Characterization of the Lipids of Mesosomal Vesicles and

Transcription

1 JOURNAL OF BACrERIOLOGY, Jan. 1975, p Copyright American Society for Microbiology Vol. 121, No. 1 Printed in U.S.A. Characterization of the Lipids of Mesosomal Vesicles and Plasma Membranes from Staphylococcus aureus P. R. BEINING,* E. HUFF, B. PRESCOTT, AND T. S. THEODORE University of Scranton, Scranton, Pennsylvania 18510, and Laboratory of Microbiology, National Institute of Allergy and Infectious Diseases, Bethesda, Maryland Received for publication 4 October 1974 Mesosomal vesicles and plasma membranes were isolated from Staphylococcus aureus ATCC 6538P by protoplasting and differential centrifugation. The lipids of each of the two membrane fractions were extracted with pyridine-acetic acid-n-butanol, and the nonlipid contaminants were removed by Sephadex treatment. The lipids were then separated by passage through diethylaminoethyl-cellulose columns and characterized by thin-layer chromatographic, chemical, and spectral analyses. The lipids were separated into four discrete diethylaminoethyl fractions: (i) vitamin K2, carotenoids, C,, isoprenoid alcohol, and monoglucosyl diglyceride; (ii) cardiolipin, carotenoids, phosphatidyl glycerol, diglucosyl diglyceride, and an unidentified ninhydrin-positive component; (iii) cardiolipid and phosphatidyl glycerol; (iv) cardiolipin, phosphatidyl glycerol, and phosphatidyl glucose. Qualitatively, no difference in lipid composition between mesosomal vesicles and plasma membranes was found. However, based on equal dry weights of membrane materials, a relative quantitative difference in the amount of specific lipids in;mesosomal vesicles and plasma membranes was observed. There are 4 times more monoglucosyl diglyceride, 2.6 times more diglucosyl diglyceride, 3.8 times more phosphatidyl glucose, 2 times more carotenoids, and 2 times more vitamin K2 found in mesosomal vesicles than in plasma membranes. The concentration of cardiolipin and phosphatidyl glycerol is 3.6 and 6 times greater, respectively, in mesosomal vesicles. The lipid content of Staphylococcus aureus has been shown to comprise 2.5% of the cell dry weight, 84% of which consisted of phosphatides and a glucolipid (M. Macfarlane, Biochem. J. 82:40 p, 1962). Other investigators have reported finding the following classes of lipids in this organism: monoglucosyl diglyceride (MGDG; 18), diglucosyl diglyceride (DGDG; 15), phosphatidyl glucose (PGL; 19), lysylphosphatidyl glycerol (LPG, 8), phosphatidyl glycerol (GPG; 11), cardiolipin (Macfarlane, Biochem. J. 82:40 p, 1962), nitrogenous phosphatides (11), and vitamin K2 derivatives (2). The respiratory pigments have been identified as cytochromes a, b, and o (21). By the use of a pressure cell, White and Frerman (31) separated a membrane fraction from a staphylococcal cell preparation and isolated the following lipids: vitamin K2, MGDG, DGDG, LPG, GPG, and cardiolipin. By similar extraction procedures, Short and White (19) recently isolated, in addition, PGL and phosphatidyl ethanolamine. By a cell wall lytic enzyme protoplasting technique and ultracentrifugation on a sucrose 137 density gradient, the plasma membrane and mesosomal vesicles have been separated and isolated from a strain of S. aureus (24). The protein and fatty acid compositions of these staphylococcal membrane fractions have been determined (23). Since the composition and functional role in the cell of the mesosome with respect to the plasma membrane are not entirely clarified, it was of interest to define more precisely the lipid differences, if any, between the two types of membrane. In the present study, mesosomal vesicles and plasma membranes of S. aureus ATCC 6538P were isolated and characterized with respect to their lipid composition. MATERIALS AND METHODS Preparation of mesosomal vesicles and plasma membranes. Mesosomal vesicles and plasma membranes were isolated from S. aureus ATCC 6538P after growth in Difco AOAC synthetic broth. Growth procedures, protoplasting techniques, and procedures for the isolation of mesosomal vesicles and plasma membranes by differential centrifugation were described previously (24). Prior to lipid extraction, mesosomal vesicles and plasma membranes were washed twice in

