Vol. 140, No. 3
JOURNAL OF BACTERIOLOGY, Dec. 1979, p. 944-948
0021-9193/79/12-0944/05$02.00/0
Carotenoids Act as Reinforcers of the Acholeplasma laidlawii Lipid Bilayer SHLOMO ROTTEM* AND ORA MARKOWITZ Biomembrane Research Laboratory, The Hebrew University-Hadassah Medical School, Jerusalem, Israel Received for publication 10 October 1979
Acholeplasma laidlawii cells grown with oleic acid produced much more colored carotenoids than did cells grown with elaidic acid. The amount of carotenoids was decreased 80 to 90% by growing the cells with 0.05 M propionate, resulting in a marked increase in the mobility of both 5-doxylstearate and 12doxylstearate incorporated into the membranes. The fatty acid composition of the propionate-grown cells differed from that of cells grown without propionate by containing odd-numbered rather than even-numbered saturated fatty acids, but the ratios of saturated to unsaturated fatty acids were the same. To determine whether the carotenoids are the cause for the restricted mobility in the membranes, the carotenoids were selectively removed from A. laidlawii membranes by incubating the membranes with phosphatidylcholine vesicles. The carotenoiddepleted membranes showed an increase in the mobility of the hydrocarbon chains of the spin-labeled fatty acids. Furthermore, the incorporation of carotenoids into artificial membrane vesicles restricted the mobility of the hydrocarbon chain. Our results support the notion that the carotenoids in A. laidlawii act as a rigid insert reinforcing the membrane bilayer.
Acholeplasma species are capable of de novo biosynthesis of carotenoids (17) localized exclusively in the cell membrane (8). The idea that in Acholeplasma, carotenoids with a planar hydrocarbon structure are essential for cell growth, fulfilling functions analogous to those of cholesterol in the Mycoplasma species, was put forward by Smith and co-workers (18-20). Accordingly, it was suggested that the low cholesterol levels in Acholeplasma species are due to high contents of carotenoids interfering with cholesterol uptake by occupying the same sites in the membrane (20). Other findings contradicted this issue mainly by showing that carotenoid biosynthesis is not essential for A. laidlawii growth and that induced changes in the carotenoid content of the cells have no effect on the amounts of cholesterol incorporated into the membrane (9). This controversial issue was recently raised again (4) when it was shown that inhibition of carotenoid biosynthesis resulted in an increase in the mobility of spin-labeled fatty acids incorporated into the membrane. The results presented in the present communication extend this study by showing that the selective removal of the carotenoids from A. laidlawii membranes results in an increase in membrane fluidity, whereas the incorporation of carotenoids into artificial membranes decreases membrane fluidity, supporting the hypothesis that polyterpen-
oids act as reinforcers of the lipid bilayer (10).
MATERIALS AND METHODS Organisms and growth conditions. A. laidlawii
(oral strain) was grown in a modified Edward medium containing 0.5% (wt/vol) bovine serum albumin (Sigma Chemical Co., St. Louis, Mo.) and 20 ,ug of either oleic or elaidic acid. The pH of the medium was adjusted to 8.5. Growth was followed by measuring the absorbance of the culture at 640 nm, using a Bausch and Lomb Spectronic 80 spectrometer. Cells were harvested at the mid-exponential phase of growth (absorbance at 640 nm, 0.15 to 0.20) by centrifugation at 9,000 x g for 15 min, washed once, and suspended in 0.25 M NaCl. Cell membranes were isolated by osmotic lysis (7). The membranes were washed twice and suspended in deionized water. Protein was determined in cell and membrane suspensions by the method of Lowry et al. (5). Lipid analyses. Lipids were extracted from isolated membranes by the method of Bligh and Dyer (1). The absorbance at 438 nm of the lipid extract was taken as a measure of the carotenoid content. For fractionation of the carotenoid pigments the lipids were passed through a silicic acid column (14). The neutral lipid fraction eluted from the column by chloroform was further fractionated on 0.25-mm layers of Silica Gel G activated by heating at 110°C for 60 min. The developing solvents included benzene-diethyl ether-ethanol-acetic acid (50:40:2:0.2, by volume) followed by hexane-diethyl ether (94:6, vol/vol). Chromatography was carried out in a nitrogen environment
944
VOL. 140, 1979
in the dark. Carotenoids were detected by fluorescence under UV light. The carotenoid band was scraped off the plates into test tubes. Ten milliliters of chloroformmethanol (1:1, vol/vol) was then added to each tube, which was vigorously shaken. The solvent, containing the carotenoids, was separated from the silica gel particles by filtration through a fiber glass GF/C flter and evaporated to dryness under N2 in the dark. The carotenoids were weighed and dissolved in ethanol until used. Methyl esters of the fatty acids were prepared by heating lipid samples in 14% boron trifluoride in methanol (Sigma) at 72°C for 15 min. The resulting methyl esters were extracted with n-hexane and subjected to gas-liquid chromatography in a Packard model 840 instrument equipped with a polar column (200 by 0.3 cm, 10% SP2340 on 100/120 chromosorb W AW). Fatty acids were identified by their retention time relative to that of standard methyl ester mixtures (Supelco Inc., Bellafonte, Pa.). Carotenoid depletion. Membrane suspensions containing 1.5 mg of membrane protein per ml were incubated with an equal volume of phosphatidylcholine vesicles at 37°C for 2 h. The incubation was in a nitrogen environment in the dark with constant shaking. For vesicle preparation, 5 mg of egg phosphatidylcholine (Makor Chemicals, Jerusalem, Israel) and 10 ml of 0.25 M NaCl solution were added to a sonication vessel and were sonicated under N2 in the cold for 20 min, using a Heat Systems model W-350 sonicator at 160 W. Upon completion of the incubation period, the membranes were collected by centrifugation at 30,000 x g for 30 min, washed twice with deionized water, assayed for carotenoid content, and analyzed by electron paramagnetic resonance spec-
CAROTENOIDS IN MYCOPLASMAS
945
Keith (3) and expressed as nanoseconds. Greater freedom of motion, indicating higher fluidity, is associated with smaller values of 2 T1 and 'To.
