keroseine bacteria

1979, 137(2):795. J. Bacteriol. D G Cooper, J E Zajic and D E Gracey

grown on kerosene.Corynebacterium lepus produced byacids and other fatty acids Analysis of corynomycolic

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Vol. 137, No. 2JOURNAL OF BACTERIOLOGY, Feb. 1979, p. 795-8010021-9193/79/02-0795/07$02.00/0

Analysis of Corynomycolic Acids and Other Fatty AcidsProduced by Corynebacterium lepus Grown on Kerosene

DAVID G. COOPER,l* JAMES E. ZAJIC,l AND D. E. FERGUS GRACEY2Biochemical Engineering, Faculty of Engineering Science, The University of Western Ontario,' and Labatt

Breweries of Canada Ltd.,2 London, Ontario, Canada N6A 5B9

Received for publication 24 November 1978

The saponifiable carboxylic acids of the extracellular product of Corynebacte-rium lepus grown on kerosene have been isolated and characterized. About 25%of these acids were a mixture of simple, saturated fatty acids ranging from C13 toC24 and including both even and odd homologues. The distribution of these acidswas bimodal, with maxima at C15 and C21. The other 75% of the acids was amixture of corynomycolic acids [R1-CH(OH)-CH(R2)-COOH] ranging fromC28 to C43. The R' alkyl fragments varied from C16 to C25, and R2 fragmentsvaried from C6 to C14. Both even and odd corynomycolic acid homologues wereobserved, and the distribution had a single pronounced maximum at C32 andC33. Bacterial utilization of the carboxylic oxidation products of the kerosenesubstrate is suggested to account for the wide distribution in chain length of thesesaturated fatty acids and for the observation of both even and odd homologues.

The scientific literature contains several pa-pers demonstrating the effect of the chain lengthof a hydrocarbon substrate on the distributionof fatty acids found in various microorganisms(16-19, 21, 25, 26, 28, 30). The characterizationof corynomycolic acids (1, 2, 12, 23, 27) and otherfB-hydroxy-a-branched carboxylic acids (4, 7, 12,20, 22) is also well documented. Many of theorganisms, including Corynebacterium, Arthro-bacterium, Nocardia, and related species, fromwhich these 83-hydroxy-a-branched fatty acidsare isolated, are known to grow well on hydro-carbons (8, 23, 27). However, there has beenlittle reported data on the effect of such sub-strates on these complex acids. Several of theseorganisms have been shown to produce surface-active lipids containing hydroxy fatty acids whengrown on hydrocarbons (9, 13, 27), but only onepaper reports a full characterization of the iso-lated corynomycolic acids (27).

Corynebacterium lepus has been recently iso-lated and characterized (8). This organism pro-duced copious amounts of a surface-active prod-uct, when growing on kerosene, which caused asubstantial decrease in the surface tension of thewhole broth. The effective surfactant in thisproduct was found to be a mixture of extractablelipids. The present paper reports the character-ization of the simple fatty acids and the cory-nomycolic acids which have been isolated fromthis C. lepus product and discusses the effect ofthe kerosene substrate on these species.

MATERIALS AND METHODSProduction and isolation of surface-active

product from C. lepus. Gerson and Zajic (8) havealready described the isolation, identification, andmaximization of growth on kerosene of C. lepus. C.lepus whole-broth samples were centrifuged to give aslurry of product and excess kerosene floating on thewater. This slurry was collected by suction and lyoph-ilized. At the end of the fermentation the yield was 2to 3 g of a friable off-white solid per liter.

