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Originally published In Press as doi:10.1074/jbc.M703536200 on May 28, 2007

J. Biol. Chem., Vol. 282, Issue 30, 21738-21745, July 27, 2007
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Identification of a Soluble Diacylglycerol Kinase Required for Lipoteichoic Acid Production in Bacillus subtilis*

Agoston Jerga{ddagger}, Ying-Jie Lu{ddagger}1, Gustavo E. Schujman§2, Diego de Mendoza§23, and Charles O. Rock{ddagger}4

From the {ddagger}Department of Infectious Diseases, St. Jude Children's Research Hospital, Memphis, Tennessee 38105-2794 and §Instituto de Biología Molecular y Celular de Rosario and Departamento de Microbiología, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Rosario, Argentina

Received for publication, April 27, 2007 , and in revised form, May 24, 2007.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Diacylglycerol kinases (DagKs) are key enzymes in lipid metabolism that function to reintroduce diacylglycerol formed from the hydrolysis of phospholipids into the biosynthetic pathway. Bacillus subtilis is a prototypical Gram-positive bacterium with a lipoteichoic acid structure containing repeating units of sn-glycerol-1-P groups derived from phosphatidylglycerol head groups. The B. subtilis homolog of the prokaryotic DagK gene family (dgkA; Pfam01219) was not a DagK but rather was an undecaprenol kinase. The three members of the soluble DagK protein family (Pfam00781) in B. subtilis were tested by complementation of an E. coli dgkA mutant, and only the essential yerQ gene possessed DagK activity. This gene was dubbed dgkB, and the soluble protein product was purified, and its DagK activity was verified in vitro. Conditional inactivation of dgkB led to the accumulation of diacylglycerol and the cessation of lipoteichoic acid formation in B. subtilis. This study identifies a soluble protein encoded by the dgkB (yerQ) gene as an essential kinase in the diacylglycerol cycle that drives lipoteichoic acid production.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Diacylglycerol kinases (DagKs)5 are key enzymes in phospholipid metabolism that function to reintroduce DAG formed from the breakdown of phospholipids into the biosynthetic pathway. In Escherichia coli, PtdGro is degraded to DAG by the transfer of the sn-glycerol-1-P head group to membrane-derived oligosaccharides (1, 2). These periplasmic oligosaccharides function in osmotic homeostasis (3), and PtdGro is used as a substrate in their biosynthesis in the periplasmic space. The DAG formed is converted to PtdOH for the resynthesis of PtdGro by DgkA (4). DgkA is an inner membrane lipid kinase that exists as a trimer with each monomer containing three membrane-spanning domains (5, 6). The physiological substrate for DgkA is DAG, but the enzyme also less efficiently phosphorylates related lipids, like ceramide, that are not found in the bacterium (7, 8). The dgkA mutants are viable under growth conditions that do not osmotically stress the bacteria and where membrane-derived oligosaccharide production is minimal (9, 10). However, dgkA is an essential gene in cells growing in an osmotically challenging environment, and lethality is induced in dgkA mutants by including arbutin in the medium, which acts as an artificial sugar acceptor of glycerol-P groups from PtdGro (11, 12). The inactivation of DgkA interrupts the diacylglycerol cycle, leading to the accumulation of neutral lipid, primarily DAG (13, 14). DgkA is the founding member of a large family of proteins that constitute the widely distributed prokaryotic type of DagK (Pfam01219). This family of integral membrane proteins contrasts with the mammalian DagK superfamily consisting of soluble lipid kinases that transiently associate with the membrane and function in regulating intracellular phospholipid signaling (15). The catalytic cores of these proteins define a distinct family of soluble lipid kinases (Pfam00781) that includes some bacterial proteins of unknown function.

It has been recognized for some time that the sn-glycerol-1-P polymer that adorn most Gram-positive LTAs arise from PtdGro (Fig. 1). LTAs are polydisperse macroamphiphiles composed of poly(sn-glycerol-1-P) attached to a glycolipid anchor and contribute to the continuum of anionic charge that forms a protective layer surrounding the bacterium (for a review, see Ref. 16). The length of the polyglycerol-P chain varies from 14 to 33 repeating units, and in the prototypical Gram-positive bacterium Bacillus subtilis, the repeating units are attached to diglucosyldiacylglycerol. The gene product that catalyzes the polymerization step is unknown. Metabolic labeling experiments identify PtdGro as the source of the glycerol-1-P groups (17–20), which means that the biosynthesis of a single LTA molecule requires the utilization of an average of 25 PtdGro molecules. Accordingly, PtdGro turnover is rapid in these bacteria (17, 19), and the large amount of DAG formed requires a DagK for its efficient reintroduction into the phospholipid biosynthetic pathway (Fig. 1). Because B. subtilis has a homolog of the E. coli dgkA gene, it has been assumed that the product of this gene is the DagK that carries out this function; however, dgkA is not an essential gene in B. subtilis (21). In light of the high demand for PtdGro in LTA biosynthesis, the finding that dgkA was dispensable was somewhat surprising and stimulated our investigation of phospholipid turnover in B. subtilis. This work shows that the B. subtilis dgkA gene does not encode a DagK but rather is an undecaprenol kinase (UdpK). The authentic DagK is identified as the product of the essential yerQ (dgkB) gene that encodes a soluble DagK belonging to the eukaryotic DagK protein superfamily (Pfam00781). DgkB is directly tied to the recycling of DAG in vivo (Fig. 1) based on the analysis of lipid metabolism and LTA formation in a conditional dgkB (yerQ) knock-out strain.