RESULTS
The amount of carotenoids synthesized by A. laidlawii cells was much higher in cells grown with cis-octadecenoic acid (oleic acid) than in cells grown with the corresponding trans isomer (elaidic acid) (Table 1). The carotenoid concentration was assessed from measurements of the absorbance at 438 nm, the optimum for neurosporene, which is the major colored pigment of A. laidlawii (21). The table also shows the very marked reduction in the carotenoid content of A. laidlawii caused by propionate. The propionate had almost no effect on the growth of A. laidlawii cells in the elaidate-containing medium, but in the oleate-containing medium cell growth was inhibited by over 50%. The fatty acid profiles of the propionategrown cells differed from those of cells grown without propionate (Table 2). The differences were mainly a high content of odd-numbered fatty acids and a low content of even-numbered fatty acids. The odd-numbered saturated fatty acids represent the products of the A. laidlawii fatty acid biosynthesis system operating with propionate as a precursor rather than with acetate. The ratios of saturated to unsaturated fatty trometry. acids in the polar lipid fraction of cells grown Electron paramagnetic resonance spectrome- with and without propionate were, however, try. Membrane preparations and multilamellar phos- nearly the same (-0.55). phatidylcholine vesicles were spin-labeled with NElectron paramagnetic resonance spectrum oxyl-4',4'-dimethyloxazoline derivatives of 5-keto- analyses were carried out with spin-labeled stearic acid (5-doxylstearate) or 12-ketostearic acid (12-doxylstearate), products of Syva (Palo Alto, membranes from oleate-enriched cells grown Calif.). For spin labeling of membranes, 2 pl of the 2.5 with or without propionate. Throughout the mM spin label solution in ethanol were added to a test temperature range tested (5 to 45°0) there was tube containing 0.1 ml of diethyl ether. The solvent no indication of heterogeneity in the spectra. evaporated, and 1 ml of a membrane suspension containing 1 mg of membrane protein was added. The tube was then gently shaken for 15 min at room temperature. For spin labeling of multilamellar phosphatidylcholine vesicles, 2 pl of the spin label solution (2.5 mM) was added to a test tube containing 0.75 mg of phosphatidylcholine and 0.125 to 0.5 mg of carotenoids in diethyl ether. The solvent was evaporated to dryness, forming a thin film on the walls of the tube. Fifty microliters of a 0.25 M NaCl solution was then added to each tube, and the tubes were vigorously shaken with a Vortex mixer. The spin-labeled, multilamellar lipid vesicles or membranes were then transferred to a disposable pipette sealed at one end, and electron paramagnetic resonance spectra were obtained in a Varian E-4 spectrometer equipped with a temperature-control unit cooled by liquid air. The freedom of motion of the spin-labeled fatty acids in the preparations was assessed from the large hyperfine splitting (2 T1, reference 12) and from the motion parameter (To) calculated according to Henry and was
TABLE 1. Effect of sodium propionate on cell growth and carotenoid content of A. laidlawii cells grown with oleic or elaidic acid' Cel yield
Cells Oleate grown Without propionate With propionate
Mem-
Cell(mg of
brane (mg
cell protam) tein
branembrane protein)
69.4 31.6
18.2 8.0
Carotenoid8b 79.0 16.5
Elaidate grown 40.1 24.9 85.8 Without propionate 19.4 11.4 78.4 With propionate 0.5% bovine serum in medium containing a Cells were grown albumin and oleic or elaidic acid (20 ,ug/ml), with or without 0.5% sodium propionate. Membranes were isolated and carotenoid content was determined as described in the text. bAbsorbance at 438 nm x 1,000 per mg of cell protein.
946
ROTTEM AND MARKOWITZ
J. BACTERIOL.
TABLE 2. Fatty acid composition of the polar lipid fraction ofA. laidlawii grown with or without propionatea Polar lipid preparation from cells grown:
Fatty acid (mol%)
13:0b
14:0
15:0
17:0
16:0
18:0
18:1
63.2 0 5.5 22.0 0 5.1 0 Without propionate 64.8 5.5 1.1 8.0 13.6 1.5 3.7 With propionate a The cells were grown in a medium containing 20 jig of oleic acid per ml with or without 0.5% sodium propionate. ' The first number indicates the number of carbon atoms, and the second number indicates the number of double bonds.
