PHOSPHOLIPIDS IN VEGETATIVE CELLS AND SPORES

Transcription

1 JOURNAL OF BACTERIOLOGY Vol. 87,rNo. 1, pp January, 1964 Copyright a 1964 by the American Society for Microbiology Printed in U.S.A. PHOSPHOLIPIDS IN VEGETATIVE CELLS AND SPORES OF BACILLUS POLYMYXA' JACK R. MATCHES,2 HOMER W. WALKER, AND JOHN C. AYRES Department of Bacteriology and Department of Dairy and Food Industry, Iowa State University of Science and Technology, Ames, Iowa Received for publication 25 July 1963 ABSTRACT MATCHES, JACK R. (Iowa State University of Science and Technology, Ames), HOMER W. WALKER, AND JOHN C. AYRES. Phospholipids in vegetative cells and spores of Bacillus polymyxa. J. Bacteriol. 87: The same types of phospholipids were recovered from both vegetative cells and spores of Bacillus polymyxa la39. Nitrogen-containing phospholipids were identified as phosphatidyl ethanolamine, lysophosphatidyl ethanolamine, lysophosphatidyl serine, and lysolecithin. Acidic phosphatidescontainingno nitrogen were identified as phosphatidic acid, phosphatidyl glycerol, and a fraction appearing to be bis (phosphatidic) acid. The major phosphatide fraction in both cells and spores was phosphatidyl ethanolamine. Smaller amounts of phosphatidyl glycerol and bis (phosphatidic) acid were present; the other acidic phospholipid components were present only in trace amounts. Heat resistance of the spore as compared to the vegetative cell could not be attributed to a specific phospholipid, since no difference in the type of phospholipids present was observed. The presence of fatty acids in the sporulating medium has been shown by Sugiyama (1951) to increase the heat resistance of spores of Clostridium botulinum. Extraction of lipids from the surface of these spores, and others as well, apparently has little effect on heat-resistance characteristics (Sugiyama, 1951; Church et al., 1956; Long and Williams, 1960). The fatty acids seem to be incorporated into the spores during sporulation. Spores formed in the presence of palmitate, for example, have greatly increased heat resistance; the addition of palmitate to the mature spore is without effect (Sugiyama, 1952). The phospholipids in sporeforming organisms have been studied to a limited extent. Long and Williams (1960) could find no cephalin or sphingomyelin in spores or vegetative cells of B. stearothermophilus; Dyer (1953) had previously reported the presence of sphingomyelin in this organism. Long and Williams believed that the phospholipids might be either lecithins or phosphatidic acids. A preponderance of the work on phospholipids in the bacilli has been restricted to vegetative cells. Some of the phospholipids isolated from the vegetative forms have been phos- Attempts to correlate lipids of bacterial spores with heat resistance have met with only limited phatidic acids from ghosts of Bacillus M (Weibull, success. Gaughran (1947) reviewed some of the 1957); neutral lipids and phospholipids from the early studies on this subject, and observed that protoplast membrane of B. megaterium (Yukin, the lipids of Bacillus subtilis decreased in quantity 1962); phosphatidyl serine, phosphatidyl ethanolamine, and an inositol phosphatide in the mem- and unsaturation as the incubation temperature was raised above the optimum; these conditions brane fractions from lysed protoplasts of B. of incubation usually resulted in greater heat stability. The lipids of an unidentified thermophile amine, phosphatidyl glycerol, lecithin, lysoleci- megaterium (Hill, 1962); phosphatidyl ethanol- remained unchanged under similar conditions. thin, lysophosphatidyl ethanolamine, and three I Journal Paper no. J-4680 of the Iowa Agricultural and Home Economics Experiment Sta- Kushner, and James, 1962); and phosphatidyl acidic phosphatides from cells of B. cereus (Kates, tion, Ames Project no. 1393, Center for Agricultural and Economic Development cooperating. (Haverkate, Houtsmuller, and Van Deenen, glycerol from cells of B. cereus and B. megaterium Part of this work was submitted by the senior author in partial fulfillment of the requirements 1962). The last authors also mentioned the presence of cardiolipin, phosphatidic acid, and phos- for the Ph.D. degree. 2 Present address: College of Fisheries, University of Washington, Seattle. these organisms. phatidyl ethanolamine in certain fractions from 16

2 VOL. 87, 1964 PHOSPHOLIPIDS IN B. POLYMYXA 17 The present study was initiated to identify the phospholipids occurring in cells and spores of B. polymyxa 1A39, and to determine whether any differences existed which might be related to the greater heat tolerance of spores. MATERIALS AND METHODS Organism. B. polymyxa 1A39 was obtained from the Department of Bacteriology, Iowa State University. Cells were grown in the G medium of Stewart and Halvorson (1953). Spores were produced in the same medium, except that the calcium chloride content was increased to 100 mg/ iter to obtain satisfactory sporulation. The inoculum for the production of both vegetative cells land spores was prepared by washing the growth from the surface of ten stock-culture agar slants and adding this to 1.5 liters of G medium. The inoculum was shaken continuously and incubated overnight at 30 C. The cells were then added to 15 liters of G medium in a 20-liter Pyrex carboy fitted with a stirrer, and sterile air was forced into the liquid medium through spargers at a rate calculated to be 3.5 liters/min. Vegetative cells were collected after 12 hr, and spores after approximately 24 hr, of incubation at 30 C. The collected cells and spores were washed four times: once with alkaline distilled water, ph 10.0; once with acidic distilled water, ph 2.5; and twice with distilled water. Extraction of cell lipids. The methods of Kates, Kushner, and James (1962), and of Folch, Lees, and Stanley (1957), were used for extraction of lipids. After extraction by either method, it was necessary to remove the solvent in vacuo and reextract with diethyl ether. This step removed a nonlipid contaminant that reduced the flow on the silicic acid column. Extraction of spore lipids. Lipid material was extracted from spores in a mixture of chloroformmethanol (2:1, v/v). The spores were suspended in the solvent as a very thick slurry (200 mg/ml), and were disrupted with a Mickle tissue disintegrator; 4 ml of the spore slurry, 6 g of glass beads (80 to 120 mesh), and 2.5 ml of solvent were added to the Mickle containers. Complete disruption of the spores was obtained in approximately 45 min. The homogenate was decanted, and the lipids were extracted from the spore material by the method of Folch, Lees, and Stanley (1957). Silicic acid column. A glass column (13 X 350 mm), fitted with a 200-ml reservoir on the top, a Teflon stopcock at the bottom, and a porous glass disc fused 2.5 cm above the stopcock, was used for making silicic acid columns. A nitrogen cylinder was connected to a ground glass ball and socket joint on top of the reservoir. A glass-wool plug was placed on the porous glass disc to support the silicic acid. A filter-paper disc was placed on top of the silicic acid to prevent disruption of the colunm surface when adding solvents. A petroleum ether slurry containing 7 g of silicic acid was added to the column. The column was washed with acetone-ether (1:1, v/v), and then with anhydrous ether. A lipid sample containing approximately 150,ug of phosphorus was carefully layered on the top surface of the column, and slight nitrogen pressure was applied. The sample was allowed to drain nearly to the surface of the silicic acid, and then additional small quantities of solvent were slowly added until the sample was completely washed onto the column. Neutral lipids were removed from the column by washing with anhydrous ether; the phospholipids were eluted from the column with chloroform containing increasing increments of methanol. An automatic fraction collector was used to collect 5-ml samples. Phosphorus determination. Phosphorus determinations were made on 0.5 ml of each fraction. After removal of the solvent by evaporation at 80 C, 0.1 ml of concentrated sulfuric acid was added. These samples were digested at 150 to 160 C for 4 hr; two drops of 30% hydrogen peroxide were then added to each tube and digestion was continued for 2 hr. If needed, further additions of peroxide were made to get complete digestion and clear solutions. The digests were diluted with 5 ml of 2 N NaOH; 2 ml of freshly prepared color reagent were added and mixed with the contents of the tubes. The color reagent was prepared by the method of Chen, Toribara, and Warner (1956). The samples were placed in a water bath at 37 C for 1.5 hr, after which absorbance at 820 m,u was determined. Separation of phosphatides. The phosphatides were separated on silicic acid-impregnated paper. Diisobutyl ketone-acetic acid-water (40:20:3, v/v) was used as the solvent system (Marinetti, 1962). Sodium silicate was prepared according to the procedure of Marinetti (1962). Whatman no. 1 paper was impregnated by the method of Hack (1961). Chromatograms were stained with rhodamine 6G for detection of phospholipid compo-

3 1818MATCHES, WALKER, AND AYRES J. BACTERIOL. nents (Marinetti and Stotz, 1956; Marinetti, Erbland, and Kochen, 1957). Tests for the detection of amino phosphatides, choline, unsaturation, and plasmalogens were those outlined by Marinetti (1962). The Dragendorif reagent (Bregoff, Roberts, and Delwiche, 1953) was also used for the detection of choline. The spray recommended by Hanes and Isherwood (1949) was used for the detection of phosphorus-containing lipids. The ~B ; 0.6- Ia C4 FIG. 1. Tracing of a typical chromatogram of phospholipids extracted from vegetative cells and spores of Bacillus polymyxa. Abbreviations: Y, yellow; B, blue; 0, orange. Identification of the spots is given in Table 1. alkaline silver nitrate reagents of Trevelyan, Procter, and Harrison (1956) were used for glycerol; and the periodate-schiff reagent, for vicinal glycol-containing lipids. Hydrolysis procedures. Acid hydrolysis of the phosphatides was performed by the method of Kaneshiro and Marr (1962). Products of hydrolysis were separated by ascending and descending chromatography on Whatman no. 1 paper with n-propanol-ammonium hydroxide-water (6:3: 1, v/v) according to the method of Kaneshiro and Marr (1962), and with n-butanol-methyl ethyl ketone-water (2:2:1, v/v), as recommended by Mizell and Simpson (1961), as the solvent systems. Alkaline hydrolysis was done by the methods of Dawson (1960) and of Benson and Strickland (1960). The deacylated phosphatides were separated by ascending chromatography on Whatman no. 1 paper with phenol-water (100:38, w/v) and with butanol-acetic acid-water (5:3:1, v/v). RESULTS AND DISCUSSION Analyses of the total lipid extracted from both vegetative cells and spores of B. polymyxa 1A39 revealed the presence of the same eight components in each. A tracing of a typical chromatogram (Fig. 1) illustrates the type of separation obtained on silicic acid-impregnated paper. The spot number, RF values, and some of the characteristics of the complex lipids from cells and spores are listed in Table 1. The results of deacylation and chromatographic separation of the products of hydrolysis of cell lipids in phenol-water (100: 38, w/v) and butanol-acetic acid-water (5:3:1, v/v) are given in Table 2. Phosphatidyl ethanolamine. The major component of vegetative cells and spores, i.e., spot 5, has been identified as phosphatidyl ethanolamine. When separated on silicic acid-impregnated paper, stained with rhodamine 6G, and viewed under ultraviolet light, this component appeared orange and had an RF value of A standard sample of phosphatidyl ethanolamine was found to have the same RF and positive reaction with ninhydrin. Other RF values reported for this compound have been 0.50 (Marinetti, 1962) and 0.57 (Kates, Sehgal, and Gibbons, 1961; Kates et al., 1962). After deacylation and chromatographic separation, a spot corresponding to glycerophosphoryl ethanolamine was detected (Table 2); it had the same RF as that obtained with the

