Two Distinct Pools of Membrane Phosphatidylglycerol in Bacillus megaterium

Transcription

1 JOURNAL OF BACTERIOLOGY, Feb. 1980, p /80/ /08$02.00/0 Vol. 141, No. 2 Two Distinct Pools of Membrane Phosphatidylglycerol in Bacillus megaterium FRANK J. LOMBARDI AND ARMAND J. FULCO* Department of Biological Ch[2istry, University of California at Los Angeles Medical School, and Laboratory of Nuclear Medicine and Radiation Biology, Los Angeles, California The predominant membrane lipid in Bacillus megaterium ATCC 14581, phosphatidylglycerol (PG), is present in two distinct pools, as shown by [3P]phosphate incorporation and chase experiments. One pool (PGJ) undergoes rapid turnover of the phosphate moiety, whereas the second pool (PG.) exhibits metabolic stability in this group. The phosphate moiety of the other major phospholipid, phosphatidylethanolamine, is stable to turnover. [3P]phosphate- and [2-3H]glycerol-equilibrated cultures yielded the following glycerolipid composition: 56 mol% PG (34 mol% PGt and 22 mol% PG.), 21 mol% phosphatidylethanolamine, 1 to 2 mol% phosphatidylserine, 20 mol% diglycerides, less than 0.5 mol% cardiolipin, and 0.2 to 0.4 mol% lysophosphatidylglycerol. Accumulation of PG was halted immediately after the addition of cerulenin, an inhibitor of de novo fatty acid synthesis, whereas phosphatidylethanolamine accumulation continued at the expense of the diglyceride and PG pools. Strikingly, initial rates of [32P]phosphate incorporation into PG were unaffected by cerulenin. In control cultures at 35 C, incorporation of[32p]phosphate into PG exhibited a biphasic time course, whereas incorporation into phosphatidylethanolamine was concave upward and lagged behind that of PG during the initial rapid phase of PG incorporation. Finally, levels of lysophosphatidylglycerol expanded rapidly after cerulenin addition at 200C, but not at 350C. Moreover, incorporation of [32P]phosphate into lysophosphatidylglycerol lagged behind incorporation into PG in both the presence and absence of cerulenin at 20 and 350C. In recent work with Bacillus megaterium ways initiated by the branch point enzymes ATCC 14581, it was found that ["4C]palmitate phosphatidylserine synthetase (13,17) and phosphatidylglycerophosphate synthetase (8), re- added to cultures growing at 20 or 350C was rapidly incorporated into the 1-acyl position of spectively. The latter activity has also been observed in extracts of Bacillus licheniformis (14). phosphatidylglycerol (PG) (2, 3). When the [I4C]palmitate addition was preceded by the addition of cerulenin (an inhibitor of fatty acid devoid of phosphatidylglycerophosphate syn- In E. coli, however, mutants which are almost synthesis), the appearance of [14C]PG was retarded at 200C and was preceded by transient reported (16). thetase but synthesize PG normally have been accumulation of 1-[14C]palmitoyl lysophosphatidylglycerol. This intermediate was gradually PG labeled with 3P lost radioactivity when the Early in vivo studies with E. coli showed that converted to 1-[14C]acyl-phosphatidylglycerol cells were transferred to unlabeled medium, by acylation with endogenous acyl groups (3). whereas the phosphate of PE was metabolically At 350C, however, transient appearance of 14C_ stable (10). The glycerophosphate moiety of PG labeled lysophosphatidylglycerol (lyso-pg) was can be transferred intact into membrane-derived not consistently observed in cerulenin-treated oligosaccharides in E. coli (25) and into the cultures (2). teichoic and lipoteichoic acids of Streptococcus The present studies were undertaken to define sanguis (5, 6). In E. coli, this transfer apparently in vivo patterns of glycerolipid formation and produces sn-1,2-diglyceride (19), a minor lipid metabolism in B. megaterium ATCC and constituent in this organism, which is metabolized at a modest rate to phosphatidic acid (18) were preparatory to further detailed investigations on the role of lyso-pg. In Escherichia coli, and thence to CDP-diglyceride (12). Moreover, extensive studies have shown that the major direct conversion of two equivalents of PG to lipids of this organism, phosphatidylethanolamine (PE) and PG are formed in vitro from a been demonstrated in E. coli (9) and Staphylo- one of cardiolipin and one of free glycerol has common precursor, CDP-diglyceride, in pathcoccus aureus (23). As shown in E. coli by 618