Extraction of C. lepus lipids. Lipids were ex-tracted from the C. lepus-lyophilized product, usingchloroform-methanol-water (25:25:4) as outlined byKates (10). The resulting yellow oil was extracted withroom-temperature acetone. An acetone-soluble frac-tion was obtained as a yellow oil after evaporation ofthe solvent. Thin-layer chromatography studies indi-cated that this fraction was neutral lipids. A typicalyield was 3% of the original weight of dried product.The acetone-insoluble fraction was a pale-yellow,waxy solid which contained polar lipids (by thin-layerchromatography). This was dissolved in chloroformand filtered, and the solvent was removed. The yieldwas normally more than 20% of the weight of the driedproduct.An alternate extraction procedure was used to ob-

tain samples of lipopeptide free from other lipids.Sufficient HC1 was added to a whole-broth sample toreduce the pH below 2, and then it was extracted withchloroform. This was filtered, the solvent was re-moved, and the residue was washed with acetone. Thelipopeptide was obtained as a white powder in yieldsup to 0.35 g/liter. This lipopeptide was free fromphosphate, carbohydrate, and neutral lipids (by thin-layer chromatography).

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796 COOPER, ZAJIC, AND GRACEY

Saponification of C. lepus products. The cory-nomycolic acids and fatty acids were extracted fromvarious C. lepus products by using a modification ofthe published method (4, 22). To 10 g of solid wereadded 40 ml of benzene and 40 ml of 2.5% (wt/vol)potassium hydroxide in methanol. This was refluxedfor 5 h. The solution was filtered hot and extractedtwice with hot benzene (40 ml). The combined extractswere evaporated to an oil and washed with acetone toremove neutral lipids.

Derivatives of corynomycolic acids and fattyacids. The free acids were obtained by neutralizationof the salts in water with hydrochloric acid, followedby extraction into hexane (10).The methyl esters of the corynomycolic acids and

the fatty acids were also synthesized from the saltsusing anhydrous hydrochloric acid in methanol (10).This reagent was prepared by adding acetyl chlorideto methanol.The trimethylsilyl ether derivatives of the methyl

esters of the corynomycolic acids were prepared withTri-Sil "Z" (Pierce Chemical Co.).The acetate derivatives of the mixed corynomycolic

anhydrides were prepared by reacting the corynomy-colic acids with acetic anhydride in pyridine (1).Chromatography of acids and methyl esters.

All the chromatography studies were monitored withan LKB 8300 Unicord II column monitor (254 nm).The free acids were chromatographed on a column (3cm2 by 30 cm) of LH20 Sephadex (5, 6) eluted with95% chloroform-5% methanol.The methyl esters were chromatographed on a col-

umn packed with alumina (20, 22). The simple fattyacid esters were obtained with hexane, but it wasnecessary to elute with chloroform to remove thecorynomycolic acids.

Instrumentation. Infrared (IR) spectra were ob-tained on a Beckman IR-20 spectrometer, using chlo-roform solutions. Mass spectra were obtained with aVarian Mat CH-7 instrument.

RESULTSIdentification of ,B-hydroxy-a-branched

carboxylic acids by using IR spectroscopy.The mixtures of corynomycolic acids were ini-tially classified as 83-hydroxy-a-branched fatty

acids by comparison of the IR spectra of variousderivatives with values in the literature (1, 2, 4,7, 20, 22, 29). Table 1 compiles the most indica-tive bands of these derivatives prepared fromthe acids extracted from various C. lepus prod-ucts. The potassium salt (4), the free acid (1, 4,20, 22, 29), the methyl ester (7, 20, 22, 29), thetrimethylsilyl ether of the methyl ester (29), andthe anhydride of the acetate (1) of 16-hydroxy-a-branched carboxylic acids have all been de-scribed previously.The three types of compounds with a hydroxyl

function (salt, free acid, and methyl ester) allexhibited a weak v0-H at 3,310 to 3,560 cm-'and -C-O at 1,100 to 1,120 cm-'. These bandswere not observed when the hydroxyl functionwas derivatized as either a trimethylsilyl etheror an acetate.The vC=O band of the carboxyl function was