The spectra revealed that the large hyperfine splitting (2 T1l) of 5-doxylstearate and 12-doxylstearate was higher in carotenoid-rich membranes obtained from cells grown without propionate than in carotenoid-poor membranes obtained from cells grown with propionate (Fig. 1). The percent change in the hyperfine splitting between the carotenoid-rich and -poor membranes was, however, larger in membranes labeled with 12-doxylstearate. Carotenoids could be removed from A. laidlawii membranes by incubating the membranes with an excess of phosphatidylcholine vesicles (Table 3). This removed about 85% of the colored carotenoids. These carotenoids were quantitatively recovered from the vesicle suspension by successive extractions with diethyl ether (not shown in the table). The treatment with the lipid vesicles did not affect the polar lipid composition of the membrane. Thus, the de novo synthesized phospho-, glyco-, and phosphoglycolipid composition of the treated membranes was the same as that of untreated membranes. Likewise, only negligible amounts of phosphatidylcholine originating from the vesicles were incorporated into the membranes. The treatment of carotenoid-rich A. laidlawii membranes with the lipid vesicles had, however, a pronounced effect on membrane fluidity. The much lower motion parameters of 12-doxylstearate in the treated membranes suggest that the freedom of motion of the hydrocarbon chains in the carotenoid-depleted membranes is much higher than in control membranes. No changes in membrane fluidity were found by treating carotenoid-poor membranes obtained from propionate-grown cells with the suspension of phosphatidylcholine vesicles. The motion parameter was calculated only from the 12-doxylstearate spectrum since only this probe moved in a nearly isotropic fashion, warranting its use for the determination of the motion parameter (3). When carotenoids were incorporated into multilamellar phosphatidylcholine vesicles, the motion parameters of 12-doxylstearate were markedly decreased (Table 4), suggesting a re-
64F N
60-
0(A 561 w z La
520
15
30
45
TEMPERATURE (C) FIG. 1. Temperature dependence of the hyperfine splitting (2 T) of spin-labeled fatty acids incorporated into A. laidlawii membranes. Symbols: 0, , 5doxylstearate; *, E 12-doxylstearate; O, , carotenoid-rich A. laidlawii membranes; 0, 0, carotenoidpoor A. laidlawii membranes.
stricted mobility of the hydrocarbon chains induced by the carotenoids. The restricted mobility detected with 12-doxylstearate was not pronounced when 5-doxylstearate was used. The large hyperfine splitting calculated from spectra of 5-doxylstearate incorporated into carotenoidcontaining vesicles was very similar to that of phosphatidylcholine vesicles not containing the carotenoid pigment. DISCUSSION Our results, that the freedom of motion of fatty acid spin labels in membranes from cells grown with propionate was higher than in membranes from cells grown without it, are in accord with those of Huang and Haug (4). These results may indicate the influence of carotenoids on the physical state of A. laidlawii membranes. When the cells are grown with propionate, colored carotenoid biosynthesis is inhibited (13), result-
CAROTENOIDS IN MYCOPLASMAS
VOL. 140, 1979
947
TABLE 3. Motion parameter of 12-doxylstearate in A. laidlawii membranes treated with lipid vesiclesa Motion parameter (ns) assayed at: Membrane prepn
Carotenoidb 300C
350C
400C
From cells grown without propionate 60.0 >10.OC 7.5 5.6 Untreated 10.0 4.7 3.9 Treated with lipid vesicles 3.5 From cells grown with propionate 15.0 5.6 3.9 Untreated 3.4 13.0 5.5 3.6 3.4 Treated with lipid vesicles a with or without sodium propionate. Cells were grown in a medium supplemented with oleic acid (20 Membranes were prepared and treated with lipid vesicles as described in the text. b Absorbance at 438 nm x 1,000 per milligram of cell protein. c The values were calculated only for correlation times which were shorter than 10 ns as the high field lines above this value showed inconsistent variability.
pg/ml)
TABLE
4. Freedom of motion of 12-doxylstearate in mixed egg phosphatidylcholine-carotenoid vesiclesa Motion parameters (ns)
assayed at:
Liposome prepn (mg/ml)
300C
350C
400C
3.6 2.9 2.4 Egg phosphatidylcholine (0.75) 5.9 4.9 3.9 Egg phosphatidylcholine (0.75) + carotenoids (0.12) 7.6 6.1 5.1 Egg phosphatidylcholine (0.75) + carotenoids (0.5) a The lipid vesicles were made by Vortex shaking of lipids in excess of 0.25 M NaCl as described in the text.
ing in an increased membrane fluidity. As the absence of colored carotenoids from propionategrown cells may have been due to inhibition of the sequential step of unsaturation rather than of the biosynthesis of the polyterpene chain (18, 19), we used two completely different approaches to show the effect of carotenoids on membrane fluidity. By incubating A. laidlawii membranes with phosphatidylcholine vesicles, membr,ane carotenoids were selectively removed, apparently in a manner similar to the removal of cholesterol from biomembranes (2). The removal of the carotenoids resulted in an increase in the freedom of motion of the spinlabeled probes. On the other hand, the addition of carotenoids to multilamellar lipid vesicles decreased the freedom of motion. The marked effect of the carotenoids on the freedom of motion in A. laidlawii membranes was observed with both 5-doxylstearate and 12doxylstearate. The nitroxide radical in 5-doxylstearate is in proximity of the carboxyl group of the fatty acid, whereas in 12-doxylstearate the radical is close to the methylene end group. Since fatty acid spin labels in biomembranes are oriented parallel to the hydrocarbon chains of membrane polar lipids (11), it seems that the carotenoids restrict the mobility of hydrocarbon chains of membrane lipids both near the polar
