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Journal of Bacteriology, August 2007, p. 5816-5824, Vol. 189, No. 16
0021-9193/07/$08.00+0     doi:10.1128/JB.00602-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Coupling of Fatty Acid and Phospholipid Synthesis in Bacillus subtilis

Luciana Paoletti,^1, Ying-Jie Lu,^2,, Gustavo E. Schujman,¹ Diego de Mendoza,¹ and Charles O. Rock^2*

Instituto de Biología Molecular y Celular de Rosario, and Departamento de Microbiología, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Rosario, Argentina,¹ Department of Infectious Diseases, St. Jude Children's Research Hospital, 332 N. Lauderdale, Memphis, Tennessee²

Received 18 April 2007/ Accepted 29 May 2007

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
plsX (acyl-acyl carrier protein [ACP]:phosphate acyltransferase), plsY (yneS) (acyl-phosphate:glycerol-phosphate acyltransferase), and plsC (yhdO) (acyl-ACP:1-acylglycerol-phosphate acyltransferase) function in phosphatidic acid formation, the precursor to membrane phospholipids. The physiological functions of these genes was inferred from their in vitro biochemical activities, and this study investigated their roles in gram-positive phospholipid metabolism through the analysis of conditional knockout strains in the Bacillus subtilis model system. The depletion of PlsX led to the cessation of both fatty acid synthesis and phospholipid synthesis. The inactivation of PlsY also blocked phospholipid synthesis, but fatty acid formation continued due to the appearance of acylphosphate intermediates and fatty acids arising from their hydrolysis. Phospholipid synthesis ceased following PlsC depletion, but fatty acid synthesis continued at a high rate, leading to the accumulation of fatty acids arising from the dephosphorylation of 1-acylglycerol-3-P followed by the deacylation of monoacylglycerol. Analysis of glycerol 3-P acylation in B. subtilis membranes showed that PlsY was an acylphosphate-specific acyltransferase, whereas PlsC used only acyl-ACP as an acyl donor. PlsX was found in the soluble fraction of disrupted cells but was associated with the cell membrane in intact organisms. These data establish that PlsX is a key enzyme that coordinates the production of fatty acids and membrane phospholipids in B. subtilis.

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Phosphatidic acid (PtdOH) is synthesized by the successive acylation of glycerol-P followed by the acylation of 1-acyl-glycerol-P, and there are two enzyme systems that carry out the first reaction in bacteria. In the Escherichia coli model system, either acyl-acyl carrier protein (ACP) or acyl-coenzyme A (CoA) thioesters are utilized by the membrane-bound PlsB acyltransferase to acylate the 1 position of glycerol-P (7, 14, 25). However, the E. coli paradigm is not universal, and the plsB gene is confined to gram-negative bacteria (primarily gammaproteobacteria) (27). Most gram-positive bacteria utilize the recently discovered PlsX/Y pathway for 1-acyl-glycerol-P formation (27). PlsX is a soluble protein that catalyzes the formation of acylphosphate (acyl-PO₄) from acyl-ACP. This activated fatty acid is then used by the membrane-bound PlsY acyltransferase to acylate the 1 position of glycerol-P (26). The second acyltransferase in PtdOH formation, PlsC, is universally expressed in bacteria and completes the synthesis of PtdOH by transferring an acyl chain from acyl-ACP (or acyl-CoA) to the 2 position of 1-acyl-glycerol-P (27). In E. coli, PlsC utilizes either acyl-ACP or acyl-CoA as an acyl donor (5), whereas in Streptococcus pneumoniae, PlsC uses only acyl-ACP as the acyl donor (27).

The PlsX and PlsY reactions were demonstrated in vitro, and their physiological roles were inferred from the biochemical analysis and the fact that they are essential genes in bacteria that lack plsB (27). Bacillus subtilis is typical of a gram-positive bacterium that lacks a plsB gene. The B. subtilis plsX gene was annotated based on its similarity to the E. coli counterpart and is an essential gene in B. subtilis (22). The E. coli plsX gene was discovered as a mutated allele required for a plsB26 mutant strain to exhibit a glycerol-P auxotrophic growth phenotype (23). The E. coli plsB26 mutant produces a PlsB acyltransferase with an elevated K_m for glycerol-P (1, 2, 19). Bioinformatic analysis of the B. subtilis genome shows that the yhdO gene corresponds to the plsC gene and that the yneS gene corresponds to the plsY gene. Both of these genes are also essential in B. subtilis (22). The goal of this study is to employ a genetic approach to investigate the roles of plsX, plsY, and plsC in gram-positive phospholipid metabolism through the analysis of conditional knockout strains for each of these three genes in the B. subtilis model system.

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Materials. Sources of supplies were American Radiolabeled Chemicals Inc. ([2-¹⁴C]acetate [specific activity, 55 Ci/mol] and [U-¹⁴C]glycerol-P [specific activity, 150 Ci/mole]), Avanti Polar Lipids, Inc. (palmitoyl-CoA and phospholipids), and Sigma Chemical Co. (glycerol-P and fatty-acid-free bovine serum albumin [BSA]); acyl-PO₄ was synthesized as described previously (27). Acyl-ACPs were prepared enzymatically by using the acyl-ACP synthase method (32, 33) using E. coli ACP as the starting material. Protein was measured by the Bradford method (3). All other chemicals were reagent grade or better.

