JouMAL OF BACTERIOLOGY, Aug. 1979, p. 448-453 0021-9193/79/08-0448/06$02.00/0

Vol. 139, No. 2

5-n-Alkylresorcinols from Encysting Azotobacter vinelandii: Isolation and Characterizationt ROSETTA N. REUSCH AND HAROLD L. SADOFF* Department of Microbiology 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 encystment was initiated by 0.2% f8-hydroxybutyrate. An examination of these compounds led to the isolation and characterization of 5-n-heneicosylresorcinol, 5-n-tricosylresorcinol, and their galactoside derivatives. We previously reported the presence of a 5substituted alkylresorcinol with a C21 side chain in the lipids of encysting Azotobacter vinelandii (17). In this communication we report the isolation and characterization of this novel resorcinol and one of its homologs and their galactoside derivatives. A. vinelandii is a large (2 by 5 ,m), gramnegative bacterium which undergoes a cyclic process of differentiation leading to the formation of spherical, metabolically dormant cysts. The cyst has a volume approximately half that of a vegetative cell and consists of a contracted, highly vacuolated cell (central body) surrounded by a capsule made up of a thin outer layer (exine) and a thicker inner layer (intine). Encystment can be chemically induced by removing glucose from exponential-phase cells and replacing it with fl-hydroxybutyrate, a natural intermediate in the metabolism of the organism (13). Polymeric ,-hydroxybutyrate frequently accumulates as large granules in stationaryphase vegetative cells and is a major component of the central body of cysts (18). We speculated that this shift to /)-hydroxybutyrate metabolism would result in the synthesis of lipids unique to encysting cells. A major portion of these new lipids proved to be 5-n-alkylresorcinols and their glycosides. The natural occurrence of 5-n-alkylresorcinols has been previously reported in the families Anacardiaceae (nut shell) (2, 19), Ginkgoaceae (fruit) (9), Graminae (wheat bran) (21), Proteaceae (seed pods and wood) (7, 16), and, most recently, Myrsinaceae (seed) (14). In this communication we report the formation of novel lipid substances during the encystment of A. vinelandii and the isolation and characterization of four of these substances. This was the first discovery of 5-n-alkylresorcinols or t Journal article no. 8941 from the Michigan Agricultural

Experiment Station.

448

5-n-alkylresorcinol glycosides in procaryotes, although isomeric monomethyl derivatives of 5-nalkylresorcinols (a- and 18-leprosol) have been isolated from Mycobacterium leprae (5, 8) and Streptomyces species contain an enzyme inhibitor (panosialin) which is a mixture of homologs ofsulfated 5-n-alkylresorcinols (11) (Fig. 1). This is also the first report of 5-n-tricosylresorcinol and the galactosides of 5-n-heneicosylresorcinol and 5-n-tricosylresorcinol from natural sources. Since our initial report, 5-n-heneicosylresorcinol and 5-n-nonodecylresorcinol have been isolated from Azotobacter chrococcum (3). MATERIALS AND METHODS Growth and encystment. A. vinelandii ATCC 12837 was used in these studies. The organism was cultured at 300C on a gyratory shaker in 500-ml Erlenmeyer flasks containing 100 ml of Burk nitrogen-free buffer plus 1% glucose (22). The inoculum was 5% of an overnight culture in the same medium. Cell growth was followed by measurement of turbidity (optical density at 620 nm) in a Gilford spectrophotometer. Generation time under these conditions was 3 h. At late exponential phase (optical density at 620 nm, -0.8) the cells were collected by centrifugation, washed twice with Burk buffer to remove all trace of glucose (13), and suspended in Burk buffer to an optical density of -0.5. Encystment was induced by adding,-hydroxybutyrate to a final concentration of 0.2%, and the cells were incubated at 300C with agitation. Encystment was followed by microscopic examination and required 5 to 7 days for completion

(13).

