JouMAL OF BACTERIOLOGY, Aug. 1979, p. 448-4530021-9193/79/08-0448/06$02.00/0

Vol. 139, No. 2

5-n-Alkylresorcinols from Encysting Azotobacter vinelandii:Isolation and CharacterizationtROSETTA N. REUSCH AND HAROLD L. SADOFF*

Department ofMicrobiology and Public Health, Michigan State University, East Lansing, Michigan 48824

Received for publication 4 June 1979

Azotobacter vinelandii was found to form novel lipid compounds when en-cystment was initiated by 0.2% f8-hydroxybutyrate. An examination of thesecompounds led to the isolation and characterization of 5-n-heneicosylresorcinol,5-n-tricosylresorcinol, and their galactoside derivatives.

We previously reported the presence of a 5-substituted alkylresorcinol with a C21 side chainin the lipids of encysting Azotobacter vinelandii(17). In this communication we report the iso-lation and characterization of this novel resor-cinol and one of its homologs and their galacto-side derivatives.A. vinelandii is a large (2 by 5 ,m), gram-

negative bacterium which undergoes a cyclicprocess of differentiation leading to the forma-tion of spherical, metabolically dormant cysts.The cyst has a volume approximately half thatof a vegetative cell and consists of a contracted,highly vacuolated cell (central body) surroundedby a capsule made up of a thin outer layer(exine) and a thicker inner layer (intine). En-cystment can be chemically induced by remov-ing glucose from exponential-phase cells andreplacing it with fl-hydroxybutyrate, a naturalintermediate in the metabolism of the organism(13). Polymeric,-hydroxybutyrate frequentlyaccumulates as large granules in stationary-phase vegetative cells and is a major componentof the central body of cysts (18). We speculatedthat this shift to /)-hydroxybutyrate metabolismwould result in the synthesis of lipids unique toencysting cells. A major portion of these newlipids proved to be 5-n-alkylresorcinols and theirglycosides.The natural occurrence of 5-n-alkylresorcinols

has been previously reported in the familiesAnacardiaceae (nut shell) (2, 19), Ginkgoaceae(fruit) (9), Graminae (wheat bran) (21), Protea-ceae (seed pods and wood) (7, 16), and, mostrecently, Myrsinaceae (seed) (14).

In this communication we report the forma-tion of novel lipid substances during the encyst-ment of A. vinelandii and the isolation andcharacterization offour ofthese substances. Thiswas the first discovery of 5-n-alkylresorcinols or

t Journal article no. 8941 from the Michigan AgriculturalExperiment Station.

5-n-alkylresorcinol glycosides in procaryotes, al-though isomeric monomethyl derivatives of 5-n-alkylresorcinols (a- and 18-leprosol) have beenisolated from Mycobacterium leprae (5, 8) andStreptomyces species contain an enzyme inhibi-tor (panosialin) which is a mixture of homologsofsulfated 5-n-alkylresorcinols (11) (Fig. 1). Thisis also the first report of 5-n-tricosylresorcinoland the galactosides of 5-n-heneicosylresorcinoland 5-n-tricosylresorcinol from natural sources.Since our initial report, 5-n-heneicosylresorcinoland 5-n-nonodecylresorcinol have been isolatedfrom Azotobacter chrococcum (3).

MATERIALS AND METHODSGrowth and encystment. A. vinelandii ATCC

12837 was used in these studies. The organism wascultured at 300C on a gyratory shaker in 500-ml Erlen-meyer flasks containing 100 ml of Burk nitrogen-freebuffer plus 1% glucose (22). The inoculum was 5% ofan overnight culture in the same medium. Cell growthwas followed by measurement of turbidity (opticaldensity at 620 nm) in a Gilford spectrophotometer.Generation time under these conditions was 3 h. Atlate exponential phase (optical density at 620 nm,-0.8) the cells were collected by centrifugation,washed twice with Burk buffer to remove all trace ofglucose (13), and suspended in Burk buffer to anoptical density of -0.5. Encystment was induced byadding,-hydroxybutyrate to a final concentration of0.2%, and the cells were incubated at 300C with agi-tation. Encystment was followed by microscopic ex-amination and required 5 to 7 days for completion(13).