Construction of conditional knockout mutant strains. The integrative plasmid pDH88 (30) or pMUTIN4 (35), containing the IPTG (isopropyl-ß-D-thiogalactopyranoside)-inducible Pspac promoter, or plasmid pGES49, a derivative of pAG58 (34) containing the xylose-inducible Pxyl promoter, was used for conditional gene expression in B. subtilis. Plasmid pLP6 (Fig. 1A) was constructed using a 250-bp DNA fragment, generated by PCR using primers YhdOpU and YhdOpL (Table 1), carrying the ribosome binding site and a 5' portion of yhdO. The amplification product was digested with HindIII and BamHI and cloned into vector pDH88, previously digested with HindIII and BglII. Plasmid pLP35 (Fig. 1B) was constructed using a 514-bp DNA fragment, generated by PCR with primers PYneSU and PYneSL (Table 1), containing the ribosome binding site and the 5' upstream region of the yneS gene. The PCR product was first cloned into PCR-Blunt II-TOPO. This plasmid was then digested with HindIII and BglII, and the resultant fragment was inserted into the HindIII and BamHI sites of vector pMUTIN4. Plasmid pLP21 (Fig. 1C) was constructed using a 546-bp DNA fragment, generated by PCR with primers PYlpcU and PYlpcL (Table 1), containing the ribosome binding site and the 5' upstream region of the plsX gene. The product of PCR was digested with HindIII and BamHI and cloned into plasmid pGES49. Plasmid pGES310 (Fig. 1C) was constructed using a 451-bp DNA fragment containing the ribosome binding site and the 5' upstream fragment of the fabD gene generated by PCR using primers FabDmutB and PplsXU (Table 1). The PCR product was digested with BamHI and EcoRI and cloned into plasmid pMUTIN4. Plasmid pLP36 was constructed using a 389-bp DNA fragment containing the ribosome binding site and the 5' upstream fragment of the yneR gene generated by PCR using primers PYneRU and PyneRL (Table 1). The PCR product was digested with BamHI and EcoRI and cloned into plasmid pMUTIN4.

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FIG. 1. Diagram of molecular constructs. (A) Initial and final genomic organizations of strain LP15 (Pspac-plsC) containing plsC (yhdO) under IPTG control. (B) Initial and final genomic organizations of strain LP61 (Pspac-plsY) containing plsY (yneS) under IPTG control. (C) Initial and final genomic organizations of strain LP39 (Pxyl-plsX) containing plsX under the control of the xylose promoter. IPTG was also required for growth of this strain to express the downstream essential fadD and fabG genes that are located in the plsX operon as indicated in the diagram.

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TABLE 1. Oligonucleotides used in this study

Strains LP15, LP39, GS311, LP61, and LP62 were generated by integration of the circular form of the plasmids pLP6, pLP35, pGES310, pLP21, and pLP36, respectively, into the B. subtilis chromosome by a single-crossover event (Fig. 1). This approach results in the conditional inactivation of the target gene whose expression can be controlled by either Pspac or Pxyl (Fig. 1). Each of these strains was checked by PCR to ensure that the plasmid had integrated into the correct site. B. subtilis strains were cultured in Luria-Bertani (LB) medium supplemented, where required, with 0.4% xylose and/or 0.2 mM IPTG. B. subtilis strains were transformed as previously described (34). Transformants were selected on LB agar supplemented, where required, with chloramphenicol (5 µg/ml), erythromycin (0.5 µg/ml), lincomycin (12.5 µg/ml), 0.2 mM IPTG, or 0.4% xylose.

The yneR gene is located just downstream of the yneS gene, and although yneR does not apparently have a role in lipid metabolism, the two genes may exist in an operon. YneR belongs to a family of proteins involved in iron-sulfur cluster biosynthesis (Pfam01521) and was not scored as being an essential protein in the screen for essential Bacillus genes (22). In strain LP61, both genes yneS and yneR were under the control of the Pspac promoter (Fig. 1B); therefore, we constructed strain LP62 to rule out an effect of yneR on the yneS phenotype. In this strain, only yneR expression depended on the presence of IPTG. Strain LP62 grew normally in the presence and absence of the inducer (data not shown), confirming that the yneR gene was not essential and that the growth defect of strain LP61 in the absence of IPTG was due to the lack of yneS expression.

Growth and metabolic labeling of mutant strains. LP39 (plsX) was grown overnight in LB medium with 0.2 mM IPTG-0.4% xylose. LP61 (plsY) and LP15 (plsC) were grown in LB medium with 0.3 mM IPTG and the corresponding antibiotic. Cells were washed twice and resuspended in LB medium. Strains LP39, LP61, and LP15 were inoculated at A₆₀₀ values of 0.06, 0.15, and 0.04, respectively, and grown in the presence or absence of inducers. Each of the constructs required a slightly different number of cell divisions to dilute out the preexisting protein in the absence of inducer. The different inoculation densities were empirically determined to provide cell cultures making the transition to PlsX-, PlsY-, or PlsC-dependent growth in 3 to 4 h at between 100 and 200 Klett units.