different for each of the derivatives. The valuesof 1,570 and 1,710 cm-' were typical of the saltsof the acids and the free acids, respectively. Theband at 1,725 cm-' is reasonable for the methylester of a ,B-hydroxy-a-branched carboxylic acidbut lower than that for the methyl esters of asimple fatty acid (10). This phenomenon hasbeen attributed to hydrogen bonding betweenthe carboxyl function and the hydroxyl on the,8-carbon (22). When the hydroxylic proton wasreplaced with a trimethylsilyl group, hydrogenbonding was no longer possible and vC O in-creased to 1,733 cm-'. The acetate derivative ofthe anhydride had two very strong bands at1,835 and 1,755 cm-' typical of an anhydridefunction (10), which obscured the vC=O of theacetate group.IR spectroscopy was also useful in identifying

the simple, saturated fatty acids extracted fromC. lepus products. The spectra of the free acids,the salts, and the methyl esters were virtuallyidentical to the corresponding spectra of penta-decanoic acid and its derivatives.

TABLE 1. IR data (in centimeters-') from chloroform solutions of derivatives of corynomycolic acids[R1-CH(OY)-CHR2-COOX] isolated from C. lepusXa K+ H CH:, CH3 Anhydrideyb H H H Si(CH3)a3 C(O)CH3

PO-HHydroxyl 3,310 3,530 3,560Carboxyl 3,200, 2,700

MC=O 1,570 1,710 1,725 1,733 1,835, 1,755vC OHydroxyl 1,100 1,100 1,120Carboxyl 1,290 1,170 1,165 1,240, 1,070

Substituents of the carboxyl function.Substituents of the hydroxyl function.Other bands observed which were indicative of a trimethylsilyl derivative were 8 SiCH3, 1,248 cm-', and v

Si-C, 840 cm-'.

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CORYNOMYCOLIC ACIDS PRODUCED BY C. LEPUS 797

Extraction of C. lepus floating product.The yield of surface-active product at the end ofa fermentation varied between 2 and 3 g (dryweight)/liter, of which up to 25% was extractablelipid. Saponification of the whole dried productresulted in a mixture of carboxylic acids (yield,ca. 10%). However, saponification of the productresidue after the chloroform-methanol-waterextraction of the lipids yielded no carboxylicacids.The lipid extract could be divided into neutral

(12%) and polar (88%) lipids on the basis ofacetone solubility. The neutral lipids containedvery little saponifiable fatty acids. Saponifica-tion of the polar lipids gave at least 42% of thetotal weight as carboxylic acids. Most of thesaponifiable fatty acids present in the originalproduct were present in the extractable, polarlipid fraction.Chromatography of the carboxylic acids

and their methyl esters. The mixtures of fattyacids obtained by neutralization of the saponifi-cation products were chromatographed on a col-umn of LH20 Sephadex, using 95%chloroform-5% methanol. Figure 1 is a typicaltrace of UV absorption versus elution volume.Essentially the same elution pattern was ob-tained from the saponification products of eitherthe whole floating product or the polar lipidextract. The void volume (40 ml) was marked bya sharp small peak. This peak and the absorptionup to about 60 ml were due to trace amounts ofrelatively strongly UV-absorbing neutral lipidresidues. The large peak centered at 73 ml wasidentified by IR spectroscopy as the /3-hy-droxy-a-branched carboxylic acids. Samplestaken in the broad multi-humped region from 90to 140 ml had IR spectra attributable to simple,saturated fatty acids. Known fatty acids, loadedon the column and eluted with the same solventOD

006-

004-

002-

_, _

40 80 120

Elution Volume ml

FIG. 1. Typical trace of a monitored elution of amixture of fatty acids and corynomycolic acids iso-lated from C. lepus from a column ofLH-20 Sephadex,using chloroform (95%o)-methanol (5%). OD, Opticaldensity.

mixture, confirmed the IR identification of thislast, broad fraction. For example, stearic acidwas eluted at 109 ml and tridecanoic acid waseluted at 123 ml.Although the chromatographs from the Seph-

adex column were useful in comparing mixturesof simple fatty acids and corynomycolic acidsfrom different sources, a much improved sepa-ration of the two types of acids was obtained bychromatography of the mixed methyl esters onalumina. Hexane elution removed only the sim-ple fatty acid esters from the column. Theseamounted to 25% (by weight) of the originalmixture of esters. The corynomycolic acid esterscould be eluted from the column with chloro-form, and this fraction accounted for the re-maining 75% of the ester mixture.