head groups and in the central lipid bilayer regions. Such restriction can best be explained by a preferred orientation of the carotenoid molecule with its amphiphilic axis parallel to the hydrocarbon chains of A. laidlawii polar lipids. A parallel orientation of the carotenoids is not necessarily the case with the multilamellar phosphatidylcholine vesicles, for which the restriction of motion was very pronounced with 12doxylstearate but not with 5-doxylstearate. The fatty acid analysis of membrane polar lipids from cells grown with propionate strongly suggests that the biosynthesis pathway in the A. laidlawii oral strain is basically similar to that described for the A. laidlawii B strain (15). The cells, when grown with propionate, synthesized odd-numbered saturated fatty acids from the three-carbon primer. The products of the biosynthetic system were fatty acids of 13 to 17 carbons. The bulk of the biosynthesis output, using the three-carbon primer, was pentadecanoic acid (>70%). The even-numbered hexadecanoic acid (C16:0) and octadecanoic acid (C18:0) found in the polar lipid fraction are apparently either exogenous fatty acids incorporated from the growth medium or represent the products of the biosynthetic system operated with acetate formed during glucose metabolism. Changing the saturated-to-unsaturated fatty acid ratio of membrane polar lipids and affecting the fatty acid chain elongation system are the two mechanisms previously described as being involved in controlling membrane fluidity in A. laidlawii (6, 11, 16). The former mechanism was found to be active when growth temperature is changed (6), whereas the latter operates in response to variation in the fatty acid composition of the growth medium (16). Our observation that as carotenoid biosynthesis was inhibited by propionate, the saturated-to-unsaturated fatty acid ratio and the average chain length of A. laidlawii polar lipids remained almost the same suggests that A. laidlawii cells do not possess a
948
ROTTEM AND MARKOWITZ
regulatory mechanism that senses carotenoid content for the maintenance of constant membrane fluidity. The absence of such mechanisms was suggested previously (9), when it was found that the inhibition of carotenoid biosynthesis has no effect on the incorporation of cholesterol into A. laidlawii membranes. On the other hand, it may be that the carotenoid biosynthesis system senses membrane fluidity. Thus, the carotenoid content in cells grown with oleate was much higher than in celLs grown with elaidate, but additional fatty acid compositions need to be studied to establish firmly such a relationship. ACKNOWLEDGMENTS We thank S. Razin for reading the manuscript and M. Wormser for technical assistance. LITERATURE CITED 1. Bligh, E. G., and W. J. Dyer. 1959. A rapid method of total lipid extraction and purification. Can. J. Biochem. Physiol. 37:911-917. 2. Bruckdorfer, V. R., P. A. Edwards, and C. Green. 1968. Properties of aqueous dispersions of phospholipid and cholesterol. Eur. J. Biochem. 4:506-511. 3. Henry, S., and A. Keith. 1971. Membrane properties of saturated fatty acid mutants of yeast revealed by spin labels. Chem. Phys. Lipids 7:245-265. 4. Huang, L, and A. Haug. 1974. Regulation of membrane lipid fluidity in Acholeplasma laidlawii. Effect of carotenoid pigment content. Biochim. Biophys. Acta 352: 361-370. 5. 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. 6. Melchior, D. L, and J. M. Stein. 1977. Control of fatty acid composition of Achokplasma laidlawii membranes. Biochim. Biophys. Acta 466:148-159. 7. Razin, S. 1963. Osmotic lysis of mycoplasma. J. Gen. Microbiol. 33:471-475. 8. Razin, S. 1969. Structure and function of mycoplasma. Annu. Rev. Microbiol. 23:317-356.
J. BACTERIOL. 9. Razin, S., and S. Rottem. 1967. Role of carotenoids and cholesterol in the growth of Mycoplasma laidlawii. J. Bacteriol. 93:1181-1182. 10. Rohmer, M., P. Bouvier, and G. Ourisson. 1979. Molecular evolution of biomembranes: structural equivalents and phylogenetic precursors of sterols. Proc. Natl. Acad. Sci. U.S.A. 76:847-851. 11. Rottem, S. 1979. Molecular organization of membrane lipids, p. 259-288. In M. F. Barile and S. Razin (ed.), The mycoplasmas, vol. 1. Academic Press Inc., New
York. 12. Rottem, S., W. L. Hubbell, L. Hayflick, and H. M. McConnell. 1970. Motion of fatty acid spin labels in the plasma membrane of mycoplasma. Biochim. Biophys. Acta 219:104-113. 13. Rottem, S., and S. Razin. 1967. Uptake and utilization of acetate by mycoplasma. J. Gen. Microbiol. 48:53-63. 14. Rottem, S., and S. Razin. 1973. Membrane lipids of Mycoplasma hominis. J. Bacteriol. 113:565-571. 15. Saito, Y., J. R. Silvius, and R. N. McElhaney. 1977. Membrane lipid biosynthesis in Acholeplasma laidlawii B: de novo biosynthesis of saturated fatty acids by growing cells. J. Bacteriol. 132:497-504. 16. Silvius, J. R., Y. Saito, and R. N. McElhaney. 1977. Membrane lipid biosynthesis in Acholeplasma laidlawii B. Investigation into the in vivo regulation of the quantity and hydrocarbon chain lengths of de novo biosynthesized fatty acids in response to ezogenously supplied fatty acids. Arch. Biochem. Biophys. 182:455464. 17. Smith, P. F. 1963. The carotenoid pigments of Mycoplasma. J. Gen. Microbiol. 32:307-319. 18. Smith, P. F. 1971. The biology of mycoplasma. Academic Press Inc., New York. 19. Smith, P. F. 1979. The composition of membrane lipids and lipopolysaccharides, p. 231-258. In M. F. Barile and S. Razin (ed.), The mycoplasmas, vol. 1. Academic Press Inc., New York. 20. Smith, P. F., and C. V. Henrikson. 1966. Growth inhibition of Mycoplasma by inhibitors of polyterpene biosynthesis and its reversal by cholesterol. J. Bacteriol. 91:1854-1858. 21. Tully, J. G., and S. Razin. 1968. Physiological and serological comparisons among strains of Mycoplasma granularum and Mycoplasma laidlawii. J. Bacteriol. 95:1504-1512.