Cells were labeled with 1 µCi/ml [¹⁴C]acetate for 30 min at the time points indicated in the figures. Cells were collected, and total lipids were extracted. Briefly, cells were resuspended in 1.8 ml of chloroform-methanol-acetic acid (1/2/0.02, vol/vol/vol) followed by 0.5 ml water, 0.9 ml of chloroform, and 0.9 ml of 2 M KCl. Samples were vortexed and centrifuged to separate the phases. The lower phase was transferred into a new tube, and the upper phase was extracted again with 0.9 ml chloroform and combined with the first extraction. The organic phase was evaporated under nitrogen and redissolved in chloroform. Lipids were analyzed using either preadsorbent Silica Gel G layers (Analtech) developed with hexane-ethyl ether-acetic acid (80/20/1, vol/vol/vol) to separate the neutral lipids or Silica Gel H layers (Analtech) developed with chloroform-methanol-ammonia-water-0.25 M EDTA (45/35/1.5/8.34/0.16, vol/vol/vol/vol/vol) to resolve the phospholipids. Radioactivity on the plates was visualized using a Typhoon 9200 PhosphorImager screen and quantified using ImageQuant software (version 5.2). Lipid species were identified by comigration with standards.

Detection of acyl-PO₄ with mass spectroscopy. LP61 cells were grown overnight on an LB plate with 12.5 µg/ml lincomycin, 0.5 µg/ml erythromycin, and 0.3 mM IPTG. Cells were scraped off the plate and used to inoculate LB medium to an A₆₀₀ of 0.03 to initiate growth at 37°C. Cells were collected by centrifugation (6,000 rpm, 4°C, 10 min) when the A₆₀₀ reached 0.5 and were immediately processed with total lipid extraction using chloroform-methanol-concentrated HCl (1/2/0.02, vol/vol/vol). The dried sample was dissolved in 50% methanol-water and analyzed by negative-ion electrospray precursor ion-scanning mass spectrometry.

Mass spectrometry of acyl-PO₄ was performed by the Hartwell Center for Bioinformatics and Biotechnology at St. Jude Children's Research Hospital. Mass spectrometry analysis was performed using a Finnigan TSQ Quantum (Thermo Electron, San Jose, CA) triple-quadrupole mass spectrometer equipped with the nanospray ion source. The instrument was operated in the negative-ion mode using precursor ion scanning to detect the loss of a phosphate group (27). Ion source parameters were as follows: a spray voltage of 1,600 V, a capillary temperature of 270°C, and a capillary offset of –35 V (tube lens offset was set by infusion of the polytyrosine tuning and calibration solution [Thermo Electron, San Jose, CA] in electrospray mode). Mass spectrometry acquisition parameters for Q1 scanning were as follows: a scan range of 225 to 500 m/z, a scan time zero of 1 s, and a peak width for Q1 of 0.7 FWHM (full width at half height). Mass spectrometry acquisition parameters for parent ion scanning were as follows: a scan range of 225 to 500 m/z, a scan time zero of 1 s, a product mass of 79 m/z, a collision energy of 15 V, a peak width for Q1 and Q3 of 0.7 FWHM, and Q2 collision-induced dissociation gas (argon) of 0.5 mTorr. Instrument control and data acquisition were performed with Finnigan Xcalibur (version 1.4 SR1) software (Thermo Electron, San Jose, CA).

PlsC activity assay. The membrane fraction of B. subtilis strain 168 was purified using sucrose gradient ultracentrifugation according to a method described previously (27). Reactions were carried out in a solution containing 100 mM Tris-HCl (pH 7.4), 150 mM NaCl, 100 µM [¹⁴C]glycerol-P, 1 mg/ml BSA, and 5 mM Na₃VO₄ (to inactive phosphatases) with 5 µg purified membrane protein. Reactions were started by adding combinations of 200 µM palmitoyl-PO₄, 50 µM palmitoyl-ACP, or 50 µM palmitoyl-CoA to the mixtures. Reaction mixtures were extracted by chloroform-methanol-concentrated HCl (1/2/0.02, vol/vol/vol) and spotted onto preadsorbent Silica Gel H layers (Analtech) developed with chloroform-methanol-ammonia-water-0.25 M EDTA (45/35/1.5/8.34/0.16), and product formation was detected with a Typhoon 9200 PhosphorImager and quantified using ImageQuant software (version 5.2).

Immunofluorescence microscopy. The strains were grown to the exponential phase under the conditions described above. Samples were processed for immunofluorescence microscopy (16). All cultures were fixed in growth medium with a final concentration of 2.6% paraformaldehyde, 0.006% glutaraldehyde, and 30 mM sodium phosphate buffer (pH 7.4) for 15 min at room temperature and 30 min on ice. The cells were washed, briefly treated with lysozyme, and affixed to poly-L-lysine-treated multiwell slides. Cells were stained with purified primary antibodies raised in rabbits against the purified target proteins. The specificity of the anti-PlsX, anti-FabF, and anti-PlsC antibodies was analyzed by Western blotting using protein extracts from wild-type strain 168. These antibodies detected a band of the correct molecular weight in the control cells (data not shown). After washing with the fluorescein isothiocyanate (FITC)-conjugated secondary antibodies, 1 µg/ml of 4',6'-diamidino-2-phenylindole (DAPI) was added with Slow Fade equilibration buffer (Slow Fade kit from Molecular Probes) for 5 min. Coverslips were mounted with Slow Fade containing glycerol. All photographs were taken using a Nikon Eclipse E800 microscope equipped with a 100x Plan Apo objective and a Nikon FDX-35 automatic camera system. Two filter sets were used, one for visualizing FITC (FITC-HYQ) and the other for visualizing DAPI (UV-2A).