Lipid extraction. Cells were collected by centrif-ugation and washed once with Burk buffer. Lipidswere extracted by the one-phase method of Bligh andDyer (4) as described by Ames (1). Cell paste wassuspended in distilled water, and 2 ml of methanol and1 ml of chloroform were added for each 0.8 ml of cellsuspension. After extraction overnight at 40C, thesolids were removed by filtration through glass wool,and 0.25 volume of water and 0.25 volume of chloro-form were added. The mixture was shaken vigorouslyon a Vortex mixer for 2 min and then spun at low

448

5-n-ALKYLRESORCINOLS IN ENCYSTING A. VINELANDII

HO NR X a H

III GalactoseR a Cm H45

ox CtaH4T

A. vineIandii

H3CO R~R a

OCHu

X a HR s CtlHa*

Cat H44

A. chrococcum

HOsSO RCitrH5s TiR a (CHzOut CH(C H3)a2CieHsi ~~Cis H3iCisHul S0r H(CHI)guCH(CHB)zOSOaM C ia HP.?

Ci* HusM. lepras StreptomycesFIG. 1. 5-n-Alkyl resorcinols and their derivatives in procaryotes.

speed to separate the layers. The chloroform layer andinterphase were washed once with 0.7% NaCl solutionand then dried to constant weight with a stream ofprepurified nitrogen. The lipids were dissolved in asmall amount of chloroform-methanol (1:1, vol/vol)and stored under nitrogen at 40C.Chromatographic methods. Column chroma-

tography. Primary lipid separation was carried outon heat-activated silicic acid (Mallinckrodt analyticreagent, 100 mesh) in a 1.5- by 25-cm column. Columnswere prepared from a suspension of silicic acid inchloroform (1 g/5- to 8-mg sample). Lipids were dis-solved in chloroform-methanol (1:1, vol/vol) and ab-sorbed onto a minimal amount of silicic acid. Afterevaporation of the solvent, the silicic acid was addedto the top of the column. This was necessary becausethe lipids were not completely soluble in chloroform,the first elution solvent. Neutral lipids were elutedwith chloroform (15 ml/g of silicic acid), glycolipidsand resorcinols were eluted with acetone (15 ml/g ofsilicic acid), and phospholipids were eluted with meth-anol (20 ml/g of silicic acid) (12). Fractions were driedto constant weight with a stream of nitrogen, sus-

pended in chloroform-methanol (1:1, vol/vol), andstored under nitrogen at 4°C.

Glycolipids and resorcinols were fractionated withFlorisil (Matheson, Coleman, and Bell, 60 to 200 mesh)in a 1.2- by 25-cm column (1 g/10-mg sample). Elutionwas stepwise beginning with chloroform, followed byincreasing concentrations of methanol in chloroformand finally by 100% methanol. The elution volume was20 ml of solvent per g of Florisil. Separation was

monitored by analytical thin-layer chromatography(TLC).TLC. Precoated Silica Gel H glass plates (Analtech

Labs) were used for separating preparative quantitiesof lipids. Precoated TLC Silica Gel 60 aluminum sheets(EM Labs) were used for analytical separation. Noheat activation was used. Development was in solventsystem (i) chloroform-methanol (85:15, vol/vol), (ii)chloroform-methanol (90:10, vol/vol), or (iii) chloro-form-methanol-water (65:25:3.8, vol/vol/vol) for lipidsand in (iv) n-butanol-methanol-water (5:3:1, vol/vol/

vol) or (v) acetone-water (90:10, vol/vol) for sugars.For preparative chromatography of resorcinols, plateswere dried under nitrogen.

Total lipids were detected by exposure to iodinevapor, organic phosphate was detected with mercury-molybdate reagent (20), sugars were detected withanisaldehyde-suluric acid reagent (10), and aminonitrogen was detected with 0.1% (wt/vol) ninhydrin inacetone. Resorcinols turned red or purple on standingin air or on exposure to iodine vapor and gave a brightred color with anisaldehyde-sulfuric acid reagent.