2 VOL. 141, 1980 Ballesta et al. (1), free glycerol can exchange with the nonacylated glycerol moiety of PG at a somewhat faster rate than the rate of phosphate turnover in this lipid. Furthermore, these authors demonstrated that only some of the PG molecules undergo this exchange reaction in E. coli, whereas the remainder are stable (1). Similarly, on the basis of differential turnover of various cardiolipin and PG moieties, White and co-workers concluded that there are two distinct pools of PG present in S. aureus (22) and Haemophilus parainfluenzae (24). In B. megaterium strain KM, recent work (20) has shown that PE is distributed asymmetrically in the cell membrane, with the cytoplasmic monolayer containing twice as much PE as the outer monolayer. Furthermore, newly synthesized PE appears first on the cytoplasmic side of the membrane and is subsequently redistributed to both inner and outer monolayers (21). In a preliminary communication (F. J. Lombardi, S. L. Chen, and A. J. Fulco, Fed. Proc. 37: 1494, 1978), we reported that B. megaterium ATCC possesses two distinct pools of PG, one (PG.) which is metabolically stable and another (PGj) which undergoes rapid metabolic turnover in the phosphate group. These findings are confirmed and extended in the present paper. In the accompanying paper (15) it is demonstrated that this second pool of PG is in near equilibrium with a large and metabolically active pool of diglycerides and, in addition, serves as a precursor for PE formation in this organism. MATERIALS AND METHODS Materials. Carrier-free [32P]orthophosphoric acid was obtained from New England Nuclear Corp., Boston, Mass. Cerulenin was a product of Makor Chemicals, Ltd., Jerusalem, Israel. Lipid standards were from Sigma Chemical Co., St. Louis, Mo. Growth and incubation of bacteria. B. megaterium ATCC was grown in a glucose-salts-amino acid medium (GCN medium) as described previously (7) or in a low-phosphate medium, S-M56-LP (20), consisting of M56-LP salts (4) supplemented with 2% glucose and 0.4% Casamino Acids. The phosphate concentration of medium S-M56-LP was calculated to be 1.1 mm, arising from the 0.3 mm phosphate present in M56-LP salts plus the 0.8 mm phosphate derived from Casamino Acids (2% phosphate by weight; Difco Laboratories, Detroit, Mich.). Incubations were carried out in constant-temperature rotary incubator water baths (7). Previous experience with this organism showed that growth under these conditions is strongly dependent on oxygenation (7), and the doubling time is a complex function of cell density, swirling rate, and geometry of the culture. Under the conditions used in the present work, it was found that the growth of the bacteria was exponential up to a cell density of about 50 Klett units (equivalent PG POOLS IN B. MEGATERIUM 619 to 1.05 g of wet packed cells per liter), with population doubling times of approximately 200 and 60 min at 20 and 35 C, respectively. Above 50 Klett units, the increase in the optical densities of cultures was approximately linear with time up to at least 300 Klett units. Various control incubations carried out between 25 and 300 Klett units revealed no significant differences in in vivo lipid metabolic patterns over this density range. Lipid extraction and analysis. After incubation, cells were harvested on glass depth filters (2), washed with 10 ml of fresh growth medium, and immediately transferred with the filters to vials containing 7.6 ml of CHC13-CH3OH-H20 (1:2:0.8, vol/vol). After vigorous mixing, each sample was filtered on a second glass depth filter and washed with 3.8 ml of CHCl3-CH30H- H20 (1:2:0.8, vol/vol), 3 ml of water, and finally 3 ml of CHCl3. The resulting filtrates separated into two phases. The lower (CHC13) phase was removed, and the upper (H20-CH30H) phase was extracted twice with 2-ml volumes of CHCL3-CH30H (2:1, vol/vol) and once with 2 ml of CHC13. The chloroform phases, which contained the major lipids, were combined, and the remaining H20-CH30H phase, which contained lyso-pg, was acidified and extracted with CHC13 to obtain this component (2). A sample of each extract was removed for determination of radioactivity by liquid scintillation counting. A second sample was mixed with nonradioactive lipid standards, spotted onto a precoated silica gel plate (5 by 20 cm; Silica Gel 60; E. Merck, Darmstadt, Germany), and developed for 2.5 h in CHC13-CH30H-H20 (65:25:4, vol/vol), or, in the case of lyso-pg-containing samples, in CHC13- CH3OH-CH3COOH-H20 (65:25:8:4, vol/vol). The lipids were visualized with iodine vapor, and the plates were scanned with a Packard model 7200 radiochromatogram scanner. Radioactivity in individual lipid components was determined from integrated peak areas and by scraping and counting. For further resolution of the solvent front lipids (see Fig. 1), the solvent front region of the plate was scraped and the lipids were extracted with CHCl3-CH3OH-H20 (10:5:1, vol/ vol) and rechromatographed in either benzene-etherethyl acetate-acetic acid (80:10:10:0.2, vol/vol) or n- pentane-ether-acetic acid (80:20:1, vol/vol). RESULTS lipid composition of B. megaterium ATCC The percent composition of the major lipids in cultures grown to isotopic equilibrium at 35 C in the presence of [ P]phosphate or [2-3H]glycerol (Fig. 1A, C, and D) was as follows: 56 mol% PG, 21 mol% PE, 1 to 2 mol% phosphatidylserine, and 20 mol% diglycerides. Cultures grown at 20 C showed a similar composition of the major lipids, although the PG/ PE ratio was slightly higher than the ratio at 35 C. The PG, PE, and phosphatidylserine peaks in Fig. 1A comigrated with their authentic standards during thin-layer chromatography in the following solvent systems: CHC13-CH30H- H20 (65:25:4, vol/vol), CHC13-CH30H-CH3-

3 620 LOMBARDI AND FULCO J. BACTERIOL. Sd"ut Frut Lipids E 1,306;V-99 F DC S F-i FIG. 1. Lipids of B. megaterium ATCC (A) A culture was grown for five population doublings at 350C in medium S-M56-LP containing [32P]phosphate, cells were harvested, and the lipids were extracted and partitioned between CHCl3 and CH30H-H20 (see text). The CHCl3 extract was 8ubjected to thin-layer chromatography in solvent system 1 (CHC13-CH30H-H20 [65:25:4, vol/voll) and scanned. (B) A similar culture was grown for five population doublings in medium S-M56-LP containing [32P]phosphate, cerulenin (20 pg/mi) was added, and the culture was incubated for an additional 2 h at 200C. The lipids werepartitioned between CHCl3 and CH30H-H20, and this was followed by acidification and CHCl3 extraction ofthe CH30H- H20 phase and chromatography of the extract in solvent system 2 (CHCI3-CH30H-CH3COOH-H20 [65:25:8: 4, vol/vol]). (C and D) A culture was grown for five population doublings at 35 C in GCN medium supplemented with [2-3Higlycerol, and after thin-layer chromatography of the CHCI3-phase lipids in solvent system 1 (C), the solvent front lipids were extracted from the plate and rechromatographed in solvent system 3(benzene-ether-ethyl acetate-acetic acid [80:10:10:0.2, vol/vol]) (D). (E and F) A culture growing at 35'C in GNC medium was labeled for 15 min with [U-g4Cglycerol, and after thin-layer chromatography ofthe CHCl3- phase lipids in solvent system 1, the solvent front lipids were extracted from the plate and portions were rechromatographed in solvent system 3 (E) or solvent system 4 (n-pentane-ether-acetic acid [80:20:1, voll vol]) (F). PS, Phosphatidylserine; DG, diglyceride. COOH-H20 (65:25:8:4, vol/vol), CHC13- CH30H-7 M NH4OH (60:35:5, vol/vol), and diisobutylketone-acetic acid-water (40:25:5, vol/ vol). Moreover, phosphatidic acid and cardiolipin, which comigrated with PG in CHC13- CH30H-H20 (65:25:4, vol/vol/vol) (Fig. 1A), were found, after reselution in CHC13-CH30H- CH3COOH (65:25:8, sol/vol/vol), to contain negligible radioactivity. As shown elsewhere (15), radioactivity from [2-3H]glycerol or [U-_4C]glycerol was incorporated solely into the glycerol moieties of B. megaterium lipids, none was found in fatty acyl, ethanolamine, or other lipid moieties, suggesting that exogenous glycerol, although incorporated efficiently into lipids, is not oxidized appreciably under these conditions. Furthermore, the ratio of PG to PE radioactivity in Fig. 1 and C, as determined by scraping and extraction of the lipids from thin-layer plates and liquid scintillation counting, was approximately twofold higher when [2-3H]glycerol was used than when [3P]phosphate was used, thus indicating that most or all of the material identified as PG in fact exhibited a ratio of glycerol to phosphate of 2:1 relative to that of PE. (Relative peak areas of 3H-lipids on radiochromatograms [Fig. 1C and D] are not necessarily proportional to radioactivity due to differential quenching ofthe various 3H-lipids adsorbed to the silica gel.)