Lipid extraction. Cells were collected by centrifugation and washed once with Burk buffer. Lipids were extracted by the one-phase method of Bligh and Dyer (4) as described by Ames (1). Cell paste was suspended in distilled water, and 2 ml of methanol and 1 ml of chloroform were added for each 0.8 ml of cell suspension. After extraction overnight at 40C, the solids were removed by filtration through glass wool, and 0.25 volume of water and 0.25 volume of chloroform were added. The mixture was shaken vigorously on a Vortex mixer for 2 min and then spun at low

5-n-ALKYLRESORCINOLS IN ENCYSTING A. VINELANDII

VOL. 139, 1979

HO NR III

a

R

a

HOsSO a

a

s

H CtlHa* Cat H44

A. chrococcum

R

TiR

CitrH5s CieHsi CisHul

OCHu

X R

H Galactose Cm H45

CtaH4T A. vineIandii

ox

H3CO R ~R

X

449

a

(CHzOut C H(C H3)a2

~~Cis H3i

S0r H(CHI)guCH(CHB)z C ia HP.?

OSOaM

Ci* Hus M. lepras

Streptomyces

FIG. 1. 5-n-Alkyl resorcinols and their derivatives in procaryotes. speed to separate the layers. The chloroform layer and vol) or (v) acetone-water (90:10, vol/vol) for sugars. interphase were washed once with 0.7% NaCl solution For preparative chromatography of resorcinols, plates and then dried to constant weight with a stream of were dried under nitrogen. Total lipids were detected by exposure to iodine prepurified nitrogen. The lipids were dissolved in a small amount of chloroform-methanol (1:1, vol/vol) vapor, organic phosphate was detected with mercurymolybdate reagent (20), sugars were detected with and stored under nitrogen at 40C. Chromatographic methods. Column chroma- anisaldehyde-suluric acid reagent (10), and amino tography. Primary lipid separation was carried out nitrogen was detected with 0.1% (wt/vol) ninhydrin in acetone. Resorcinols turned red or purple on standing on heat-activated silicic acid (Mallinckrodt analytic reagent, 100 mesh) in a 1.5- by 25-cm column. Columns in air or on exposure to iodine vapor and gave a bright red color with anisaldehyde-sulfuric acid reagent. were prepared from a suspension of silicic acid in Lipids of interest were removed from plates with a chloroform (1 g/5- to 8-mg sample). Lipids were dissolved in chloroform-methanol (1:1, vol/vol) and ab- Brinkmann spot collector and eluted from the adsorbsorbed onto a minimal amount of silicic acid. After ent with chloroform-methanol (1:1, vol/vol), followed evaporation of the solvent, the silicic acid was added by chloroform-methanol (1:2, vol/Vol). Solvent was to the top of the column. This was necessary because removed wtih a stream of nitrogen, and samples were the lipids were not completely soluble in chloroform, stored under nitrogen at 4°C. the first elution solvent. Neutral lipids were eluted Gas chromatography. A Varian Aerograph 1440 with chloroform (15 ml/g of silicic acid), glycolipids gas chromatograph equipped with a flame ionization and resorcinols were eluted with acetone (15 ml/g of detector was used. The carrier gas was helium. Colsilicic acid), and phospholipids were eluted with meth- umns were 6 feet by 0.125 inch (ca. 183 by 0.32 cm), anol (20 ml/g of silicic acid) (12). Fractions were dried stainless steel, packed with either 1.5% SE-30 on to constant weight with a stream of nitrogen, sus- Chrom G A/W (100/120 mesh) or 3% Carbowax-20M pended in chloroform-methanol (1:1, vol/vol), and on Chrom G A/W DMCS (100/120 mesh). Sugar stored under nitrogen at 4°C. trimethylsilyl (TMS) derivatives were chromatoGlycolipids and resorcinols were fractionated with graphed at 200°C on SE30 or at 160°C on CarbowaxFlorisil (Matheson, Coleman, and Bell, 60 to 200 mesh) 20M. Resorcinols were chromatographed on SE-30 in a 1.2- by 25-cm column (1 g/10-mg sample). Elution with temperature programming from 200 to 325°C at was stepwise beginning with chloroform, followed by 6°C/min. increasing concentrations of methanol in chloroform High-pressure liquid chromatography. The diand finally by 100% methanol. The elution volume was rect-phase high-pressure liquid chromatography uti20 ml of solvent per g of Florisil. Separation was lized a stainless-steel column (4-mm inside diameter monitored by analytical thin-layer chromatography by 30 cm) packed with 10-,um fully porous silica par(TLC). ticles (utPorasil, Waters Associates). Reverse-phase TLC. Precoated Silica Gel H glass plates (Analtech high-pressure liquid chromatography was done on a Labs) were used for separating preparative quantities stainless-steel column (4-mm inside diameter by 30 of lipids. Precoated TLC Silica Gel 60 aluminum sheets cm) packed with 10-rIm Bondapak C18 (Waters Asso(EM Labs) were used for analytical separation. No ciates). The sample injector was a Rheodyne model heat activation was used. Development was in solvent 7120 (Anspec Co.). Pressure was applied with a Milton system (i) chloroform-methanol (85:15, vol/vol), (ii) Roy model 396 minipump. Detection was with an chloroform-methanol (90:10, vol/vol), or (iii) chloro- Altex Analytical (model 153) UV-VIS detector at 280 form-methanol-water (65:25:3.8, vol/vol/vol) for lipids nm. Mobile-phase solvents were chloroform-methanol and in (iv) n-butanol-methanol-water (5:3:1, vol/vol/ (90:10, vol/vol) for direct-phase and methanol-water

