Biosynthesis of Saturated and Unsaturated Fatty Acids by a

JOURNAL OF BACTERIOLOGY, Oct. 1976, p. 170-173 Copyright C 1976 American Society for Microbiology

Vol. 128, No. 1 Printed in U.S.A.

Biosynthesis of Saturated and Unsaturated Fatty Acids by a T-Strain Mycoplasma (Ureaplasma) NINO ROMANO,' SHLOMO ROTTEM, AND SHMUEL RAZIN* Department of Clinical Microbiology, The Hebrew University-Hadassah Medical School, Jerusalem, Israel Received for publication 1 July 1976

A human T mycoplasma (Ureaplasma urealyticum) incorporated radioactivity into its lipids from [1-'4C]acetate in the growth medium. Methanolysis of the lipids showed the label to be confined almost entirely to the methyl esters of the fatty acids. About 80% of the label was associated with the methyl esters of the saturated fatty acids, and the rest was found in the unsaturated methyl ester fraction. Gas-liquid chromatography of the saturated methyl esters showed the label to be present in the peaks of palmitate, myristate, and stearate, whereas in the unsaturated methyl ester fraction most of the radioactivity emerged in the peak of palmitoleate. The addition of either oleic or palmitic acid to the growth medium markedly decreased the organisms' incorporation of radioactivity from acetate. It is concluded that the T mycoplasma strain is capable of de novo synthesis of both saturated and unsaturated fatty acids, in this respect differing from all of the Mycoplasma and Acholeplasma strains investigated to date.

The ability of mycoplasmas to synthesize membrane lipids is restricted, and several lipids and lipid precursors, essential for membrane structure and function, have to be supplied exogenously (8). None of the Mycoplasma species tested so far is able to synthesize longchain fatty acids or to elongate, desaturate, or interconvert any preformed fatty acid added to the growth medium (3, 10, 11, 15, 19). Acholeplasma species are capable of utilizing acetate for fatty acid synthesis (3, 15) and exhibit acyl carrier protein activity (13). However, the Acholeplasma species tested so far were found capable of synthesizing only saturated fatty acids (6, 15). The T mycoplasmas are distinguished from all other known mycoplasmas by their possession of urease and were recently given a status of a new genus, Ureaplasma, in the family of the sterol-requiring Mycoplasmataceae (18). Little is known at present about the lipid-synthesizing ability of the T mycoplasmas. Like other mycoplasmas and acholeplasmas, the T mycoplasmas can incorporate exogenous palmitic and oleic acids into their cell lipids (12). Nevertheless, the addition of various saturated and unsaturated fatty acids to a lipid-poor medium supplemented with cholesterol did not stimulate their growth, leading to the suggestion that these organisms, unlike all other mycoplasmas, do not require fatty acids for growth (14). However, since the medium could have I Permanent address: Instituto d'Igiene dell' Universita di Palermo, 90141 Palermo, Italy.

contained fatty acids in amounts sufficient for the rather limited growth characterizing the T mycoplasmas, no definite conclusion could be drawn from these experiments (18). The present communication provides direct evidence for the ability of a T mycoplasma to synthesize both saturated and unsaturated long-chain fatty acids from acetate. MATERIALS AND METHODS Organisms and growth conditions. The T mycoplasma studied was strain P108 isolated from a human vagina in the laboratories of the Institute of Hygiene, University of Palermo, Palermo, Italy. This strain can be classified as Ureaplasma urealyticum (18) on the basis of its habitat, cultural and biochemical properties, and serological cross-reactivity with serotype VI (Pi, ATCC 27818) of U. urealyticum. Acholeplasma laidlawii (oral strain) and Mycoplasma hominis (ATCC 15056) were included in our study for comparison. The organisms were grown in a modified Edward medium (7) supplemented with 5% horse serum. For the growth of the T mycoplasma the medium was also supplemented with 0.05% urea and its pH was adjusted to 6.0, whereas for the growth of M. hominis the medium was supplemented with 20 mM L-arginine and its pH was adjusted to 6.5. Sodium [1-_4C]acetate (41 mCi/ mmol) or [1-14C]oleic acid (59.7 mCi/mmol), purchased from the Radiochemical Centre, Amersham, England, were added to the growth medium in amounts of 1 /iCi/100 ml of medium. The organisms were harvested after incubation at 37'C for 16 to 24 h by centrifugation at 25,000 x g for 30 min. The sedimented organisms were washed twice in 0.25 M NaCl. Extraction and fractionation of cell lipids. 170