Interpretation of mass spectral data. Theidentification of saturated fatty acids by IR spec-troscopy was confirmed by mass spectra. Sam-ples taken from the last broad fraction from theSephadex column (i.e., between 90 and 140 ml)were esterified and mass spectra were obtained.In every sample only methyl esters of saturatedfatty acids were observed. These were identifiedby their typical fragmentation patterns (15), andthe molecular formulas of several parent ionsand fragments were verified by accurate massmeasurements.The fraction obtained from the hexane eluant

of the alumina column was also verified as amixture of saturated fatty acid methyl esters(C13 to C24). These spectra were used to esti-mate the relative abundance of each homologuefrom the height of the parent ion peaks (Table2). This tabulation was obtained by averagingthe intensities of several spectra at probe tem-peratures ranging from 30 to 90C, all of whichshowed a similar distribution. The estimation of

TABLE 2. Fatty acids isolated from C. lepus asdetermined by the mass spectra of the mixture of

methyl estersSaturated fatty P Relative abun-

acid Parent ion dance (%)aC13 228 3.8C14 242 6.4C15 256 14.4C16 270 9.9C17 284 8.3C18 298 7.6C19 312 26.2C20 326 15.4C21 340 13.3C22 354 2.3C23 368 2.0C24 382 0.3

a Estimated from several spectra obtained over aprobe temperature range from 30 to 90C.

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798 COOPER, ZAJIC, AND GRACEY

the abundance of the lowest-molecular-weighthomologues was obscured due to the fragmen-tation patterns of the heavier esters, and theseare not reported. There did not appear to beappreciable amounts of esters of acids shorterthan tridecanoic acid. Both even and odd hom-ologues were observed, and more than 50% ofthe fatty acid content was due to acids with anodd number of carbon atoms. The distributioi1of fatty acids isolated is illustrated by a plot ofestimated relative abundance versus number ofcarbon atoms (Fig. 2). There were two maximain this plot. The larger was centered on nona-decanoic acid, which accounted for about 25% ofthe saturated fatty acids. There was also a sec-ond smaller maximum at pentadecanoic acid.The mass spectral studies of ,8-hydroxy-a-

branched corynomycolic acids also confirmedthe IR assignments and characterized the twohydrocarbon branches, R1 and R2 [R1-CH(OH)-CH(R2)-COOH]. Two derivativeswere used, the methyl esters and the trimethyl-silyl ethers of the methyl esters. Although frag-mentation patterns of both of these compoundswere dominated by cleavage of the carbon-car-bon bonds on either side of the fl-carbon, theirspectra contained significantly different types ofions. Figure 3 demonstrates the major types offragment ions observed. These ions have beenverified by accurate mass measurements and areconsistent with the observed further fragmen-tation. The spectra were obtained at probe tem-peratures varying from 90 to 130C. For each

type of ion a wide range of homologues wasobserved, including both even and odd numbersof carbon atoms. For many of these series of ionsthe less abundant members were obscured, es-pecially the lightest end of a series, by otherfragmentation patterns.

Parent ions were insignificant for both deriv-atives, but an abundant series of ions was ob-served for the trimethylsilyl ethers due to theloss of a methyl group (Table 3). From these M-15 ions 16 different corynomycolic acids could

OH +OH

R-CHCH-COOCH+-'- IICH-CH-COOCH3+ _R

IR2 R2OHP1-CHO+ R-CIHC-OCH3

R'-CHO + R'-CH = C-OCH3

OSi (CH 3 )3 +OSi(CH 3 )3R'-CH-CH--COOCH'- CH-CH-COOCH3 + pR

1 R2 2

R'- CH =OSi(CH3)3++ R2-CH-COOCH3

+O=Si(CH3 )2R'-CH-CH-COOCH 3 + -CH 3

R2FIG. 3. Possible mass spectral fragmentations giv-

ing rise to the major series of ions observed for themethyl esters and the trimethylsilyl ether of themethyl esters of the C. lepus corynomycolic acids.These assignments were based on studies of similarfunctional groups (15), accurate mass measurements,and secondary fragmentation patterns.