4 VOL. 141, 1980 A major lipid fraction which migrated near the solvent front in solvent system 1 (Fig. 1C) and containing little or no phosphorus (Fig. 1A) was found on subsequent chromatography in solvent system 3 to contain predominantly 1,2- diglyceride and 1,3-diglyceride (Fig. 1D, E, and F) in a ratio of about 1:2. When 1,2- and 1,3- diglyceride peak areas were separately extracted and put through the entire lipid extraction and purification procedure, each was reisolated largely in unchanged form. In all analyses reported here and in the accompanying paper (15), the two components were pooled and treated as total diglycerides. Another phosphorus-deficient component(s) which migrated with the diglycerides in solvent system 1 (Fig. 1C) but remained stationary in solvent system 3 was prominent in [2-3H]glycerol-equilibrated cultures (Fig. 1D, peak SF-1) but contained only miniimal radioactivity after a 15-min pulse with [U-_4C]glycerol (Fig. 1E). The unknown lipid(s) in peak SF-1 was also labeled rapidly with L-[U-_4C]serine in the presence and absence of cerulenin (data not shown). Cultures equilibrated with [32P]phosphate and then treated with cerulenin at 200C accumulated [32P]lyso-PG up to levels of 3 to 4 mol% (Fig. 1B). This component is extracted from the aqueous methanol phase after acidification (2) and is accompanied by residual PG and 32pcontaining nonlipid contaminants, which remain at the origin in thin-layer chromatography (Fig. 1B). Radioactive lyso-pg has also been isolated from cerulenin-treated cultures incubated with [U-_4C]palnitate (2, 3, 16) and [2-3H]- or [U- 14C]glycerol (data not shown) and from control cultures equilibrated with [32P]phosphate at 200C (Fig. 2B) and 350C (see Fig. 5B), in which the endogenous levels were approximately 0.2 to 0.4 mol%. The results in Fig. 1 suggest the presence of additional minor lipid components and do not exclude the possibility of phosphorus-deficient lipids not labeled by exogenous [2-3H]- or [U- 14C]glycerol. Effect of cerulenin on phospholipid synthesis. The addition of cerulenin (20,ug/ml) to a 32P-equilibrated culture growing at 200C produced rapid and complete inhibition of net PG accumulation (Fig. 2A). In contrast, PE accumulation continued in the presence of cerulenin for 30 to 40 min at the control rate; after 40 min net PE levels fell increasingly below those of the control culture (Fig. 2A). As shown below (Fig. 2B) and elsewhere (15), cerulenin exerts its characteristic effects on lipid metabolism in B. megaterium very rapidly (3 to 5 min) after its addition. Thus, the modest net increase in PE ob- =,,10 PG POOLS IN B. MEGATERIUM 621 C. a PG(+CER) 20- PE(-CER). -,,,._ A, PE(+ CER) Q.50-i B 0 /," LYSO-PG 0Q25-C,(-ER) 01/ TIME(MIN.) FIG. 2. Effect of cerulenin (CER) on phospholipid levels at 20 C. A culture was grown for five population doublings to a cell density of 50 Klett units in medium S-M56-LP containing [32PJphosphate. At zero time, cerulenin (20 pg/ml) was added to aportion ofthe culture; incubation was continued at 200C, and samples were withdrawn at intervals for 32P-lipid analysis. (A) Symbols: 0, PG, no cerulenin; *, PG, +cerulenin; A, PE, no cerulenin; A, PE, +cerulenin. (B) Symbols: O, lyso-pg, no cerulenin; *, lyso-pg, +cerulenin. served in the presence of cerulenin was not due to a delayed action of the inhibitor, but instead occurred largely at the expense of the diglyceride pool at 200C (data not shown) or of the diglyceride plus PG pools at 350C (15). As Fig. 2B shows, cerulenin addition caused rapid expansion of the lyso-pg pool at 200C. Lyso-PG levels attained in the presence of the inhibitor were elevated at least 10-fold over endogenous values at 2 h. Furthermore, steadystate levels of lyso-pg accumulation in cerulenin-treated cultures exhibited a marked temperature dependence. Thus, although the lyso-pg pool rapidly expanded over 10-fold after cerulenin addition at 200C, the addition of the inhibitor at 350C produced little or no expansion of the lyso-pg pool above endogenous levels. Stability of[32p]pe and [32P]PG. [32P]phos-

