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JOURNAL OF BACTERIOLOGY, Feb., 1967, p. 636-641 Copyright © 1967 American Society for Microbiology Vol. 93, No. 2 Printed in U.S.A. Synthesis of Saturated Long Chain Fatty Acids from Sodium Acetate-li-C4 by Mycoplasmal J. D. POLLACK2 AND M. E. TOURTELLOTTE Departments of Bacteriology and Animal Diseases, University of Connecticut, Storrs, Connecticut Received for publication 22 October 1966 ABSTRACT Three strains of Mycoplasma, M. laidlawii A and B, and Mycoplasma sp. A60549, were grown in broth containing sodium acetate-i-C'4. The methyl esters of the phospholipid fatty acids of harvested radioactive cells were prepared and identified by comparison of their mobilities to known radioactive fatty acid methyl esters by use of a modified reversed-phase partition-thin layer chromato- graphic technique. No radioactive methyl oleate or methyl linoleate was detected. Compounds migrating as radioactive methyl myristate, stearate, palmitate, and, with less certainty, laurate and octanoate were detected. The qualitative findings for all three organisms appeared similar. M. laidlawii B synthesized a radioactive substance, presumably a saturated fatty acid detected as the methyl ester deriva- tive, which migrated in a position intermediate to methyl myristate-J-C'4 and methyl palmitate-l-C'4. This work indicates that M. laidlawii A and B and Mycoplasma sp. A60549 are capable, in a complex medium containing fatty acids, of synthesizing saturated but not unsaturated fatty acids entirely or in part from acetate. Growth of Mycoplasma laidlawii in a semi- defined medium is promoted by oleic acid (8), and it has been suggested that this organism is incapable of synthesizing unsaturated fatty acids (8, 9). In a defined medium, M. laidlawii B ap- parently requires saturated fatty acids for growth, a requirement which was not replaced by acetate (16). Growth of M. mycoides var. mycoides in a semidefined medium requires additions of both saturated and unsaturated fatty acids (10, 11). Further, the saturated and unsaturated fatty acid content of the membrane of M. laidlawii B mimics the fatty acid composition of the growth medium (9, 13). This organism also incorporates radioactive palmitic or stearic acids added to the growth medium almost exclusively into the mem- brane phospholipid (9). These data, however, do not constitute direct proof of the inability of M. laidlawii B to syn- thesize long chain fatty acids, an inability which has already been questioned (9). This communication presents the results of a I Scientific contribution no. 210 from the Agri- cultural Experiment Station, University of Con- necticut, Storrs. 2 Present address: Department of Clinical Micro- biology, Hadassah Medical School, The Hebrew Uni- versity, Jerusalem, Israel. study which demonstrates synthesis of saturated long chain fatty acids by M. laidlawii B, M. laidlawii A, and Mycoplasma sp. A60549 from sodium acetate-i-C14. MATERIALS AND METHODS Organisms. M. laidlawii A (PG8) and B (PG9) ob- tained from D. G. ff. Edward (Wellcome Research Laboratories, Beckenham, Kent, England) and the saprophytic Mycoplasma sp. A60549 isolated by us from the vagina of a healthy cow at the University of Connecticut were used in this study. Growth media. The basal medium was tryptose broth of the following composition (per liter): tryp- tose (Difco), 20 g; NaCl, 5 g; glucose, 7 g; penicillin G (crystalline), 100,000 units. The pH of the medium was 8.2 to 8.4 without adjustment. A solution of sodium acetate-i-C'4 (0.50 mc/0.73 mg, New England Nu- clear Corp., Boston, Mass.) was sterilized by filtration and was added to the basal medium to give a final concentration of 0.50 ,uc/ml for growth of M. laidlawii B and 0.25 ,uc/ml for both M. laidlawii A and Myco- plasma sp. A60549. For the growth of the latter two organisms, PPLO Serum Fraction (Difco) was added to the basal medium to give a final concentration of 1% (v/v). Cells were grown in 200-ml quantities of medium in screw-capped flasks after inoculation with 2 to 6 ml of 18-hr cultures. The flasks were incubated statically for 18 to 24 hr at 37 C. The organisms were collected 636 on January 27, 2020 by guest http://jb.asm.org/ Downloaded from

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JOURNAL OF BACTERIOLOGY, Feb., 1967, p. 636-641Copyright © 1967 American Society for Microbiology

Vol. 93, No. 2Printed in U.S.A.