4 VOL. 87, 1964 PHOSPHOLIPIDS IN B. POLYMYXA 19 TABLE 1. Characteristics of complex lipids extracted from cells and spores of Bacillus polymyxa* Average Color with Ninhy- Periodate- Choline Iett fcmoet Spot no RF value rhodamine 6G drin Schiff stain Identity of components stain stain Yellow (w) Lysolecithin Blue (w) Lysophosphatidyl serine Orange (w) Lysophosphatidyl ethanolamine Blue (s) Phosphatidyl glycerol Orange (s) Phosphatidyl ethanolamine Blue (s) Bis (phosphatidic) acid Blue (w) Phosphatidic acid 8 Orange (s) Neutral lipids * Abbreviations: w, weak; s, strong; +, positive; - negative. deacylated standard sample. These spots also reacted with the ninhydrin stain for amino groups, the periodate-schiff stain for vicinal glycol-containing lipids, and the Hanes-Isherwood stain for phosphorus-containing lipids. Acid hydrolysis of the total lipid extracted from vegetative cells released ethanolamine which could be detected with the ninhydrin stain. A sample of acid-hydrolyzed standard phosphatidyl ethanolamine and a sample of ethanolamine served as references in identifying the ethanolamine from bacterial cells. Phosphatidyl ethanolamine has also been identified as a major phosphatide in several other bacteria, namely, B. cereus, Micrococcus halodenitrificans, Azotobacter agilis, Agrobacterium tumefaciens, Escherichia coli, and Clostridium butyricum (Kates et al., 1961, 1962; Kaneshiro and Marr, 1961, 1962; Goldfine, 1962). Asselineau and Lederer (1960), in a review of the literature, could find no reference to phosphatidyl ethanolamine in the mycobacteria. Later work has shown this phosphatide to be present in four Mycobacterium species, two of which are pathogenic (Subrahmanyam, Nandedkar, and Viswanathan, 1962). Lysophosphatidyl ethanolamine. Another closely related phospholipid, lysophosphatidyl ethanolamine, was found in both vegetative cells and spores (spot 3). The reactions of this compound were similar to those obtained with the parent compounds. Upon treatment with rhodamine 6G, an orange fluorescent spot having an RF of 0.36 was obtained. This component was present in low concentrations and could be detected with the ninhydrin stain only when a large sample was used. Lysophosphatidyl ethanolamine has also been observed in M. halodenitrificans (RF of 30) and B. cereus (RF of 40; Kates et al., 1961, 1962). TABLE 2. RF values of hydrolysis products of phospholipids extracted from Bacillus polymyxa* Spot RF PW Rp BAW Identified as I Glycerophosphate II Glycerophosphoryl glycerol III Glycerophosphoryl ethanolamine IV Glycerol V Glycerophosphoryl choline * Abbreviations: PW, phenol-water (100:38, w/v); BAW, butanol-acetic acid-water (5:3:1, v/v). Phosphatidyl glycerol. Spot 4 has been identified as phosphatidyl glycerol on the basis of blue fluorescence in the presence of rhodamine 6G, RF of 0.45, positive reaction with the periodate- Schiff reagent, and the presence of glycerophosphoryl glycerol in the hydrolysate. Other workers have reported a blue fluorescence and RF values of 0.45 (Kates et al., 1961), 0.49 (Sehgal, Kates, and Gibbons, 1962), and 0.51 (Kates et al., 1962). Since then, phosphatidyl glycerol has been found as a component of the phospholipids of M. halodenitrificans, B. cereus, Halobacterium cutirubrum, and Ml. lysodeikticus (Kates et al., 1962; Sehgal et al., 1962; Macfarlane, 1961). Bis (phosphatidic) acid. Spot 6 has been tentatively identified as bis (phosphatidic) acid. RF values varying from 0.57 to 0.69 were obtained; these values are very close to the range of 0.57 to 0.67 reported for the synthetic compound (Marinetti, 1962). This spot gave a blue fluorescence upon treatment with rhodamine 6G.

5 20 MATCHES, WALKER, AND AYRES J. BACTERIOL. Amino nitrogen could not be detected with ninhydrin, and a negative periodate-schiff test was obtained. Glycerophosphoryl glycerol was detected when the sample was deacylated. A standard sample of this compound was not available for comparison, but the acidic nature and large RF value of this compound suggests that it might have been a polyglycerol phosphatide. After deacylation, glycerophosphoryl glycerol was detected and a spot corresponding to 1, 3-diglycerophosphoryl glycerol was not detected. This would indicate that the compound was not a polyglycerol phosphatide. Bis (phosphatidic) acid has seldom been isolated from natural sources. It has been found in the lipids of cod and haddock by Garcia, Lovern, and Olley (1956); reports of its occurrence in other tissues have not been found. Phosphatidic acid. Spot 7 has been identified as phosphatidic acid as a result of the large RF when separated on silicic acid-impregnated paper, blue fluorescence in the presence of rhodamine 6G, and comparison with synthetic phosphatidic acid. The RF value of 0.75 is very close to that of 0.78 reported by Marinetti (1962). After deacylation, a spot corresponding to glycerophosphate could be detected with the Hanes and Isherwood spray. This phosphatidic component occurred in low concentration and was difficult to detect because of weak color development. Phosphatidic acids have been reported to occur in spores of B. stearothermophilus (Long and Williams, 1960) and in other bacilli (Weibull, 1957). They may be present as such in the intact cell, or may result from the action of phosphatidases. Harverkate, Houtsmuller, and Van Deenen (1962) showed that phosphatidase D could attack phosphatidyl glycerol with the release of glycerol and phosphatidic acid. They also reported the occurrence of phosphatidic acid in lipids extracted from B. cereus and B. megaterium. Lysolecithin. Spot 1 has been identified as lysolecithin. This compound gave a yellow fluorescence with rhodamine 6G and an average RF value of Marinetti (1962) reported an RF of 0.18 and a yellow fluorescence for lysolecithin; however, Kates et al. (1962) and Sehgal et al. (1962) reported blue-orange and yellow fluorescence and RF values of 0.32 and 0.25, respectively. A choline-containing spot was detected in the deacylated lipids from cells as well as from spores when the Dragendorff reagent and the phosphomolybdic acid-stannous chloride reagents were used. Further evidence of its identity was obtained by comparing the unknown with a standard containing both lecithin and lysolecithin. Lysophosphatidyl serine. Spot 2 has been tentatively identified as lysophosphatidyl serine on the basis of its blue fluorescence in the presence of rhodamine 6G, RF value of 0.30, and positive reaction with ninhydrin. Purified lysophosphatidyl serine was reported by Marinetti (1962) to fluoresce blue and to have an RF of After deacylation, glycerophosphoryl serine was not detected; the low levels at which this compound occurred may explain the inability to find it. Lysolecithin and lysophosphatidyl serine were detected in very low concentration in cells and spores of B. polymyxa, but the parent compounds, lecithin and phosphatidyl serine, could not be detected. Lyso compounds have been reported in animal tissue, but it was not until the work of Kates and his co-workers (1961, 1962) that lyso compounds were reported in bacterial lipids. They could also detect the parent compounds. Neutral lipids. Spot 8 was assumed to be neutral lipid; further identification was not attempted. This spot readily gave a positive test for unsaturation when potassium dichromate or iodine vapors were used. The only other component to give a positive test for unsaturation was phosphatidyl ethanolamine. Long and Williams (1960) found no unsaturated lipids in their analysis of the phospholipids of B. stearothe? mophilus. The products of hydrolyses in methanolic HCI of the total lipids contained ethanolamine. This compound, when stained with ninhydrin, showed a large intense spot on paper within a very few minutes. If the chromatogram were held at room temperature overnight, two other very faint spots became visible. One of these spots appeared to be serine; the other was not identified. Glycerol was also detected in the hydrolysates; no carbohydrates were detected under these conditions. Acid and alkaline hydrolyses were made only on the lipid of vegetative cells. The quantity of available spores and the percentage of lipid in the spores limited the amount of material avail-

6 'VOL. 87, 1964 PHOSPHOLIPIDS IN B. POLYMYXA 21 able for this type of analysis. The spore phospholipids were identified by specific spot tests on paper chromatograms, by chromatographing unknown fractions with the identified cell phospholipids, and by comparisons with standard.compounds. Column separation of phospholipids. Lipid samples extracted from cells of B. polymyxa were separated into individual phospholipid,components on a silica gel column; elution was followed by phosphorus determinations on each fraction (Fig. 2). The phospholipid components eluted by the solvent mixtures and identified on silicic acid-impregnated paper are recorded in Table 3. The average recovery of phosphorus from the column was 104%. Difficulties similar to those reported by Harverkate et al. (1962), Hornstein, Crowe, and Heimberg (1961), and Wren and Mitchell (1959) were encountered in which complete and sharp separation on the silicic acid column was not obtained. Failure to obtain well-defined separation may be the result of oxidation of the samples during extraction or chromatographic analysis. Wren (1960) pointed out that precautions must be taken because phospholipids are susceptible to autoxidation, and that it may be desirable to chromatograph them anaerobically. Infrared spectra of cell and spore lipids. Infrared spectra of the lipids representing each of the peaks in Fig. 2 showed absorptions at 2950 to 2910, 2870 to 2850, 1735 to 1725, 1455 to 1450, and 1365 cm7l. Wren and Mitchell (1959) reported these to be characteristic of lipid absorption. A peak corresponding to lecithin or lyso- TUBE NUMBER (5 ML FRACTION) FIG. 2. Separation by means of a silicic acid column of phospholipids extracted from cells of Bacillus polymyxa. C = chloroform; M = methanol. Identification of the phospholipids in these fractions is s3hown in Table S. TABLE 3. Phospholipids eluted from silicic acid columns by various mixtures of chloroform and methanol Solvent mixture Volume Phospholipid components ml CHCl3 35 Neutral lipids CHCl3-MeOH 95 Bis (phosphatidic) acid (20:1) Phosphatidic acid CHCl3-MeOH 105 Phosphatidyl glycerol (9:1) Lysophosphatidyl ethanolamine CHCl3-MeOH 105 Phosphatidyl ethanolamine (4:1) Lysophosphatidyl serine Phosphatidyl glycerol CHCl3-MeOH 90 Lysolecithin (3:2) CHCl3-MeOH 50 Nothing detected (1:4) MeOH 50 Nothing detected lecithin was obtained at 3400 cm-', but the band at 970 cm-l characteristic of lecithins (Smith and Freeman, 1959; Schwarz et al., 1957) could not be detected. The spectrum of CHCla- MeOH (4:1) fraction (Fig. 2), which was composed mainly of phosphatidyl ethanolamine, was compared with the spectrum of a standard sample. These two spectra, although not identical, were similar. The fatty acids present in these phospholipids have not been identified and may be different in each phospholipid component. Also, any oxidation or degradation of the fatty acids would produce an altered infrared spectrum and would complicate interpretation of the absorption curves. The infrared spectra have not been shown because the purity of the fractions was not known. Quantitative estimates of the phospholipids in spores were not made because disruption in the Mickle disintegrator resulted in loss of material. cells was estimated on the basis of phosphorus content and was calculated to be 0.15%. The size of samples required, and the size of spots on chro- The amount of phospholipid in vegetative

7 22 MATCHES, WALKER, AND AYRES J. BACTERIOL. matograms, would indicate little difference between cells and spores in phospholipid content. Even if all the phospholipids were extracted and identified, differences in heat resistance between the spore and vegetative cell could not be attributed to the type of phospholipid. However, as was pointed out previously, the fatty acids associated with these compounds have not been identified. The role that fatty acids may have has not been evaluated. A number of functions have been attributed to the phospholipids in the cell (Beveridge, 1956; Cantarow and Schepartz, 1962). Since the spore is relatively inactive metabolically, the structural function would probably be the primary one. Denstedt (1956), in discussing the structural or physicochemical role of phospholipids in cells, stated that little is known about the distribution of the phospholipids in the cell. He pointed out that such components as cell walls, membranes, nuclei, and mitochondria contain phospholipids, and that the phospholipids play an important role in the semipermeability of cell membranes. However, with the present information, no further conclusions can be made concerning the role of phospholipids in the bacterial endospore. ACKNOWLEDGMENTS This investigation was supported in part by research grant EF from the National Institutes of Health, U.S. Public Health Service, Division of Environmental Engineering and Food Protection. The authors express their appreciation to Erich Baer, Banting Institute, University of Toronto, for a sample of the disodium salt of a-stearoyl, f3-oleoyl-l-a-glycerylphosphoric acid. LITERATURE CITED ASSELIN1hEAU, J., AND E. LEDERER Chemistry and metabolism of bacterial lipides, p In K. Bloch [ed.], Lipide metabolism. John Wiley & Sons, Inc., New York. BENSON, A. A., AND E. H. STRICKLAND Plant phospholipids. III. Identification of diphosphatidyl glycerol. Biochim. Biophys. Acta 41: BEVERIDGE, J. M. R The function of phospholipids. Can. J. Biochem. Physiol. 34:361- S69.' BREGOFF, H. M., E. ROBERTS, AND C. C. DEL- WICHE Paper chromatography of quaternary ammonium bases and related compounds. J. Biol. Chem. 205: CANTAROW, A., AND B. SCHEPARTZ Biochemistry. W. B. Saunders Co., Philadelphia. CHEN, P. S., JR., T. Y. TORIBARA, AND H. WARNER Microdetermination of phosphorus. Anal. Chem. 28: f58. CHURCH, B. D., H. HALVORSON, D. S. RAMSEY, AND R. S. HARTMAN Population heterogeneity in the resistance of aerobic spores to ethylene oxide. J. Bacteriol. 72: DAWSON, R. M. C A hydrolytic procedure for the identification and estimation of individual phospholipids in biological samples. Biochem. J. 75: DENSTEDT, 0. F The function of phospholipids. Discussion. Can. J. Biochem. Physiol. 34: DYER, D. L The lipids of a thermophilic bacterium. M.S. Thesis, University of Nebraska, Lincoln. FOLCH, J., M. LEES, AND G. H. S. STANLEY A simple method for the isolation and purification of total lipids from animal tissues. J. Biol. Chem. 226: GARCIA, M. D., J. A. LOVERN, AND J. OLLEY The lipids of fish. VI. The lipids of cod flesh. Biochem. J. 62: GAUGHRAN, E. R. L The thermophilic microorganisms. Bacteriol. Rev. 11: GOLDFINE, H The characterization and biosynthesis of an N-methyl ethanolamine phospholipid from Clostridium butyricum. Biochim. Biophys. Acta 59: HACK, M. H The chromatography of phosphatides on silicic acid impregnated filter paper. J. Chromatog. 5: HANES, C. S., AND F. A. ISHERWOOD Separation of the phosphoric ester on the filter paper chromatogram. Nature 164: HAVERKATE, F., U. M. T. HOUTSMULLER, AND L. L. M. VAN DEENEN The enzymic hydrolysis and structure of phosphatidyl glycerol. Biochim. Biophys. Acta 63: HILL, P. B Incorporation of orthophosphate into phospholipid by isolated membrane fractions of bacteria. Biochim. Biophys. Acta 57: HORNSTEIN, I., P. F. CROWE, AND M. J. HEIM- BERG Fatty acid composition of meat tissue lipids. Food Sci. 26: KANESHIRO, T., AND A. G. MARR Cis-9, 10- methylene hexadecanoic acid from the phospholipids of Escherichia coli. J. Biol. Chem. 236: KANESHIRO, T., AND A. G. MARR Phospholipids of Azotobacter agilis, Agrobacterium tu-

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