TABLE 3. Corynomycolic acids isolated from C.lepus as determined by the mass spectra of the

mixture ofR'-CH(OSi(CH3)3)-CHR2-COOCH3derivatives

30~

10 20 30Number of Carbon Atoms

FIG. 2. Estimated distributions, according to num-ber of carbon atoms, of the components of the kero-sene substrate (O) (31), the simple fatty acids (0), andcorynomycolic acids (l) isolated from C. lepus andthe precursors of these corynomycolic acids(R'-COOH [0] and R2-CH2-COOH []).

No. of carbons in Parent ion' Relative abun-acid minus 15 dance (%)28 511 229 525 2.530 539 6.831 553 13.832 567 21.533 581 20.834 595 15.035 609 8.436 623 4.237 637 2.338 651 1.339 665 0.840 679 Tr41 693 Tr42 707 Tr43 721 Tr

Ions due to the loss of one methyl group from thetrimethylsilyl function.

30/

15

0. 4 CS

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be definitely identified, having from 28 to 43carbon atoms. This size range is reasonable forthe classification ofthese 46-hydroxy-a-branchedcarboxylic acids as corynomycolic acids (2). Thedistribution of the estimated relative abun-dances of these acids exhibited a single maxi-mum at about 32 or 33 carbon atoms (Fig. 2).The four acids having 31, 32, 33, or 34 carbonatoms accounted for more than 70% of the totalcorynomycolic acids isolated.Three prominent series of fragment ions con-

tained the R2 alkyl chain but not the R' alkylchain. These could be used to calculate the rangeof lengths of R2 present. Two of these wereobserved in the spectra of the ester. The ions[R2CH2COOCH3]+ varied from mass 172 to 270,which indicated 7 to 14 carbon atoms in R2. Theseries of [HOCHCHR2COOCH3]' varied frommass 187 to 299, which indicated 6 to 14 carbonatoms in R2. The trimethylsilyl ether derivativehad one major series of ions useful in determin-ing R2, [(CH3)3SiOCHCHR2COOCH3]+. Themass varied from 287 to 357, which indicated 8to 13 carbon atoms in R2.A single series of ions in the spectra of the

trimethyl ether derivatives, [R1CHOSi(CH3)3]+,was suitable to characterize the R' alkyl group.These ions ranged from mass 327 to 453, andhence R1 varied from 16 to 25 carbon atoms.

Corynomycolic acids are thought to be syn-thesized by bacteria by the condensation of twofatty acids, R'-COOH and R2-CH2-COOH,to give a f8-keto acid, R'-C(O)-CH(R2)-COOH, which is then reduced to the 46-hydroxyacid (2, 22). Thus, it was possible to calculatethe relative amounts of the various fatty acidsused by C. lepus as precursors of corynomycolicacids from the estimated relative abundances ofR' and R2. These data are plotted in Fig. 2. Thisresulted in two separate distributions of fattyacids. The maximum of the R2-CH2-C00Hacids was at dodecanoic acid, and that of theR' COOH acids was at heneicosanoic acid.

DISCUSSION

The surface-active floating product producedby C. lepus when growing on kerosene was foundto contain up to 25% (by weight) extractablelipid and 10% saponifiable saturated fatty acidsand corynomycolic acids. However, these twofractions were not mutually exclusive, and abouthalf the weight of the polar lipid was a similarmixture of simple and corynomycolic acids. Infact, the polar lipid included most of the sapon-ifiable acids of the original product. No acidswere obtained from the residue of the productafter lipid extraction.

Figure 2 represents the distribution of satu-rated fatty acids and corynomycolic acids iso-lated from C. lepus, as well as an estimateddistribution of the saturated hydrocarbons pres-ent in the kerosene substrate (31). The distri-bution of fatty acids used by C. lepus as precur-sors of corynomycolic acids are also included inFig. 2. In all of these distributions there weresubstantial amounts of odd, as well as even,carbon number homologues.The distribution of saponifiable saturated

fatty acids exhibited two maxima. The singlemost plentiful fatty acid was nonadecanoic acid,and the second smaller maximum was centeredaround pentadecanoic acid. The distribution ofthe saturated fatty acid precursors of the cory-nomycolic acids also contained two maxima.However, these two bimodal plots were not su-perimposable. One maximum of the precursoracids was centered at heneicosanoic acid, andthese acids were incorporated exclusively intothe R' alkyl substituent of the corynomycolicacids. This distribution of the R1 precursor acidswas similar to that of the high-molecular-weightsaponifiable acids but shifted slightly towardshigher numbers of carbon atoms.The fatty acid precursofs of the R2 alkyl group

comprised the lowest-molecular-weight fattyacids observed, and the distribution was shiftedto significantly shorter carbon chains than thesecondary maximum in the saponifiable fattyacids. However, the combined distribution of theR2 precursor acids and the lower-molecular-weight saponified acids is similar to the distri-bution of saturated, normal hydrocarbons ob-served in the substrate kerosene. This suggeststhat C. lepus utilizes oxidation products of thesehydrocarbons for at least some of its fatty acidswhich it incorporates into various lipid products,including the corynomycolic acids. This is con-sistent with the observed distribution of thesefatty acids and provides a reasonable explana-tion for the presence of unusually large amountsof odd carbon number fatty acids. Microbialsynthesis of fatty acids by oxidation of hydro-carbon substrates by Micrococcus cerificans(16-18) and other organisms (19, 21, 28, 30) hasbeen previously invoked. In these studies, directcorrelations were observed between the chainlengths of various pure hydrocarbon substratesand the major cellular fatty acids isolated. Stew-art, Kallio, and co-workers (25, 26) have studieda gram-negative coccus in which the hydrocar-bon substrate was reflected in both the fattyacid and the alcohol of an extracellular wax esterproduced by the organism. Fermentations in O18-enriched environments indicated that the com-ponents of cetyl palmitate produced on a hexa-

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800 COOPER, ZAJIC, AND GRACEYdecane substrate were oxidation products of thehydrocarbon.The R' precursor fatty acids and the higher-

molecular-weight saponified fatty acids were alllonger than the hydrocarbon substrates, butthey also included substantial amounts of oddcarbon number homologues, which again impli-cates the kerosene substrate. This could be ex-plained if these fatty acids were the result ofelongation of the shorter fatty acids formed byhydrocarbon oxidation. It has been shown thatvarious types of exogenous fatty acids are usedby microorganisms as primers for fatty acid syn-thetase enzyme complexes (3, 11, 14, 24). Theseprimer acids included odd carbon number fattyacids as well as reasonably long fatty acids.The final plot in Fig. 2 is the distribution of

the corynomycolic acids isolated. The total num-ber of carbon atoms in a corynomycolic acid isa direct consequence of the two precursor fattyacids, and the estimated distribution of coryno-mycolic acids of C. lepus does reflect those ofthe Rl and R2 precursor fatty acids. For example,the peak of the number of carbons in the cory-nomycolic acids (C32) is the sum of the maximaof the R' (C20) and R' (C12) fatty acids. How-ever, for most of the corynomycolic acids ob-served there are more than one combinationi ofR' and R2 which yield the appropriate totalnumber of carbon atoms. The mass spectrum ofa mixture of corynomycolic acids cannot be usedto elucidate the distribution of the componentsof each member because of the overlap of frag-mentation patterns. So far, it has not been pos-sible to isolate individual corynomycolic acids ofC. lepus.Most of the reported, characterized coryno-

mycolic acids contained an even number of car-bon atoms, the most common being the C32 acidcondensed from two molecules of palmitic acid(2). Although the C. lepus corynomycolic aciddistribution is at a maximum at 32 carbon atoms,these acids are unusual in several respects. Thewide range of homologues and the significantnumber of odd numbers of carbon atoms can beattributed to the kerosene substrate as discussedabove. Another unusual feature was that the R'alkyl chains are significantly longer than the R2alkyl chains. There is one other report in theliterature of similar acids. Suzuki et al. (27) haveisolated corynomycolic acids from Arthrobacterparaffineus growing on kerosene. This is alsothe only reported example of the characteriza-tion of ,B-hydroxy-a-branched acids isolatedfrom microorganisms grown on hydrocarbons.Although relative abundances were not esti-mated, the authors did report a mixture of cor-ynomycolic acids similar to those isolated from