5 622 LOMBARDI AND FULCO phate previously incorporated into PE was stable during prolonged growth at 350C in the presence of unlabeled phosphate (Fig. 3). On the other hand, more than one-half of the 3P was rapidly lost from [3P]PG during 40 to 60 min of incubation in unlabeled phosphate; however, after that time the amount of radioactivity in this phospholipid attained a plateau level. These results indicate the presence of two distinct populations of PG molecules in B. megaterium ATCC 14581; in one pool (designated PG8, comprising about 40% of the total PG) the phosphate group is stable to turnover, and in the second pool (PGt, about 60% of total PG) the phosphate moiety is subject to relatively rapid metabolic turnover. As Fig. 3 shows, cerulenin had little or no effect on the stability of [32P]PE or [3P]PG. or on the rate of 32p loss from PGt. However, the loss of 32p from PGt at 350C began almost immediately in the presence of cerulenin, but lagged by approximately 5 min in the control culture. On the other hand, 32p loss from PGt at 200C showed no clear lag in either the presence \-\PC(-CER) \ PG (+ CE R) F50*a.0 -w TIME (MIN.) FIG. 3. Stability of32p-equilibrated PG and PE at 35 C. A culture was grown for five population doublings to a cell density of 155 Klett units in medium S-MA56-LP containing 1.1 mm [32P]phosphate; cerulenin (CER) (20 pg/ml) was added to one-half of the culture, and both portions were then diluted threefold with fresh medium (35 C) containing 38.5 mm unlabeledphosphate (zero time). Incubation was continued at 35 C, and 5-ml samples were withdrawn at intervals for 32P-lipid analysis. Symbols: 0, PG, no cerukenin; 0, PG, +cerulenin; A, PE, no cerulenin; A, PE, +cerulenin. or absence of cerulenin and exhibited the same time course in both cultures (data not shown). Phosphate incorporation studies. [32P]- phosphate incorporation into PE lagged substantially behind incorporation into PG in cultures growing at 200C (Fig. 4A) or 350C (Fig. 5A). Moreover, the incorporation curve for PE at 350C (and probably also at 2000) was concave upward. During the initial 30 min, incorporation into PE was less than 3% of that into PG for both 20 and 350C cultures. As observed for net PE synthesis (Fig. 2A), initial rates of [32P]phosphate incorporation into PE were not inhibited by cerulenin (Fig. 4A and 5A), but incorporation eventually fell below that of the control cultures, especially at 350C. Incorporation of [32P]phosphate into PG at 350C exhibited a biphasic time course in the control culture after a brief initial lag of about 5 min (Fig. 5A). The modest rate elevation observed between 5 and 40 min coincided with the A PG(-CER) a- CL / \ 200- / PC(+CER) N 100- PE(-CER) J. BACTRIOL. 38- B 20- LYSO-PG (+ CER), TIME (MIN.) FIG. 4. Incorporation of[32pjphosphate into phospholipids at 20 C. A culture growing in medium S- M56-LP at a ceu density of 150 Klett units was divided into two equal portions. Cerulenin (CER) (20 pg/ml) was added to oneportion, and [32P]phosphate (2.73 ftci/ml) was then added to both portions (zero time). Incubation was continued at 201C, and 5-mi samples were withdrawn at intervals for 32P-lipid analysis. (A) Symbols: 0, PG, no cerulenin; 0, PG, + cerulenin; A, PE, no cerulenin; A, PE, +cerulenin. (B) Symbols: O, Jyso-PG, no cerulenin; *, lyso-pg, +cerulenin.