JOURNAL OF BACTERIOLOGY, Dec. 1979, p. 944-948
0021-9193/79/12-0944/05$02.00/0
Carotenoids Act as Reinforcers of the Acholeplasma laidlawii Lipid Bilayer SHLOMO ROTTEM* AND ORA MARKOWITZ Biomembrane Research Laboratory, The Hebrew University-Hadassah Medical School, Jerusalem, Israel Received for publication 10 October 1979
Acholeplasma laidlawii cells grown with oleic acid produced much more colored carotenoids than did cells grown with elaidic acid. The amount of carotenoids was decreased 80 to 90% by growing the cells with 0.05 M propionate, resulting in a marked increase in the mobility of both 5-doxylstearate and 12doxylstearate incorporated into the membranes. The fatty acid composition of the propionate-grown cells differed from that of cells grown without propionate by containing odd-numbered rather than even-numbered saturated fatty acids, but the ratios of saturated to unsaturated fatty acids were the same. To determine whether the carotenoids are the cause for the restricted mobility in the membranes, the carotenoids were selectively removed from A. laidlawii membranes by incubating the membranes with phosphatidylcholine vesicles. The carotenoiddepleted membranes showed an increase in the mobility of the hydrocarbon chains of the spin-labeled fatty acids. Furthermore, the incorporation of carotenoids into artificial membrane vesicles restricted the mobility of the hydrocarbon chain. Our results support the notion that the carotenoids in A. laidlawii act as a rigid insert reinforcing the membrane bilayer.
Acholeplasma species are capable of de novo biosynthesis of carotenoids (17) localized exclusively in the cell membrane (8). The idea that in Acholeplasma, carotenoids with a planar hydrocarbon structure are essential for cell growth, fulfilling functions analogous to those of cholesterol in the Mycoplasma species, was put forward by Smith and co-workers (18-20). Accordingly, it was suggested that the low cholesterol levels in Acholeplasma species are due to high contents of carotenoids interfering with cholesterol uptake by occupying the same sites in the membrane (20). Other findings contradicted this issue mainly by showing that carotenoid biosynthesis is not essential for A. laidlawii growth and that induced changes in the carotenoid content of the cells have no effect on the amounts of cholesterol incorporated into the membrane (9). This controversial issue was recently raised again (4) when it was shown that inhibition of carotenoid biosynthesis resulted in an increase in the mobility of spin-labeled fatty acids incorporated into the membrane. The results presented in the present communication extend this study by showing that the selective removal of the carotenoids from A. laidlawii membranes results in an increase in membrane fluidity, whereas the incorporation of carotenoids into artificial membranes decreases membrane fluidity, supporting the hypothesis that polyterpen-
oids act as reinforcers of the lipid bilayer (10).
MATERIALS AND METHODS Organisms and growth conditions. A. laidlawii
(oral strain) was grown in a modified Edward medium containing 0.5% (wt/vol) bovine serum albumin (Sigma Chemical Co., St. Louis, Mo.) and 20 ,ug of either oleic or elaidic acid. The pH of the medium was adjusted to 8.5. Growth was followed by measuring the absorbance of the culture at 640 nm, using a Bausch and Lomb Spectronic 80 spectrometer. Cells were harvested at the mid-exponential phase of growth (absorbance at 640 nm, 0.15 to 0.20) by centrifugation at 9,000 x g for 15 min, washed once, and suspended in 0.25 M NaCl. Cell membranes were isolated by osmotic lysis (7). The membranes were washed twice and suspended in deionized water. Protein was determined in cell and membrane suspensions by the method of Lowry et al. (5). Lipid analyses. Lipids were extracted from isolated membranes by the method of Bligh and Dyer (1). The absorbance at 438 nm of the lipid extract was taken as a measure of the carotenoid content. For fractionation of the carotenoid pigments the lipids were passed through a silicic acid column (14). The neutral lipid fraction eluted from the column by chloroform was further fractionated on 0.25-mm layers of Silica Gel G activated by heating at 110°C for 60 min. The developing solvents included benzene-diethyl ether-ethanol-acetic acid (50:40:2:0.2, by volume) followed by hexane-diethyl ether (94:6, vol/vol). Chromatography was carried out in a nitrogen environment
944
VOL. 140, 1979
in the dark. Carotenoids were detected by fluorescence under UV light. The carotenoid band was scraped off the plates into test tubes. Ten milliliters of chloroformmethanol (1:1, vol/vol) was then added to each tube, which was vigorously shaken. The solvent, containing the carotenoids, was separated from the silica gel particles by filtration through a fiber glass GF/C flter and evaporated to dryness under N2 in the dark. The carotenoids were weighed and dissolved in ethanol until used. Methyl esters of the fatty acids were prepared by heating lipid samples in 14% boron trifluoride in methanol (Sigma) at 72°C for 15 min. The resulting methyl esters were extracted with n-hexane and subjected to gas-liquid chromatography in a Packard model 840 instrument equipped with a polar column (200 by 0.3 cm, 10% SP2340 on 100/120 chromosorb W AW). Fatty acids were identified by their retention time relative to that of standard methyl ester mixtures (Supelco Inc., Bellafonte, Pa.). Carotenoid depletion. Membrane suspensions containing 1.5 mg of membrane protein per ml were incubated with an equal volume of phosphatidylcholine vesicles at 37°C for 2 h. The incubation was in a nitrogen environment in the dark with constant shaking. For vesicle preparation, 5 mg of egg phosphatidylcholine (Makor Chemicals, Jerusalem, Israel) and 10 ml of 0.25 M NaCl solution were added to a sonication vessel and were sonicated under N2 in the cold for 20 min, using a Heat Systems model W-350 sonicator at 160 W. Upon completion of the incubation period, the membranes were collected by centrifugation at 30,000 x g for 30 min, washed twice with deionized water, assayed for carotenoid content, and analyzed by electron paramagnetic resonance spec-
CAROTENOIDS IN MYCOPLASMAS
945
Keith (3) and expressed as nanoseconds. Greater freedom of motion, indicating higher fluidity, is associated with smaller values of 2 T1 and 'To.