Synthesis of Saturated Long Chain Fatty Acids fromSodium Acetate-li-C4 by Mycoplasmal

J. D. POLLACK2 AND M. E. TOURTELLOTTEDepartments of Bacteriology and Animal Diseases, University of Connecticut, Storrs, Connecticut

Received for publication 22 October 1966

ABSTRACT

Three strains of Mycoplasma, M. laidlawii A and B, and Mycoplasma sp.

A60549, were grown in broth containing sodium acetate-i-C'4. The methyl estersof the phospholipid fatty acids of harvested radioactive cells were prepared andidentified by comparison of their mobilities to known radioactive fatty acidmethyl esters by use of a modified reversed-phase partition-thin layer chromato-graphic technique. No radioactive methyl oleate or methyl linoleate was detected.Compounds migrating as radioactive methyl myristate, stearate, palmitate, and,with less certainty, laurate and octanoate were detected. The qualitative findingsfor all three organisms appeared similar. M. laidlawii B synthesized a radioactivesubstance, presumably a saturated fatty acid detected as the methyl ester deriva-tive, which migrated in a position intermediate to methyl myristate-J-C'4 andmethyl palmitate-l-C'4. This work indicates that M. laidlawii A and B andMycoplasma sp. A60549 are capable, in a complex medium containing fatty acids,of synthesizing saturated but not unsaturated fatty acids entirely or in part fromacetate.

Growth of Mycoplasma laidlawii in a semi-defined medium is promoted by oleic acid (8),and it has been suggested that this organism isincapable of synthesizing unsaturated fatty acids(8, 9). In a defined medium, M. laidlawii B ap-parently requires saturated fatty acids for growth,a requirement which was not replaced by acetate(16). Growth of M. mycoides var. mycoides in asemidefined medium requires additions of bothsaturated and unsaturated fatty acids (10, 11).

Further, the saturated and unsaturated fattyacid content of the membrane of M. laidlawii Bmimics the fatty acid composition of the growthmedium (9, 13). This organism also incorporatesradioactive palmitic or stearic acids added to thegrowth medium almost exclusively into the mem-brane phospholipid (9).These data, however, do not constitute direct

proof of the inability of M. laidlawii B to syn-thesize long chain fatty acids, an inability whichhas already been questioned (9).

This communication presents the results of aI Scientific contribution no. 210 from the Agri-

cultural Experiment Station, University of Con-necticut, Storrs.

2 Present address: Department of Clinical Micro-biology, Hadassah Medical School, The Hebrew Uni-versity, Jerusalem, Israel.

study which demonstrates synthesis of saturatedlong chain fatty acids by M. laidlawii B, M.laidlawii A, and Mycoplasma sp. A60549 fromsodium acetate-i-C14.

MATERIALS AND METHODS

Organisms. M. laidlawii A (PG8) and B (PG9) ob-tained from D. G. ff. Edward (Wellcome ResearchLaboratories, Beckenham, Kent, England) and thesaprophytic Mycoplasma sp. A60549 isolated by usfrom the vagina of a healthy cow at the University ofConnecticut were used in this study.