2 138 BEINING ET AL. J. BACTERIOL M tris(hydroxymethyl)aminomethane buffer (ph 7.4), and dry weights were determined gravimetrically. Lipid extraction. The mesosomal vesicles and plasma membranes were depleted of their lipids by extraction with pyridine-acetic acid-n-butanol (PAB; 22.2:31.1:100, vol/vol/vol) as described by Huff et al. (9). A 4-ml distilled-water suspension each of mesosomal vesicles (66.5 mg [dry weightyml) and plasma membranes 54.5 mg [dry weightyml) was treated with 20 ml of PAB for 2 h at 20 C. PAB-treated material was sedimented by centrifugation at 15,000 x g for 20 min, appropriate supernatants were combined, and lipids were evaporated with a stream of N, in a 60 C water bath. Dry weights on these and all subsequent fractions were determined as described above. Column chromatography. The PAB extract of each membrane fraction was suspended in chloroform-methanol-water (60:30:4.5, vol/vol/vol) and passed through Sephadex G25 fine columns to remove nonlipid contaminants (30). Sephadex-treated lipids were then fractionated by diethylaminoethyl ()-cellulose chromatography, as described by Plackett et al. (14). Five specific lipid subfractions were eluted from the columns with 500 ml of each of the following solvents: fraction 1 (F 1), chloroform-methanol (100:1, vol/vol); fraction 2 (F 2), chloroform-methanol (7:3, vol/vol); fraction 3 (F 3), chloroform-methanol-ammonium hydroxide (140: 60:4, vol/vol/vol); fraction 4 (F 4), chloroformmethanol-ammonium hydroxide-ammonium acetate (140:60:4:800 mg, vol/vol/vol/wt); and fraction 5 (F 5), methanol. All fractions were dried under a stream of N2, weighed, suspended to 10 mg/ml in chloroformmethanol (2:1, vol/vol), and stored at -20 C. Thin-layer chromatography. Uniplates, obtained from Analtech, Inc., Newark, Del., were used throughout this study. These plates (20 by 20 cm) were precoated with silica gel plus 1% sodium borate to a thickness of 500 um. Before use, plates were activated with the appropriate solvent, heated for 2 h at 100 C, and stored in a desiccator over KOH for no longer than 24 h. For identification, 100-Ag amounts of the various lipid fractions were chromatographed in a Shandon S/P Chromatank for a distance of 100 mm. The solvent used for vitamin K, and C., isoprenoid alcohol detection was ethyl acetate-n-heptane (10:90, vol/vol) (26). The solvent for chromatography of all other lipids in this study was chloroform-methanolwater (60:25:4, vol/vol/vol). Lipids were detected with the following sprays: iodine vapor (12) and 45% aqueous sulfuric acid (16) for general lipid detection, acid molybdate for phosphate (4), ninhydrin for amino nitrogen (12), periodate-schiff (17) and diphenylamine (29) for glycolipids, and Dragendorff reagent for choline (28). Recovery of Upids from thin-layer plates. Controls and test materials were chromatographed on the same thin-layer plates. Controls alone were stained for the appropriate lipid components. Corresponding test spots, located by chromatography with the known standards, were removed by scraping, and lipid was eluted twice from the silica gel with chloroformmethanol (2:1, vol/vol). Extracted lipids were removed from the silica gel by centrifugation (6,000 x g for 20 min) and were evaporated with a stream of N,. Chemical analysis. After acid hydrolysis of the lipid in 2 N HCl for 4 days at 100 C under argon and subsequent neutralization with NaOH, glycerol was assayed enzymatically with a glycerol Stat-Pack (Calbiochem, Los Angeles, Calif.). Glucose was determined colorimetrically by the Glucostat test (Worthington Biochemicals Corp., Freehold, N.J.) after acid hydrolysis (2 N HCl for 1 h at 100 C). Total phosphate was determined colorimetrically by the micromethod of Chen et al. (3) in conjunction with the Mg(NO,), ashing method of Ames and Dubin (1). Carotenoids were measured spectrophotometrically by their absorbancy at 430 nm in a Beckman DU spectrophotometer. Vitamin K, was assayed by the procedure of Lester et al. (10). RESULTS Extraction of lipids. Table 1 shows the results of the lipid extraction through PAB and Sephadex treatment of mesosomal vesicles and plasma membranes of S. aureus. Ten liters of culture produced 6 g (dry weight) of cells. From these 6 g of cells, 61 mg of mesosomal vesicles and 447 mg of plasma membranes were obtained. These figures correspond to approximately 1 and 7% of the dry weight of the staphylococcal cells, respectively. PAB-treated mesosomal vesicles and plasma membranes were found to contain 29 and 132 mg of lipids, respectively. After subsequent Sephadex fractionation, the mesosomal vesicles and plasma membranes were found to contain 22 and 89 mg of lipid, respectively. The total yield of lipid is greater in the larger plasma membrane fraction than in the mesosomal vesicles. However, after Sephadex treatment, 36.1% of the mesosomal vesicle starting material was found to be lipid TABLE 1. Extraction of lipids from mesosomal vesicles and plasma membranes of Staphylococcus aureus ATCC 6538P Determination Dry wt (mg) Dry wt of starting material (%) Meso- Plasma Meso- Plasma somal mem- somal memvesicles branes vesicles branes Starting material.. 61 (25)b 447 (250)1 PAB lipid extract Sephadex 1, lipid fraction (S 1) Sephadex 2, nonlipid fraction (S 2) a Mesosomal vesicles and plasma membranes isolated from 10 liters of culture (6 g [dry weight I of cells). "Protein content.