Lipids of interest were removed from plates with aBrinkmann spot collector and eluted from the adsorb-ent with chloroform-methanol (1:1, vol/vol), followedby chloroform-methanol (1:2, vol/Vol). Solvent wasremoved wtih a stream of nitrogen, and samples werestored under nitrogen at 4°C.Gas chromatography. A Varian Aerograph 1440

gas chromatograph equipped with a flame ionizationdetector was used. The carrier gas was helium. Col-umns were 6 feet by 0.125 inch (ca. 183 by 0.32 cm),stainless steel, packed with either 1.5% SE-30 onChrom G A/W (100/120 mesh) or 3% Carbowax-20Mon Chrom G A/W DMCS (100/120 mesh). Sugartrimethylsilyl (TMS) derivatives were chromato-graphed at 200°C on SE30 or at 160°C on Carbowax-20M. Resorcinols were chromatographed on SE-30with temperature programming from 200 to 325°C at6°C/min.

High-pressure liquid chromatography. The di-rect-phase high-pressure liquid chromatography uti-lized a stainless-steel column (4-mm inside diameterby 30 cm) packed with 10-,um fully porous silica par-ticles (utPorasil, Waters Associates). Reverse-phasehigh-pressure liquid chromatography was done on astainless-steel column (4-mm inside diameter by 30cm) packed with 10-rIm Bondapak C18 (Waters Asso-ciates). The sample injector was a Rheodyne model7120 (Anspec Co.). Pressure was applied with a MiltonRoy model 396 minipump. Detection was with anAltex Analytical (model 153) UV-VIS detector at 280nm. Mobile-phase solvents were chloroform-methanol(90:10, vol/vol) for direct-phase and methanol-water

449VOL. 139, 1979

450 REUSCH AND SADOFF

(99:1, vol/vol) for reverse-phase chromatography.Spectrometric methods. UV spectra were taken

in 95% ethanol in a Perkin-Elmer double-beam- spec-trophotometer. Infrared spectra were taken as mullsor dilute solutions in chloroform in a Perkin-Elmer700 infrared spectrophotometer. Mass spectra wererun in a Varian MAT CH5/DF mass spectrometerwith a direct probe at 3-kV acceleration voltage and1,000 resolution. Peak matching was done with a res-olution of 10,000. The data system was Digital Equip-ment Corp. PDP 11/05 and PDP 11/40. The samplesize was 3,g. Proton magnetic resonance spectra weretaken on a Varian T 60 spectrometer with tetramethylsilone as the internal standard.

Derivatives. lhe dimethyl ether of AR, was pre-pared by adding 7 mg of the compound to 300 mg ofdimethyl sulfate and 2 g of potassium carbonate in 20ml of dry acetone and refluxing the mixture for 24 h.After filtration the-solvent was removed with a streamof dry nitrogen, leaving 7.5 mg of a pale yellow solid(melting point, 53 to 65°C).For TMS derivatives of sugars, 1 to 2 mg of the

solid sugar was added to 1.0 ml of Tri-sil Z (PierceChemical Co.) in a small screw-cap septum vial. Mix-tures were shaken and heated to 70°C for 10 min.After cooling, the solution was injected into the gaschromatograph.

RESULTS AND DISCUSSIONLipid composition. Lipids extracted from

vegetative cells of A. vinelandii (Bligh-Dyerprocedure) were compared with those extractedfrom cells during the course of encystment (Fig.2). Only a small fraction of the polymeric f,-hydroxybutyrate, a lipid component of late-ex-ponential-phase vegetative cells and ofencystingcells, was extracted by this procedure. Totallipid extracted from exponential-phase vegeta-tive cells accounted for 10.6% of the dry weightofthe cells. During encystment the cells' contentof lipids rapidly increased and ultimatelyreached 20.2% of the dry weight of the maturecyst. Upon fractionation of the lipids on silicicacid, it was seen that the increase was due almostentirely to lipids in the acetone fraction (glyco-lipids and resorcinols) (Fig. 3). This fraction wasonly 7% of vegetative cell lipids but was 82% ofthe lipids in mature cysts. The weight of thechloroforn fraction (neutral lipids) and themethanol fraction (phospholipids), which com-prised 93% of the lipids of exponential-phasevegetative cells, changed only slightly duringencystment.Lipids of the acetone fraction. TLC of the