RESULTS
The amount of carotenoids synthesized by A. laidlawii cells was much higher in cells grown with cis-octadecenoic acid (oleic acid) than in cells grown with the corresponding trans isomer (elaidic acid) (Table 1). The carotenoid concentration was assessed from measurements of the absorbance at 438 nm, the optimum for neurosporene, which is the major colored pigment of A. laidlawii (21). The table also shows the very marked reduction in the carotenoid content of A. laidlawii caused by propionate. The propionate had almost no effect on the growth of A. laidlawii cells in the elaidate-containing medium, but in the oleate-containing medium cell growth was inhibited by over 50%. The fatty acid profiles of the propionategrown cells differed from those of cells grown without propionate (Table 2). The differences were mainly a high content of odd-numbered fatty acids and a low content of even-numbered fatty acids. The odd-numbered saturated fatty acids represent the products of the A. laidlawii fatty acid biosynthesis system operating with propionate as a precursor rather than with acetate. The ratios of saturated to unsaturated fatty trometry. acids in the polar lipid fraction of cells grown Electron paramagnetic resonance spectrome- with and without propionate were, however, try. Membrane preparations and multilamellar phos- nearly the same (-0.55). phatidylcholine vesicles were spin-labeled with NElectron paramagnetic resonance spectrum oxyl-4',4'-dimethyloxazoline derivatives of 5-keto- analyses were carried out with spin-labeled stearic acid (5-doxylstearate) or 12-ketostearic acid (12-doxylstearate), products of Syva (Palo Alto, membranes from oleate-enriched cells grown Calif.). For spin labeling of membranes, 2 pl of the 2.5 with or without propionate. Throughout the mM spin label solution in ethanol were added to a test temperature range tested (5 to 45°0) there was tube containing 0.1 ml of diethyl ether. The solvent no indication of heterogeneity in the spectra. evaporated, and 1 ml of a membrane suspension containing 1 mg of membrane protein was added. The tube was then gently shaken for 15 min at room temperature. For spin labeling of multilamellar phosphatidylcholine vesicles, 2 pl of the spin label solution (2.5 mM) was added to a test tube containing 0.75 mg of phosphatidylcholine and 0.125 to 0.5 mg of carotenoids in diethyl ether. The solvent was evaporated to dryness, forming a thin film on the walls of the tube. Fifty microliters of a 0.25 M NaCl solution was then added to each tube, and the tubes were vigorously shaken with a Vortex mixer. The spin-labeled, multilamellar lipid vesicles or membranes were then transferred to a disposable pipette sealed at one end, and electron paramagnetic resonance spectra were obtained in a Varian E-4 spectrometer equipped with a temperature-control unit cooled by liquid air. The freedom of motion of the spin-labeled fatty acids in the preparations was assessed from the large hyperfine splitting (2 T1, reference 12) and from the motion parameter (To) calculated according to Henry and was
TABLE 1. Effect of sodium propionate on cell growth and carotenoid content of A. laidlawii cells grown with oleic or elaidic acid' Cel yield
Cells Oleate grown Without propionate With propionate
Mem-
Cell(mg of
brane (mg
cell protam) tein
branembrane protein)
69.4 31.6
18.2 8.0
Carotenoid8b 79.0 16.5
Elaidate grown 40.1 24.9 85.8 Without propionate 19.4 11.4 78.4 With propionate 0.5% bovine serum in medium containing a Cells were grown albumin and oleic or elaidic acid (20 ,ug/ml), with or without 0.5% sodium propionate. Membranes were isolated and carotenoid content was determined as described in the text. bAbsorbance at 438 nm x 1,000 per mg of cell protein.
946
ROTTEM AND MARKOWITZ
J. BACTERIOL.
TABLE 2. Fatty acid composition of the polar lipid fraction ofA. laidlawii grown with or without propionatea Polar lipid preparation from cells grown:
Fatty acid (mol%)
13:0b
14:0
15:0
17:0
16:0
18:0
18:1
63.2 0 5.5 22.0 0 5.1 0 Without propionate 64.8 5.5 1.1 8.0 13.6 1.5 3.7 With propionate a The cells were grown in a medium containing 20 jig of oleic acid per ml with or without 0.5% sodium propionate. ' The first number indicates the number of carbon atoms, and the second number indicates the number of double bonds.