Figure 1
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  		FIGURE 1.
Proposed role of DagK in lipoteichoic acid biosynthesis. LTA is formed by the sequential addition of glycerol-1-P groups transferred from PtdGro to the LTA glycolipid anchor (diglucosyldiacylglycerol; Glc2DAG) by an enzyme(s) that has not been identified (?). This process for DAG, which is converted to PtdOH by the soluble DAG kinase DgkB (YerQ), is the subject of this paper. PtdGro is formed by the sequential action of CDP-DAG synthase (Cds), PtdGroP synthase (PgsA), and PtdGroP phosphatase (PgpP). The average number of glycerol-1-P residues on LTA is ~25; thus, 25 mol of PtdGro are consumed to produce 1 mol of LTA.

 

   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials—Sources of supplies were as follows: phospholipids, 1,2-dioleoyl-sn-glycerol, 1,2-dioctanoyl-sn-glycerol, N-acetoyl-D-erythro-sphingosine, N-palmitoyl-D-erythro-sphingosine, D-erythro-sphingosine, ceramide (brain), and ceramide (egg) (Avanti%20Polar%20Lipids">Avanti Polar Lipids Inc.); l-3-phosphatidylinositol (porcine liver) (Doosan Serdary Research Laboratories); undecaprenol, 1,2-[14C]dipalmitoylphosphatidic acid, and [{gamma}-32P]ATP (American Radiolabeled Chemicals Inc.); [32P]orthophosphate (Amersham Biosciences); 1-oleoyl-rac-glycerol (Sigma); anti-His antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA); QuikChange site-directed mutagenesis kit (Stratagene); and restriction enzymes (Promega). LB medium consisted of 10 g of tryptone, 5 g of yeast extract, and 10 g of NaCl per liter. All other chemicals were of reagent grade or better.

Construction of the Conditional Knock-out yerQ Mutant—The integrative plasmid pMUTIN4 (22) containing the IPTG-inducible Pspac promoter was used for conditional expression of yerQ in B. subtilis. Plasmid pGES434 (Fig. 2) was constructed using a 393-bp DNA fragment, generated by PCR using primers YerQMutUp (5'-CATAATTAAAGCTTGTTAGTGTAAAAATGGATC; HindIII site underlined) and YerQMutLow (5'-ATGACAGGATCCGCCGCTTTTAAAATATC; BamHI site underlined), carrying the ribosome binding site and a 5' portion of yerQ. The amplified product was digested with HindIII and BamHI and cloned into pMUTIN4 previously digested with the same restriction enzymes. Strain GS435 was generated by integration of the circular form of plasmid pGES434 into the B. subtilis chromosome by a single crossover event (Fig. 2). This approach resulted in conditional inactivation of the target gene whose expression was controlled by the Pspac promoter via IPTG supplementation of the medium (Fig. 2).


Figure 2
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  		FIGURE 2.
Construction and structure of the B. subtilis conditional yerQ (dgkB) knock-out strain. A, the essential yerQ gene was placed under control of the Pspac promoter as outlined under "Experimental Procedures," and the structure of the chromosomal DNA in strain GS435 is diagramed. B, B. subtilis strain GS435 dgkB conditional knock-out was grown in liquid medium supplemented with (•) or without ({circ}) IPTG, resulting in either a normal growth pattern (•) or growth arrest ({circ}) when the cellular level of DkgB became limiting. The arrow indicates the point in cell growth where the metabolic labeling experiments were performed (see Fig. 5). The growth characteristics of strain GS435 were very reproducible. The error bars were smaller than the symbols on the graph.