acetone fraction of encysting cells in chloroform-methanol (85:15, vol/vol) revealed the presenceof two major and several minor substances thatwere not present in extracts from exponential-phase vegetative cells. The two major sub-stances which were phenolic were designatedAR, (Rf 0.70) and AR2 (Rf 0.15) (Fig. 4).

300

0

I

31.

20(

100

1 2 3 4 5

TIME - DAYS

FIG. 2. Dry weight (0) and total extractable lpidweight (0) (see text) ofA. vinelandii as a function oftime after the initiation of encystment by 0.2% 1?-

hydroxybutyrate. Cells were pregrown in Burk bufferplus 1% glucose.

I

60-*

45 /OI-~~~~~~~~I

30

'U~~~~~~~.

TIME - DAYSFIG. 3. Composition of the total extractable lipids

(see text) as determined by chromatographic separa-tion on silica get as a function of time after theinitiation of encystment by 0.2% ,B-hydroxybutyrate.Symbols: total extractable lipids (0); neutral lipids(chloroform fraction) (0); glycolipids and resorcinols(acetone fraction) (0); phospholipids (methanol frac-tion) (U). Cells werepregrown in Burk buffer plus 1%glucose.

Isolation ofAR. and AR2. The acetone frac-tion from silicic acid separation of the lipidsextracted from encysting cells was chromato-graphed on Florisil. AR, was eluted with chlo-roform-methanol (95:5, vol/vol) and AR2 was

eluted with 100%/ methanol. Purification was

I--

_ ,I

J. BACTERIOL.

5-n-ALKYLRESORCINOLS IN ENCYSTING A. VINELANDII 451

ARi..-..--AR2

FIG. 4. Thin-layer chromatogram of the glycolip-ids and resorcinols extracted from A. vinelandii 22 hafter the initiation of encystment by 0.2% fi-hydroxy-butyrate. Total lipids extracted from 40 mg (dryweight) of cells were chromatographed on a silicicacid column. Neutral lipids uwre eluted with chloro-form, glycolipids and resorcinols were eluted withacetone, and phospholipids were eluted with metha-nol. Solvent was completely evaporated from the ace-

tone fraction, and the residue was dissolved in 1 mlofchloroform-methanol (1:1, vol/vol). A 10-1l amount

completed by TLC on Silica Gel H. Solventsystem ii was used for AR, (Rf 0.48) and solventsystem iii was used for AR2 (Rf 0.43). The sub-stances were eluted from the adsorbant throughsintered-glass filters with chloroform-methanol(1:1, vol/vol) and, after concentration to a smallvolume, were stored under nitrogen at 40C.Characterization ofAR1. AR,, a major com-

ponent of the lipids of encysting cells, was awhite crystalline solid of melting point 90 to92°C. On exposure to air it turned a fawn color.It was insoluble in pure chloroform or puremethanol but was readily soluble in any mixtureof these two solvents.The UV spectrum of AR, in 95% ethanol had

maxima at 275 nm (loge = 3.25) and 281 nm (loge= 3.24). Addition of alkali caused a batho-chromic shift and an increase in the molecularextinction coefficient, which is characteristic ofphenols.A ferric chloride test for phenols was negative,

but a mercuric nitrate test and a vanillan testfor phenols were both positive. This reactionpattern was consistent with AR1 being a 5-n-alkylresorcinol (6).The infrared spectrum has absorption bands

at 3,600 and 3,350 cm-' (OH), 2,955 and 2,890cm-' (aliphatic C-H), 1,605 and 1,475 cm-'(aromatic ring), 1,155 cm-' (phenol C-OH), and840 cm-' (benzene ring substitution).

