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• W2016872786 abstract "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. 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. Diacylglycerol kinases (DagKs) 5The abbreviations used are: DagK, diacylglycerol kinase; DAG, diaclyglycerol; PtdGro, phosphatidylglycerol; UdpK, undecaprenol kinase; LTA, lipoteichoic acid; LB, Luria broth; IPTG, isopropyl β-d-1-thiogalactopyranoside; MOPS, 4-morpholinepropanesulfonic acid.5The abbreviations used are: DagK, diacylglycerol kinase; DAG, diaclyglycerol; PtdGro, phosphatidylglycerol; UdpK, undecaprenol kinase; LTA, lipoteichoic acid; LB, Luria broth; IPTG, isopropyl β-d-1-thiogalactopyranoside; MOPS, 4-morpholinepropanesulfonic acid. 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 (1Schulman H. Kennedy E.P. J. Biol. Chem. 1977; 252: 4250-4255Abstract Full Text PDF PubMed Google Scholar, 2Goldberg D.E. Rumley M.K. Kennedy E.P. Proc. Natl. Acad. Sci. U. S. A. 1981; 78: 5513-5517Crossref PubMed Scopus (23) Google Scholar). These periplasmic oligosaccharides function in osmotic homeostasis (3Kennedy E.P. Proc. Natl. Acad. Sci. U. S. A. 1982; 79: 1092-1095Crossref PubMed Scopus (123) Google Scholar), 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 (4Raetz C.R.H. Newman K.F. J. Bacteriol. 1979; 137: 860-868Crossref PubMed Google Scholar). DgkA is an inner membrane lipid kinase that exists as a trimer with each monomer containing three membrane-spanning domains (5Smith R.L. O'Toole J.F. Maguire M.E. Sanders II, C.R. J. Bacteriol. 1994; 176: 5459-5465Crossref PubMed Google Scholar, 6Oxenoid K. Sonnichsen F.D. Sanders C.R. Biochemistry. 2002; 41: 12876-12882Crossref PubMed Scopus (44) Google Scholar). 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 (7Schneider E.G. Kennedy E.P. J. Biol. Chem. 1973; 248: 3739-3741Abstract Full Text PDF PubMed Google Scholar, 8Schneider E.G. Kennedy E.P. Biochim. Biophys. Acta. 1976; 441: 201-212Crossref PubMed Scopus (38) Google Scholar). The dgkA mutants are viable under growth conditions that do not osmotically stress the bacteria and where membrane-derived oligosaccharide production is minimal (9Rumley M.K. Therisod H. Weissborn A.C. Kennedy E.P. J. Biol. Chem. 1992; 267: 11806-11810Abstract Full Text PDF PubMed Google Scholar, 10Lacroix J.M. Loubens I. Tempete M. Menichi B. Bohin J.P. Mol. Microbiol. 1991; 5: 1745-1753Crossref PubMed Scopus (34) Google Scholar). 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 (11Jackson B.J. Bohin J.P. Kennedy E.P. J. Bacteriol. 1984; 160: 976-981Crossref PubMed Google Scholar, 12Fiedler W. Rotering H. J. Biol. Chem. 1985; 260: 4799-4806Abstract Full Text PDF PubMed Google Scholar). The inactivation of DgkA interrupts the diacylglycerol cycle, leading to the accumulation of neutral lipid, primarily DAG (13Raetz C.R.H. Newman K.F. J. Biol. Chem. 1978; 253: 3882-3887Abstract Full Text PDF PubMed Google Scholar, 14Bielawska A. Perry D.K. Hannun Y.A. Anal. Biochem. 2001; 298: 141-150Crossref PubMed Scopus (76) Google Scholar). 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 (15Topham M.K. Prescott S.M. J. Biol. Chem. 1999; 274: 11447-11450Abstract Full Text Full Text PDF PubMed Scopus (254) Google Scholar). 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. 16Neuhaus F.C. Baddiley J. Microbiol. Mol. Biol. Rev. 2003; 67: 686-723Crossref PubMed Scopus (738) Google Scholar). 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 (17Koch H.U. Haas R. Fischer W. Eur. J. Biochem. 1984; 138: 357-363Crossref PubMed Scopus (104) Google Scholar, 18Cabacungan E. Pieringer R.A. J. Bacteriol. 1981; 147: 75-79Crossref PubMed Google Scholar, 19Taron D.J. Childs III, W.C. Neuhaus F.C. J. Bacteriol. 1983; 154: 1110-1116Crossref PubMed Google Scholar, 20Koga Y. Nishihara M. Morii H. Biochim. Biophys. Acta. 1984; 793: 86-94Crossref PubMed Scopus (11) Google Scholar), 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 (17Koch H.U. Haas R. Fischer W. Eur. J. Biochem. 