3 VOL. 121, 1975 and only 19.9% of the plasma membrane starting material was lipid. Based on comparative percentages of starting material, there is an 80% greater concentration of lipid in the mesosomal vesicles than in the plasma membranes. The ratio of protein to PAB lipid-extractable material was 0.83 for mesosomal vesicles and 1.89 for plasma membranes. For comparison, when chloroform-methanol (2:1) was used as the lipid extractant, we obtained a protein-lipid ratio of 1.21 and 2.24 for mesosomal vesicles and plasma membranes, respectively (23). -celiulose column chromatography. The fractionation on -cellulose columns of Sephadex-treated lipids from mesosomal vesicles and plasma membranes is shown in Table 2. When 100 mg of each of the isolated mesosomal vesicle and plasma membrane materials was treated with PAB and then fractionated on Sephadex G25 fine columns, 36.1 mg of mesosomal vesicle lipid and 20 mg of plasma membrane lipid were obtained. The amounts of lipids isolated from the -cellulose chromatography are listed in the first column of Table 2. Based on the amounts of starting material, the percentage of lipid yield for mesosomal vesicles and plasma membranes in each fraction is shown in the second column of this Table. The yield percentages in mesosomal vesicles and plasma membranes were roughly identical for the first, second, and fifth fractions. However, there is approximately an 80 and 60% greater concentration of mesosomal vesicle lipid in the third and fourth fractions, respectively. In addition to dry weights and percentage yields, column 3 of TABLE 2. LIPIDS OF S. AUREUS MESOSOMAL VESICLES Table 2 shows the specific lipids found within the various fractions. The existence and location of these specific lipids were detected with one-dimensional, thin-layer chromatography (Fig. 1). Most of the neutral lipids and all of the MGDG were found in F 1. All the DGDG was extracted in F 2. A slight ninhydrin-positive spot (R, 0.17) was also observed in both mesosomal vesicles and plasma membranes in this same fraction. Lacking a pure LPG standard, it was impossible to identify this compound as the LPG seen by White and Frerman (31) in staphylococci. All of the PGL was found in F 4. Cardiolipin and GPG were detectable in the second, third, and fourth fractions. Apparently, the greater concentration of lipid in mesosomal vesicles, as seen in Table 1 and as shown for F 3 and F 4, is due to the relatively greater concentration of cardiolipin, GPG, and PGL in mesosomal vesicles. Characterization of lipids. The lipids contained in the staphylococcal mesosomal vesicles and plasma membranes were separated by means of a one-dimensional, thin-layer chromatographic analysis of 100,g of the respective Sephadex lipid and fractions. A composite, diagrammatic representation of several thin-layer chromatograms of these lipids according to their R, values is shown in Fig. 1. Known standards were also chromatographed along with the various lipid fractions. Identification of the staphylococcal lipids, their Rt values, and the R, values of known standards are given in Table 3. It can be seen from this table and from Fig. 1 that F 1 contains all of the Fractionation of lipids from mesosomal vesicles and plasma membranes of Staphylococcus aureus ATCC 6538P on -cellulose columns Dry wt (mg)a % S 1 fraction Determination Meso- Plasma Meso- Plasma Lipid components somal mem- somal memvesicles branes vesicles branes Sephadex 1, lipid fraction (S 1) fraction 1 (F 1) Vitamin K,, carotenoids, C55 isoprenoid alcohol, MGDG fraction 2 (F 2) DGDG, cardiolipin, GPG fraction 3 (F 3) Cardiolipin, GPG fraction 4 (F 4) Cardiolipin, GPG, PGL fraction 5 (F 5) None detectable % Recovery (F 1 - F5)/S a Based on 100 mg [dry weight] of starting material. 139