J. BACTERIOL.

C. lepus. The range of the acids from A. paraf-fineus was smaller with R' from C18 to C23 andR2 from C7 to C12, but R' was significantlylonger than R2 and both even and odd homo-logues were observed.The corynomycolic acids isolated from orga-

nisms growing on hydrocarbons seem to reflectthe nature of the substrate. With C. lepus, andprobably A. paraffineus, the oxidation productsof the hydrocarbon appear to be precursors ofthe R2 alkyl group and, after being lengthenedby an average of 10 methylene groups, of the R'alkyl group. The distribution of the saponifiable,simple fatty acids also reflects some direct oxi-dation products, but to a greater extent theyreflect elongated homologues generated fromthese fatty acids.

ACKNOWLEDGMENTSThis work was supported by the department of Energy,

Mines and Resources of the Government of Canada.We express our appreciation to D. S. Montgomery for

useful and interesting discussions.

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6. Downey, W. K., R. F. Murphy, and M. K. Keogh. 1970.Separation of fatty acids, phospholipids and chloroplastpigments on Sephadex LH-20. J. Chromatogr. 46:120-124.

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8. Gerson, D. F., and J. E. Zajic. 1977. Surfactant produc-tion from hydrocarbons by Cotvnebacteriuxrnl lepus sp.nov. and Pseudomonas asphaltenicus sp. nov. Dev.Ind. Microbiol. 19:577-599.

9. Iguchi, T., I. Takeda, and H. Ohsawa. 19(69. Emulsify-ing factor of hydrocarbon produced by a hydrocarbon-assimilating yeast. Agric. Biol. Chem. 33:16557-1658.

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11. Ingram, L. O., L. S. Chevalier, E. J. Gablay, K. D.Ley, and K. Winters. 1977. Propionate-induced syn-thesis of odd-chain-length fatty acids by Escherichi'acoli. J. Bacteriol. 131:1023-1025.

12. Ioneda, T., E. Lederer, and J. Rozanis. 1970. Sur lastructure des diesters de trehalore ('cord factors") pro-duits par Nocordio asteroildes et Nocardlio rhocloch-rotis. Cherm. Phsys. L,ipids 4:375-392.


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13. Itoth, S., and T. Suzuki. 1972. Effects of rhamnolipidson growth of Pseudomonas aeruginosa mutant defi-cient in n-paraffin-utilizing ability. Agric. Biol. Chem.36:2233-2235.

14. Lynen, F. 1972. Enzyme systems for fatty acid synthesis.Curr. Trends Biochem. Lipids 35:5-26.

15. McCloskey, J. A. 1969. Mass spectrometry of fatty acidderivatives. Topics Lipid Chem. 1:369-440.

16. Makula, R., and W. R. Finnerty. 1968. Microbial assim-ilation of hydrocarbons. I. Fatty acids derived fromnormal alkanes. J. Bacteriol. 95:2102-2107.

17. Makula, R., and W. R. Finnerty. 1968. Microbial assim-ilation of hydrocarbons. II. Fatty acids derived from 1-alkenes. J. Bacteriol. 95:2108-2111.

18. Makula, R. A., and W. R. Finnerty. 1972. Microbialassimilation of hydrocarbons: cellular distribution offatty acids. J. Bacteriol. 112:398-407.

19. Makula, R. A., P. J. Lockwood, and W. R. Finnerty.1975. Comparative analysis of the lipids of Acinetobac-ter species grown on hexadecane. J. Bacteriol. 121:250-258.

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