Growth media. The basal medium was tryptosebroth of the following composition (per liter): tryp-tose (Difco), 20 g; NaCl, 5 g; glucose, 7 g; penicillin G(crystalline), 100,000 units. ThepH of the medium was8.2 to 8.4 without adjustment. A solution of sodiumacetate-i-C'4 (0.50 mc/0.73 mg, New England Nu-clear Corp., Boston, Mass.) was sterilized by filtrationand was added to the basal medium to give a finalconcentration of 0.50 ,uc/ml for growth of M. laidlawiiB and 0.25 ,uc/ml for both M. laidlawii A and Myco-plasma sp. A60549. For the growth of the latter twoorganisms, PPLO Serum Fraction (Difco) was addedto the basal medium to give a final concentration of1% (v/v).

Cells were grown in 200-ml quantities of medium inscrew-capped flasks after inoculation with 2 to 6 ml of18-hr cultures. The flasks were incubated staticallyfor 18 to 24 hr at 37 C. The organisms were collected

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by centrifugation at 9,000 X g for 20 min. The sedi-mented cells were washed four times in 100 volumes ofsaline to remove entrapped sodium acetate-l-C'4.

Extraction and separation of phospholipids. Lipidswere extracted from the washed sedimented cells withchloroform-methanol (2: 1) by the filtration method ofTourtellotte et al. (15). The lipid extract in chloro-form-methanol was evaporated to dryness under nitro-gen, and the residue, redissolved in chloroform, wasapplied to a silicic acid column (1). The column wasfirst eluted with chloroform to remove neutral lipids,and then it was eluted with methanol to extract phos-pholipids (9, 15). The phospholipid fraction wasreduced to dryness under nitrogen and the residuewas used for all further analyses.

Analysis ofphospholipids. Methyl esters of the fattyacids of the phospholipid fractions were prepared byincubation of the phospholipid residues at room tem-perature in acidified anhydrous methanol (3). To thesereaction mixtures after 12 hr of incubation, 1 to 2 mlof saline was added. This aqueous solution was thenextracted in the cold with methylbutane. Methylbutaneextracts were pooled, and the solvent was removed byevaporation under a gentle stream of nitrogen (3 ml/min). Particular care was taken to prevent hazardousloss of radioactive methyl esters of fatty acids withcarbon numbers of approximately 10 or less. Theresidues were used as the C'4-fatty acid methyl esterfractions.

Analysis of saturated fatty acid methyl esters. Thereversed-phase partition-thin layer chromatographic(RP-PTLC) two-dimensional technique of Bergelsonet al. (2) was modified and used for the identificationof the methyl esters. For the one-dimensional separa-tion of satuwated fatty acid methyl esters, 20 X 20 cmplates were spread to a thickness of 250 , with aslurry of Silica-gel G (Brinkmann Instruments Inc.,Great Neck, N.Y.), then were dried at room temper-ature, and were immediately heated at 105 C for 1 hr.When the cooling plates were barely warm to thetouch, they were very slowly immersed (0.5 cm/sec)in an impregnating solution composed of 170 ml ofdodecane and 1 liter of hexane. After removal of thehexane by a current of warm air, plates were usedwithin 18 hr, although they could be stored for 5 daysin the absence of desiccant without apparent deteriora-tion of resolving power. Methyl esters were applied asbenzene solutions and resolved with an acetone-acetonitrile (1:1) solvent saturated with dodecane(approximately 6 to 8 ml of dodecane per 100 ml ofacetone-acetonitrile). In this modification, a secondand slower front appeared which was assumed to bethe lower limit of a zone ot excess dodecane. Reducingthe level of dodecane in the impregnating solutionreduced the size of the zone and, in our hands, theresolution of the methyl esters as well. RF values werecalculated by use of this second front and are approxi-mately 10% lower than those originally published (2).

The methyl-C'4 ester of arachidic acid was preparedby reaction of arachidic acid (Applied Science Labo-ratories, Inc., State College, Pa.) with diazomethane-C14, produced from N-methyl-C'4-N-nitroso-p-tolu-enesulfonamide (New England Nuclear Corp., Boston,Mass.), as described by Mangold (5).