4 140 BEINING ET AL. J. BACTERIOL. Sephadex lipid fraction Solvent front 1,2 1,2 fraction I 6I,2 91,2 fraction 2 fraction 3 fraction 4 A3 3. * 3. 3 t9.9 J4 5 0 A4 A7 A * A4 A4 A4 &4 Origin FIG. 1. Diagrammatic illustration of thin-layer chromatograms used to separate the lipids from mesosomal vesicles and plasma membranes of Staphylococcus aureus. Thin-layer plates of silica gel were prepared, and chromatographed in one direction. Chromatographs of C,, isoprenoids are developed in ethyl acetate-heptane (10:90, vol/vol). Chromatographs of all other lipids were developed in chloroform-methanol-water (60:25:4, vol/vol/vol). Spots were detected with iodine, followed by staining with acid molybdate for phosphate and periodate-schiff for glycolipids. Numbers refer to the lipids in Table 3. TABLE 3. A4 A A 6 7 _ 8,9 -_8,9 *10 "lo -6 _6 Thin-layer chromatography of lipids from staphylococcal mesosomal vesicles and plasma membranes Lipid component Rt Solvent system 1. Vitamin K (0.98) a Chloroform-methanol-water (60:25:4, vovvol/vol) 2. Carotenoids (0.98)a Chloroform-methanol-water (60:25:4, vol/vol/vol) 3. MGDG (0.74)b Chloroform-methanol-water (60:25:4, vol/vol/vol) 4. Cardiolipin (0.69)a Chloroform-methanol-water (60:25:4, vol/vol/vol) 5. DGDG (0.53)1 Chloroform-methanol-water (60:25:4, vol/vol/vol) 6. Vitamin K (0.59)a Ethyl acetate-heptane (10:90, vol/vol) 7. GPG (0.47)0 Chloroform-methanol-water (60:25:4, vol/vol/vol) 8. PGL.0.31 (0.32)a Chloroform-methanol-water (60:25:4, vol/vol/vol) 9. C56 isoprenoid alcohol (0.34)c Ethyl acetate-heptane (10:90, vol/vol) 10. Ninhydrin spot Chloroform-methanol-water (60:25:4, vol/vol/vol) ar, values of known standards which were chromatographed with test materials. ' R, values of monogalactosyl diglyceride and digalactosyl diglyceride standards. Rt values of known standards taken from literature (26). 7 A 7 '8 *8 vitamin K2, isoprenoids, and MGDG, and essentially all of the carotenoids present in the mesosomal vesicles and plasma membranes. DGDG was detected only in F 2. PGL was found only in F 4. Cardiolipin and GPG were present in F 2, F 3, and F 4. The unequal intensity of chromatographic spots of cardiolipin and GPG in F 3 and F 4 and of PGL in F 4 reflects the greater concentration of lipid in mesosomal vesicles over plasma membranes (Table 2). With the amounts used, no lipids were detected in F 5. A slight ninhydrin-positive spot (R, 0.17) was observed in both the mesosomal vesicles and plasma membranes only within F 2. This ninhydrin-positive spot was not evident on chromatograms stained with acid molybdate. A pure LPG standard was unobtainable. In F 4 a spot was observed on several chromatograms that was significantly more prominent in mesosomal vesicles than in plasma membranes. This spot was positive for phosphate and glycolipid and had an R, value of The spot was identified as PGL on the basis of close relationship in Rt value (0.32) with a known standard.