VOL. 128, 1976

BIOSYNTHESIS OF FATTY ACIDS BY UREAPLASMA

Washed, packed organisms were extracted twice for 1-h periods with chloroform-methanol (2:1) at room temperature. The lipids were separated into major classes by passage through a column (0.8 by 3 cm) of activated silicic acid (100 mesh; Mallinckrodt Chemical Works, St. Louis, Mo.). The neutral lipids were eluted with 20 ml of chloroform, and the polar lipids were subsequently eluted with 20 ml of methanol. Preparation of methyl esters of fatty acids. Cell lipids extracted with chloroform-methanol were subjected to methanolysis by heating in 5% HCI in anhydrous methanol for 4 h at 80 to 100°C in tubes fitted with Teflon-lined screw caps. The methyl esters were extracted with hexane, and the extract was chromatographed on a silicic acid column (0.8 by 3 cm). Elution from the column was carried out successively with 10 ml of hexane, 20 ml of 3% diethyl ether in hexane, and 10 ml of 8% diethyl ether in hexane. The fraction eluted with 3% diethyl ether in hexane contained the purified methyl esters of the fatty acids (2). Separation of methyl esters of saturated and unsaturated fatty acids. The purified methyl ester fraction was chromatographed on a column (0.8 by 3.5 cm) of silica gel impregnated with AgNO3 (2). Elution was carried out with 15-ml portions of increasing concentrations of diethyl ether (1 to 8%) in hexane. The saturated methyl esters are eluted with 1 to 2% diethyl ether in hexane, whereas the unsaturated methyl esters are eluted with 4 to 8% diethyl ether in hexane (2). The efficiency of the chromatographic method was checked and confirmed with the methyl esters of labeled palmitic and oleic acids. Nearly all of the methyl ester of palmitic acid was eluted from the column with 1% diethyl ether in hexane, whereas the methyl ester of oleic acid was eluted with 4 to 6% diethyl ether in hexane. Hydrogenation of methyl esters of the unsaturated fatty acids was carried out by the method of Brian and Gardner (1). Gas-liquid chromatography. Methyl esters of the fatty acids of T mycoplasma were examined by capillary gas-liquid chromatography (5) in a Perkin Elmer model 990 apparatus, using a polar column (1.50 feet by 0.01 inch) [ca. 45.72 by 0.02 cm] coated with Carbowax K-20 plus V-93 (99:1). Fatty acids were then identified by their retention time relative to that of standard methyl ester mixtures (Applied

171

Science Laboratories, College Park, Pa.). Pooled fractions of the methyl esters of the saturated and of the unsaturated fatty acids of the T mycoplasma grown with [1-'4C]acetate were chromatographed on a packed column (200 by 0.3 cm; 15% of diethylene glycol adipate on Chromosorb W) as previously described (16). Fatty acid fractions were collected into scintillation vials containing 10 ml of dioxane-toluene scintillation liquor (17) and counted. Analytical methods. Cell protein was determined by the method of Lowry et al. (4) with crystalline bovine serum albumin as standard. Radioactivity in cells or in lipid preparations was determined in a Packard Tri-Carb liquid scintillation spectrometer, using dioxane-toluene scintillation liquor (17).

RESULTS

The ability of the T mycoplasma to incorporate radioactivity from [1-_4C]acetate and [1'4Cloleate into its lipids is compared with that of A. laidlawii and M. hominis (Table 1). The results demonstrate that oleic acid was incorporated into the lipids of all three organisms, although the values for the T mycoplasma were lower. The values for radioactivity derived from acetate were much higher for the T mycoplasma and A. laidlawii than for M. hominis. Moreover, the ratio of radioactivity derived from acetate to that derived from oleate was the highest for the T mycoplasma. Table 2 shows that the radioactivity derived from [1-'4C]acetate was about equally distributed between the neutral and polar lipid fractions of the T mycoplasma, whereas in A. laidlawii over 85% of the radioactivity was found in the polar lipid fraction. Methanolysis of the [1-''C]acetate-labeled lipids and purification of the methyl esters of the fatty acids by silicic acid column chromatography resulted in the recovery of over 80% of the radioactivity in the purified methyl ester fraction of both the T mycoplasma and A. laidlawii. Separation of the methyl esters of the saturated and unsaturated fatty acids by chro-

TABLE 1. Incorporation of radioactivity from [1-'4C]acetate and [1-14C]oleate by growing mycoplasmas adioactivity (cpm/mg of protein) OtRYield of organ compound Yield(m ofpora-_____________ Labeledadded Organism Whole orgaTotal lipids tein) nisms

isse(mionpo

T mycoplasma (U. urealyticum)

Oleate Acetate

1.0 0.9

18,720 1,650

17,600 1,555

A. laidlawii

Oleate Acetate

63.0 59.0

38,078 1,940

36,500 1,810

6.0 Oleate 28,362 510 5.0 Acetate a Grown in 500 ml of Edward medium containing 5% horse serum and 5 ,Ci of sodium 4C]oleic acid. The organisms were harvested after 18 h of incubation at 37°C. M. hominis

27,030 375

[1-'4C]acetate or [1-

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J. BBACTERIOL.