 
Construction of Expression Plasmids—The following strains were used to obtain genomic DNA: E. coli, DH1; B. subtilis, P31K6; Staphylococcus aureus, RN4220; Streptococcus pneumoniae, T4. The E. coli and B. subtilis dgkA genes were amplified by PCR using forward primers that contained coding sequence for six histidines at the NH2 terminus and an NcoI site at the initiating codon and the reverse primers with a BamHI site downstream of the stop codon. The S. aureus SAR1989 and SAR780 genes were amplified by PCR using primer pairs containing an XhoI site upstream of the initiating codon and a BamHI site after the stop codon to create an amino-terminal His-tagged protein. Primers for the amplification of B. subtilis yerQ, bmrU, ytlR, and SP1045 included an NcoI site at the initiating codon and a coding sequence for six histidines at the carboxyl terminal codon followed by a BamHI site downstream of the engineered stop codon. The inserts were then transferred to either pET15b (Novagen) or pPJ131, a plasmid that was derived from pBluescript II KS+ by deleting its XhoI site, followed by inserting the XbaI-BamHI fragment of pET15b into the multiple cloning site. In plasmids pAJ001–pAJ006 and pAJ011, the G nucleotide directly downstream of the ATG (part of the NcoI site) was mutated using QuikChange (Stratagene) to a nucleotide that corresponded to the gene sequence. The sequences were verified by the Hartwell Center for Bioinformatics and Biotechnology at St. Jude Children's Research Hospital. Plasmid pAJ018 was constructed using PCR to amplify the nucleotide sequence in pAJ001 including the ribosomal binding site and tagged E. coli dgkA. The forward primer contained a BamHI site, and the reverse primer contained an XbaI site downstream of the stop codon, and the product was ligated into the B. subtilis expression vector pHCMC02 (23).

Complementation Experiments—Electrocompetent cells of strains FB21625 or GS435 were transformed with the indicated plasmids, and the transformants were recovered on LB plates supplemented with 100 µg/ml carbenicillin or LB plates supplemented with 100 µg/ml carbenicillin, 0.5 µg/ml erythromycin, 12.5 µg/ml lincomycin, and 250 µM IPTG. Individual clones were isolated and streaked on LB plates either with or without 90 mM arbutin to test for complementation of the E. coli dgkA mutant phenotype (11). Alternately, the cells were streaked on LB plates supplemented with 100 µg/ml carbenicillin, 0.5 µg/ml erythromycin, and 12.5 µg/ml lincomycin either in the presence or absence of IPTG to test for complementation of the B. subtilis dgkB growth phenotype.

DagK Assays—The DagK assay was modeled after the assay developed by Walsh and Bell (24). Assays contained 50 mM MOPS, pH 7.0, 0.5 mM dithiothreitol, 10 mM MgCl2, 1 mM EGTA, 150 mM LiCl, 5 mM [{gamma}-32P]ATP (specific activity = 0.02 Ci/mmol), and 4 mol % (1 mM) in the micelle phase of 50 mM octyl-beta-D-glucopyranoside (critical micellar concentration = 25 mM) in a final volume of 100 µl. The reactions were initiated by the addition of protein and incubated at 25 °C for 30 min. Reactions were quenched by the addition of 700 µl of chloroform/methanol/HCl (1:2:0.03, v/v/v), and the lipids were extracted after adding 350 µl of water and 100 µl of chloroform (100 µl). Both long- and short-chain PtdOH were efficiently extracted by this method. Radioactivity in the organic layer was quantified by a liquid scintillation counter, and the lipids were separated by thin layer chromatography on Silica Gel H layers developed with chloroform, methanol, water, ammonium hydroxide, 250 mM EDTA (45:35:8.4:1.5:0.16, v/v/v/v/v).

Purification of B. subtilis DgkB—The E. coli Rosetta strain (Novagen) harboring the His-tagged B. subtilis DgkB expression plasmid pAJ011 was grown in LB medium, supplemented with 100 µg/ml carbenicillin and 30 µg/ml chloramphenicol, at 37 °C with rotary shaking until the A600 reached 0.8. Then 400 µM IPTG was added to induce expression of B. subtilis DgkB, and rotary shaking of the culture was continued at 25 °C for 16 h. Cells were harvested by centrifugation (6000 x g for 15 min), resuspended in 20 mM Tris, pH 7.9, 500 mM NaCl, 1 mM beta-mercaptoethanol, 10 mM imidazole, 10% (v/v) glycerol, and protease inhibitor mixture (Roche Applied Science). Bacterial lysis was achieved by a lysozyme (1 mg/ml) digest for 10 min at 4 °C followed by one freeze-thaw cycle of cells in the presence of 0.1% (w/v) Triton X-100. The viscosity of the cell-free extract was reduced by the addition of 0.2 mg of DNase along with 2 mM CaCl2 and 2 mM MgCl2. After the insoluble debris was removed by centrifugation (20,000 x g for 40 min), the extract was loaded onto an Ni2+-NTA affinity column (Qiagen). The resin was washed with 10 column volumes of 20 mM Tris, pH 7.9, 500 mM NaCl, 1 mM beta-mercaptoethanol, 10 mM imidazole, and 10% (v/v) glycerol, followed by 10 column volumes of the same buffer containing 50 mM imidazole. B. subtilis DgkB was eluted from the column in the same buffer containing 500 mM imidazole. The protein was concentrated to 3.0 mg/ml using a centrifugal filter device (Amicon) and dialyzed against 20 mM Tris, pH 7.9, 100 mM NaCl, 1 mM EDTA, and 1 mM beta-mercaptoethanol. Protein purity was assessed by SDS gel electrophoresis. Aliquots (150 µl) were flash-frozen in liquid nitrogen and stored at –80 °C. Protein was measured by the Bradford method (25).