Direct-probe mass spectroscopy showed thecharacteristic spectrum of 5-n-alkylresorcin-ols (15). The base peak was at m/e 124[(OH)2C8H3+], and there were two apparent mo-lecular ions, a major one (87%) at mle 404 anda minor one (13%) at mWe 432 (Fig. 5). Peakmatching gave molecular formulas of C27H4802and CnH5202 for the major and minor compo-nents, respectively. The side chains are lost asalkenes (C2oH4o and C22H44) to give the basepeak. This occurs by,8-cleavage with hydrogentransfer, which is the predominant method ofcleavage when the oxygen is meta to the longalkyl group. AR1, then, appears to be a mixtureoftwo homologous 5-n-alkylresorcinols with sidechains of C21H43 and C23H47.The homologs of AR1 were separated on two

high-pressure liquid chromatographic columns.Direct-phase chromatography (uPorasil) withchloroform-methanol (90:10, vol/vol) eluted theminor homolog first, whereas reverse-phase

was spotted at the origin on a TLC Silica Gel 60aluminum sheet. Development was with chloroform-methanol (85:15, vol/vol). Resorcinols turned red onexposure to air. (-.*) Presence ofa small amount of alipid substance which was not found in exponential-phase vegetative cells ofA. vinelandii.

VOL. 139, 1979

452 REUSCH AND SADOFF

z IX2 Xe

FIG. 5.(DMs4pcrmo04.2 stebspazo 5nakleocnl (H)C,(H .44i h

z IW 548

galactosew1 576

0 100 200 300 400 500 600

MASS NUMBERFIG. 5. (A) Mass spectrum ofARi. 124 is the base peak of 5-n-alikylresorcinols [(OH)2C6,H4(CH)3~j.404 is the

molecular ion of5-n-heneicosylresorcinol. 432 is the molecular ion of5-n-tricosylresorcinoL (B) Mass spectrumofAR2. 548 is 5-n-heneicosylresorcinol galactose with loss oftwo water molecules. 576 is 5-n-tricosylresorcinolgalactose with loss of two water molecules.

high-pressure liquid chromatography (uBonda-pak C18) with methanol-water (99:1, vol/vol)eluted the major homolog first. The slightlymore polar major homolog was collected andupon mass spectrometry yielded one molecularion at mWe 404.Nuclear magnetic resonance spectroscopy in

CDC13 with a trace of CH3OH (necessary forsolution) indicated three benzylic protons at 6.15ppm and approximately 40 methylene protonsat 1.25 ppm. The hydroxyl protons were notdetectable because of the presence of methanol.The dimethyl ether derivative had the samepeaks and, in addition, six aromatic methoxyprotons at 3.65 ppm.The C27H4802 component identified as 5-n-he-

neicosylresorcinol was reported previously byWenkert et al. (21) in the non-saponifiable lipidsextracted from wheat bran. The C27H6202 hom-olog, 5-n-tricosylresorcinol, has not been re-ported in natural substances.Characterization of AR,. AR2 was a minor

component of the lipids ofA. vinelandii in earlyencystment, but it is present in increasinglylarger amounts as encystment progresses, and,in the mature cyst, AR1 and AR2 were presentin approximately equal quantities.AR2 was a white solid of melting point 100 to

1020C which darkened quickly on exposure toair. The UV spectrum in 95% ethanol had amaximum at 279 to 281 nm (log e = 320) and aminiimum at 256 to 257 nm. Addition of alkaligave the bathochromic shift and increase inmolecular extinction coefficient expected ofphe-nols.The infrared spectrum had absorption bands

at 3,300 cm-l (OH), 1,730 cm- (lactone), 1,630

and 1,580 cm-' (aromatic ring), and 725 cm-'(aromatic ring substituent).AR2 was suspected to be a derivative of AR,.