The spectra revealed that the large hyperfine splitting (2 T1l) of 5-doxylstearate and 12-doxylstearate was higher in carotenoid-rich membranes obtained from cells grown without propionate than in carotenoid-poor membranes obtained from cells grown with propionate (Fig. 1). The percent change in the hyperfine splitting between the carotenoid-rich and -poor membranes was, however, larger in membranes labeled with 12-doxylstearate. Carotenoids could be removed from A. laidlawii membranes by incubating the membranes with an excess of phosphatidylcholine vesicles (Table 3). This removed about 85% of the colored carotenoids. These carotenoids were quantitatively recovered from the vesicle suspension by successive extractions with diethyl ether (not shown in the table). The treatment with the lipid vesicles did not affect the polar lipid composition of the membrane. Thus, the de novo synthesized phospho-, glyco-, and phosphoglycolipid composition of the treated membranes was the same as that of untreated membranes. Likewise, only negligible amounts of phosphatidylcholine originating from the vesicles were incorporated into the membranes. The treatment of carotenoid-rich A. laidlawii membranes with the lipid vesicles had, however, a pronounced effect on membrane fluidity. The much lower motion parameters of 12-doxylstearate in the treated membranes suggest that the freedom of motion of the hydrocarbon chains in the carotenoid-depleted membranes is much higher than in control membranes. No changes in membrane fluidity were found by treating carotenoid-poor membranes obtained from propionate-grown cells with the suspension of phosphatidylcholine vesicles. The motion parameter was calculated only from the 12-doxylstearate spectrum since only this probe moved in a nearly isotropic fashion, warranting its use for the determination of the motion parameter (3). When carotenoids were incorporated into multilamellar phosphatidylcholine vesicles, the motion parameters of 12-doxylstearate were markedly decreased (Table 4), suggesting a re-
64F N
60-
0(A 561 w z La
520
15
30
45
TEMPERATURE (C) FIG. 1. Temperature dependence of the hyperfine splitting (2 T) of spin-labeled fatty acids incorporated into A. laidlawii membranes. Symbols: 0, , 5doxylstearate; *, E 12-doxylstearate; O, , carotenoid-rich A. laidlawii membranes; 0, 0, carotenoidpoor A. laidlawii membranes.
stricted mobility of the hydrocarbon chains induced by the carotenoids. The restricted mobility detected with 12-doxylstearate was not pronounced when 5-doxylstearate was used. The large hyperfine splitting calculated from spectra of 5-doxylstearate incorporated into carotenoidcontaining vesicles was very similar to that of phosphatidylcholine vesicles not containing the carotenoid pigment. DISCUSSION Our results, that the freedom of motion of fatty acid spin labels in membranes from cells grown with propionate was higher than in membranes from cells grown without it, are in accord with those of Huang and Haug (4). These results may indicate the influence of carotenoids on the physical state of A. laidlawii membranes. When the cells are grown with propionate, colored carotenoid biosynthesis is inhibited (13), result-
CAROTENOIDS IN MYCOPLASMAS
VOL. 140, 1979
947
TABLE 3. Motion parameter of 12-doxylstearate in A. laidlawii membranes treated with lipid vesiclesa Motion parameter (ns) assayed at: Membrane prepn
Carotenoidb 300C
350C
400C
From cells grown without propionate 60.0 >10.OC 7.5 5.6 Untreated 10.0 4.7 3.9 Treated with lipid vesicles 3.5 From cells grown with propionate 15.0 5.6 3.9 Untreated 3.4 13.0 5.5 3.6 3.4 Treated with lipid vesicles a with or without sodium propionate. Cells were grown in a medium supplemented with oleic acid (20 Membranes were prepared and treated with lipid vesicles as described in the text. b Absorbance at 438 nm x 1,000 per milligram of cell protein. c The values were calculated only for correlation times which were shorter than 10 ns as the high field lines above this value showed inconsistent variability.
pg/ml)
TABLE
4. Freedom of motion of 12-doxylstearate in mixed egg phosphatidylcholine-carotenoid vesiclesa Motion parameters (ns)
assayed at:
Liposome prepn (mg/ml)
300C
350C
400C
3.6 2.9 2.4 Egg phosphatidylcholine (0.75) 5.9 4.9 3.9 Egg phosphatidylcholine (0.75) + carotenoids (0.12) 7.6 6.1 5.1 Egg phosphatidylcholine (0.75) + carotenoids (0.5) a The lipid vesicles were made by Vortex shaking of lipids in excess of 0.25 M NaCl as described in the text.
ing in an increased membrane fluidity. As the absence of colored carotenoids from propionategrown cells may have been due to inhibition of the sequential step of unsaturation rather than of the biosynthesis of the polyterpene chain (18, 19), we used two completely different approaches to show the effect of carotenoids on membrane fluidity. By incubating A. laidlawii membranes with phosphatidylcholine vesicles, membr,ane carotenoids were selectively removed, apparently in a manner similar to the removal of cholesterol from biomembranes (2). The removal of the carotenoids resulted in an increase in the freedom of motion of the spinlabeled probes. On the other hand, the addition of carotenoids to multilamellar lipid vesicles decreased the freedom of motion. The marked effect of the carotenoids on the freedom of motion in A. laidlawii membranes was observed with both 5-doxylstearate and 12doxylstearate. The nitroxide radical in 5-doxylstearate is in proximity of the carboxyl group of the fatty acid, whereas in 12-doxylstearate the radical is close to the methylene end group. Since fatty acid spin labels in biomembranes are oriented parallel to the hydrocarbon chains of membrane polar lipids (11), it seems that the carotenoids restrict the mobility of hydrocarbon chains of membrane lipids both near the polar
