Vol. 124, No. 2
JOURNAL OF BACTERIOLOGY, Nov. 1975, p. 602-605 Copyright ©) 1975 American Society for Microbiology
Printed in U.S.A.
Whole-Cell and Membrane Lipids of the Methylotrophic Bacterium Methylosinus trichosporium T. L. WEAVER,* M. A. PATRICK, AND P. R. DUGAN
Laboratory of Microbiology, Cornell University, Ithaca, New York 14853 * and Department of Microbiology, Ohio State University, Columbus, Ohio 43210 Received for publication 5 June 1975
The lipid composition of the methylotrophic bacterium Methylosinus trichosporium was examined. Whole-cell lipid distribution was 39.1% neutral lipids, 34.5% polar lipids, and 26.4% poly-beta-hydroxybutyric acid. Membrane lipids were 83% phospholipids, with phosphatidylethanolamine and phosphatidylglycerol accounting for over 94% of the total. All the phospholipids had similar fatty acid compositions, with 18:1 accounting for about 87% of the total and most of the rest consisting of 16:1. Similarities between the lipid composition of this bacterium and other bacteria are discussed. The predominant ultrastructural characteris- in nitrogen-flushed screw-top tubes to prevent degratic of methane-oxidizing bacteria is the pres- dation. Dry weight of cells and membranes were ence of extensive arrays of intracytoplasmic obtained by weighing representative samples in alupans after drying 16 h at 60 C. membranes (21). In this regrad, these bacteria minum Poly-beta-hydroxyPoly-beta-hydroxlybutyrate. are similar to other membranous prokaryotic butyrate was precipitated from lipid extracts by addmicroorganisms such as photosynthetic bacte- ing 2 volumes of methanol and holding at -10 C for ria (12), blue-green bacteria (5), and nitrifying 2 to 4 h. The samples was then pelleted by centrifubacteria (20), which also possess internal mem- gation, and the poly-beta-hydroxybutyrate-free sambrane systems. The role of lipids, particularly ple was concentrated by vacuum evaporation at phospholipids, in membrane structure is well 40 C. Poly-beta-hydroxybutyrate was quantitated documented, and the important physiological by the method of Law and Slepecky (11). Silicic acid column chromatography. A concensignificance of lipids in membranes is recogtrated containing 10 to 100 mg of lipid was nized (17). Although the lipids of all the other appliedsample to a 10-g column of silicic acid that was membranous types of prokaryotes previously prewashed and by heating at 110 C for 12 mentioned have been investigated (1, 10, 14), h. Neutral lipidsactivated were eluted with chloroform, and only one species of methane oxidizer, Methano- polar lipids were eluted with methanol. Both lipid monas methanoxidans, has been subjected to fractions were dried in a stream of nitrogen and lipid examination and this was restricted to quantitated gravimetrically. Thin-layer chromatography. Phospholipids were fatty acid examination (19). The present study was undertaken to examine the lipids of the separated on silica gel (D-O, Camag) thin-layer methane oxidizer, Methylosinus trichospo- plates. The plates were prewashed in acetone-petroleum ether (1:3, vol/vol) and developed in either rium. chloroform-methanol-water (65:25:4 vol/vol/vol) or MATERIALS AND METHODS Microorganism. M. trichosporium (OB3b isolate of R. Whittenbury) was grown in mineral salts CM under methane and air as described previously (21). Membranes from these cells were prepared by differential centrifugation as reported previously (22). This membrane preparation was relatively devoid of cell wall materials, as indicated by carbohydrate analysis, and consisted mainly of intracytoplasmic membranes. Lipid extraction. Wet cell pellets and membrane preparations were extracted routinely with chloroform-methanol (2:1 vol/vol) by shaking for 18 h at 25 C (4). The extracts were filtered through several thicknesses of chloroform-methanol-washed Whatman no. 1 filter paper and washed by the method of Radin (16). Lipid extracts were handled and stored