450

REUSCH AND SADOFF

J. BACTERIOL.

(99:1, vol/vol) for reverse-phase chromatography. 300 Spectrometric methods. UV spectra were taken in 95% ethanol in a Perkin-Elmer double-beam- specI-trophotometer. Infrared spectra were taken as mulls or dilute solutions in chloroform in a Perkin-Elmer 0 700 infrared spectrophotometer. Mass spectra were run in a Varian MAT CH5/DF mass spectrometer I 20( with a direct probe at 3-kV acceleration voltage and 1,000 resolution. Peak matching was done with a resolution of 10,000. The data system was Digital Equipment Corp. PDP 11/05 and PDP 11/40. The sample size was 3,g. Proton magnetic resonance spectra were 31. 100 taken on a Varian T 60 spectrometer with tetramethyl silone as the internal standard. Derivatives. lhe dimethyl ether of AR, was prepared by adding 7 mg of the compound to 300 mg of dimethyl sulfate and 2 g of potassium carbonate in 20 ml of dry acetone and refluxing the mixture for 24 h. ,I _ After filtration the -solvent was removed with a stream 4 5 1 2 3 of dry nitrogen, leaving 7.5 mg of a pale yellow solid (melting point, 53 to 65°C). TIME - DAYS For TMS derivatives of sugars, 1 to 2 mg of the FIG. 2. Dry weight (0) and total extractable lpid solid sugar was added to 1.0 ml of Tri-sil Z (Pierce weight (0) (see text) of A. vinelandii as a function of Chemical Co.) in a small screw-cap septum vial. Mix- time after the initiation of encystment by 0.2% tures were shaken and heated to 70°C for 10 min. hydroxybutyrate. After cooling, the solution was injected into the gas plus 1% glucose. Cells were pregrown in Burk buffer chromatograph. 1?-

RESULTS AND DISCUSSION Lipid composition. Lipids extracted from vegetative cells of A. vinelandii (Bligh-Dyer procedure) were compared with those extracted from cells during the course of encystment (Fig. 2). Only a small fraction of the polymeric f,hydroxybutyrate, a lipid component of late-exponential-phase vegetative cells and of encysting cells, was extracted by this procedure. Total lipid extracted from exponential-phase vegetative cells accounted for 10.6% of the dry weight of the cells. During encystment the cells' content of lipids rapidly increased and ultimately reached 20.2% of the dry weight of the mature cyst. Upon fractionation of the lipids on silicic acid, it was seen that the increase was due almost entirely to lipids in the acetone fraction (glycolipids and resorcinols) (Fig. 3). This fraction was only 7% of vegetative cell lipids but was 82% of the lipids in mature cysts. The weight of the chloroforn fraction (neutral lipids) and the methanol fraction (phospholipids), which comprised 93% of the lipids of exponential-phase vegetative cells, changed only slightly during encystment. Lipids of the acetone fraction. TLC of the acetone fraction of encysting cells in chloroformmethanol (85:15, vol/vol) revealed the presence of two major and several minor substances that were not present in extracts from exponentialphase vegetative cells. The two major substances which were phenolic were designated AR, (Rf 0.70) and AR2 (Rf 0.15) (Fig. 4).

I

60

-*

45 I-~~~~~~~~I

/O

30

'U~~~~~~~.

TIME - DAYS FIG. 3. Composition of the total extractable lipids (see text) as determined by chromatographic separation on silica get as a function of time after the initiation 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 fraction) (U). Cells were pregrown in Burk buffer plus 1% glucose.