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Depletion of PlsX, PlsY, and PlsC. B. subtilis contains three open reading frames that have homology with enzymes predicted to be involved in the biosynthesis of PtdOH based on their in vitro enzymatic activities (27). Two of these genes, plsX and plsY (yneS), are related to the PlsX/Y pathway for the first acylation of glycerol-P. The third gene, plsC (yhdO), is predicted to encode an acyltransferase catalyzing the acylation of 1-acyl-glycerol-P to give PtdOH. These three genes were flagged as being essential based on the systematic genome-wide inactivation of B. subtilis genes (22). Therefore, we constructed strains LP15, LP39, and LP61 that expressed each of these genes under the control of a regulated promoter (Fig. 1). Strains LP15 and LP61 contain the plsC or the plsY gene under the control of the IPTG-inducible Pspac promoter, respectively, while in strain LP39, plsX is controlled by the xylose-inducible Pxyl promoter (Fig. 1). In strain LP39, the essential fabD and fabG genes located downstream of plsX were driven by Pspac to alleviate the potential polar effect caused by the generation of the Pxyl-plsX chromosomal fusion (Fig. 1). Strain LP39 consistently grew to higher densities than the other two strains due to the supplementation of the medium with xylose that not only induced plsX expression but also served as a carbon source.

Each of the disruption strains was able to grow in the presence of the appropriate inducer (IPTG for strains LP15 and LP61 or xylose plus IPTG for strain LP39), and their removal resulted in no cell growth on agar plates, indicating that plsX, plsY, and plsC were indeed essential. Unlike temperature-sensitive mutants, the removal of inducer did not result in the immediate inactivation of the protein or cessation of cell proliferation, but rather, cell growth continued until the preexisting protein was diluted out by subsequent cell divisions as illustrated by the growth curves shown in Fig. 2. There were three phases to cell growth in the absence of inducer. First, there was a log phase that was identical to that of the wild type, representing the period of time where the particular gene product was present in sufficient quantities to permit normal growth. Second, there was a transition phase where the cell culture continued to increase in density but at a continually slower rate than the wild type. This phase was when the cellular content of the particular gene product was becoming limiting for growth. Third, the cell density reached a plateau, and the production of all cell constituents was arrested. Our bacterial growth procedures were established to metabolically label the cells during the second phase of growth, corresponding to the transition from log- to stationary-phase growth (Fig. 2). These data established the growth parameters for analyzing the effect of depleting each of these proteins on lipid metabolism.

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FIG. 2. Essentiality and growth phenotypes of PlsX, PlsY, and PlsC mutants. Strain LP39 (plsX) was grown overnight in LB medium with 0.3 mM IPTG-0.4% xylose, and strains LP61 (plsY) and LP15 (plsC) were grown in LB medium with 0.3 mM IPTG. Cells were washed twice and resuspended in LB medium. Strains LP39, LP61, and LP15 were inoculated to A600 values of 0.06, 0.15, and 0.04, respectively, and grown in the presence (open symbols) or absence (closed symbols) of inducers. Strain LP39 (PlsX depletion) was grown with () or without (•) 0.4% xylose; strain LP61 (PlsY depletion) was grown with () or without () 0.3 mM IPTG; strain LP15 (PlsC depletion) was grown with () or without () 0.3 mM IPTG. Strain LP39 grew to a higher cell density due to the presence of xylose as an inducer, which is also used as a carbon source.

Lipid metabolism in PlsX-, PlsY-, and PlsC-depleted strains. The effect of eliminating each of these gene products on lipid metabolism was assessed by metabolic labeling with [¹⁴C]acetate during the time periods when the gene products were becoming limiting to growth, as indicated in Fig. 2. Acetate incorporation into the lipid fraction of strain LP39 (Pxyl-plsX) in the absence of inducer was about 10% of the rate in the presence of IPTG (Table 2). In strain LP61 (Pspac-plsY), the comparison of acetate incorporation with inducer and that without inducer showed that the acetate incorporation was approximately the same. Surprisingly, the result with strain LP15 (Pspac-plsC) showed that the absence of inducer resulted in a 250% increase in acetate labeling (Table 2). The effect of the defects in lipid metabolism on general cell metabolic activity was assessed by the incorporation of [¹⁴C]acetate into nonlipid (essentially protein) cell components. In all cases, the incorporation of acetate into protein was comparable. In a separate experiment, we normalized the incorporation of [¹⁴C]acetate into cells and normalized the results to total cell protein. The pattern was essentially the same with the PlsX-depleted strain, incorporating 10.6% of the control, the PlsY-depleted cells, incorporating 98%, and the PlsC-depleted cells, incorporating 202%. These data show that the depletion of PlsX resulted in the cessation of fatty acid synthesis, while the inactivation of PlsY or PlsC did not.