butanol-acetic acid-water (60:20:20, vol/vol/vol). Phospholipids were routinely detected by using the molybdate spray reagent of Goswami and Frey (7). Phospholipids were identified by relative Rf with standards (Supelco. Inc., Bellefonte, Pa.), acid hydrolysis and mild alkaline hydrolysis products (3), periodate oxidation to detect vicinal hydroxyls (23), argentation chromatography (18), and by various selective spray reagents including the orcinol reagent for glycolipids, ninhydrin reagent for amino groups, and the Reitsema test for plasmalogens (18). Quantitation of phospholipids. A 1-mCi amount of 32P as orthophosphate (Amersham/Searle, Arlington Heights, Ill.) was added to 2 liters of CM medium that contained 1.4 mmol of unlabeled phosphate per liter. Cells were grown and harvested as described previously (21). After membrane fractions were prepared and phospholipids were extracted and 02
VOL. 124, 1975
LIPIDS OF M. TRICHOSPORIUM
603
separated, spots were scraped from the thin-layer chromatrography plate and counted in a standard toluene-based cocktail containing Cabosil (Packard Instrument Co., Inc., Downers Grove, Ill.). Phospholipid fatty acid analysis. Methyl esters of phospholipid fatty acids were prepared by the mild methanolysis procedure of White (24). Methyl esters were analyzed by using a Varian model 200 gas chromatograph equipped with 10% diethyleneglycol succinate on a Chromosorb-W column (8 feet by 1/8 inch [ca. 2.44 m by 0.32 cm]) and a flame ionization detector. The injector and detector were 200 C while the column was maintained at 170 C. Nitrogen carrier gas was adjusted to 25 ml/min. Relative retention times were compared with methyl ester standards (Applied Science Laboratory, State College, Pa.). Unsaturated and cyclopropane fatty acids were differentiated by the method of Brian and Gardner (2).
TABLE 2. Scintillation spectrophotometric quantitation of 32P-labeled membrane phospholipids from a thin-layer chromatography plate
RESULTS
pholipids were present in such small quantities that they could not be tested by hydrolysis. Argentation thin-layer chromatography through silver nitrate-impregnated silica gel yielded no additional spots. The fatty acid composition of the major phospholipids is listed in Table 3. The 16:1 and 18:1 fatty acids hydrogenated completely under the mild conditions of Brian and Gardner (2), revealing that the fatty acids are monounsaturates and not the cyclopropane derivatives.
Extraction of wet cells (778 mg dry weight) and wet membranes (84 mg dry weight) yielded 71.6 and 23.5 mg of lipids, respectively. A summary of the lipid distribution in these extracts is provided in Table 1. Table 2 lists the distribution of phospholipids in the membranes. It was found that the same relative phospholipid distribution was also observed with whole cells as with the isolated membranes. Each phospholipid chromatographed identically with a known standard in both solvents; all reacted with Goswami reagent, and phosphatidylethanolamine (PE) and phosphatidylserine reacted with ninhydrin. All tested negatively for plasmalogen and glycolipid. As further confirmation of the identity of the two major phospholipids, the water-soluble, mild alkaline hydrolysis product of PE chromatographed with a similarly hydrolyzed standard, the acid-hydrolyzed product of PE chromatographed with ethanolamine, and the periodate oxidation of the phosphatidylglycerol (PG) yielded formaldehyde as it also did with standard. The other minor phosTABLE 1. Lipid components of whole cells and membranes of M. trichosporium Calculated Total
Total
Lipid
dry
dry
drywto to
dry
wt tal e wt of cells b of membl (% (% wt/wt) /wt)lipid( wb
tlt)
tl
11
extract-
able membrane lipids
%,
w
wtlwt)
PHBa
2.4
0.11
26.4
0.4
Neutral lipid Polar lipid
3.6
4.5
39.1
16.2
3.2
23.3
34.5
83.4
a
PHB, Poly-beta-hydroxybutyrate.
Phospholipid
CounWmin
% Total membrane
Diphosphatidylglycerol PE PG Phosphatidylserine Phosphatidylcholine
2,956 150,198 229,583 16,862 2,419
0.4a 37.5 57.3 4.2 0.6
phospholipids
a Since diphosphatidylglycerol contains two phosphorus atoms, this figure was based upon (0.5) (2,956 counts/min)/total counts per minute in all five fractions.