6 VOL. 141, 1980 filling of the PGt pool, as indicated in the cerulenin-treated culture (Fig. 3), and was followed in the control by a lower incorporation rate into PG, which was concomitant with accelerated [32P]PE formation. The rate of 32P incorporation into PG during the 5- to 40-min time period was about twofold greater than that observed at later times when allowance was made for growth. Similar results were obtained at 20 C (Fig. 4A). Strikingly, the 3P incorporation rate into PG was unaffected by cerulenin during the initial 60 min at 200C and during the initial 20 min at 35 C (Fig. 4A and 5A). At 200C, [3P]phosphate was incorporated into lyso-pg at a slow rate and only after a 20- min lag (Fig. 4B) in both the presence and absence of cerulenin. Moreover, expansion of the lyso-pg pool in the cerulenin-treated culture relative to the control was evident at this temperature. At 350C (Fig. 5B), incorporation of [32P]phosphate into lyso-pg again lagged behind that of [3 P]PG in both the control and cerule- "A a- me TIME (MIN.) FIG. 5. Incorporation of[32p]phosphate intophospholipids at 35 C. Experimental conditions were as described in the legend to Fig. 4 except that the temperature was 350C and the cell density at zero time was 68 Klett units. (A) Symbols: 0, PG, no cerulenin (CER); *, PG, + cerulenin; A, PE, no cerulenin; A, PE, +cerulenin. (B) Symbols: O, lyso- PG, no cerukenin; *, lyso-pg, +cerulenin. PG POOLS IN B. MEGATERIUM 623 nin-treated cultures. In this case, however, there was no apparent expansion of the lyso-pg pool in the presence of cerulenin. Indeed, the plateau level of [32P]lyso-PG was higher in the absence of cerulenin, probably due to continued growth in the control culture. DISCUSSION In B. megaterium ATCC 14581, the major membrane lipid, PG (70% of total phospholipids), is present in two distinct pools (Fig. 3). One pool (PGS, 27% of total phospholipids) exhibits metabolic stability of the phosphate group, whereas the second pool (PGt, 43% of total phospholipids) undergoes rapid turnover in this moiety. As shown elsewhere (15), the metabolic stability of PG8 extends to the nonacylated glycerol moiety as well, whereas turnover and/ or exchange of the nonacylated glycerol in PGt can be even more rapid than phosphate turnover in this pool. Evidence for two distinct PG pools has been reported previously for E. coli (1), S. aureus (22), and H. parainfluenzae (24), whereas two topologically distinct populations of PE have been demonstrated in B. megaterium KM (20, 21). The basis of the PG heterogeneity that we observed in B. megaterium ATCC is unknown. As pointed out by Rothman and Kennedy (20), it is unlikely that phospholipid heterogeneity is directly related to the presence of mesosomes in B. megaterium, since the lipid compositions of the mesosomes and plasma membranes are indistinguishable (D. J. Ellar, T. D. Thomas, and J. A. Postgage, Biochem. J. 122:44P-45P, 1971), as presumably are their topological distributions (20). On the other hand, one possible role for PGt which would be consistent with its observed labeling characteristics would be as a donor of glycerophosphate units for teichoic and/or lipoteichoic acid synthesis, as demonstrated in S. sanguis (5, 6). The effects of the fatty acid synthesis inhibitor cerulenin on the metabolic patterns of the major phospholipids were of interest. Although PG accumulation halted immediately after cerulenin addition (Fig. 2A), PE formation continued unabated for a time (Fig. 2, 4, and 5) at the expense of the diglyceride (2000) or diglyceride plus PG pools (3500) (15). Furthernore, turnover of the phosphate moiety in the PGt pool was not significantly affected by cerulenin (Fig. 3 through 5). The long lags observed for [32P]phosphate incorporation into PE as compared with PG (Fig. 4 and 5), as well as the concave-upward appearance of the incorporation curves into PE, would not be predicted on the basis of branching