Analysis of unsaturated fatty acid methyl esters.The technique of Bergelson et al. (2) was also modifiedand used for the one-dimensional separation of theunsaturated fatty acid methyl esters. Plates were pre-pared through the activation step as described above.When cool, they were sprayed with a 50% (w/v)aqueous solution of AgNO3, air-dried, reactivated,cooled, and used immediately. In some experiments,silver nitrate-impregnated silica gel (ADN-1, AppliedScience Laboratories Inc., State College, Pa.) replacedSilica-gel G and the need for the AgNO3 spray. Inother experiments, commercially prepared plates con-taining AgNO3 (Analtech, Inc., Wilmington, Del.)were used successfully. In practice, benzene solutionscontaining unsaturated and saturated fatty acid methylesters were applied to these plates, which were thenresolved with n-propyl ether-hexane (2:3). In thissystem, saturated esters traveled together near thesolvent front (RF, 100-96), whereas unsaturated esterstrailed behind. Replacement of this solvent system withn-butyl ether-hexane (1:9) gave better separation ofpalmitoleic and oleic methyl esters from each otherand from the saturated methyl esters (T. A. Lostyand J. D. Pollack, unpublished data).

The acetoxymercuri-methyl adducts (AMM) ofmethyl oleate-l-C'4 and methyl linoleate-l-C'4 wereprepared according to Jantzen and Andreas (4).

Radioautography. Thin-layer chromatography(TLC) plates were exposed to No-Screen MedicalX-Ray Safety Film (Eastman Kodak Co., Rochester,N.Y.) for periods up to 50 days. After 50 days of ex-posure, approximately 10-6, c were detected per mm2of the TLC area containing the chromatographed spotof either methyl palmitate-J-C'4 or methyl oleate-J-C'4.This represents a sample of approximately 2 X 10-'uc. Generally, qualitative visualization of componentsin unknown mixtures derived from M. laidlawii B waseffected by an exposure of 5 to 36 hr; further incuba-tion, up to 50 days, failed to reveal additional com-ponents.

Subsequent to development of X-ray films, photo-graphic contact prints were made of the radioauto-graphs, and such "reversed" prints are presented inthis work. Sections of the radioautographs were alsocut out and mounted in masks for examination andtranslation to a line recording in a Spinco-RB Analy-trol (Beckman Instruments, Inc., Palo Alto, Calif.),equipped with a Scan-A-Tron logarithmic cam andmotor, a 500 mp nominal peak filter, and a 0.5-mmslit width.

Standards. Radioactive methyl esters were preparedfrom carboxyl-labeled free fatty acids or salts asdescribed above. Mixtures of radioactive methylesters were chromatographed adjacent to experimentalsamples. Occasionally, methyl-C'4 arachidate was in-corporated into unknown mixtures as an internalstandard, since the free acid had not been detected inM. laidlawii B grown under similar conditions (9).

Nonradioactive methyl esters were synthesized asdescribed from their free fatty acids (Applied ScienceLaboratories Inc., State College, Penn.). The individ-ual C"2-esters were readily visualized by charring atthe 50-,Ag level (2).

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POLLACK AND TOURTELLOTTE

REsULTSWe were unable to obtain adequate resolution

of the saturated methyl esters with the two-dimensional procedure of Bergelson et al. (2).The saturated esters, resolved in the first dimen-sion and subsequently traveling near and parallelto the solvent front of the second dimensionsolvent, "smeared." The omission of the seconddimension resulted in a confluence of the methylesters of oleic and palmitic acids which did notcompletely resolve in the first dimension.

It was possible to separate the unsaturatedesters and to remove them by scraping afterformation of their AMM derivatives by prepara-tive TLC on AgNO3-impregnated silica gel in ann-propyl ether-hexane system. However, recoverywas generally poor, ranging between 75 and 95%.Figure 1, plate A, shows that in this system amixture of six radioactive saturated fatty acidmethyl esters (lanes 1 and 8) could be clearlyresolved from the migrating methyl oleate-l-C'4(lane 3) and methyl linoleate-l-C'4 (lane 6), fromtheir immobile free acids (lanes 2 and 5, respec-tively), and from AMM derivatives (lanes 4 and7, respectively).