1984; 138: 357-363Crossref PubMed Scopus (104) Google Scholar, 19Taron D.J. Childs III, W.C. Neuhaus F.C. J. Bacteriol. 1983; 154: 1110-1116Crossref PubMed Google Scholar), 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 (21Kobayashi K. Ehrlich S.D. Albertini A. Amati G. Andersen K.K. Arnaud M. Asai K. Ashikaga S. Aymerich S. Bessieres P. Boland F. Brignell S.C. Bron S. Bunai K. Chapuis J. Christiansen L.C. Danchin A. Debarbouille M. Dervyn E. Deuerling E. Devine K. Devine S.K. Dreesen O. Errington J. Fillinger S. Foster S.J. Fujita Y. Galizzi A. Gardan R. Eschevins C. Fukushima T. Haga K. Harwood C.R. Hecker M. Hosoya D. Hullo M.F. Kakeshita H. Karamata D. Kasahara Y. Kawamura F. Koga K. Koski P. Kuwana R. Imamura D. Ishimaru M. Ishikawa S. Ishio I. Le Coq D. Masson A. Mauel C. Meima R. Mellado R.P. Moir A. Moriya S. Nagakawa E. Nanamiya H. Nakai S. Nygaard P. Ogura M. Ohanan T. O'Reilly M. O'Rourke M. Pragai Z. Pooley H.M. Rapoport G. Rawlins J.P. Rivas L.A. Rivolta C. Sadaie A. Sadaie Y. Sarvas M. Sato T. Saxild H.H. Scanlan E. Schumann W. Seegers J.F.M.L. Sekiguchi J. Sekowska A. Seror S.J. Simon M. Stragier P. Studer R. Takamatsu H. Tanaka T. Takeuchi M. Thomaides H.B. Vagner V. van Dijl J.M. Watabe K. Wipat A. Yamamoto H. Yamamoto M. Yamamoto Y. Yamane K. Yata K. Yoshida K. Yoshikawa H. Zuber U. Ogasawara N. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 4678-4683Crossref PubMed Scopus (1132) Google Scholar). 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.EXPERIMENTAL PROCEDURESMaterials—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 Polar Lipids Inc.); l-3-phosphatidylinositol (porcine liver) (Doosan Serdary Research Laboratories); undecaprenol, 1,2-[14C]dipalmitoylphosphatidic acid, and [γ-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 (22Vagner V. Dervyn E. Ehrlich S.D. Microbiology. 1998; 144: 3097-3104Crossref PubMed Scopus (571) Google Scholar) 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 2Construction 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 (○) IPTG, resulting in either a normal growth pattern (•) or growth arrest (○) 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.View Large Image Figure ViewerDownload Hi-res image Download (PPT)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 (23Nguyen H.D. Nguyen Q.A. Ferreira R.C. Ferreira L.C. Tran L.T. Schumann W. Plasmid. 2005; 54: 241-248Crossref PubMed Scopus (114) Google Scholar).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 (11Jackson B.J. Bohin J.P. Kennedy E.P. J. Bacteriol. 1984; 160: 976-981Crossref PubMed Google Scholar). 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 (24Walsh J.P. Bell R.M. J. Biol. Chem. 1986; 261: 6239-6247Abstract Full Text PDF PubMed Google Scholar). Assays contained 50 mm MOPS, pH 7.0, 0.5 mm dithiothreitol, 10 mm MgCl2, 1 mm EGTA, 150 mm LiCl, 5 mm [γ-32P]ATP (specific activity = 0.02 Ci/mmol), and 4 mol % (1 mm) in the micelle phase of 50 mm octyl-β-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 × g for 15 min), resuspended in 20 mm Tris, pH 7.9, 500 mm NaCl, 1 mm β-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 × 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 β-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 β-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 (25Noto T. Miyakawa S. Oishi H. Endo H. Okazaki H. J. Antibiot. (Tokyo). 1982; 35: 401-410Crossref PubMed Scopus (117) Google Scholar).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 β-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 × 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 [γ-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 (26Behr T. Fischer W. Peter-Katalinic J. Egge H. Eur. J. Biochem. 