This suspicion was confirmed by solid-probemass spectroscopy which showed all the majorpeaks of AR,, including the base peak at mle124 and the peaks at m/e 404 and 432. It alsohad peaks at mle 548 (86%) and 576 (14%) (Fig.5). Peak matching of these highest peaks gaveformulas of C33H5606 and C35H6006. AR2 ap-peared to be AR1 with a C611804 substituent.Since water is easily eliminated from sugars inmass spectrometrv, the substituent was believedto be a C6H1206 sugar.

Identification of the sugar in AR,. Theanisaldehyde-sulfuric acid spray reagent ofStahl, when applied to AR2 on silica gel thin-layer plates, gave a green-grey color character-istic of galactose (10).AR2 was hydrolyzed in 3 N HCI in sealed

tubes at 700C for 12 h. After cooling, the hy-drolysate was extracted four times with ether.The HCI was removed from the aqueous layerby repeated evaporation under a stream of nitro-gen. The resulting solid material was dissolvedin a small quantity of distilled water and chro-matographed on thin-layer silica gel plates withtwo ascents, using solvent systems iv and v.Several known hexoses were run at the sametime as the standards. The AR2 sugar had thesame Rf as galactose in both systems (Table 1).The TMS derivative was prepared and sub-jected to gas chromatography on 3% Carbowax-20M at 1600C and on 1.5% SE-30 at 2000C. Theretention time on both columns was identical tothat of D-galactose TMS (Table 1). AR2 sugarTMS and D-galactose TMS cochromatographed

J. BACTERIOL.

5-n-ALKYLRESORCINOLS IN ENCYSTING A. VINELANDII 453

TABLE 1. Chromatographic properties of the carbohydrate moiety ofAR2 and of monosaccharide standardsTLC Gas chromatography

Sugar Anisaldehyde-sulfuric acid Column1dColum 2dreagent (10) Solvent iva Solvent vb (retention (retention(R() (Rf) timne [min]) time [min])

D-Fructose Violet 0.41 0.42 6.8 3.6D-Galactose Green-grey 0.35 0.29 7.5 5.6D-Glucose Light blue 0.39 0.37 8.8 5.1D-Mannose Green 0.46 0.43 7.1 4.1AR2 sugar Green-grey 0.35 0.29 7.5 5.6

an-Butanol-methanol-water (5:3:1, vol/vol/vol); two ascents on precoated Silica Gel 60 aluminum sheets.b Acetone-water (90:10, vol/vol); two ascents on precoated Silica Gel 60 aluminum sheets.e 1.5% SE-30 (6 feet by 0.125 inch) at 2000C.d 3% Carbowax-20M (6 feet by 0.125 inch) at 1600C.

on both columns with symmetrical peaks. Fi-nally, the AR1 sugar TMS and D-galactose TMSgave identical mass spectra.Role of resorcinolic substances. The role

of resorcinolic substances in encystment is notunderstood. However, the facts that they formgalactosides and that the exine contains appre-ciable quantities of both resorcinol and resorci-nol galactosides indicate that they may have astructural role. Preliminary studies of the resor-cinol properties indicate that it produces someinhibition of the growth of gram-positive bacte-ria. Since both the resorcinol and the resorcinolgalactoside are excreted into the culture me-dium, a protective role may also exist.

ACKNOWLEDGMENTISWe gratefully acknowledge the National Institutes of

Health mass spectrometry facility at Michigan State Univer-sity for the mass spectra and William Reusch of the ChemistryDepartment at Michigan State University for the nuclearmagnetic resonance spectra.

This work was supported by Public Health Service grantAI 01863 from the National Institute of Allergy and InfectiousDiseases.

LITERATURE CITED1. Ames, G. F. 1968. Lipids of Sabnonella typhimurium and

Escherichia coli: structure and metabolism. J. Bacte-riol. 90:833-843.

2. Backer, H. J., and N. J. Haack. 1941. Composants dulalex de l'Anacardium occidentale Linn. Recl. Trav.Chim. Pays-Bas 60:661-677.

3. Batrakov, S. G., N. N. Predochena, E. D. Kruglyak,and E. D. Novogrudskaya. 1977. Phenolic lipid fromAzotobacter chrococcum. Khim. Prir. Soedin. 4:494-499.