Later, it was found that M. laidlawii B pro-

duced no detectable radioactive oleate (Fig. 1,plate B, lanes 1 and 2), as evidenced by the ab-sence of a spot comparable to a methyl oleate-l-C14 standard (lane 3) after 50 days of exposure.In the same figure, methyl oleate-J-C'4 and methyllinoleate-l-C"4 were resolved in lane 4. Lane 5contained the same sample of impure methyllinoleate-J-C'4, and lane 6, methyl palmitate-l-C14. In another solvent system, ethyl ether-hexane(1:1), the ability of methyl linoleate to migratewas demonstrated, and with the same samplesfrom M. laidlawii B, its presence could not bedetected after 45 days of exposure. Earlier in-vestigation did not demonstrate the presence ofthis unsaturated acid in M. laidlawii B under simi-lar growth conditions, but oleic acid as the methylester was found (9).The total radioactivity of the unseparated C14-

fatty acid methyl ester fraction of M. laidlawil Bwas 10-1 tic. This is a conversion of 0.1% of the100 ,uc of acetate-i-C'4 added to the growthmedium. As no methyl oleate-l-C'4 was detectedafter 50 days of exposure, its presence did notexceed 2 X 10-' AC (see Materials and Methods,Radioautography). This amount, 2 X 10-' Mc,therefore, represents a portion which is consid

FIG. 1. Thin-layer chromatography offatty acid methyl esters. Plates: Silica-gel G sprayed with aqueous silvernitrate. Solvent: n-propyl ether-hexane (2:3). Detection: radioautography. Plate A: 36 hr of exposure, 1O-3 PC ofeach standard. Lanes I and 8: methyl octanoate-l-C'4, methyl laurate-i-C14, methyl myristate-i-C14, methylpalmitate-i-C14, methyl stearate-i-C'4, methyl-C'4 arachidate. Lane 2: oleic acid-i-C14. Lane 3: methyl oleate-l-C14. Lane 4: acetoxymercuri-methoxy methyl oleate-i-C'4. Lane 5: linoleic acid-i-C'4. Lane 6: methyl linoleate-l-C14. Lane 7: acetoxymercuri-methoxy methyl linoleate-i-C14. Plate B: 50 days of exposure, 4 X 10-4 jC of eachstandard. Lanes I and 2: C'4-fatty acid methyl ester fraction of Mycoplasma laidlawii B. Lane 3: methyl oleate-l-C14. Lane 4: methyl oleate-i_-C4 and methyl linoleate-i-C'4. Lane 5: methyl linoleate-l-C'4. Lane 6: methyl palmi-tate-_-C'4.

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erably less than one-thousandth of the total radio-activity, 10-1 ,tc, of the C'4-fatty acid methyl esterfraction.

Figure 2 shows a typical separation andAnalytrol translation of the radioautographedmethyl esters of octanoic, lauric, myristic, pal-mitic, stearic, and arachidic acids (left to right)by use of the solvent system of acetonitrile-acetone (1:1) saturated with dodecane. Themethyl esters of behenic and the slower movinglignoceric acids were separable from each otherand the above esters by the same system (J. D.Pollack and M. E. Tourtellotte, unpublished data).These latter two esters followed methyl arachi-date. Hence, methyl esters of the even-numberedfatty acids from C8 to C24, excluding C,O, wereresolvable in one dimension.