Preparation of Extracts Containing DgkA—Strain FB21625 (dgkA::Tn5) harboring plasmids pAJ001 (E. coli DgkA), pAJ002 (B. subtilis DgkA), or pPJ131 (control) were grown overnight in 5 ml of LB medium supplemented with 100 µl of carbenicillin. The cells were harvested and resuspended in 0.5 ml of 20 mM Tris, pH 7.9, 500 mM NaCl, 1 mM beta-mercaptoethanol, 5 mM imidazole, 10% (v/v) glycerol, and protease inhibitor mixture (Roche Applied Science). Cells were lysed with lysozyme (1 mg/ml for 10 min at 4 °C), followed by freeze-thawing the samples in the presence of 0.1% (w/v) Triton X-100. Insoluble debris was removed by centrifugation at 5000 x g for 20 min.

Radiolabeling of Lipids and LTA—Strain GS435 was grown in LB medium (5 ml), supplemented with 0.5 µg/ml erythromycin and 12.5 µg/ml lincomycin and 250 µM IPTG, at 37 °C with rotary shaking. At A600 = 0.4, two aliquots of 80 µl of culture were filtered on a 0.45-µm membrane filter (Millipore), and after two rinses with 3 ml of medium, the filter disks were transferred to 5 ml of LB medium supplemented with the above antibiotics either with or without IPTG. Cell growth was monitored, and at the time indicated in the figures, cells were labeled either with 0.5 mCi of [1-14C]acetate (55 mCi/mmol) for 10 min or 2 mCi of [{gamma}-32P]orthophosphate (6000 Ci/mmol) for 30 min. Cells were filtered and resuspended in nonradioactive medium, followed by removal of 0.5-ml aliquots periodically. Cells were harvested by centrifugation, the lipids were extracted, and the total radioactivity incorporated was determined by liquid scintillation counting. Diacylglycerol and polar lipids were separated on Silica Gel G layers with hexane/ether/acetic acid (80: 20:1), and the radioactive content of the bands was determined by scraping and scintillation counting. Orthophosphate-labeled phospholipids were separated on Silica Gel H layer using a mixture of chloroform, methanol, water, ammonium hydroxide, 250 mM EDTA (45:35:8.4:1.5:0.16, v/v/v/v/v), and activities in each phospholipid fraction were quantified using the Typhoon 9600 PhosphorImager. All results were normalized to 0.5-ml samples of radiolabeled culture. LTA was extracted from 5 ml of culture after labeling cells for 30 min with [32P]orthophosphate and purified on octyl-Sepharose column (Vc = 1 ml) using an isopropyl alcohol gradient between 5 and 80% (v/v) and collecting 3-ml fractions (26).


Figure 3
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  		FIGURE 3.
DgkA and DgkB Sequence alignments. A, alignment of four representative Gram-negative and Gram-positive members of the prokaryotic diacylglycerol kinase (DgkA) superfamily, Pfam01219. Residues that are common to three of four Gram-negative DgkA sequences are highlighted in both groups of proteins, and the position of the aligned sequences in the structure of E. coli DgkA are shown diagrammatically above the alignments. The E{Phi}LNSAIEAVVD sequence defines the proteins as members of Pfam01219. B, alignment of six prokaryotic members of the soluble DagK superfamily, Pfam00781. The proteins are separated into two groups, with three examples of DgkBs (two are experimentally known to have DagK activity) and three other family members that are experimentally known not to have DagK activity. The top alignment shows the similarities between these proteins that group them in Pfam00781 (GGDGT{Phi}NEVVXG) and probably defines the ATP binding site. The lower group of sequences shows the alignment of a conserved sequence in the DgkBs (KGXE{Phi}LPYD) that is not found in the prokaryotic family members that lack DagK activity. {Phi}, a variable hydrophobic amino acid.