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TABLE 2. [14C]Acetate labeling of strains deficient in PlsX, PlsY, or PlsCa

The next experiments examined the labeled products that were found in the depleted cells. The analysis of the lipid products is illustrated in Fig. 3, and the distribution of radioactivity is shown in Table 2. Depletion of the plsX gene product resulted in the inhibition of total lipid synthesis without accumulation of fatty acids or other intermediates, indicating that in the absence of PlsX, the syntheses of both fatty acid and phospholipid were severely compromised. There was a trace of fatty acid in xylose-supplemented strain LP39 that was not detected in the PlsX-depleted cells. PlsY depletion also blocked phospholipid synthesis, although the synthesis of fatty acid was still active, as evidenced by the accumulation of fatty acid in PlsY-deprived strain LP61 (Fig. 3 and Table 2). Depletion of PlsC also compromised phospholipid biosynthesis and led to the incorporation of acetate into lipids at increased rates. The accumulation of monoacylglycerol and a large amount of fatty acid was noted following the inactivation of PlsC. Monoacylglycerol arising from the dephosphorylation of acyl-glycerol-P was noted previously in acyltransferase assays using either E. coli (31) or Rhodopseudomonas spheroids (28) membranes as the in vitro enzyme source. The inactivation of PlsC in E. coli plsC(Ts) mutants led to a large accumulation of phosphatidic acid; however, the experiments would not have detected monoacylglycerol if it was formed (4). In summary, our data indicate that PlsX depletion inhibits total lipid biosynthesis, while the depletion of either PlsY or PlsC affects only phospholipid biosynthesis. Thus, the formation of acyl-PO₄ by PlsX is the critical step in the initiation of the phospholipid biosynthetic pathway, coupling the biosynthesis of fatty acids with phospholipid synthesis.

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FIG. 3. [14C]Acetate labeling profiles of PlsX-, PlsY-, and PlsC-depleted cells. Cells were labeled with 1 µCi/ml [14C]acetate for 30 min during the transition from log- to stationary-phase growth (Fig. 2). Cells were harvested, and total lipids were extracted and analyzed by thin-layer chromatography on preadsorbent Silica Gel G layers (Analtech) developed with hexane-ethyl ether-acetic acid (80/20/1, vol/vol/vol). The radioactive lipid species were visualized using a PhosphorImager screen, and the bands were identified by their comigration with standards. FA, fatty acid; DAG, diacylglycerol; MAG, monoacylglycerol; PL, phospholipid. The example shown is typical of three experiments performed in both laboratories.

PlsX is responsible for the formation of acyl-PO₄. Despite repeated attempts, we were unable to detect the presence of acyl-PO₄ intermediates in wild-type cells either by metabolic labeling and thin-layer chromatography or by mass spectrometry. These negative experiments were not completely unexpected, because acyl-PO₄ intermediates were not detected in S. pneumoniae wild-type cells either (27). However, we were able to detect acyl-PO₄ peaks in samples of strain LP61 (Pspac-plsY) grown in the absence of IPTG during the transition from log-phase growth to growth stasis (Fig. 4). These mass spectrometry results clearly showed the presence of 15 and 17 carbon acyl-PO₄s, reflecting the major chain lengths of fatty acids produced by the B. subtilis fatty acid biosynthetic pathway. A small 19-carbon peak was also detected (Fig. 4), indicating that some excess elongation of acyl chains occurred in the absence of utilization. This observation was reminiscent of a report that the blockade of phospholipid synthesis in E. coli at the acyltransferase step using either plsB or gpsA mutants gave rise to the formation of abnormally long fatty acid products (9). This technology cannot distinguish between iso and anteiso fatty acids. Mass spectrometry is a very sensitive method to detect biomolecules, but one drawback is that it is not a quantitative analysis. A standard curve generated with pure 16:0-PO₄ was linear, but spiking these standards into the lipid extract did not give linear responses. Thus, we could not directly determine the concentration of acyl-PO₄ with this method. However, acyl-PO₄ peaks were not observed in extracts of wild-type cells or cells grown in the presence of inducer. Also, the thin-layer chromatography analysis of [¹⁴C]acetate-labeled cells using the phospholipid system did not reveal the accumulation of a radiolabeled spot at the R_f for acyl-PO₄ (not shown). These data indicate that the cellular content of acyl-PO₄ was lower than the detection limit using ¹⁴C-radiolabeling technology. Thus, cells depleted of PlsX do accumulate low levels of acyl-PO₄, a minor component of which hydrolyzes to fatty acid.

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FIG. 4. Accumulation of acyl-PO4 in strain LP61. Strain LP61 was grown overnight in LB medium with 0.3 mM IPTG. Cells were inoculated into LB medium at an A600 of 0.03 and grown at 37°C until the A600 reached 0.5. Cells were collected by centrifugation (6,000 rpm, 4°C, 10 min), and the lipids were immediately extracted using chloroform-methanol-concentrated HCl (1:2:0.02, vol/vol/vol). The dried sample was dissolved in 50% methanol-water and analyzed by negative-ion electrospray precursor ion-scanning mass spectrometry as described in Materials and Methods.