Figure 3 shows a reversed print of the radio-autograph of three levels of the C'4-methyl esterfraction of M. laidlawii B (sample A, A', and B)and the methyl esters of carboxyl-labeled oc-tanoic, lauric, myristic, palmitic, and stearic acids(sample C, spots a, b, c, d, e, respectively). Figure4 shows an Analytrol translation of the radio-autograph used for preparing Fig. 3. Sections A,B, and C of Fig. 4 are the translation of theequivalently designated samples of Fig. 3. It isseen in both figures that there are areas within theresolved M. laidlawii B samples that migrate asC'4-methyl myristate, palmitate, and stearate insample C. Also in Fig. 4, sample A, a peak, notedwith an arrow, migrated as methyl octanoate(Fig. 4, sample C, peak a). In Fig. 4, sample B, apeak, also noted with an arrow, migrated be-tween the apparent methyl myristate and pal-mitate peaks. The shoulders in Fig. 4, samples Aand B, to the left of the myristate peak, suggestthe presence of an ester with a lower carbonnumber, perhaps methyl laurate (see Fig. 4,sample C, peak b).A C'4-fatty acid methyl ester fraction was also

isolated from M. laidlawii A and Mycoplasma sp.

f ~ ~ , -,/\\

FIG. 2. Translation of RP-PTLC radioautograph ofC14-saturated fatty acid methyl esters. Solvent: aceto-nitrile-acetone (1:1) saturated with dodecane; migrationfrom right to left. Left to right: methyl octanoate-1_-C'4,RF 0.91; methyl laurate-l-C'4, RF 0.79; methyl my-ristate-1-C14, RF 0.63; methylpalmitate-l-C'4, RF 0.44;methyl stearate-_-C14, RF 0.28; methyl-C'4 arachidate,RF 0.13.

FIG. 3. RP-PTLC of fatty acid methyl esters.Plates: Silica-gel G, impregnation with dodecane.Solvent: acetonitrile-acetone (1:1) saturated withdodecane. Detection: radioautography. Lanes A, A',and B reveal C'4-fatty acid methyl ester fraction ofMycoplasma laidlawii B. Lane C reveals methyl octa-noate-J-C'4 (a), methyl laurate-l-C'4 (b), methylmyristate-1-C'4 (c), methyl palmitate-l-C'4, (d) methylstearate-l-C'4 (e).

A60549. After subjection to the RP-PTLC tech-nique described above, both organisms demon-strated areas with the same values for RF as thosefound in M. laidlawii B, except for the absence ofthe material noted by an arrow in Fig. 4, sampleB. The qualitative findings for all three organismsappeared similar. However, with M. laidlawii Aand Mycoplasma sp. A60549 only about 0.001 %of the acetate-i-C'4 radioactivity added to thegrowth medium was found in the unseparatedC'4-fatty acid methyl ester fraction.

DISCUSSIONThe ability of M. laidlawii B to incorporate

radioactivity from sodium acetate-l-C'4 into anonsaponifiable lipid fraction was first demon-strated by Smith and Rothblat (14). Later Smithand Henrikson (12) demonstrated the incorpora-tion of acetyl coenzyme A (CoA)-C'4 into s-hydroxy-,3-methyl-glutaryl CoA by condensation

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POLLACK AND TOURTELLOTTE

E0-

0

!q

Su

z

so0

FIG. 4. Translation of RP-PTLC radioautograph ofthe C'4-fatty acid methyl ester fraction of Mycoplasmalaidlawii B. Sections A, B, and C represent translationsoflanes A, B, and C of Fig. 3. Sections A and B repre-

sent C14-fatty acid methyl ester fraction ofM. laidlawiiB. Lane C represents methyl octanoate-1-C"4 (a),methyl laurate-1-C'4 (b), methyl myristate-J-C"4 (c),methyl palmitate-_-C'4 (d), methyl stearate-J-C'4 (e).

into acetoacetyl CoA and the incorporation ofradioactivity from sodium acetate-2-C'4 intomevalonic acid by the same organism. Further,the high lipid content of Mycoplasma spp. iscompatible with the demonstrated need for co-

enzyme A of M. laidlawii B (16). Our work showsthat this organism can also incorporate C14-acetate into long chain fatty acids, identified as

myristic, palmitic, and stearic acids. With lesscertainty, lauric and octanoic acids were alsodetected.