 

   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Identification of the B. subtilis DagK by Genetic Complementation—The biochemical pathway for LTA formation requires a DagK to salvage the released DAG (Fig. 1), and this activity was most likely encoded by the B. subtilis dgkA gene. B. subtilis DgkA is clearly related to E. coli DgkA, which is a bona fide DagK. B. subtilis DgkA has three predicted transmembrane helices and regions corresponding to the two signature sequence motifs shared by members of the Gram-negative group of DgkAs and the Pfam01219 protein family (Fig. 3). These motifs (VE{Phi}LNSAIEXVVDR and HXLSXXAKDMGSAA) are located in the second cytoplasmic loop and extend into the third transmembrane domain (Fig. 3A). Therefore, we tested the activity of B. subtilis DgkA using a genetic complementation strategy based on the sensitivity of E. coli strain FB21625 (dgkA::Tn5) to arbutin. Although E. coli DgkA complemented the arbutin-sensitive growth phenotype of strain FB21625, expression of B. subtilis DgkA did not (Table 1). The DagK constructs were His-tagged, and protein expression was verified by Western blotting using anti-His antibody (not shown). These data indicated that B. subtilis DgkA was not a DagK.


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  		TABLE 1
Complementation of E. coli dgkA and B. subtilis dgkB knockouts

 
A bioinformatic analysis of the B. subtilis genome revealed three genes (yerQ, bmrU, and ytlR) that belonged to the soluble DagK protein family, Pfam00781 (Fig. 3B). Although the overall sequence identity among the family members is low, the group is defined by the presence of the common GGDGT{Phi}NEVVXG motif that may represent a shared ATP binding sequence. Therefore, we tested each of these genes in the complementation assay and found that only yerQ complemented the arbutin-sensitive growth phenotype of strain FB21625 (Table 1). These data indicated that yerQ encoded a DagK; therefore, we have renamed the gene dgkB to indicate this fact. We also tested a few other Gram-positive members of Pfam00781. Of these, SAR1989 of S. aureus complemented the dgkA mutant, indicating that it was also a dgkB gene. However, the other Pfam00781 member in S. aureus (SAR0780), the two other members in B. subtilis (BmrU and YtlR), and the only member of this family in S. pneumoniae (SPR1045) did not complement the dgkA mutant phenotype (Table 1). All of these proteins were expressed in strain FB21625, based on a Western blot with anti-His antibody. The His-tagged BmrU, YtlR, SP1045, and SAR0780 proteins were purified through the affinity chromatography step. Each was recovered as a soluble protein, and none of these purified proteins exhibited any DagK activity in vitro (not shown). These genetic and biochemical studies lead us to conclude that of none of these proteins function as DagKs.

The dgkB gene was flagged as an essential gene based on a systematic genome-wide inactivation of B. subtilis genes (21). Therefore, we constructed strain GS435 that expressed dgkB under the control of an IPTG-regulated promoter (Fig. 2A). Strain GS435 was able to grow in the presence of IPTG but did not form colonies on plates that lacked the inducer, confirming the essential nature of the dgkB gene (Table 1). Unlike temperature-sensitive mutants, the removal of inducer in liquid cultures 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. 2B. There were three phases to cell growth in the absence of inducer. First, a log phase that was nearly identical to the IPTG-supplemented culture representing the period of time when DgkB 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 rate continually slower than that of wild type. This phase was when the cellular content of DgkB was becoming limiting for growth. Third, there is a final plateau phase, where the cells ceased proliferation. E. coli DgkA complemented the IPTG-dependent growth phenotype of strain GS435 (Table 1). These data confirmed a requirement for DagK activity for the viability of B. subtilis and indicated that the subcellular localization (integral membrane versus soluble) was not critical to the function of the enzyme in vivo.

Biochemical Activities of B. subtilis DgkA and B. subtilis DgkB—The biochemical activity of B. subtilis DgKB was evaluated by expressing the His-tagged protein in E. coli and purifying the protein by affinity and gel filtration chromatography as described under "Experimental Procedures." These methods yielded a pure protein as judged by SDS gel electrophoresis with an apparent molecular mass that reflected the 34-kDa mass calculated from the predicted amino acid sequence (Fig. 4A). B. subtilis DgkB exhibited robust DagK activity in a mixed micelle assay and was more active toward long-chain DAG compared with short-chain DAG (Fig. 4B). We confirmed that our assay extraction conditions efficiently removed the short-chain PtdOH from the reaction for accurate quantitation. The substrate specificity of B. subtilis DgkB was assessed with a panel of phosphoacceptors, and the enzyme only phosphorylated DAG (Fig. 4C). Phosphorylation of monoacylglycerol, ceramide, or undecaprenol was not detected in these experiments (<0.01%). These data establish B. subtilis DgkB as a DAG-specific kinase with selectivity for long-chain DAG that would arise from membrane phospholipid hydrolysis.

The biochemical activities of the B. subtilis DgkA were compared with E. coli DgkA by the analysis of the kinase substrate specificity in extracts from strain FB21625 (dgkA::Tn5) harboring either plasmid pAJ001 expressing E. coli DgkA or plasmid pAJ002 expressing B. subtilis DgkA (Fig. 4). As expected, E. coli DgkA readily phosphorylated DAG, and to a lesser extent, monoacylglycerol (5%), phosphatidylinositol (0.7%), and ceramide (0.4%) were also substrates (Fig. 4D). In contrast, B. subtilis DgkA exhibited no detectable activity against DAG, monoacylglycerol, or ceramide (<0.01%) but readily phosphorylated undecaprenol (Fig. 4E). This experiment established B. subtilis DgkA as a UdpK, and not a DagK, and clearly distinguished the Gram-positive and Gram-negative DgkAs as separate enzymes.


Figure 4
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  		FIGURE 4.
Substrate specificities of B. subtilis DagKA and B. subtilis DagKB. A, SDS gel electrophoresis and silver staining illustrates the purity of B. subtilis DgkB. B. subtilis DgkB was expressed as a His-tagged protein and purified by affinity and gel filtration chromatography as described under "Experimental Procedures." B, B. subtilis DgKB kinase activity as a function of protein concentration using either 1,2-dioleoyl-sn-glycerol (diC18:1), or 1,2-dioctanoyl-sn-glycerol (diC8) as substrate. The specific activity of DgkB with diC18:1 as substrate was 89 ± 2.4 nmol/min/mg compared with 4.8 ± 0.7 nmol/min/mg using diC8 as substrate (S.E.). C, substrate specificity of B. subtilis DgkB. Strain FB21625 (dgkA::Tn5) was transformed with vectors expressing either E. coli or B. subtilis dgkA genes, the membrane fractions were isolated, and each of the lipid kinases were assayed using a panel of substrates. D, E. coli DgKA; E, B. subtilis DgKA; F, empty vector control. Reactions contained [{gamma}-32P]ATP and the indicated phosphoacceptor present as 4 mol % (1 mM) in the micellar phase of 50 mM octyl glucoside. Assays were performed, and products were detected following extraction and thin layer chromatography conditions described under "Experimental Procedures." The Rf of PtdOH acid was 0.60; the Rf of undecaprenol-P was 0.69; and the Rf of lyso-PtdOH was 0.44 on Silica Gel H layers developed with chloroform, methanol, water, ammonium hydroxide, 250 mM EDTA (45:35:8.4:1.5:0.16, v/v/v/v/v).

 
Disruption of Lipid Metabolism and LTA Synthesis in the Absence of DgkB—The effect of eliminating DgkB activity on B. subtilis lipid metabolism was assessed in metabolic labeling experiments (Fig. 5). Strain GS435 was grown in the presence of IPTG, labeled with [32P]orthophosphate for 30 min, and chased with unlabeled medium (Fig. 5A). PtdGro was the most rapidly labeled phospholipid during the pulse, and the radioactivity rapidly exited the PtdGro pool during the chase. The apparent half-life for PtdGro calculated from these data was 15 min. Other membrane phospholipids exhibited insignificant turnover in this experiment. These experiments confirmed the earlier reports that PtdGro undergoes very rapid head group turnover in B. subtilis, as was reported in other Gram-positive bacteria (17–19).

The next experiment was to determine how the absence of the B. subtilis DgkB activity impacted phospholipid metabolism by pulse-labeling strain GS435 with [14C]acetate for 10 min 1 h after the growth fork either in the presence or absence of IPTG (see Fig. 2B) and then to follow the change in the distribution of the label between DAG and phospholipid as a function of time (Fig. 5B). In the presence of inducer, the DAG/phospholipid ratio remained constant during the chase, indicating that the DAG formed from PtdGro turnover was returned to the phospholipid pool. However, in the absence of inducer, this ratio steadily increased, reflecting both a decrease in labeled phospholipid and an increase in labeled DAG. Unlike in E. coli, DAG is an easily measurable component of wild-type B. subtilis that accounts for between 10 and 15% of the total lipid (27). Strain GS435 contained 13.1% DAG based on the analysis of cells grown in medium containing [14C]acetate plus IPTG and harvested in midlog phase of growth. The DAG content rose to 36.3% of the total labeled lipid in the same steady-state labeling experiment of strain GS435 cells that were grown in the absence of IPTG and harvested 1 h after the growth fork (see Fig. 2B). We also measured DgkB-dependent LTA production beginning at the same time as the acetate labeling experiment. Strain GS435 was labeled with [32P]orthophosphate for 30 min at 1 h after the growth fork (see Fig. 2B) either in the presence or absence of IPTG inducer. In the presence of inducer, LTA production was easily detected, whereas in the absence of inducer, LTA formation was not detected (Fig. 5C). These data show that PtdGro turnover was rapid in B. subtilis, and the inactivation of B. subtilis DgkB resulted in the accumulation of DAG and the cessation of LTA formation.


Figure 5
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  		FIGURE 5.
Analysis of pulse-chase labeling of phospholipids and LTA in B. subtilis strain GS435. A, strain GS435 was cultured in the presence of IPTG and labeled with [32P]orthophosphate (4 mCi) for 30 min when the cells reached an A600 of 0.3. The label was removed, and the cells were cultured in unlabeled medium for the times indicated on the x axis. Aliquots of the cultures were collected, lipids were extracted, and the distribution of label among the membrane phospholipid classes was quantified by thin layer chromatography as described under "Experimental Procedures." Phospholipid classes were PtdGro (•), phosphatidylethanolamine ({blacksquare}), lysylphosphatidylglycerol ({circ}), and cardiolipin ({square}). B, pulse-chase labeling of diacylglycerol and phospholipid labeling with [1-14C]acetate. Strain GS435 grown in either the presence (•) or absence ({circ}) of IPTG inducer was pulse-labeled for 10 min at the time point indicated in Fig. 2B, aliquots of the cells were collected at 10-min intervals, and the lipids were extracted. DAG was separated from phospholipid fractions by thin layer chromatography on Silica Gel G layers developed with hexane/ether/acetic acid (80:20:1, v/v/v). Bands corresponding to the phospholipid and DAG fractions were scraped from the plate and quantitated by liquid scintillation counting. C, strain GS435 was grown in the presence (•) or absence ({circ}) of IPTG and labeled for 30 min with [32P]orthophosphate at the time the growth rates of the cultures separated, as indicated in Fig. 2B. LTA was separated from other soluble labeled metabolites (primarily nucleotides) by hydrophobic interaction chromatography on an octyl-Sepharose column as described under "Experimental Procedures." The LTA fraction eluted in the 3-ml fraction between 18 and 22% isopropyl alcohol (2-PrOH), and radioactivity was quantified by liquid scintillation counting. Data were derived from triplicate determinations, and the error bars show S.E.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
This study identifies DgkB (YerQ) as a soluble DagK that is involved in the production of LTA as outlined in Fig. 1. The disappearance of PtdGro, the accumulation of DAG, and the cessation of LTA production following the inactivation of dgkB (yerQ) ties the kinase activity of DgkB to the turnover of PtdGro linked to LTA formation. B. subtilis DgkB is essential for growth, but it is not clear whether this is due to the accumulation of DAG in the bacterial membranes or to the absence of LTA. B. subtilis mutants that are unable to synthesize the glycolipid anchor have morphological and growth phenotypes under defined culture conditions but are viable and do not have a growth phenotype in rich medium (28). They do produce an LTA-like molecule by polymerizing glycerol-1-P onto DAG rather than the normal glycolipid anchor. Thus, it is unknown whether the phenotypes are due to the absence of glycolipid or a bona fide LTA molecule. The experience with the E. coli model does suggests that DAG accumulation is deleterious to bacterial growth. The addition of arbutin to dgkA mutants links DAG accumulation to growth arrest (11). The growth phenotype is not due to the absence of membrane-derived oligosaccharides, because the arbutin-resistant dgkA bypass mutants lack the mdoB activity that produces glycerol-P-containing oligosaccharides (11, 12). The gene(s) that polymerize the glycerol-1-P chain on the Gram-positive glycolipid anchor is not known; therefore, we do not know if the absence of LTA is essential for cell viability under laboratory conditions.