5 VOL. 121, 1975 When synthetic vitamin K2 isoprenologues of C25, C30, C35, C40, and C45 side chains were chromatographed with the staphylococcal quinone, all isoprenologues were found in F 1 and in similar concentration in the mesosomal vesicles and plasma membranes. Only traces of isoprenologues of C25, C30, C40, and C4,5 side chains were detected. The major quinone in mesosomal vesicles and plasma membrane appears to be vitamin K2 [C35]. A major lipid component (R, 0.34) was detected in the first fraction and tentatively identified from Thorne and Kodicek's chromatographic data (26) as a C55 isoprenoid alcohol (Bactoprenol). No lipids were detected in Sephadex 2, the nonlipid fraction. Chemical analysis of staphylococcal lipids. Table 4 shows the chemical analysis of the staphylococcal lipids from an initial 10-mg (dry weight) sample of membrane and mesosome materials that were fractionated on Sephadex and -cellulose columns. The data in the first column of this table show that mesosomal vesicles possess four times more glucose in F 1, which contains MGDG, and 3.8 times more glucose in F 4, which contains PGL, than did the plasma membranes. Ninety-four percent of all mesosomal vesicle glucose and 80% of all plasma membrane glucose were associated with DGDG. Previously it was shown that the glycerol-containing compounds were found in F 2, F 3, and F 4. From the glycerol analysis data in the second column of Table 4, it is seen that in F 2, F 3, and F 4, respectively, there are 1.5, 4.5, and 9.4 times as much glycerol in mesosomal vesicles as in plasma membranes. The glycerol in these three fractions accounts for 98% of the total mesosomal vesicle TABLE 4. LIPIDS OF S. AUREUS MESOSOMAL VESICLES glycerol and 78% of the total plasma membrane glycerol found in the Sephadex-lipid material. Column 3 of this table shows a similar significantly higher concentration of phosphate in the mesosomal vesicles over plasma membranes in those fractions where phospholipids have been detected by thin-layer chromatography. As shown in columns 4 and 5, twice as many optical density units of carotenoids and vitamin K2 were detected in mesosomal vesicles as in plasma membranes. Table 5 is a summation chart. Using 10 mg (dry weight) of the particular membrane and mesosomal material and determining micromolar concentrations, there is found in pertinent fractions four times more MGDG, 2.6 times more DGDG, and 3.8 times more PGL in mesosomal vesicles than in plasma membranes. Likewise using the -fractioned material, but detecting optical density units, there is found two times more carotenoids and vitamin K2 in mesosomal vesicles than in plasma membranes. Analysis of isolated, thin-layer chromatographic spot material showed that mesosomal vesicles had six times more GPG and 3.6 times more cardiolipin than the plasma membranes. For purposes of comparison, the results in Table 5 also show the lipid content based on 10 mg of protein from the starting material. DISCUSSION In the analysis of the aggregate membrane system of S. aureus, it has been demonstrated that the following lipids are present: MGDG, DGDG, PGL, cardiolipin, GPG, LPG, carotenoids, and vitamin K2 (31). Using isolated membrane components, this study established qualitatively that the lipid composition of mesosomal vesicles and plasma membranes of S. Chemical analysis of staphylococcal lipids fractionated on -cellulose columns Glucosea Glycerola Phosphate0a Carotenoids" Vitamin K,c Fraction Meso- Plasma Meso- Plasma Meso- Plasma Meso- Plasma Meso- Plasma somal mem- somal mem- somal mem- somal mem- somal memvesicles branes vesicles branes vesicles branes vesicles branes vesicles branes 141 S d _ - - F F NDe ND F ND ND ND ND F ND ND ND ND F 5 ND ND ND ND ND ND % Recovery (F 1 - F5)/S a Glucose, glycerol, and phosphate concentrations expressed as micromoles/10 mg (dry weight) of starting material. bcarotenoid concentration expressed as optical density units at 430 nm/10 mg (dry weight) of starting material. c Vitamin K, concentration expressed as optical density units (262 to 246 nm)/10 mg (dry weight) of starting material. dnot done. ' None detected.