Isolation of AR. and AR2. The acetone fraction from silicic acid separation of the lipids extracted from encysting cells was chromatographed on Florisil. AR, was eluted with chloroform-methanol (95:5, vol/vol) and AR2 was eluted with 100%/ methanol. Purification was

VOL. 139, 1979

.-

5-n-ALKYLRESORCINOLS IN ENCYSTING A. VINELANDII 451 completed by TLC on Silica Gel H. Solvent system ii was used for AR, (Rf 0.48) and solvent system iii was used for AR2 (Rf 0.43). The substances were eluted from the adsorbant through sintered-glass filters with chloroform-methanol (1:1, vol/vol) and, after concentration to a small volume, were stored under nitrogen at 40C. Characterization of AR1. AR,, a major comof the lipids of encysting cells, was a ponent ARi white crystalline solid of melting point 90 to 92°C. On exposure to air it turned a fawn color. It was insoluble in pure chloroform or pure methanol but was readily soluble in any mixture of these two solvents. The UV spectrum of AR, in 95% ethanol had maxima at 275 nm (log e = 3.25) and 281 nm (log

e= 3.24). Addition of alkali caused a bathochromic shift and an increase in the molecular extinction coefficient, which is characteristic of

phenols. A ferric chloride test for phenols was negative, but a mercuric nitrate test and a vanillan test for phenols were both positive. This reaction pattern was consistent with AR1 being a 5-nalkylresorcinol (6). The infrared spectrum has absorption bands at 3,600 and 3,350 cm-' (OH), 2,955 and 2,890 cm-' (aliphatic C-H), 1,605 and 1,475 cm-' (aromatic ring), 1,155 cm-' (phenol C-OH), and 840 cm-' (benzene ring substitution). Direct-probe mass spectroscopy showed the characteristic spectrum of 5-n-alkylresorcinols (15). The base peak was at m/e 124 [(OH)2C8H3+], and there were two apparent molecular ions, a major one (87%) at mle 404 and a minor one (13%) at mWe 432 (Fig. 5). Peak matching gave molecular formulas of C27H4802 and CnH5202 for the major and minor components, respectively. The side chains are lost as AR2 alkenes (C2oH4o and C22H44) to give the base peak. This occurs by,8-cleavage with hydrogen transfer, which is the predominant method of cleavage when the oxygen is meta to the long alkyl group. AR1, then, appears to be a mixture of two homologous 5-n-alkylresorcinols with side chains of C21H43 and C23H47. The homologs of AR1 were separated on two high-pressure liquid chromatographic columns. FIG. 4. Thin-layer chromatogram of the glycolip- Direct-phase chromatography (uPorasil) with ids and resorcinols extracted from A. vinelandii 22 h chloroform-methanol (90:10, vol/vol) eluted the after the initiation of encystment by 0.2% fi-hydroxy- minor homolog first, whereas reverse-phase

butyrate. Total lipids extracted from 40 mg (dry weight) of cells were chromatographed on a silicic acid column. Neutral lipids uwre eluted with chloroform, glycolipids and resorcinols were eluted with acetone, and phospholipids were eluted with methanol. Solvent was completely evaporated from the acetone fraction, and the residue was dissolved in 1 ml of chloroform-methanol (1:1, vol/vol). A 10-1l amount

was spotted at the origin on a TLC Silica Gel 60 aluminum sheet. Development was with chloroformmethanol (85:15, vol/vol). Resorcinols turned red on exposure to air. (-.*) Presence of a small amount of a lipid substance which was not found in exponentialphase vegetative cells of A. vinelandii.

452

REUSCH AND SADOFF

J. BACTERIOL.

Xe

IX2

z z W

I

548

2 stebspazo 5nakleocnl (H)C,(H .44i h FIG. 5. (DMs4pcrmo04. w1 galactose 576 0

100

300

200

400

500

600

MASS NUMBER

FIG. 5. (A) Mass spectrum of ARi. 124 is the base peak of 5-n-alikylresorcinols [(OH) 2C6,H4(CH)3~j.404 is the molecular ion of 5-n-heneicosylresorcinol. 432 is the molecular ion of 5-n-tricosylresorcinoL (B) Mass spectrum of AR2. 548 is 5-n-heneicosylresorcinol galactose with loss of two water molecules. 576 is 5-n-tricosylresorcinol galactose with loss of two water molecules.

high-pressure liquid chromatography (uBondapak C18) with methanol-water (99:1, vol/vol) eluted the major homolog first. The slightly more polar major homolog was collected and upon mass spectrometry yielded one molecular ion at mWe 404. Nuclear magnetic resonance spectroscopy in CDC13 with a trace of CH3OH (necessary for solution) indicated three benzylic protons at 6.15 ppm and approximately 40 methylene protons at 1.25 ppm. The hydroxyl protons were not detectable because of the presence of methanol. The dimethyl ether derivative had the same peaks and, in addition, six aromatic methoxy protons at 3.65 ppm. The C27H4802 component identified as 5-n-heneicosylresorcinol was reported previously by Wenkert et al. (21) in the non-saponifiable lipids extracted from wheat bran. The C27H6202 homolog, 5-n-tricosylresorcinol, has not been reported in natural substances. Characterization of AR,. AR2 was a minor component of the lipids ofA. vinelandii in early encystment, but it is present in increasingly larger amounts as encystment progresses, and, in the mature cyst, AR1 and AR2 were present in approximately equal quantities. AR2 was a white solid of melting point 100 to 1020C which darkened quickly on exposure to air. The UV spectrum in 95% ethanol had a maximum at 279 to 281 nm (log e = 320) and a miniimum at 256 to 257 nm. Addition of alkali gave the bathochromic shift and increase in molecular extinction coefficient expected of phenols. 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-probe mass spectroscopy which showed all the major peaks of AR,, including the base peak at mle 124 and the peaks at m/e 404 and 432. It also had peaks at mle 548 (86%) and 576 (14%) (Fig. 5). Peak matching of these highest peaks gave formulas of C33H5606 and C35H6006. AR2 appeared to be AR1 with a C611804 substituent. Since water is easily eliminated from sugars in mass spectrometrv, the substituent was believed to be a C6H1206 sugar. Identification of the sugar in AR,. The anisaldehyde-sulfuric acid spray reagent of Stahl, when applied to AR2 on silica gel thinlayer plates, gave a green-grey color characteristic of galactose (10). AR2 was hydrolyzed in 3 N HCI in sealed tubes at 700C for 12 h. After cooling, the hydrolysate was extracted four times with ether. The HCI was removed from the aqueous layer by repeated evaporation under a stream of nitrogen. The resulting solid material was dissolved in a small quantity of distilled water and chromatographed on thin-layer silica gel plates with two ascents, using solvent systems iv and v. Several known hexoses were run at the same time as the standards. The AR2 sugar had the same Rf as galactose in both systems (Table 1). The TMS derivative was prepared and subjected to gas chromatography on 3% Carbowax20M at 1600C and on 1.5% SE-30 at 2000C. The retention time on both columns was identical to that of D-galactose TMS (Table 1). AR2 sugar TMS and D-galactose TMS cochromatographed

VOL. 139, 1979

5-n-ALKYLRESORCINOLS IN ENCYSTING A. VINELANDII

453

TABLE 1. Chromatographic properties of the carbohydrate moiety of AR2 and of monosaccharide standards TLC Gas chromatography Sugar

acid Anisaldehyde-sulfuric reagent (10)

Solvent iva

Solvent vb

(R()

(Rf)

Column

1d Colum 2d (retention

(retention timne [min])

time

[min])

D-Fructose Violet 0.41 0.42 6.8 3.6 D-Galactose 0.35 0.29 Green-grey 7.5 5.6 D-Glucose 0.39 0.37 Light blue 8.8 5.1 D-Mannose Green 0.46 0.43 7.1 4.1 AR2 sugar 0.35 0.29 7.5 Green-grey 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. Finally, the AR1 sugar TMS and D-galactose TMS gave identical mass spectra. Role of resorcinolic substances. The role of resorcinolic substances in encystment is not understood. However, the facts that they form galactosides and that the exine contains appreciable quantities of both resorcinol and resorcinol galactosides indicate that they may have a structural role. Preliminary studies of the resorcinol properties indicate that it produces some inhibition of the growth of gram-positive bacteria. Since both the resorcinol and the resorcinol galactoside are excreted into the culture medium, a protective role may also exist.

Grasby, G. H. Green, J. Mock, S. Nimigirawath, R. W. Read, R. Ritchie, W. C. Taylor, A. Vadasz, and

8.

9.

10.

11.

ACKNOWLEDGMENTIS We gratefully acknowledge the National Institutes of

12.