Biochemistry of PlsC. Membranes were isolated from wild-type B. subtilis strain 168 and assayed for acyltransferase activities using three acyl donors, and the 1-acyl-glycerol-P and PtdOH reaction products were separated by thin-layer chromatography and quantified (Fig. 5A and B). In the presence of acyl-PO₄, only 1-acyl-glycerol-P was formed. Acyl-PO₄ plus acyl-CoA led to a significant reduction in 1-acyl-glycerol-P formation. This finding was consistent with the in vitro characterization of long-chain acyl-CoA as a potent noncompetitive inhibitor of PlsY utilization of acyl-PO₄ (26). In the presence of acyl-PO₄ plus acyl-ACP, both 1-acyl-glycerol-P and PtdOH were formed. Acyl-ACP did not yield any acyltransferase products, illustrating that the first acylation step is acyl-PO₄ specific. These data were consistent with B. subtilis membranes containing acyl-PO₄-specific PlsY and acyl-ACP-specific PlsC acyltransferases. Acyl-CoA was not a substrate for either acyltransferase.

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FIG. 5. PlsC is an acyl-ACP-specific acyltransferase. Cell membranes of B. subtilis strain 168 were purified using sucrose gradient ultracentrifugation and used to assay the acyltransferases. Reactions were carried out in a solution containing 100 mM Tris-HCl (pH 7.4), 150 mM NaCl, 100 µM [14C]glycerol-P, 1 mg/ml BSA, and 5 mM Na3VO4 (to inactivate phosphatases) with 5 µg membrane protein. Reactions were initiated by adding either 200 µM acyl-PO4, 50 µM palmitoyl-ACP, 50 µM palmitoyl-CoA, or combinations thereof to the mixture as indicated in the figure. Reactions were terminated after 30 min, and mixtures were extracted with chloroform-methanol-concentrated HCl (1/2/0.02, vol/vol/vol). The formation of [14C]acyl-glycerol-P (A) and [14C]PtdOH (B) was quantified following separation by thin-layer chromatography on Silica Gel H layers developed with chloroform-methanol-ammonia-water-0.25 M EDTA (45/35/1.5/8.34/0.16). Products were visualized following exposure to a PhosphorImager screen and quantified using ImageQuant software. (C) PlsC acyltransferase assays. The left two bars are the activities in E. coli strain SJ361 [plsC(Ts)] transformed with either an empty vector or one containing the B. subtilis plsC gene. The right two bars are PlsC activities in wild-type B. subtilis and strain LP15 grown in the absence of an inducer. The arrows and gene symbols along the y axis indicate the steps in PtdOH formation that were being measured in the experiment.

The plsC gene was cloned into the expression plasmid pPJ131, a modified pBluescript vector, to create plasmid pLP19. Transformation of E. coli strain SJ361 [plsC(Ts)] resulted in the rescue of the temperature-sensitive growth phenotype of the E. coli strain. Acyltransferase assays at 42°C using membranes isolated from strain SJ361/pLP19 (plsC) and SJ361/pPJ131 (control) using palmitoyl-ACP as a substrate showed that membranes from the strain harboring plasmid pLP19 exhibited robust 1-acyl-glycerol-P acyltransferase activity compared to the control. Membranes isolated from wild-type B. subtilis also had high 1-acyl-glycerol-P acyltransferase activity using the same assay, and membranes isolated from strain LP15 (Pspac-plsC) 2 h after growth ceased exhibited defective 1-acyl-glycerol-P acyltransferase activity. We also tested these membranes for PlsC activity using acyl-CoA as a substrate and were unable to detect activity above the background (not shown). Thus, B. subtilis PlsC is an acyl-ACP-specific 1-acylglycerol-P acyltransferase.

Localization of PlsX. The acyltransferases systems described to date are uniformly membrane bound. However, PlsX was purified as a soluble protein (27), whereas PlsY (26) and PlsC (4, 30) were clearly typical intrinsic membrane proteins. The localization of PlsX within the cell was examined by immunofluorescence microscopy to detect PlsX expressed in B. subtilis wild-type strain JH642. The PlsX antibody exhibited a punctate pattern of staining and a peripheral localization that was most closely associated with the membrane (Fig. 6A). This pattern of distribution was distinct from the homogenous appearance of the antibody to an established cytoplasmic protein, FabF (Fig. 6B), and was most similar to the distribution of the PlsC antibody (Fig. 6C), an established membrane protein (30). We tested whether its membrane localization was due to an interaction with PlsY by examining if there was a change in PlsX localization in cells depleted of PlsY. Strain LP61 (Pspac-plsY) was grown for 2 h after reaching the stationary phase of growth to maximally deplete the PlsY protein, and the cells were imaged for the distribution of the PlsX protein. The depletion of PlsY did not significantly alter the distribution of the PlsX antibody (Fig. 6D) compared to cells containing PlsY (Fig. 6E). This experiment also illustrated the point that the depleted cells did not have a significantly altered overall size or shape compared to their wild-type counterpart. These imaging data indicated that PlsX was an extrinsic membrane protein and that the association of PlsX with the membrane in vivo did not involve a specific interaction with PlsY. PlsX may associate with an unidentified membrane protein or with the phospholipids themselves.