Our bioinformatic analysis suggests a tool that can be used to distinguish DagKs from other prokaryotic members of this family with other activities. B. subtilis possesses two other genes that are members of the soluble DagK protein family (bmrU and ytlR), but neither of these genes is essential, they do not encode a DagK, and their biochemical functions are unknown. E. coli and other Gram-negatives also contain members of the Pfam00781 superfamily that are not DagKs. For example, E. coli YegS, whose x-ray structure is known (29), phosphorylates the headgroup of phosphatidylglycerol, but it is not known whether this is its actual physiological function. It remains to be determined if these proteins are actually lipid kinases or phosphorylate a nonlipid metabolic intermediate or protein. Both groups contain Motif-1, which places them in Pfam00781 and probably corresponds to the common ATP-binding region of the proteins (Fig. 3B). The prokaryotic clade of enzymes that contain B. subtilis DgkB and S. aureus DgkB (SAR1989) also possess a highly conserved region downstream of the ATP binding site that we designate Motif-2 (Fig. 3). Motif-2, which includes the highly conserved KGXE{Phi}LPYD region, can be used to separate DgkBs from other prokaryotic members of Pfam00781 (Fig. 3B). Mycobacterium tuberculosis Rv2253 encodes a member of Pfam00781 and most efficiently phosphorylates ceramide of the substrates tested. DAG is a poor substrate for this family member (30).

The identification of B. subtilis DgkA as a UdpK identifies the gene responsible for an activity that has been known for some time to exist in Gram-positive bacteria (31). B. subtilis DgkA did not phosphorylate DAG but readily phosphorylated undecaprenol, leading us to conclude that this is the physiological substrate. Pfam02673 defines a group of hydrophobic membrane proteins that is incorrectly annotated as the family of Gram-positive UdpKs based on the hypothesis that the bacitracin resistance gene of E. coli (bacA) phosphorylated undecaprenol (32). However, BacA is clearly an undecaprenolpyrophosphate phosphatase (uppP) (33); therefore, Pfam02673 is actually a family of integral membrane lipid phosphatases. The Gram-positive members of the prokaryotic DagK superfamily (Pfam01219) form a distinct evolutionary subset of DgkA proteins that probably represent UdpK enzymes rather than DagKs (Fig. 3A). The conserved sequence motifs that define Pfam01219 are primarily the residues that specify ATP binding (34), and little is known about the key residues that are important for substrate specificity. We do note that Arg81 is found in all Gram-negative DagKs we examined, whereas this residue is replaced by a leucine in the Gram-positive UdpKs. Extensive mutagenesis of E. coli DgkA (35) shows that Arg81 could only be replaced by Gly, Ser, or His without the complete loss of activity, indicating that R81L substitution is not compatible with DagK function. Because the substrates (DAG or undecaprenol) are membrane-bound, the major determinants of substrate specificity may reside in the residues that comprise the transmembrane helices. The similarities in transmembrane helix-2 of the Gram-negative DgkAs are not reflected in the same helical segment in the Gram-positive DgkAs, and vice versa (Fig. 3A). However, the sequence diversity within this large protein family makes signaling a few diagnostic residues somewhat problematic.

Previous work with B. subtilis dgkA mutants and their effect of sporulation were interpreted assuming that the Bacillus dgkA encodes a functional DagK (36). Our work shows that this is not correct and that the phenotypes associated with the B. subtilis dgkA mutants most likely arise from a deficiency in UdpK activity. The dgkA homolog in Streptococcus mutans plays a role in acid stress response and in expression of the lantibiotic mutacin II (38), but it also appears to encode a UdpK and cannot phosphorylate DAG (39, 40). The role of UdpK in cell wall metabolism Gram-positive organisms is not clear. However, analytical work establishes that S. aureus contains quantities of undecaprenol (41) and has a membrane-bound UdpK activity (31, 37) that would function to produce undecaprenolphosphate to support peptidoglycan production. There are no udpK-like genes in E. coli, suggesting that Gram-negative bacteria do not form undecaprenol as an intermediate in peptidoglycan biosynthesis.


   FOOTNOTES
 
Note Added in Proof—After submission of our paper, we became aware of a much more relevant and recent study (29).

* This work was supported by National Institutes of Health Grant GM34496 (to C. O. R.), Cancer Center Support Grant CA 21765, Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET, Argentina), Agencia de Promoción Científica y Tecnológica (FONCYT, Argentina), and the American Lebanese Syrian Associated Charities. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

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

2 Career Investigator at CONICET. Back

3 An International Research Scholar of the Howard Hughes Medical Institute. Back

4 To whom correspondence should be addressed: Dept. of Infectious Diseases, St. Jude Children's Research Hospital, 332 N. Lauderdale, Memphis, TN 38105-2794. Tel.: 901-495-3491; Fax: 901-495-0399; E-mail: charles.rock{at}stjude.org.

5 The abbreviations used are: DagK, diacylglycerol kinase; DAG, diaclyglycerol; PtdGro, phosphatidylglycerol; UdpK, undecaprenol kinase; LTA, lipoteichoic acid; LB, Luria broth; IPTG, isopropyl beta-D-1-thiogalactopyranoside; MOPS, 4-morpholinepropanesulfonic acid. Back



   REFERENCES
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 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

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