is the pro-totype can be further identified by the charac-teristically high content of residue phosphorus inthe spores. Thus, from these criteria, i. e., sporecontent of DNA and residue phosphorus, itappears that B. cereus var. alesti and B. sottomay not be as closely related to B. thuringiensisas previously thought by others (Delaporte andBeguin, 1955; Heimpel and Angus, 1958). Forconvenience in comparisons of this type we
consider all these crystal-bearing and derivednoncrystal-bearing organisms as variants of B.cereus as already suggested by Smith et al.(1952) for B. thuringiensis. In addition to pos-sessing an exosporium, as B. cereus, cultures ofthese organisms on egg yolk agar showed thepositive reaction due to lecithinase production(Colmer, 1948).The spores of the three strains of B. megaterium
studied possess similar amounts of DNA. Thestrains of this species, however, can be furtherseparated by the presence or absence of a
phosphorus-containing spore coat residue. Onthe other hand, Bacillus 350, in keeping with itsnutritional requirements, is distinguishable fromtypical strains of B. megaterium by its doubledamount of DNA.
Recently, Woese (1958) reported that spores
of B. subtilis, Bacillus mesentericus, and Bacillusbrevis exhibited "single hit" inactivation param-
eters on exposure to X-rays whereas spores ofB. cereus, B. cereus var. mycoides, and threespecies of B. megaterium showed "multiple hit"inactivation curves. To explain these differences,the author suggested that the genetic materialin the multiple hit spores is doubled in comparisonto the single hit spores. It is interesting that ofthese spores, B. subtilis belongs in the group withthe lowest content of DNA and that this amountis indeed doubled in B. cereus and in the threeB. megaterium species analyzed, but tripled inB. cereus var. mycoides. An interpretation of thepresent results, in the light of Woese's data, isthat 5 X 1016 g DNA-P represents a singlechromosome set and those spores with multiplesof this amount of DNA possess 2, 3, and 4similar units.
It must be emphasized, however, that noincrease in number of "chromosome sets" canbe observed cytologically when spores with theseincreasing amounts of DNA are compared. Allof these spores, regardless of DNA content,contained one nuclear body, i. e., a single con-tinuum of DNA as discerned microscopically,and all of the vegetative cells contained twochromatin bodies in some stage of division(Fitz-James, 1955b; Young and Fitz-James, 1959).However, the content of DNA was reflected bythe size of the stained nuclear bodies. Thus, theincreased amount of DNA between organismsrepresents an increase in DNA per chromatinbody and not an increase in number of chromatinbodies per cell. It is interesting to note that asimilar increase in DNA per discrete nuclearbody was encountered by Ogg and Zelle (1957)during the derivation of a strain of giant cellsof Escherichia coli by treatment with camphor.With this increase Zelle and Ogg (1957) observeda shift from a single to a multiple hit irradia-tion survival curve.
Further studies on other species may revealthat the DNA content of the spores of this genusform a more closely integrated scale of valuesthan the stepwise pattern encountered so far.The shorter step from B. cereus strain N to B.thuringiensis (groups B to C, figure 12) may beso explained. Nevertheless, the grouping of thevarious organisms in figure 12 suggests that achanging DNA content is part of the process ofvariation in the evolution of this genus.
ACKNOWLEDGMENTS
The authors wish to thank Dr. C. F. Robinowfor his interest, encouragement, and helpfuldiscussions throughout the course of these studies.We are grateful to MIrs. Sheila Newton for hercareful technical assistance.
SUMMARY
The size and phosphorus fractions of sporesof an assortment of Bacillus species have beencompared. Noncrystal-forming mutants wereisolated from and compared to parent crystal-forming strains of Bacillus sotto, Bacillus thu-ringiensis, and Bacillus cereus var. alesti. These,like B. cereus, B. cereus var. mycoides, andB. cereus var. anthracis and others, all possess adistinctive exosporium, on which basis they weregrouped. The B. cereus group could be furtherdivided into 4 subgroups depending on the
spore content of deoxyribonucleic acid and, insome instances, of residue phosphorus. Themedium used and the presence or absence ofcrystal-forming ability did not alter these twoparameters, but on a limiting sporulation mediumthe ribonucleic acid content of spores of crystal-formers was lower. Spores of three Bacillusmegaterium strains possessed identical contents ofdeoxyribonucleic acid, but the quantity of resi-due phosphorus varied markedly. The spores ofBacillus subtilis and the heavy, square-coatedspores of Bacillus apiarius had a deoxyribonu-cleic acid content half that found in B. cereus.When the values for the average deoxyribo-
nucleic acid per spore of all the cultures studiedwere assembled, except for one group, all werenear multiples of the amount found in thesrnallest spores.
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