head groups and in the central lipid bilayer regions. Such restriction can best be explained by a preferred orientation of the carotenoid molecule with its amphiphilic axis parallel to the hydrocarbon chains of A. laidlawii polar lipids. A parallel orientation of the carotenoids is not necessarily the case with the multilamellar phosphatidylcholine vesicles, for which the restriction of motion was very pronounced with 12doxylstearate but not with 5-doxylstearate. The fatty acid analysis of membrane polar lipids from cells grown with propionate strongly suggests that the biosynthesis pathway in the A. laidlawii oral strain is basically similar to that described for the A. laidlawii B strain (15). The cells, when grown with propionate, synthesized odd-numbered saturated fatty acids from the three-carbon primer. The products of the biosynthetic system were fatty acids of 13 to 17 carbons. The bulk of the biosynthesis output, using the three-carbon primer, was pentadecanoic acid (>70%). The even-numbered hexadecanoic acid (C16:0) and octadecanoic acid (C18:0) found in the polar lipid fraction are apparently either exogenous fatty acids incorporated from the growth medium or represent the products of the biosynthetic system operated with acetate formed during glucose metabolism. Changing the saturated-to-unsaturated fatty acid ratio of membrane polar lipids and affecting the fatty acid chain elongation system are the two mechanisms previously described as being involved in controlling membrane fluidity in A. laidlawii (6, 11, 16). The former mechanism was found to be active when growth temperature is changed (6), whereas the latter operates in response to variation in the fatty acid composition of the growth medium (16). Our observation that as carotenoid biosynthesis was inhibited by propionate, the saturated-to-unsaturated fatty acid ratio and the average chain length of A. laidlawii polar lipids remained almost the same suggests that A. laidlawii cells do not possess a
948
ROTTEM AND MARKOWITZ
regulatory mechanism that senses carotenoid content for the maintenance of constant membrane fluidity. The absence of such mechanisms was suggested previously (9), when it was found that the inhibition of carotenoid biosynthesis has no effect on the incorporation of cholesterol into A. laidlawii membranes. On the other hand, it may be that the carotenoid biosynthesis system senses membrane fluidity. Thus, the carotenoid content in cells grown with oleate was much higher than in celLs grown with elaidate, but additional fatty acid compositions need to be studied to establish firmly such a relationship. ACKNOWLEDGMENTS We thank S. Razin for reading the manuscript and M. Wormser for technical assistance. LITERATURE CITED 1. Bligh, E. G., and W. J. Dyer. 1959. A rapid method of total lipid extraction and purification. Can. J. Biochem. Physiol. 37:911-917. 2. Bruckdorfer, V. R., P. A. Edwards, and C. Green. 1968. Properties of aqueous dispersions of phospholipid and cholesterol. Eur. J. Biochem. 4:506-511. 3. Henry, S., and A. Keith. 1971. Membrane properties of saturated fatty acid mutants of yeast revealed by spin labels. Chem. Phys. Lipids 7:245-265. 4. Huang, L, and A. Haug. 1974. Regulation of membrane lipid fluidity in Acholeplasma laidlawii. Effect of carotenoid pigment content. Biochim. Biophys. Acta 352: 361-370. 5. 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. 6. Melchior, D. L, and J. M. Stein. 1977. Control of fatty acid composition of Achokplasma laidlawii membranes. Biochim. Biophys. Acta 466:148-159. 7. Razin, S. 1963. Osmotic lysis of mycoplasma. J. Gen. Microbiol. 33:471-475. 8. Razin, S. 1969. Structure and function of mycoplasma. Annu. Rev. Microbiol. 23:317-356.
J. BACTERIOL. 9. Razin, S., and S. Rottem. 1967. Role of carotenoids and cholesterol in the growth of Mycoplasma laidlawii. J. Bacteriol. 93:1181-1182. 10. Rohmer, M., P. Bouvier, and G. Ourisson. 1979. Molecular evolution of biomembranes: structural equivalents and phylogenetic precursors of sterols. Proc. Natl. Acad. Sci. U.S.A. 76:847-851. 11. Rottem, S. 1979. Molecular organization of membrane lipids, p. 259-288. In M. F. Barile and S. Razin (ed.), The mycoplasmas, vol. 1. Academic Press Inc., New
York. 12. Rottem, S., W. L. Hubbell, L. Hayflick, and H. M. McConnell. 1970. Motion of fatty acid spin labels in the plasma membrane of mycoplasma. Biochim. Biophys. Acta 219:104-113. 13. Rottem, S., and S. Razin. 1967. Uptake and utilization of acetate by mycoplasma. J. Gen. Microbiol. 48:53-63. 14. Rottem, S., and S. Razin. 1973. Membrane lipids of Mycoplasma hominis. J. Bacteriol. 113:565-571. 15. Saito, Y., J. R. Silvius, and R. N. McElhaney. 1977. Membrane lipid biosynthesis in Acholeplasma laidlawii B: de novo biosynthesis of saturated fatty acids by growing cells. J. Bacteriol. 132:497-504. 16. Silvius, J. R., Y. Saito, and R. N. McElhaney. 1977. Membrane lipid biosynthesis in Acholeplasma laidlawii B. Investigation into the in vivo regulation of the quantity and hydrocarbon chain lengths of de novo biosynthesized fatty acids in response to ezogenously supplied fatty acids. Arch. Biochem. Biophys. 182:455464. 17. Smith, P. F. 1963. The carotenoid pigments of Mycoplasma. J. Gen. Microbiol. 32:307-319. 18. Smith, P. F. 1971. The biology of mycoplasma. Academic Press Inc., New York. 19. Smith, P. F. 1979. The composition of membrane lipids and lipopolysaccharides, p. 231-258. In M. F. Barile and S. Razin (ed.), The mycoplasmas, vol. 1. Academic Press Inc., New York. 20. Smith, P. F., and C. V. Henrikson. 1966. Growth inhibition of Mycoplasma by inhibitors of polyterpene biosynthesis and its reversal by cholesterol. J. Bacteriol. 91:1854-1858. 21. Tully, J. G., and S. Razin. 1968. Physiological and serological comparisons among strains of Mycoplasma granularum and Mycoplasma laidlawii. J. Bacteriol. 95:1504-1512.