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FIG. 6. Cellular localization of PlsX. (A) PlsX immunolocalization in strain JH642. FabF (B) and PlsC (C) were immunolocalized in strain JH642 as controls for the distribution expected for soluble and membrane-bound proteins, respectively. (D and E) Immunofluorescent images of PlsX in strain LP61 (Pspac-plsY) grown with (D) or without (E) IPTG. The photographs are of cells immunostained with antibodies raised against purified B. subtilis PlsX, PlsC, or FabF protein and visualized with a secondary FITC-conjugated antibody.

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Our experiments verify the proposed biochemical roles of PlsX, PlsY, and PlsC in vivo and assign a key role for PlsX in coupling fatty acid synthesis and phospholipid synthesis (Fig. 7). The inactivation of PlsX leads to the cessation of both fatty acid synthesis and phospholipid synthesis (Fig. 3). The lack of accumulation of lipid products in PlsX-depleted cells means that acyl-ACP phosphotransacylase activity controls both the fatty acid and phospholipid biosynthetic pathways. The inability to detect fatty acids following plsX inactivation means that B. subtilis does not possess an active acyl-ACP thioesterase that degrades the long-chain acyl-ACP end products of fatty acid synthesis. In fact, the cessation of acetate incorporation into the lipid fraction in the absence of PlsX activity suggests that PlsX is the only substantive route for the utilization of acyl-ACP in B. subtilis. PlsY-depleted cells do not have the same stringent effect on controlling the fatty acid biosynthetic pathway. PlsY is the only known enzyme that utilizes acyl-PO₄, and its consumption ceases in PlsY-depleted cells. Normally, acyl-PO₄ is not detected in growing cells (27), but detectable amounts of this ephemeral intermediate are observed in PlsY-depleted B. subtilis cells by mass spectrometry (Fig. 4). The PlsX reaction is freely reversible (27), so the acyl-PO₄ that is not hydrolyzed can be converted to acyl-ACP in the presence of nonesterified ACP. Also, fatty acids accumulate following PlsY inactivation in [¹⁴C]acetate metabolic labeling experiments due to the hydrolysis of acyl-PO₄. These high-energy intermediates are not stable in water and spontaneously hydrolyze to fatty acids (24). However, because long-chain acyl-PO₄ has a half-life of 12 h at a neutral pH and at 37°C (24), this is a relatively slow process, and the accumulation of fatty acid in our experiments may be due to catalytic hydrolysis by an unknown enzyme capable of hydrolyzing acyl-PO₄ as a side reaction. There is a very significant accumulation of fatty acid in PlsC-depleted strains (Fig. 3). In this case, membrane phospholipid synthesis is also halted, but the accumulation of the anticipated 1-acyl-glycerol-P intermediate was not observed. Rather, monoacylglycerol was detected by thin-layer chromatography, illustrating that B. subtilis has an active 1-acyl-glycerol-P phosphatase that rapidly forms monoacylglycerol, which is subsequently degraded by an esterase to fatty acid. This alternate-use pathway for acyl-ACP utilization leads to the uncoupling of the synthesis of fatty acid and phospholipid and the acceleration of acetate incorporation into fatty acid.

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FIG. 7. Coupling of fatty acid and phospholipid syntheses in B. subtilis. Long-chain acyl-ACPs are the end products of the bacterial dissociated type II fatty acid synthase system (FAS II). B. subtilis uses PlsX to convert these acyl-ACPs to acyl-PO4 using a phosphotransacylase-type reaction. The PlsX step is reversible. The next step is catalyzed by the membrane-associated PlsY (acylglycerol-P acyltransferase) encoded by the yneS gene that transfers the acyl moiety to the 1 position of glycerol-P to form acyl-G3P. Acylation of the 2 position is catalyzed by PlsC (YhdO), a membrane-bound 1-acyl-glycerol-P acyltransferase that specifically uses acyl-ACP as the acyl donor to form PtdOH. PlsX inhibition results in not only the blockade of phospholipid but also fatty acid synthesis via the feedback regulation of the type II fatty acid synthase system by acyl-ACP at the acetyl-CoA carboxylase, ß-ketoacyl-ACP synthase III, and enoyl-ACP reductase steps. Blockade of the PlsY step leads to the hydrolysis of acyl-PO4 to fatty acid (FA), which partially uncouples fatty acid and phospholipid syntheses by providing an alternate fate for acyl-ACP. Inactivation of PlsC causes runaway fatty acid formation due to the rapid dephosphorylation of acyl-glycerol-P to monoacylglycerol (MAG) and its subsequent hydrolysis to fatty acid.