4. Bligh, E. G., and W. J. Dyer. 1959. A rapid method oftotal lipid extraction and purification. Can. J. Biochem.Physiol. 37:911-917.

5. BuLock, J. D., and A. T. Hudson. 1969. ,8-Leprosol: theidentification of a trialkylresorcinol from bacterial lip-ids. J. Chem. Soc. 6:61-63.

6. Butenandt, A., and F. H. Stodola. 1939. Zur Kenntisvon a- and f-Leprosol. Ann. Chem. 539:40-57.

7. Cirigottis, K. A., L. Cleaver, J. E. T. Come, R. G.

Grasby, G. H. Green, J. Mock, S. Nimigirawath, R.W. Read, R. Ritchie, W. C. Taylor, A. Vadasz, andW. R. G. Webb. 1974. Chemical studies of the Protea-ceae. VII. An examination of the woods of 17 species forresorcinol derivatives. Aust. J. Chem. 27:345-355.

8. Crowder, J. A., F. H. Stodola, and R. J. Anderson.1936. The chemistry of the lipids of tubercle bacilli.XLV. Isolation of a and P leprosol. J. Biol. Chem. 114:431-439.

9. Furukawa, S. 1935. Constituents of Ginkgobiloba L.fruits. IV. Sci. Pap. Inst. Phys. Chem. Res. (Tokyo) 26:178-185.

10. Krebs, K. G., D. Heusser, and H. Wimmer. 1969. Sprayreagents, p. 854-909. In E. Stahl (ed.), Thin-layer chro-matography: a laboratory handbook, 2nd ed. Springer-Verlag, New York.

11. Kumagai, M., Y. Suhara, T. Aoyagi, and H. Vme-zawa. 1971. An enzyme inhibitor, Panosialin, producedby Streptomyces. II. Chemistry of panosialin, 5-alkyl-benzene-1,3-disulfates. J. Antibiot. 24:870-875.

12. Langworthy, T. A., P. F. Smith, and W. R. Mayberry.1972. Lipids of Thermoplasma acidophilum. J. Bacte-riol. 112:1193-1200.

13. Lin, L. P., and H. L. Sadoff. 1968. Encystment andpolymer production by Azotobacter vinelandii in thepresence of 8-hydroxybutyrate. J. Bacteriol. 95:2336-2343.

14. Madrigal, R. V., G. F. Spencer, R. D. Plattner, and C.R. Smith, Jr. 1977. Alkyl- and alkenylresorcinols inRapanea laetevirens seed lipids. Lipids 12:402-406.

15. Occolowitz, J. L. 1964. Mass spectrometry of naturallyoccurring alkenyl phenols and their derivatives. Anal.Chem. 36:2177-2181.

16. Occolowitz, J. L, and A. S. Wright. 1962. 5-(10-Pen-tadecenyl) resorcinol from Grevilleapyramidalis. Aust.J. Chem. 15:855-861.

17. Sadoff, H. L 1975. Encystment and germination in Azo-tobacter vinelandii. Bacteriol. Rev. 39:516-539.

18. Stevenson, L H., and M. D. Socolofsky. 1966. Cystformation and poly-fl-hydroxybutyric acid accumula-tion in Azotobacter. J. Bacteriol. 91:304-310.

19. Tyman, J. H. P. 1967. The identification ofa novel phenolin cashew nut-shell liquid. Chem. Commun., p. 982.

20. Vaskovsky, V. E., and E. Y. Kostetsky. 1968. Improvedspray for detection of phospholipids on thin-layer chro-matograms. J. Lipid Res. 9:396.

21. Wenkert, W., E. M. Loeser, S. N. Mahapatra, F.Schenker, and E. M. Wilson. 1964. Wheat bran phe-nols. J. Org. Chem. 29:435-439.

22. Wilson, P. W., and S. G. Knight. 1952. Experiments inbacterial physiology. Burgess Publishing Co., Minne-apolis.

VOL. 139, 1979