Since the inability to detect any C14 from acetatein unsaturated fatty acids in our experiments issupported by other data (8, 9, 16) demonstratinga nutritional requirement for oleic acid, it wouldappear that M. laidlawii B is lacking in the abilityto synthesize unsaturated fatty acids from acetate.

In this light, the presumable presence of a C14-fatty acid ester, noted by an arrow in Fig. 4,sample B, which migrates between methylmyristate and palmitate is worth noting. O'Leary(6), working with another Mycoplasma sp.,detected three compounds migrating betweenmethyl myristate and palmitate: a C16 unsatu-rate; probably a C15 cyclopropane saturate; and,identified with less certainty, a C15 unsaturate.Presently, we have eliminated the first possibilitysince no radioactive spot could be detected ondodecane or AgNO3-impregnated silica-gel platesmigrating as methyl palmitoleate. The possibilitythat this acid is a C15 saturate also cannot be ig-nored.The observation (16) that M. laidlawii B would

not grow in medium devoid of saturated fattyacids is difficult to reconcile with data presentedhere. Recent work (R. N. McElhaney and M. E.Tourtellotte, unpublished data), however, maygive insight into this seeming paradox. M. laid-lawii B, when grown in the presence of shortchain fatty acids (C6, C8, and C10), showed signifi-cant increases in palmitic acid, suggesting thatthe carbon chains were being elongated. The pos-sibility thus arises that M. laidlawil B is capableof adding C2 units to shorter chain fatty acids butis incapable of effecting de novo synthesis fromacetate alone.The ability of M. laidlawii A and Mycoplasma

sp. A60549 also to incorporate acetic acid intolong chain fatty acids demonstrates that thisbiosynthetic ability is not peculiar to M. laidlawiiB. However, since both of these additional strainsare also saprophytes, caution should be used inrelating these findings to all Mycoplasma. Pre-liminary work suggests that at least some of thecholesterol-requiring strains are incapable of in-corporating acetic acid into long chain fattyacids.The RP-PTLC procedure of Bergelson et al.

(2), as modified by us, was uniformly successfuland reproducible, requiring only simple equip-ment and modest expenditures of manipulativetime and supplies. Further, because it was non-degradative, the technique allowed for recovery ofsamples, though not quantitatively. Recently,Paulose (7) has developed a one-dimensionalprocedure to resolve unsaturated and saturatedfatty acid methyl esters; however, this techniquedoes not resolve methyl laurate and presumablyshorter methyl esters from methyl oleate.

ACKNOWLEDGMENTS

This investigation was supported by Public HealthService Research Grant Al 05992 from the NationalInstitute of Allergy and Infectious Diseases and by

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MYCOPLASMA SYNTHESIS OF FATLY ACIDS

Training Grant 5-TIGM-317 (to J. D. P.) from theNational Institutes of Health.

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8. RAZIN, S., AND S. ROTTEM. 1963. Fatty acid re-

quirements of Mycoplasma laidlawii. J. Gen.Microbiol. 33:459-470.

9. RAZIN, S., M. E. TOURTELLOTTE, R. N. McEL-HANEY, AND J. D. POLLACK. 1966. Influence oflipid components of Mycoplasma laidlawlimembranes on osmotic fragility of cells. J.Bacteriol. 91:609-616.

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12. SMITH, P. F., AND C. V. HENRIKSON. 1965. Com-parative biosynthesis of mevalonic acid byMycoplasma. J. Bacteriol. 89:146-153.

13. SMITH, P. F., W. L. KOOSTRA, AND C. V. HENRIK-SON. 1965. Diphosphatidyl glycerol in Myco-plasma laidlawii. J. Bacteriol. 90:282-283.

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15. TOURTELLOTTE, M. E., R. G. JENSEN, G. W.GANDER, AND H. J. MOROWITZ. 1963. Lipidcomposition and synthesis in the pleuropneu-monia-like organism Mycoplasma gallisepticum.J. Bacteriol. 86:370-379.

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