DISCUSSION The cell and membrane lipid distribution in Table 1 reveals that only about 9.2% of the cell dry weight was extracted as lipid whereas 27.9% of the membrane dry weight was extracted as lipid. This lipid enrichment in the membrane fraction would be expected since membranes are basically lipid-protein structures. The quantity of lipids observed in the membranes of M. trichosporium compares closely with previous reports of 24% lipid in inner mitochondrial membrane and 25% lipid in bacterial plasma membrane; however, these are very different from the 43% lipid in erythrocyte and liver cell membranes (8). The relatively large amount of poly-beta-hydroxybutyrate found in whole cells (26.4% of the total lipid) suggests that this compound must play an important role in the physiology of this bacterium. Presumably, the trace of poly-beta-hydroxybutyrate in the membrane sample was due to contamination. The polar lipid content increased from the 34.5% found in whole cells to 83.4% found in the membranes. This is expected since membranes normally contain relatively large quantities of phospholipids (13). The observation that the same five phospholipids in about the same ratios were observed whether whole cells or membranes were ana-
604
WEAVER, PATRICK, AND DUGAN
TABLE 3. Fatty acid compositions of the membrane phospholipids of M. trichosporium
J. BACTERIOL.
vannielii has PE/PG < 1, and of 23 grampositive bacteria, only Bacillus cereus, B. subtilus, and Micrococcus cerificans had PE/PG > 1. % Total fatty acid Again, only the singularity of this distribution Phospholipid is obvious. 16:0 16:1 18:1 The other three phospholipids, diphosphatiDiphosphatidylglycerol 0 13 87 dylglycerol, phosphatidylserine, and phosphatiPE 0.2 10.9 88.9 dycholine, account for only 5.2% of the phosphoPG 1 16 83 lipids. Diphosphatidylglycerol (or cardiolipin) Phosphatidylserine 0 13 87 frequently occurs in bacteria with PG since they are related in synthesis (6). Similarly, phosphatidylserine is an intermediate in PE lyzed was not surprising since nearly all cellu- synthesis and is often detected in bacteria with lar phospholipids are in the membranes (13). PE. Phosphatidylcholine is usually not a major This is also reassuring since it demonstrates component of bacterial phospholipids even that the phospholipid distribution observed though it is a very important component in with the membrane preparation was not a re- eukaryotic microorganisms (6). The results of the fatty acid analyses of these sult of working with small amounts of material. Furthermore, each phospholipid is homoge- phospholipids are shown in Table 3. These reneous, at least in the degree offatty acid unsatusults are similar to the report of Smith and ration, since the argentation chromatography Ribbons (19) concerning phospholipids from M. methanoxidans, where over 90% of the esteriyielded no further separation. The data in Table 2 reveal that over 94% of fied fatty acid was 18:1. A lack of diversity of the phospholipids consisted of PE and PG. Al- the fatty acids and a preponderance of one parthough these are two quite common phospho- ticular fatty acid are not particularly characterlipids among bacteria, there are some interest- istic of bacteria. For example, White (24) deing features in the distribution found for M. scribed 36 relatively evenly distributed fatty trichosporium. For example, although it is not acids ranging from 12 to 22 carbons in the phosunusual for these two phospholipids to comprise pholipids of Haemophilus parainfluenzae. 30 to 85% of the total phospholipids in bacteria, Once again of interest are similarities between the magnitude of predominance found in this M. trichosporium and the photosynthetic and study is relatively greater than normally ob- nitrifying bacteria. The ammonia-oxidizing bacserved (6). High PE and PG levels have also teria show a preponderance of 16:1 esterified been reported for other bacteria with extensive fatty acid on the phospholipids, whereas the intracytoplasmic membrane development. For nitrite-oxidizing bacteria have mostly 18:1 fatty example, PE plus PG accounts for over 90% of acid (1). Among the photosynthetic bacteria, R. the phospholipids of Chromatium sp., Rhodo- vannielii has 90% 18:1 fatty acid, and Rhodopseudomonas gelatinosa, Rhodospirillum cap- pseudomonas spheroides has 76.8% 18:1 fatty sulatus, Nitrocystis oceanus, and Nitrosomo- acid in its phospholipids (12). The similarity of nas europea (6, 9, 12). The distribution is not fatty acid composition of phospholipids could be limited to bacteria with intracytoplasmic mem- advantageous to an organism by facilitating branes, however, because many of the Entero- the synthesis of the various phosphatides via a bacteriaceae including Salmonella, Enterobac- common precursor. This could be especially imter, and Escherichia contain over 90% PE plus portant to an organism whose metabolism is PG (6). Thus, other than the fact that high PE very dependent upon complex membrane sysplus PG is more typical of gram-negative bacte- tems, and it may explain the lack of diversity ria than of gram-positive bacteria and that among the phospholipid fatty acids in M. trichomany of the bacteria containing intracytosporium. In view of the similar lipid composiplasmic membranes (which are also gram-nega- tion of M. trichosporium to M. methanoxidans, tive) have high PE plus PG, the significance of even though they are otherwise quite different, this observation is not apparent. one wonders whether there is some fundamenAnother somewhat unusual feature of the tal physiological reason why these bacteria oxiphospholipid distribution of M. trichosporium dizing methane gas both contain a preponderis the PE/PG ratio, 0.65. Gram-negative bacte- ance of 18:1 fatty acids. This is the longest fatty ria typically have PE/PG > 1, whereas gram- acid (with respect to the simple saturated and positive bacteria usually have PE/PG < 1 (6). monounsaturated fatty acids normally found in For example, of the 30 gram-negative bacteria bacteria) that would remain liquid at normal listed by Goldfine (6), only Thiobacillus thiooxi- physiological temperatures; however, why only dans, T. ferrooxidans, and Rhodomicrobium certain organisms have large amounts of 18:1
LIPIDS OF M. TRICHOSPORIUM
VOL. 124, 1975
fatty acid is unknown. Although methane solubility should increase with increasing fatty acid chain length, substrate solubility could not explain the similar membrane compositions in the photosynthetic and nitrifying bacteria. Furthermore, bacteria oxidizing hydrocarbons other than methane, which might also be expected to exhibit similar fatty acid distributions in their phospholipids if simple solubility and melting point were the answer, have recently been shown to synthesize a range of fatty acids from C,0 to C20 (15). Thus, although the phospholipids of M. trichosporium exhibit some singular characteristics, the biochemical significance relative to the function of the intracytoplasmic membranes remains to be elucidated. ACKNOWLEDGMENTS This research was supported by grant A-027-OHIO from the Office of Water Resources Research, Department of the Interior, and by federal grant funds received under the Hatch Act. LITERATURE CITED 1. Blumer, M., T. Chase, and S. W. Watson. 1969. Fatty acids in the lipids of marine and terrestrial nitrifying bacteria. J. Bacteriol. 99:366-370. 2. Brian, B. L., and E. W. Gardner. 1969. A simple procedure for detecting the presence of cyclopropane fatty acids in bacterial lipids. Appl. Microbiol. 16:549-552. 3. Dittmer, J., and M. Wells. 1969. Quantitative and qualitative analysis of lipids and lipid components, p. 482530. In J. M. Lowenstein (ed.), Methods in enzymology, vol. 14. Academic Press Inc., New York. 4. Folch, J., M. Lees, and G. H. Sloane-Stanley. 1957. A simple method for the isolation and purification of total lipids from animal tissues. J. Biol. Chem. 226:497-509. 5. Gantt, E., and S. F. Conti. 1969. Ultrastructure of bluegreen algae. J. Bacteriol. 97:1486-1493. 6. Goldfine, H. 1972. Comparative aspects of bacterial lipids, p. 1-58. In A. Rose and D. Tempest (ed.), Advances in microbial physiology, vol. 8. Academic Press Inc., New York. 7. Goswami, S. K., and C. F. Frey. 1971. Spray detection
8. 9.
10. 11. 12.
13.
14.
15. 16. 17.
18.
19. 20. 21. 22. 23. 24.
605
of phospholipids in thin layer chromatograms. J. Lipid Res. 12:509-510. Guidotti, G. 1972. The composition of biological membranes. Arch. Intern. Med. 129:194-201. Hagen, P. 0. 1966. Phospholipids of bacteria with extensive intracytoplasmic membranes. Science 151:1546. Kenyon, C. N. 1972. Fatty acid composition of unicellular strains of blue-green algae. J. Bacteriol. 109:827834. Law, J. H., and R. A. Slepecky. 1961. Assay of polybeta-hydroxybutyric acid. J. Bacteriol. 82:33-36. Oelze, J., and G. Drews. 1972. Membranes of photosynthetic bacteria. Biochim. Biophys. Acta 265:209-239. Op DenKamp, J., and L. VanDennen. 1969. Bacterial phospholipids and membranes, p. 227-325. In Structural aspects of lipoproteins in living systems. Academic Press Inc., New York. Park, C.-E., and L. R. Berger. 1967. Complex lipids of Rhodomicrobium vannielii. J. Bacteriol. 93:221-229. Patrick, M., and P. Dugan. 1974. Influence of hydrocarbons and derivatives on the polar lipid fatty acids of an Acinetobacter isolate. J. Bacteriol. 119:76-81. Radin, N. 1969. Preparation of lipid extracts, p. 245254. In J. M. Lowenstein (ed.), Methods in enzymology, vol. 14. Academic Press Inc., New York. Singer, S. J., and G.L. Nicolson. 1972. The fluid mosaic model of the structure of cell membranes. Nature (London) 175:720-731. Skipsi, V., and M. Barclay. 1969. Thin layer chromatography of lipids, p. 520-598. In J. M. Lowenstein (ed.), Methods in enzymology, vol. 14. Academic Press Inc., New York. Smith, U., and D. W. Ribbons. 1970. Fine structure of Methanomonas methanoxidans. Arch. Mikrobiol. 74:116-122. Watson, S. W. 1965. Characteristics of a marine nitrifying bacterium, Nitrocystis oceanus. Limnol. Oceanogr. 10:274-289. Weaver, T. L., and P. R. Dugan. 1975. Ultrastructure of Methylosinus trichosporium as revealed by freeze etching. J. Bacteriol. 121:704-710. Weaver, T. L., and P. R. Dugan. 1975. Methylotrophic enzyme distribution in Methylosinus trichosporium. J. Bacteriol. 122:433-436. White, D., and F. Frerman. 1967. Extraction, characterization, and cellular localization of the lipids of Staphylococcus aureus. J. Bacteriol. 94:1854-1867. White, D. C. 1968. Lipid composition of the electron transport membrane of Haemophilus parainfluenzae. J. Bacteriol. 96:1159-1170.
JOURNAL OF BACTERIOLOGY, Nov. 1975, p. 602-605 Copyright ©) 1975 American Society for Microbiology
Printed in U.S.A.
Whole-Cell and Membrane Lipids of the Methylotrophic Bacterium Methylosinus trichosporium T. L. WEAVER,* M. A. PATRICK, AND P. R. DUGAN
Laboratory of Microbiology, Cornell University, Ithaca, New York 14853 * and Department of Microbiology, Ohio State University, Columbus, Ohio 43210 Received for publication 5 June 1975
The lipid composition of the methylotrophic bacterium Methylosinus trichosporium was examined. Whole-cell lipid distribution was 39.1% neutral lipids, 34.5% polar lipids, and 26.4% poly-beta-hydroxybutyric acid. Membrane lipids were 83% phospholipids, with phosphatidylethanolamine and phosphatidylglycerol accounting for over 94% of the total. All the phospholipids had similar fatty acid compositions, with 18:1 accounting for about 87% of the total and most of the rest consisting of 16:1. Similarities between the lipid composition of this bacterium and other bacteria are discussed. The predominant ultrastructural characteris- in nitrogen-flushed screw-top tubes to prevent degratic of methane-oxidizing bacteria is the pres- dation. Dry weight of cells and membranes were ence of extensive arrays of intracytoplasmic obtained by weighing representative samples in alupans after drying 16 h at 60 C. membranes (21). In this regrad, these bacteria minum Poly-beta-hydroxyPoly-beta-hydroxlybutyrate. are similar to other membranous prokaryotic butyrate was precipitated from lipid extracts by addmicroorganisms such as photosynthetic bacte- ing 2 volumes of methanol and holding at -10 C for ria (12), blue-green bacteria (5), and nitrifying 2 to 4 h. The samples was then pelleted by centrifubacteria (20), which also possess internal mem- gation, and the poly-beta-hydroxybutyrate-free sambrane systems. The role of lipids, particularly ple was concentrated by vacuum evaporation at phospholipids, in membrane structure is well 40 C. Poly-beta-hydroxybutyrate was quantitated documented, and the important physiological by the method of Law and Slepecky (11). Silicic acid column chromatography. A concensignificance of lipids in membranes is recogtrated containing 10 to 100 mg of lipid was nized (17). Although the lipids of all the other appliedsample to a 10-g column of silicic acid that was membranous types of prokaryotes previously prewashed and by heating at 110 C for 12 mentioned have been investigated (1, 10, 14), h. Neutral lipidsactivated were eluted with chloroform, and only one species of methane oxidizer, Methano- polar lipids were eluted with methanol. Both lipid monas methanoxidans, has been subjected to fractions were dried in a stream of nitrogen and lipid examination and this was restricted to quantitated gravimetrically. Thin-layer chromatography. Phospholipids were fatty acid examination (19). The present study was undertaken to examine the lipids of the separated on silica gel (D-O, Camag) thin-layer methane oxidizer, Methylosinus trichospo- plates. The plates were prewashed in acetone-petroleum ether (1:3, vol/vol) and developed in either rium. chloroform-methanol-water (65:25:4 vol/vol/vol) or MATERIALS AND METHODS Microorganism. M. trichosporium (OB3b isolate of R. Whittenbury) was grown in mineral salts CM under methane and air as described previously (21). Membranes from these cells were prepared by differential centrifugation as reported previously (22). This membrane preparation was relatively devoid of cell wall materials, as indicated by carbohydrate analysis, and consisted mainly of intracytoplasmic membranes. Lipid extraction. Wet cell pellets and membrane preparations were extracted routinely with chloroform-methanol (2:1 vol/vol) by shaking for 18 h at 25 C (4). The extracts were filtered through several thicknesses of chloroform-methanol-washed Whatman no. 1 filter paper and washed by the method of Radin (16). Lipid extracts were handled and stored
butanol-acetic acid-water (60:20:20, vol/vol/vol). Phospholipids were routinely detected by using the molybdate spray reagent of Goswami and Frey (7). Phospholipids were identified by relative Rf with standards (Supelco. Inc., Bellefonte, Pa.), acid hydrolysis and mild alkaline hydrolysis products (3), periodate oxidation to detect vicinal hydroxyls (23), argentation chromatography (18), and by various selective spray reagents including the orcinol reagent for glycolipids, ninhydrin reagent for amino groups, and the Reitsema test for plasmalogens (18). Quantitation of phospholipids. A 1-mCi amount of 32P as orthophosphate (Amersham/Searle, Arlington Heights, Ill.) was added to 2 liters of CM medium that contained 1.4 mmol of unlabeled phosphate per liter. Cells were grown and harvested as described previously (21). After membrane fractions were prepared and phospholipids were extracted and 02
VOL. 124, 1975
LIPIDS OF M. TRICHOSPORIUM
603
separated, spots were scraped from the thin-layer chromatrography plate and counted in a standard toluene-based cocktail containing Cabosil (Packard Instrument Co., Inc., Downers Grove, Ill.). Phospholipid fatty acid analysis. Methyl esters of phospholipid fatty acids were prepared by the mild methanolysis procedure of White (24). Methyl esters were analyzed by using a Varian model 200 gas chromatograph equipped with 10% diethyleneglycol succinate on a Chromosorb-W column (8 feet by 1/8 inch [ca. 2.44 m by 0.32 cm]) and a flame ionization detector. The injector and detector were 200 C while the column was maintained at 170 C. Nitrogen carrier gas was adjusted to 25 ml/min. Relative retention times were compared with methyl ester standards (Applied Science Laboratory, State College, Pa.). Unsaturated and cyclopropane fatty acids were differentiated by the method of Brian and Gardner (2).
TABLE 2. Scintillation spectrophotometric quantitation of 32P-labeled membrane phospholipids from a thin-layer chromatography plate
RESULTS
pholipids were present in such small quantities that they could not be tested by hydrolysis. Argentation thin-layer chromatography through silver nitrate-impregnated silica gel yielded no additional spots. The fatty acid composition of the major phospholipids is listed in Table 3. The 16:1 and 18:1 fatty acids hydrogenated completely under the mild conditions of Brian and Gardner (2), revealing that the fatty acids are monounsaturates and not the cyclopropane derivatives.
Extraction of wet cells (778 mg dry weight) and wet membranes (84 mg dry weight) yielded 71.6 and 23.5 mg of lipids, respectively. A summary of the lipid distribution in these extracts is provided in Table 1. Table 2 lists the distribution of phospholipids in the membranes. It was found that the same relative phospholipid distribution was also observed with whole cells as with the isolated membranes. Each phospholipid chromatographed identically with a known standard in both solvents; all reacted with Goswami reagent, and phosphatidylethanolamine (PE) and phosphatidylserine reacted with ninhydrin. All tested negatively for plasmalogen and glycolipid. As further confirmation of the identity of the two major phospholipids, the water-soluble, mild alkaline hydrolysis product of PE chromatographed with a similarly hydrolyzed standard, the acid-hydrolyzed product of PE chromatographed with ethanolamine, and the periodate oxidation of the phosphatidylglycerol (PG) yielded formaldehyde as it also did with standard. The other minor phosTABLE 1. Lipid components of whole cells and membranes of M. trichosporium Calculated Total
Total
Lipid
dry
dry
drywto to
dry
wt tal e wt of cells b of membl (% (% wt/wt) /wt)lipid( wb
tlt)
tl
11
extract-
able membrane lipids
%,
w
wtlwt)
PHBa
2.4
0.11
26.4
0.4
Neutral lipid Polar lipid
3.6
4.5
39.1
16.2
3.2
23.3
34.5
83.4
a
PHB, Poly-beta-hydroxybutyrate.
Phospholipid
CounWmin
% Total membrane
Diphosphatidylglycerol PE PG Phosphatidylserine Phosphatidylcholine
2,956 150,198 229,583 16,862 2,419
0.4a 37.5 57.3 4.2 0.6
phospholipids
a Since diphosphatidylglycerol contains two phosphorus atoms, this figure was based upon (0.5) (2,956 counts/min)/total counts per minute in all five fractions.
DISCUSSION The cell and membrane lipid distribution in Table 1 reveals that only about 9.2% of the cell dry weight was extracted as lipid whereas 27.9% of the membrane dry weight was extracted as lipid. This lipid enrichment in the membrane fraction would be expected since membranes are basically lipid-protein structures. The quantity of lipids observed in the membranes of M. trichosporium compares closely with previous reports of 24% lipid in inner mitochondrial membrane and 25% lipid in bacterial plasma membrane; however, these are very different from the 43% lipid in erythrocyte and liver cell membranes (8). The relatively large amount of poly-beta-hydroxybutyrate found in whole cells (26.4% of the total lipid) suggests that this compound must play an important role in the physiology of this bacterium. Presumably, the trace of poly-beta-hydroxybutyrate in the membrane sample was due to contamination. The polar lipid content increased from the 34.5% found in whole cells to 83.4% found in the membranes. This is expected since membranes normally contain relatively large quantities of phospholipids (13). The observation that the same five phospholipids in about the same ratios were observed whether whole cells or membranes were ana-
604
WEAVER, PATRICK, AND DUGAN
TABLE 3. Fatty acid compositions of the membrane phospholipids of M. trichosporium
J. BACTERIOL.
vannielii has PE/PG < 1, and of 23 grampositive bacteria, only Bacillus cereus, B. subtilus, and Micrococcus cerificans had PE/PG > 1. % Total fatty acid Again, only the singularity of this distribution Phospholipid is obvious. 16:0 16:1 18:1 The other three phospholipids, diphosphatiDiphosphatidylglycerol 0 13 87 dylglycerol, phosphatidylserine, and phosphatiPE 0.2 10.9 88.9 dycholine, account for only 5.2% of the phosphoPG 1 16 83 lipids. Diphosphatidylglycerol (or cardiolipin) Phosphatidylserine 0 13 87 frequently occurs in bacteria with PG since they are related in synthesis (6). Similarly, phosphatidylserine is an intermediate in PE lyzed was not surprising since nearly all cellu- synthesis and is often detected in bacteria with lar phospholipids are in the membranes (13). PE. Phosphatidylcholine is usually not a major This is also reassuring since it demonstrates component of bacterial phospholipids even that the phospholipid distribution observed though it is a very important component in with the membrane preparation was not a re- eukaryotic microorganisms (6). The results of the fatty acid analyses of these sult of working with small amounts of material. Furthermore, each phospholipid is homoge- phospholipids are shown in Table 3. These reneous, at least in the degree offatty acid unsatusults are similar to the report of Smith and ration, since the argentation chromatography Ribbons (19) concerning phospholipids from M. methanoxidans, where over 90% of the esteriyielded no further separation. The data in Table 2 reveal that over 94% of fied fatty acid was 18:1. A lack of diversity of the phospholipids consisted of PE and PG. Al- the fatty acids and a preponderance of one parthough these are two quite common phospho- ticular fatty acid are not particularly characterlipids among bacteria, there are some interest- istic of bacteria. For example, White (24) deing features in the distribution found for M. scribed 36 relatively evenly distributed fatty trichosporium. For example, although it is not acids ranging from 12 to 22 carbons in the phosunusual for these two phospholipids to comprise pholipids of Haemophilus parainfluenzae. 30 to 85% of the total phospholipids in bacteria, Once again of interest are similarities between the magnitude of predominance found in this M. trichosporium and the photosynthetic and study is relatively greater than normally ob- nitrifying bacteria. The ammonia-oxidizing bacserved (6). High PE and PG levels have also teria show a preponderance of 16:1 esterified been reported for other bacteria with extensive fatty acid on the phospholipids, whereas the intracytoplasmic membrane development. For nitrite-oxidizing bacteria have mostly 18:1 fatty example, PE plus PG accounts for over 90% of acid (1). Among the photosynthetic bacteria, R. the phospholipids of Chromatium sp., Rhodo- vannielii has 90% 18:1 fatty acid, and Rhodopseudomonas gelatinosa, Rhodospirillum cap- pseudomonas spheroides has 76.8% 18:1 fatty sulatus, Nitrocystis oceanus, and Nitrosomo- acid in its phospholipids (12). The similarity of nas europea (6, 9, 12). The distribution is not fatty acid composition of phospholipids could be limited to bacteria with intracytoplasmic mem- advantageous to an organism by facilitating branes, however, because many of the Entero- the synthesis of the various phosphatides via a bacteriaceae including Salmonella, Enterobac- common precursor. This could be especially imter, and Escherichia contain over 90% PE plus portant to an organism whose metabolism is PG (6). Thus, other than the fact that high PE very dependent upon complex membrane sysplus PG is more typical of gram-negative bacte- tems, and it may explain the lack of diversity ria than of gram-positive bacteria and that among the phospholipid fatty acids in M. trichomany of the bacteria containing intracytosporium. In view of the similar lipid composiplasmic membranes (which are also gram-nega- tion of M. trichosporium to M. methanoxidans, tive) have high PE plus PG, the significance of even though they are otherwise quite different, this observation is not apparent. one wonders whether there is some fundamenAnother somewhat unusual feature of the tal physiological reason why these bacteria oxiphospholipid distribution of M. trichosporium dizing methane gas both contain a preponderis the PE/PG ratio, 0.65. Gram-negative bacte- ance of 18:1 fatty acids. This is the longest fatty ria typically have PE/PG > 1, whereas gram- acid (with respect to the simple saturated and positive bacteria usually have PE/PG < 1 (6). monounsaturated fatty acids normally found in For example, of the 30 gram-negative bacteria bacteria) that would remain liquid at normal listed by Goldfine (6), only Thiobacillus thiooxi- physiological temperatures; however, why only dans, T. ferrooxidans, and Rhodomicrobium certain organisms have large amounts of 18:1
LIPIDS OF M. TRICHOSPORIUM
VOL. 124, 1975
fatty acid is unknown. Although methane solubility should increase with increasing fatty acid chain length, substrate solubility could not explain the similar membrane compositions in the photosynthetic and nitrifying bacteria. Furthermore, bacteria oxidizing hydrocarbons other than methane, which might also be expected to exhibit similar fatty acid distributions in their phospholipids if simple solubility and melting point were the answer, have recently been shown to synthesize a range of fatty acids from C,0 to C20 (15). Thus, although the phospholipids of M. trichosporium exhibit some singular characteristics, the biochemical significance relative to the function of the intracytoplasmic membranes remains to be elucidated. ACKNOWLEDGMENTS This research was supported by grant A-027-OHIO from the Office of Water Resources Research, Department of the Interior, and by federal grant funds received under the Hatch Act. LITERATURE CITED 1. Blumer, M., T. Chase, and S. W. Watson. 1969. Fatty acids in the lipids of marine and terrestrial nitrifying bacteria. J. Bacteriol. 99:366-370. 2. Brian, B. L., and E. W. Gardner. 1969. A simple procedure for detecting the presence of cyclopropane fatty acids in bacterial lipids. Appl. Microbiol. 16:549-552. 3. Dittmer, J., and M. Wells. 1969. Quantitative and qualitative analysis of lipids and lipid components, p. 482530. In J. M. Lowenstein (ed.), Methods in enzymology, vol. 14. Academic Press Inc., New York. 4. Folch, J., M. Lees, and G. H. Sloane-Stanley. 1957. A simple method for the isolation and purification of total lipids from animal tissues. J. Biol. Chem. 226:497-509. 5. Gantt, E., and S. F. Conti. 1969. Ultrastructure of bluegreen algae. J. Bacteriol. 97:1486-1493. 6. Goldfine, H. 1972. Comparative aspects of bacterial lipids, p. 1-58. In A. Rose and D. Tempest (ed.), Advances in microbial physiology, vol. 8. Academic Press Inc., New York. 7. Goswami, S. K., and C. F. Frey. 1971. Spray detection
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