These data are consistent with the key regulatory role for acyl-ACP as a feedback regulator of type II fatty acid synthesis established using the E. coli model system. Long-chain acyl-ACP end products inhibit FabI via a product inhibition mechanism to reduce the rate of the fatty acid elongation cycle and inhibit the activity of FabH, thus blocking the initiation of new acyl chains (18, 20). Acetyl-CoA carboxylase is another important acyl-ACP target (11), and the inhibition of this enzyme deprives the pathway of the malonyl-CoA required to continue the extension of fatty acid chains. The importance of acyl-ACP in feedback control is highlighted by experiments that provide an alternate fate for the fatty acid synthase end products. When E. coli cells are modified to overexpress active acyl-ACP thioesterases from either plants (36) or E. coli (21), there is runaway fatty acid synthesis that results in the copious production of fatty acids. Therefore, the inhibition of synthesis of fatty acid and phospholipid in PlsX-depleted cells is attributed to the accumulation of acyl-ACP due to the block in its conversion to acyl-PO₄, and it feeds back on the type II system to arrest fatty acid production (Fig. 7). Fatty acid synthesis inhibition is not stringent in PlsY-depleted cells where the breakdown of acyl-PO₄ to fatty acid provides an alternate fate for acyl-ACP and uncouples fatty acid from phospholipid synthesis. Likewise, fatty acid synthesis continues in PlsC-depleted cells due to the degradation of 1-acyl-glycerol-P to fatty acids. Thus, acyl-ACP feedback regulation is a common feature of type II fatty acid synthesis systems in gram-negative and gram-positive bacteria. It is also clear from our work that regulation at the PlsX step is the only point where fatty acid synthesis and phospholipid synthesis can be coordinately regulated. In E. coli, the PlsB acyltransferase plays an important role in regulating the utilization of acyl-ACP for phospholipid synthesis (17), and our results suggest that PlsX has a similar role in bacteria that lack PlsB and utilize the PlsX/Y/C pathway to PtdOH. However, the mechanism(s) for the regulation of PlsX activity remains a major unanswered question in bacterial biochemistry.

B. subtilis PlsC is the second example of a biochemically characterized gram-positive 1-acyl-glycerol-P acyltransferase. Like S. pneumoniae PlsC (27), B. subtilis PlsC uses only acyl-ACP as the acyl donor. This is in contrast to the E. coli enzyme, which is capable of using acyl-CoA as the acyl donor (4), as do the plant (15) and mammalian (13) homologs. The S. pneumoniae genome does not contain homologs of the enzymes for the synthesis of acyl-CoA or ß-oxidation, and therefore, a PlsC capable of using acyl-CoA is of no practical value for this organism. In contrast, B. subtilis has two homologs of acyl-CoA synthetases (LcfA and YhfL) and a complement of genes predicted to constitute a fatty acid ß-oxidation pathway whose expression is regulated by YsiA (29). YsiA is an acyl-CoA-responsive transcriptional repressor analogous to FadR (8) that functions in controlling the fatty acid ß-oxidation regulon in E. coli. The accumulation of fatty acids, particularly in the PlsC-depleted cells, indicates that this pathway is not rapidly induced by the appearance of intracellular fatty acids under the growth conditions the we employed. There are reports of exogenous fatty acid incorporation into phospholipids in B. subtilis (10) and Bacillus megaterium (12), and our data raise the question of the pathway for exogenous fatty acid incorporation into phospholipids. Acyl-CoAs may be formed, but they could not serve as substrates for either the PlsY or PlsC acyltransferases. There are several possible explanations for these observations. There may be fatty acid turnover in membrane phospholipids, and the reacylation enzyme may use acyl-CoA as well as acyl-ACP. One such acyltransferase has been biochemically characterized in E. coli, but this lysophosphatidylethanolamine acyltransferase is acyl-ACP specific (6). B. subtilis may possess either an acyl-ACP synthetase or a fatty acid kinase. In this regard, the two putative acyl-CoA synthetase homologs of B. subtilis have not been biochemically characterized. This leaves open the possibility that one of them may be an acyl-ACP synthetase that is a component of the YsiA regulon whose function is to channel exogenous fatty acids into the phospholipid biosynthetic pathway. Fatty acid kinases are unknown to biochemistry, but this postulated activity would function to introduce exogenous fatty acids into the phospholipid biosynthetic pathway downstream of PlsX. The accumulation of fatty acids in our experiments suggests that neither of these postulated enzymes is operating in our experimental setting; however, this may be due to the depletion of ACP acceptors for the activated fatty acids when the pathway is arrested.

 
This work was supported by NIH grant GM34496 (C.O.R.), Cancer Center Support grant CA 21765, the Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET, Argentina), the Agencia de Promoción Científica y Tecnológica (FONCYT, Argentina), and the American Lebanese Syrian Associated Charities. L.P. is a fellow and G.E.S. and D.D.M. are Career Investigators at CONICET. D.D.M. is an International Research Scholar of the Howard Hughes Medical Institute.

 
* Corresponding author. Mailing address: Department of Infectious Diseases, St. Jude Children's Research Hospital, 332 N. Lauderdale, Memphis, TN 38105-2794. Phone: (901) 495-3491. Fax: (901) 495-0399. E-mail: charles.rock{at}stjude.org

Published ahead of print on 8 June 2007.

L.P. and Y.-J.L. contributed equally to the research.

Present address: Division of Infectious Diseases, Children's Hospital, Boston, MA 02115.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 

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Journal of Bacteriology, August 2007, p. 5816-5824, Vol. 189, No. 16
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