7 624 LOMBARDI AND FULCO biosynthetic pathways for PG and PE formation from CDP-diglyceride, particularly in view of the small phosphatidylserine pool size in this strain (Fig. 1). In addition, the biphasic time course of [mp]phosphate incorporation observed in PG at 350C (Fig. 5A) and the finding that initial rates of phosphate incorporation into PG were unaffected by cerulenin at either 20 or 350C (Fig. 4 and 5) have important implications for phospholipid metabolism in this organism. These aspects are discussed more fully in the accompanying paper (15). The lipid composition determined for B. megaterium ATCC is substantially different from that of the KM strain. Rothman and Kennedy (20) reported a composition in the latter strain of 69 mol% PE, approximately 30 mol% PG, about 1 mol% cardiolipin, and only traces of neutral glycerolipids. In strain we found approximately 21 mol% PE, 56 mol% PG, 1 to 2 mol% phosphatidylserine, 20 mol% diglycerides, and no detectable cardiolipin (Fig. 1). Thus, the only points of similarity in lipid composition between the two strains appear to be the small or negligible pools of phosphatidylserine and cardiolipin. Interestingly, the composition observed in strain is similar in some respects to that of Bacillus subtilis W23 (11), where levels of 21 mol% PE, 58 mol% PG, and 13 mol% diglyceride were found. On the other hand, the low levels of cardiolipin and lysylphosphatidylglycerol present in B. subtilis (11) were not detected in B. megaterium ATCC under the present growth conditions. Figure 2B demonstrates that lyso-pg is a normal lipid constituent of B. megaterium in either the presence or absence of cerulenin and of exogenously added fatty acid. After cerulenin addition at 200C, the lyso-pg pool rapidly expanded more than 10-fold, reaching levels of 3 to 4 mol%. At 350C, however, the levels of lyso- PG in cerulenin-treated cultures remained at only 0.3 to 0.5 mol% (Fig. 5B). The marked temperature dependence of lyso-pg levels in cerulenin-treated cultures is probably responsible for our earlier inability to detect ["4C]palmitate-labeled lyso-pg at 350C (2). Since the [14C]lyso-PG is converted quantitatively to PG plus diglyceride in the presence of cerulenin (2, 15), the much lower lyso-pg pool size at 350C might be due to more rapid acylation with endogenous fatty acyl groups at the higher temperature (3). In any event, the results obtained for [32P]lyso-PG are consistent with the working hypothesis advanced earlier (3) which suggests that the first acylation step for PG synthesis might occur with either the fatty acyl carrier protein or the fatty acyl coenzyme A derivative, J. BACTERIOL. whereas the second acylation would specifically require fatty acyl carrier protein. Although the endogenous lyso-pg pool expanded rapidly after cerulenin addition at 200C (Fig. 2B) and incorporation of ['4C]palmitate into this pool preceded incorporation into [I4C]PG (2, 3, 15), nevertheless incorporation of [mp]phosphate into lyso-pg lagged behind incorporation into PG in the presence and absence of cerulenin at both 20 and 350C (Fig. 4B and 5B). Indeed, the incorporation curve into [32p]_ lyso-pg was concave upward during a portion of each time course and was similar in some respects to that of PE. Present in vivo and in vitro work is directed toward the precursor-product relationships between PG and lyso-pg. ACKNOWLEDGMENI We thank Edmund Yang and Sue Hill for excellent technical assistance in this work and Elin James for the preparation of the figures. These studies were supported in part by contract DE- AM03-76-SF00012 between the Department of Energy and the University of California and by Public Health Service research grant AI from the National Institute of Allergy and Infectious Diseases. LITERATURE CITED 1. Ballesta, J. P. G., C. L. de Garcia, and M. Schaechter Tumover of phosphatidylglycerol in Escherichia coli. J. Bacteriol. 116: Chen, S. L, and A. J. Fulco The cerulenin-induced formation of 1-acyl-lysophosphatidylglycerol in Bacillus megaterium. Biochem. Biophys. Res. Commun. 80: Chen, S. L, F. J. Lombardi, and A. J. Fulco The temperature-mediated metabolism of 1-acyl-lysophosphatidylglycerol in cerulenin-treated cultures of Bacillus megaterium. Biochem. Biophys. Res. Commun. 80: Cronan, J. E., Jr., C. H. Birge, and P. R. Vagelos Evidence for two genes specifically involved in unsaturated fatty acid biosynthesis in Escherichia coli. J. Bacteriol. 100: Emdur, L. I., and T. H. 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