1992; 207: 1063-1075Crossref PubMed Scopus (121) Google Scholar).RESULTSIdentification 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 (VEΦLNSAIEXVVDR 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.FIGURE 3DgkA 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 EΦLNSAIEAVVD 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 (GGDGTΦNEVVXG) and probably defines the ATP binding site. The lower group of sequences shows the alignment of a conserved sequence in the DgkBs (KGXEΦLPYD) that is not found in the prokaryotic family members that lack DagK activity. Φ, a variable hydrophobic amino acid.View Large Image Figure ViewerDownload Hi-res image Download (PPT)TABLE 1Complementation of E. coli dgkA and B. subtilis dgkB knockoutsStrainaStrains used were E. coli strain FB21625 (dgkA::Tn5) and B. subtilis strain GS435 containing a conditional dgkB (yerQ) expression controlled by the Pspac promoter (Fig. 2).PlasmidbAll expression plasmids were derivatives of pPJ131 and were constructed to express His-tagged proteins as described under “Experimental Procedures.”Expressed geneGrowth on agar platescComplementation tests were carried out as described under “Experimental Procedures.” We did not note a visible difference in the growth rate of the strains that did grow on the arbutin-supplemented plates compared with LB alone.LBLB + arbutinFB21625pPJ131NoneYesNoFB21625pAJ001E. coli dgkAYesYesFB21625pAJ002B. subtilis dgkAYesNoFB21625pAJ003B. subtilis dgkB (yerQ)YesYesFB21625pAJ004B. subtilis bmrUYesNoFB21625pAJ005B. subtilis ytlRYesNoFB21625pAJ006S. pneumoniae SP1045YesNoFB21625pAJ007S. aureus SAR1989YesYesFB21625pAJ008S. aureus SAR0780YesNoStrainPlasmidExpressed geneGrowth on agar platesLBLB + IPTGGS435pHCMC02NoneNoYesGS435pAJ018E. coli dgkAYesYesa Strains used were E. coli strain FB21625 (dgkA::Tn5) and B. subtilis strain GS435 containing a conditional dgkB (yerQ) expression controlled by the Pspac promoter (Fig. 2).b All expression plasmids were derivatives of pPJ131 and were constructed to express His-tagged proteins as described under “Experimental Procedures.”c Complementation tests were carried out as described under “Experimental Procedures.” We did not note a visible difference in the growth rate of the strains that did grow on the arbutin-supplemented plates compared with LB alone. Open table in a new tab 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 GGDGTΦNEVVXG 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 (21Kobayashi K. Ehrlich S.D. Albertini A. Amati G. Andersen K.K. Arnaud M. Asai K. Ashikaga S. Aymerich S. Bessieres P. Boland F. Brignell S.C. Bron S. Bunai K. Chapuis J. Christiansen L.C. Danchin A. Debarbouille M. Dervyn E. Deuerling E. Devine K. Devine S.K. Dreesen O. Errington J. Fillinger S. Foster S.J. Fujita Y. Galizzi A. Gardan R. Eschevins C. Fukushima T. Haga K. Harwood C.R. Hecker M. Hosoya D. Hullo M.F. Kakeshita H. Karamata D. Kasahara Y. Kawamura F. Koga K. Koski P. Kuwana R. Imamura D. Ishimaru M. Ishikawa S. Ishio I. Le Coq D. Masson A. Mauel C. Meima R. Mellado R.P. Moir A. Moriya S. Nagakawa E. Nanamiya H. Nakai S. Nygaard P. Ogura M. Ohanan T. O'Reilly M. O'Rourke M. Pragai Z. Pooley H.M. Rapoport G. Rawlins J.P. Rivas L.A. Rivolta C. Sadaie A. Sadaie Y. Sarvas M. Sato T. Saxild H.H. Scanlan E. Schumann W. Seegers J.F.M.L. Sekiguchi J. Sekowska A. Seror S.J. Simon M. Stragier P. Studer R. Takamatsu H. Tanaka T. Takeuchi M. Thomaides H.B. Vagner V. van Dijl J.M. Watabe K. Wipat A. Yamamoto H. Yamamoto M. Yamamoto Y. Yamane K. Yata K. Yoshida K. Yoshikawa H. Zuber U. Ogasawara N. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 4678-4683Crossref PubMed Scopus (1132) Google Scholar). 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." @default.
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• W2016872786 title "Identification of a Soluble Diacylglycerol Kinase Required for Lipoteichoic Acid Production in Bacillus subtilis" @default.
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