6 142 BEINING ET AL. J. BACTERIOL. TABLE 5. Distribution of lipids in mesosomal vesicles and plasma membranes of Staphylococcus aureus ATCC 6538P Lipids Mesosomal Plasma Lipids vesiclesa membranesa MGDG (0.029)b (0.005) DGDG (0.166) (0.046) PGL (0.037) (0.007) GPG (4.651) (0.546) Cardiolipin (1.993) (0.400) Carotenoids (0.644) (0.236) Vitamin K (1.585) (0.571) a MGDG, DGDG, PGL, GPG, and cardiolipin concentrations expressed as gmol/10 mg (dry weight) of starting material. Vitamin K2 and carotenoid concentrations expressed as optical density units (262 to 246 nm and 430 nm, respectively)/10 mg (dry weight) of starting material. b Lipid content based on per 10 mg of protein from starting material. aureus ATCC 6538P is identical. MGDG, DGDG, and PGL account for 98% of the lipid glucose in both the membrane and mesosome fractions. Similarly, PGL, GPG, and cardiolipin comprise 98% of the lipid phosphate found. In addition to vitamin K2 and carotenoids, a C55 isoprenoid alcohol has been identified tentatively in both the mesosome and membrane fractions. As reported earlier (23), there is found to be a distinct quantitative difference between mesosomal vesicles and plasma membranes in gross lipid composition. In this study, after Sephadex treatment, 36% of the dry weight of mesosomal vesicles was found to be lipid, whereas only 20% of the plasma membrane dry weight is lipid. In comparing the concentration of specific lipid components within mesosomal vesicles and plasma membranes, mesosomal vesicles contained twice as much vitamin K2 and carotenoids and six times more GPG than did the plasma membranes (Table 5). In addition to the above lipids, PE and LPG have been reported to be present in staphylococcal membrane preparations (19). Our protoplasting procedure prevented the acidic conditions reported to be necessary for the detection of LPG. In the chromatography studies, a minor ninhydrin-positive spot was detected in both the mesosomal vesicles and plasma membranes ( F 2), which did not chromatograph with a PE standard. Lacking a known LPG standard, we could not conclude that this ninhydrin spot (R, 0.17) is a trace of LPG. The solvent system chosen for the extraction of lipids was PAB. This system, which extracts lipids from aqueous samples with a minimal interference of their physical integrity, was found to be more efficient lipid extractant than chloroform-methanol (2: 1). In the latter system, 34% of the dry weight of mesosomal vesicles and 25% of the plasma membranes were lipid (24); whereas with PAB 47% of the dry weight of mesosomal vesicles and 30% of the plasma membranes appeared to be lipid. In addition, PAB has been reported to be the most efficient solvent system for extraction of C55 isoprenoid compounds (20, 27). On the basis of thin-layer chromatography and comparison with known R, values, C55 isoprenoid alcohol (Bactoprenol) was tentatively identified as a component of the first fraction of mesosomal vesicles and plasma membranes. This isoprenoid compound appears to be present in similar amounts of both mesosome and membrane fractions. The total amount of mesosomal vesicle and plasma membrane lipid, isolated from 1 liter of culture (0.6 g [dry weight] of cells), indicates a two- to three-fold greater concentration of lipids in the plasma membrane. This is consistent with our finding of seven times more membrane than mesosomal vesicles per bacterial cell. However, based on identical dry weights of each membrane fraction, the mesosomal vesicles contained from two to six times more lipid than did the plasma membrane. These results suggest that the mesosomal vesicle may be involved in lipid biosynthesis. This would be consistent with the findings of Fitz-James (6), who demonstrated preferential incorporation of labeled phosphate and acetate in mesosomal vesicles of Bacillus megatherium. To the contrary, Patch and Landman (13), using B. subtilis, and Thomas and Ellar (25), using Micrococcus lysodeikticus, have reported no preferential incorporation of lipid precursors in mesosomal vesicles. Recent pulse-labeling experiments have shown that GPG is the precursor of lipoteichoic acid in both S. aureus (7) and Streptococcus sanguis (5). Our studies, which show a sixfold greater concentration of GPG in mesosomal vesicles, as well as the demonstration of increased levels of glycerol phosphate (22) which now has been shown to be lipoteichoic acid (9), present added evidence, although indirect, that mesosomal vesicles may play an active role in lipid metabolism. Present studies are directed towards pulse-labeling experiments and the assessment of the specific activity of enzymes involved in lipid synthesis. LITERATURE CITED 1. Ames, B. N., and D. T. Dubin The role of polyamines in the neutralization of bacteriophage de-