Health mass spectrometry facility at Michigan State University for the mass spectra and William Reusch of the Chemistry Department at Michigan State University for the nuclear magnetic resonance spectra. This work was supported by Public Health Service grant AI 01863 from the National Institute of Allergy and Infectious Diseases.

13.

14. 15.

LITERATURE CITED 1. Ames, G. F. 1968. Lipids of Sabnonella typhimurium and Escherichia coli: structure and metabolism. J. Bacteriol. 90:833-843. 2. Backer, H. J., and N. J. Haack. 1941. Composants du lalex 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 from Azotobacter chrococcum. Khim. Prir. Soedin. 4:494499. 4. Bligh, E. G., and W. J. Dyer. 1959. A rapid method of total lipid extraction and purification. Can. J. Biochem.

Physiol. 37:911-917. 5. BuLock, J. D., and A. T. Hudson. 1969. ,8-Leprosol: the identification of a trialkylresorcinol from bacterial lipids. J. Chem. Soc. 6:61-63. 6. Butenandt, A., and F. H. Stodola. 1939. Zur Kenntis von a- and f-Leprosol. Ann. Chem. 539:40-57. 7. Cirigottis, K. A., L. Cleaver, J. E. T. Come, R. G.

16.

17. 18. 19.

20. 21.

22.

W. R. G. Webb. 1974. Chemical studies of the Proteaceae. VII. An examination of the woods of 17 species for resorcinol derivatives. Aust. J. Chem. 27:345-355. 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. Furukawa, S. 1935. Constituents of Ginkgobiloba L. fruits. IV. Sci. Pap. Inst. Phys. Chem. Res. (Tokyo) 26: 178-185. Krebs, K. G., D. Heusser, and H. Wimmer. 1969. Spray reagents, p. 854-909. In E. Stahl (ed.), Thin-layer chromatography: a laboratory handbook, 2nd ed. SpringerVerlag, New York. Kumagai, M., Y. Suhara, T. Aoyagi, and H. Vmezawa. 1971. An enzyme inhibitor, Panosialin, produced by Streptomyces. II. Chemistry of panosialin, 5-alkylbenzene-1,3-disulfates. J. Antibiot. 24:870-875. Langworthy, T. A., P. F. Smith, and W. R. Mayberry. 1972. Lipids of Thermoplasma acidophilum. J. Bacteriol. 112:1193-1200. Lin, L. P., and H. L. Sadoff. 1968. Encystment and polymer production by Azotobacter vinelandii in the presence of 8-hydroxybutyrate. J. Bacteriol. 95:23362343. Madrigal, R. V., G. F. Spencer, R. D. Plattner, and C. R. Smith, Jr. 1977. Alkyl- and alkenylresorcinols in Rapanea laetevirens seed lipids. Lipids 12:402-406. Occolowitz, J. L. 1964. Mass spectrometry of naturally occurring alkenyl phenols and their derivatives. Anal. Chem. 36:2177-2181. Occolowitz, J. L, and A. S. Wright. 1962. 5-(10-Pentadecenyl) resorcinol from Grevilleapyramidalis. Aust. J. Chem. 15:855-861. Sadoff, H. L 1975. Encystment and germination in Azotobacter vinelandii. Bacteriol. Rev. 39:516-539. Stevenson, L H., and M. D. Socolofsky. 1966. Cyst formation and poly-fl-hydroxybutyric acid accumulation in Azotobacter. J. Bacteriol. 91:304-310. Tyman, J. H. P. 1967. The identification of a novel phenol in cashew nut-shell liquid. Chem. Commun., p. 982. Vaskovsky, V. E., and E. Y. Kostetsky. 1968. Improved spray for detection of phospholipids on thin-layer chromatograms. J. Lipid Res. 9:396. Wenkert, W., E. M. Loeser, S. N. Mahapatra, F. Schenker, and E. M. Wilson. 1964. Wheat bran phenols. J. Org. Chem. 29:435-439. Wilson, P. W., and S. G. Knight. 1952. Experiments in bacterial physiology. Burgess Publishing Co., Minne-

apolis.

5-n-Alkylresorcinols from encysting Azotobacter vinelandii: isolation and characterization.

JouMAL OF BACTERIOLOGY, Aug. 1979, p. 448-453 0021-9193/79/08-0448/06$02.00/0 Vol. 139, No. 2 5-n-Alkylresorcinols from Encysting Azotobacter vinela...
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