ROMANO, ROTTEM, AND RAZIN

ester fraction emerged with the methyl ester of palmitate, whereas the remainder emerged with the methyl esters of myristate and stearate. Most of the radioactivity of the, unsaturated methyl ester fraction emerged with a peak of methyl hexadecenoate, whereas little radioactivity was found in the peak of methyl tetradecenoic acid. The hexadecenoic and tetradecenoic methyl esters were further identified by capillary gas-liquid chromatography as cis9-hexadecenoate (palmitoleate) and cis-7-tetradecenoate, respectively. After hydrogenation of the unsaturated methyl ester fraction, both the TABLz 2. Distribution of radioactivity from [14C]acetate in the neutral and polar lipid fractions of mass and radioactivity moved to the 16:0 and 14:0 position. the T mycoplasma and A. laidlawii Since it was shown that the T mycoplasma Radioactivity (cpm) utilized acetate for fatty acid synthesis, it beNeutral Polar Organism Total came of interest to determine whether fatty lipids lipids lipids acids added to the growth medium will repress acetate utilization, as was demonstrated for A . T mycoplasma (U. 3,330 2,950 6,640a laidlawii by Rottem and Razin (15). Table 3 urealyticum) A. laidlawii 703 4,000 5,200b shows that palmitate and, even more so, oleate most significantly decreased the incorporation a Extracted from a cell pellet containing 5 mg of of radioactivity from acetate into the cell lipids protein. b Extracted from a cell pellet containing 4 mg of of the T mycoplasma. protein. DISCUSSION Our experiments demonstrate the ability of a 6 human T mycoplasma strain to synthesize o A. LAIDLAWII long-chain fatty acids from acetate. This ability distinguishes the T mycoplasma from all other 5 sterol-requiring mycoplasmas tested for this property. Resembling A. laidlawii (6), the major saturated fatty acids synthesized by the T mycoplasma were palmitate, myristate, and stearate. However, in marked contrast with the E findings for A. laidlawii, a minor but significant portion of the label derived from acetate 0 was detected in the unsaturated fatty acid fraction of the T mycoplasma. Gas-liquid chroma-

matography on silica gel impregnated with silver nitrate showed the label to be confined exclusively to the saturated methyl esters in A. laidlawii, whereas about 20% of the label was recovered in the unsaturated methyl ester fraction of the T mycoplasma (Fig. 1). Figure 2 shows the distribution of radioactivity in the methyl esters of the fatty acids of the T mycoplasma grown with [1-'4C]acetate, as resolved by gas-liquid chromatography. About 60% of the radioactivity of the saturated methyl

-

-

3

2-

-J

9

0

4 0

ILi. 0 I-

0r

w C')

z 0 a.

CL) w a:

w 1OU

6 2 4 8 PERCENTAGE OF DIETHYL ETHER IN HEXANE FIG. 1. Distribution of radioactivity in methyl esters of fatty acids eluted from a column of silica gel

impregnated with AgNO3. The saturated methyl esters are eluted with 1 to 2% diethyl ether in hexane, whereas the unsaturated methyl esters are eluted with 4 to 6% diethyl ether in hexane. The organisms were grown with [1-l4C]acetate.

0

a:0C.) a:-w

5r FIG. 2. Distribution of radioactivity in fatty acids of the T mycoplasma grown with [1- 14C]acetate. The unsaturated and saturated methyl ester fractions (see Fig. 1) were resolved by packed column gas-liquid chromatography (15). The rods show the radioactivity in the fatty acid methyl ester fractions.

BIOSYNTHESIS OF FATTY ACIDS BY UREAPLASMA

VOL. 128, 1976

tography showed almost all of the radioactivity in this fraction to be associated with palmitoleate. The addition of oleate and palmitate to the growth medium was shown to markedly inhibit the incorporation of radioactivity from acetate by the T mycoplasma (Table 3). Since the growth medium utilized contained substantial quantities of fatty acids, mostly from the horse serum component, it is possible that the fatty acid-synthesizing capacity of the T mycoplasma could not be fully expressed under our experimental conditions. The label from [1-'4C]acetate was distributed about equally between the neutral and polar lipid fractions of the T mycoplasma, whereas in A. laidlawii almost all of the label was found in the polar lipid fraction. This finding is in accord with the much higher content of free fatty acids, sterol esters, and di- and triglycerides in the T mycoplasma (12). When grown with radioactive oleate, the ratio of incorporated radioactivity to cell protein was much lower for the T mycoplasma than for A. laidlawii and M. hominis. This should not, however, be taken to indicate a lesser capacity of the T mycoplasma to incorporate exogenous fatty acids. The lower ratio in the T mycoplasma could result from the presence of serum proteins contaminating the sedimented organisms. Since the yield of T mycoplasmas is so low, the small amounts of proteins from the medium that precipitate during growth and sediment together with the organisms on centrifugation may constitute a large percentage of the total protein in the pellet (9) and thus artificially reduce the values for labeled oleate incorporation or acetate utilization when expressed as radioactivity per milligram of protein. Even though the complete potential for fatty acid synthesis of the T mycoplasma was, most probably, not expressed under our experimental conditions, our results suffice to distinguish the T mycoplasma strain we tested from all of the Mycoplasma and Acholeplasma species examined for fatty acid synthesis ability, lending TABLE 3. Inhibition of incorporation of radioactivity from [1-'4C]acetate into lipids of the T mycoplasma by long-chain fatty acids Cell yield

Fatty acid addeda

(mg of protein)

Radioactivity in cell lipids (cpm/ mg of protein)

1.02 None 1,480 Palmitic acid 0.95 838 Oleic acid 0.82 296 a The organisms were grown in 500-ml volumes of Edward medium containing 5% horse serum and 5 ACi of sodium [1-'4C]acetate. Fatty acids were added to the medium to a final concentration of 10 ,ug/ml.

173

support to the recent inclusion of the T mycoplasmas in a separate genus. It must be stressed, however, that more strains of T mycoplasmas should be tested for this property before any generalizations can be made. ACKNOWLEDGMENT We are grateful to C. Panos for his help with the capillary gas-liquid chromatography analyses and for stimulating discussions. LITERATURE CITED 1. Brian, L. B., and E. W. Gardner. 1968. Fatty acids from Vibrio cholera lipids. J. Infect. Dis. 118:47-53. 2. Fulco, A. J. 1970. The biosynthesis of unsaturated fatty acids by bacilli. J. Biol. Chem. 245:2985-2990. 3. Herring, P. K., and J. D. Pollack. 1974. Utilization of [1-'4C]-acetate in the synthesis of lipids by acholeplasmas. Int. J. Syst. Bacteriol. 24:73-78. 4. Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193:265-275. 5. Panos, C., and S. Rottem. 1970. Incorporation and elongation of fatty acid isomers by Mycoplasma laidlawii A. Biochemistry 9:407-412. 6. Pollack, J. D., and M. E. Tourtellotte. 1967. Synthesis of saturated long chain fatty acids from sodium acetate-1-C'4 by Mycoplasma. J. Bacteriol. 93:636-641. 7. Razin, S. 1963. Osmotic lysis of mycoplasma. J. Gen. Microbiol. 33:471-475. 8. Razin, S. 1973. Physiology of mycoplasmas, p. 1-80. In A. H. Rose and D. W. Tempest (ed.), Advances in microbial physiology, vol. 10. Academic Press Inc., London. 9. Razin, S., J. Valdesuso, R. H. Purcell, and R. M. Chanock. 1970. Electrophoretic analysis of cell proteins of T-strain mycoplasmas isolated from man. J. Bacteriol. 103:702-706. 10. Rodwell, A. W. 1968. Fatty acid composition of Mycoplasma lipids: a biomembrane with only one fatty acid. Science 160:1350-1351. 11. Rodwell, A. W., and J. E. Peterson. 1971. The effect of straight-chain saturated, monoenoic and branchedchain fatty acids on growth and fatty acid composition of Mycoplasma strain Y. J. Gen. Microbiol. 68:173-186. 12. Romano, N., P. F. Smith, and W. R. Mayberry. 1972. Lipids of a T strain of Mycoplasma. J. Bacteriol. 109:565-569. 13. Rottem, S., 0. Muhsam-Peled, and S. Razin. 1973. Acyl carrier protein in mycoplasmas. J. Bacteriol. 113:586591. 14. Rottem, S., E. A. Pfendt, and L. Hayflick. 1971. Sterol requirements of T-strain mycoplasmas. J. Bacteriol. 105:323-330. 15. Rottem, S., and S. Razin. 1967. Uptake and utilization of acetate by mycoplasma. J. Gen. Microbiol. 48:5363. 16. Rottem, S., and S. Razin. 1973. Membrane lipids of Mycoplasma hominis. J. Bacteriol. 113:565-571. 17. Rottem, S., 0. Stein, and S. Razin. 1968. Reassembly of mycoplasma membranes disaggregated by detergents. Arch. Biochem. Biophys. 125:46-56. 18. Shepard, M. C., C. D. Lunceford, D. K. Ford, R. H. Purcell, D. Taylor-Robinson, S. Razin, and F. T. Black. 1974. Ureaplasma urealyticum gen. nov., sp. nov.: proposed nomenclature for the human T (Tstrain) mycoplasmas. Int. J. Syst. Bacteriol. 24:160171. 19. Smith, P. F. 1971. The biology of mycoplasmas. Academic Press Inc., New York.