7 VOL. 121, 1975 LIPIDS OF S. AUREUS M[ESOSOMAL VESICLES 143 oxyribonucleic acid. J. Biol. Chem. 235: Bishop, D. H., K. P. Pandya, and H. K. King Ubiquinone and vitamin K, in bacteria. Biochem. J. 83: Chen, P. S., T. Y. Toribara, and H. Warner Microdetermination of phosphorus. Anal. Chem. 28: Dittmer, J. C., and R. L. Lester A simple, specific spray for the detection of phospholipids on thin layer chromatograms. J. Lipid Res. 5: Emdur, L. I., and T. H. Chou Turnover of phosphatidylglycerol in Streptococcus sanguis. Biochem. Biophys. Res. Commun. 59: Fitz-James, P. C The collection of mesosome vesicles extruded during protoplasting, p In L. B. Guze (ed.), Microbial protoplasts, spheroplasts and L-forms. The Williams & Wilkins Co., Baltimore. 7. Glaser, L., and B. Lindsay The synthesis of lipotechoic acid carrier. Biochem. Biophys. Res. Commun. 59: Houtsmuller, J. M. T., and L. L. M. van Deenen On the amino acid esters of phosphatidly glycerol from bacteria. Biochim. Biophys. Acta 106: Huff, E., R. M. Cole, and T. S. Theodore Lipoteichoic acid localization in mesosomal vesicles of Staphylococcus aureus. J. Bacteriol. 120: Lester, R. L., D. C. White, and S. L. Smith The 2-desmethyl vitamin K,'s. A new group of naphthoquinones isolated from Hemophilus parainfluenzae. Biochemistry 3: Macfarlane, M Characterization of lipoaminoacids as o-amino-acid esters of phosphatidyl-glycerol. Nature (London) 196: Marinetti, G. V Chromatographic separation, identification and analysis of phosphatides. J. Lipid Res. 3: Patch, C. T., and 0. L. Landman Comparison of the biochemistry and rates of synthesis of mesosomal and peripheral membranes in Bacillus subtilis. J. Bacteriol. 107: Plackett, P., B. P. Marmion, E. J. Shaw, and R. M. Lemcke Immunochemical analysis of Mycoplasma pneumoniae. Aust. J. Exp. Biol. Med. Sci. 47: Polonoviski, J., R. Wald, and M. Paysant-Diament Les Lipids de Staphylococcus aureus. Ann. Inst. Pasteur Paris 103B: Rouser, G., G. Kritchevsky, and A. Yamamoto Lipid chromatographic analysis, vol. I. Marcel Dekker, Inc., New York. 17. Shaw, N The detection of lipids on thin-layer chromatograms with the periodate-schiff reagents. Biochim. Biophys. Acta 164: Shaw, N., and J. Baddiley Structure and distribution of glycosyl diglycerides in bacteria. Nature (London) 217: Short, S. A., and D. C. White Metabolism of the glucosyl diglycerides and phosphatidylglucose of Staphylococcus aureus. J. Bacteriol. 104: Stone, K. J., and J. L. Strominger Isolation of C55-isoprenylpyrophosphate from Micrococcus lysodeikticus. J. Biol. Chem. 247: Taber, A. W., and M. Morrison Electron transport in Staphylococci. Properties of a particle preparation from exponential phase Staphylococcus aureus. Arch. Biochem. Biophys. 105: Theodore, T. S., R. M. Cole, and E. Huff Localization of glycerol phosphate in mesosomal vesicles of Staphylococcus aureus. Biochem. Res. Commun. 59: Theodore, T. S., and C. Panos Protein and fatty acid composition of mesosomal vesicles and plasma membranes of Staphylococcus aureus. J. Bacteriol. 116: Theodore, T. S., T. J. Popkin, and R. M. Cole The separation and isolation of plasma membranes and mesosomal vesicles from Staphylococcus aureus. Prep. Biochem. 1: Thomas, T. D., and D. J. Ellar Properties of plasma and mesosomal membranes isolated from Micrococcus lysodeikticus: rates of synthesis and characterization of lipids. Biochim. Biophys. Acta 316: Thorne, K. J. I., and E. Kodicek The structure of Bactoprenol, a lipid formed by lactobacilli from mevalonic acid. Biochem. J. 99: Umbreit, J. N., and J. L. Strominger Isolation of the lipid intermediate in peptidoglycan biosynthesis from Escherichia coli. J. Bacteriol. 94: Wagner, H., L. Horhammer, and P. Wolff Dunnschichtchromatographie von phosphatiden und glykolipiden. Biochem. Z. 334: Waldi, D Spray reagents for thin layer chromatography, p In E. Stahl (ed.), Thin layer chromatography. Academic Press Inc., New York. 30. Wells, M. A., and J. C. Dittmer The use of Sephadex for the removal of nonlipid contaminants from lipid extracts. Biochemistry 2: White, D. C., and F. E. Frerman Extraction, characterization, and cellular localization of the lipids of Staphylococcus aureus. J. Bacteriol. 94: