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• W2057989817 abstract "The chemotaxis machinery of Bacillus subtilis is similar to that of the well characterized system ofEscherichia coli. However, B. subtilis contains several chemotaxis genes not found in the E. coli genome, such as cheC and cheD, indicating that theB. subtilis chemotactic system is more complex. In B. subtilis, CheD is required for chemotaxis; the cheDmutant displays a tumbly phenotype, has abnormally methylated chemoreceptors, and responds poorly to most chemical stimuli. Homologs of B. subtilis CheD have been found in chemotaxis-like operons of a large number of bacteria and archaea, suggesting that CheD plays an important role in chemotactic sensory transduction for many organisms. However, the molecular function of CheD has remained unknown. In this study, we show that CheD catalyzes amide hydrolysis of specific glutaminyl side chains of the B. subtilis chemoreceptor McpA. In addition, we present evidence that CheD deamidates other B. subtilis chemoreceptors including McpB and McpC. Previously, deamidation of B. subtilis receptors was thought to be catalyzed by the CheB methylesterase, as is the case for E. coli receptors. Because cheD mutant cells do not respond to most chemoattractants, we conclude that deamidation by CheD is required forB. subtilis chemoreceptors to effectively transduce signals to the CheA kinase. The chemotaxis machinery of Bacillus subtilis is similar to that of the well characterized system ofEscherichia coli. However, B. subtilis contains several chemotaxis genes not found in the E. coli genome, such as cheC and cheD, indicating that theB. subtilis chemotactic system is more complex. In B. subtilis, CheD is required for chemotaxis; the cheDmutant displays a tumbly phenotype, has abnormally methylated chemoreceptors, and responds poorly to most chemical stimuli. Homologs of B. subtilis CheD have been found in chemotaxis-like operons of a large number of bacteria and archaea, suggesting that CheD plays an important role in chemotactic sensory transduction for many organisms. However, the molecular function of CheD has remained unknown. In this study, we show that CheD catalyzes amide hydrolysis of specific glutaminyl side chains of the B. subtilis chemoreceptor McpA. In addition, we present evidence that CheD deamidates other B. subtilis chemoreceptors including McpB and McpC. Previously, deamidation of B. subtilis receptors was thought to be catalyzed by the CheB methylesterase, as is the case for E. coli receptors. Because cheD mutant cells do not respond to most chemoattractants, we conclude that deamidation by CheD is required forB. subtilis chemoreceptors to effectively transduce signals to the CheA kinase. His6-tagged McpA cytoplasmic fragment ribosome binding site glutathione S-transferase Chemotactic prokaryotes respond behaviorally to chemoeffectors by altering the flux of phosphoryl groups through the two-component system composed of the histidine kinase CheA and its cognate response regulator CheY (1Stock J.B. Surette M.G. Neidhardt F.C. Curtiss III, R. Ingraham J.L. Lin E.C.C. Low K.B. Magasanik B. Reznikoff W.S. Riley M. Schaechter M. Umbarger H.E. Escherichia coli and Salmonella: Cellular and Molecular Biology. American Society for Microbiology Press, Washington D. C.1996: 1103-1129Google Scholar, 2Armitage J.P. Adv. Microb. Physiol. 1999; 41: 229-289Crossref PubMed Google Scholar). In Escherichia coli, transmembrane chemoreceptors are coupled by CheW to the CheA kinase to elevate phosphoryl group flux through the system during the excitation response to repellent stimuli (3Hess J.F. Oosawa K. Kaplan N. Simon M.I. Cell. 1988; 53: 79-87Abstract Full Text PDF PubMed Scopus (396) Google Scholar, 4Liu J.D. Parkinson J.S. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 8703-8707Crossref PubMed Scopus (101) Google Scholar, 5Borkovich K.A. Simon M.I. Cell. 1990; 63: 1339-1348Abstract Full Text PDF PubMed Scopus (140) Google Scholar). Adaptation to repellent stimuli depends on dephosphorylation of CheY-P, enhanced by oligomeric CheZ (3Hess J.F. Oosawa K. Kaplan N. Simon M.I. Cell. 1988; 53: 79-87Abstract Full Text PDF PubMed Scopus (396) Google Scholar, 6Blat Y. Eisenbach M. J. Biol. Chem. 1996; 271: 1232-1236Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar, 7Wang H. Matsumura P. Mol. Microbiol. 1996; 19: 695-703Crossref PubMed Scopus (64) Google Scholar). The chemoreceptors are covalently modified by methylesterification of Glu side chains (8Springer W.R. Koshland D.E., Jr. Proc. Natl. Acad. Sci. U. S. A. 1977; 74: 533-537Crossref PubMed Scopus (189) Google Scholar). Modulation of the net methylation level of the receptors provides a simple chemical “memory,” allowing responses to continuously changing concentration gradients (9Blair D.F. Annu. Rev. Microbiol. 1995; 49: 489-522Crossref PubMed Google Scholar). Receptor methylation levels are dynamically controlled through the action of two modification enzymes, a methyltransferase, CheR, and a methylesterase, CheB. CheR methylesterifies specific Glu side chains on receptors usingS-adenosylmethionine as a methyl donor (8Springer W.R. Koshland D.E., Jr. Proc. Natl. Acad. Sci. U. S. A. 1977; 74: 533-537Crossref PubMed Scopus (189) Google Scholar); CheB catalyzes the hydrolysis of these glutamyl methyl esters, releasing methanol and regenerating a Glu (10Stock J.B. Koshland D.E., Jr. Proc. Natl. Acad. Sci. U. S. A. 1978; 75: 3659-3663Crossref PubMed Scopus (143) Google Scholar). CheR and CheB are the only known chemoreceptor modification enzymes. Chemoreceptor modification occurs at specific, cytoplasmic Glu residues located within a well defined consensus sequence originally determined using methylated receptors derived from E. coli (11Le Moual H. Koshland D.E., Jr. J. Mol. Biol. 1996; 261: 568-585Crossref PubMed Scopus (151) Google Scholar, 12Terwilliger T.C. Wang J.Y. Koshland D.E., Jr. J. Biol. Chem. 1986; 261: 10814-10820Abstract Full Text PDF PubMed Google Scholar). This consensus sequence has been successfully used to identify methylation sites on receptors of the distantly related organismBacillus subtilis (13Zimmer M.A. Tiu J. Collins M.A. Ordal G.W. J. Biol. Chem. 2000; 275: 24264-24272Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar, 14Hanlon D.W. Ordal G.W. J. Biol. Chem. 1994; 269: 14038-14046Abstract Full Text PDF PubMed Google Scholar). The consensus exhibits some slight variability; receptors are often, but not exclusively, synthesized with Glu at the site of modification. In some instances, the modified site is encoded as Gln rather than Glu in the corresponding receptor gene. When this situation occurs on an E. coli receptor, the CheB methylesterase first acts as a deamidase; amide hydrolysis of Gln residues produces Glu at the corresponding position in the mature protein. These Glu residues are then subject to cycles of reversible methylation (15Kehry M.R. Bond M.W. Hunkapiller M.W. Dahlquist F.W. Proc. Natl. Acad. Sci. U. S. A. 1983; 80: 3599-3603Crossref PubMed Scopus (77) Google Scholar). Because the B. subtilis methylesterase complemented an E. coli cheB strain in swarm assays (16Kirsch M.L. Peters P.D. Hanlon D.W. Kirby J.R. Ordal G.W. J. Biol. Chem. 1993; 268: 18610-18616Abstract Full Text PDF PubMed Google Scholar), it has been assumed that the B. subtilis methylesterase possesses an equivalent glutaminase activity. Modification (methylation, demethylation, or deamidation) of theE. coli chemoreceptors has substantial effects on the electrophoretic mobility of the molecules during SDS-PAGE. Incorporation of methyl groups via CheR causes the receptor to migrate more rapidly through the acrylamide matrix (or at a lower apparentM r), whereas demethylation or deamidation via CheB causes the receptor to migrate more slowly through the acrylamide matrix (or at a higher apparent M r) (15Kehry M.R. Bond M.W. Hunkapiller M.W. Dahlquist F.W. Proc. Natl. Acad. Sci. U. S. A. 1983; 80: 3599-3603Crossref PubMed Scopus (77) Google Scholar,17Boyd A. Simon M.I. J. Bacteriol. 1980; 143: 809-815Crossref PubMed Google Scholar, 18Chelsky D. Dahlquist F.W. Proc. Natl. Acad. Sci. U. S. A. 1980; 77: 2434-2438Crossref PubMed Scopus (35) Google Scholar, 19Rollins C. Dahlquist F.W. Cell. 1981; 25: 333-340Abstract Full Text PDF PubMed Scopus (23) Google Scholar, 20Kehry M.R. Dahlquist F.W. Cell. 1982; 29: 761-772Abstract Full Text PDF PubMed Scopus (34) Google Scholar). B. subtilis utilizes a similar complement of proteins for chemotaxis, including homologs of the transmembrane chemoreceptors, CheW coupler, CheA kinase, CheY regulator, and CheR/CheB receptor-modification enzymes (21Kirby J.R. Kristich C.J. Saulmon M.M. Zimmer M.A. Garrity L.F. Zhulin I.B. Ordal G.W. Mol. Microbiol. 2001; 42: 573-585Crossref PubMed Scopus (52) Google Scholar, 22Garrity L.G. Ordal G.W. Pharmacol. Ther. 1995; 68: 87-104Crossref PubMed Scopus (56) Google Scholar). However, chemotaxis by B. subtilis deviates from the E. coli paradigm in many ways. For example, B. subtilis lacks a homolog of the CheZ phosphatase present in E. coli and other closely related γ-proteobacteria. Moreover, B. subtilis possesses several chemotaxis genes that have no counterparts in E. coli, including cheC, cheD, and cheV.The roles of CheC, CheD, and CheV in B. subtilis chemotaxis have not been well characterized. Recent work suggests roles for CheV and CheC during adaptation to chemotactic stimuli (21Kirby J.R. Kristich C.J. Saulmon M.M. Zimmer M.A. Garrity L.F. Zhulin I.B. Ordal G.W. Mol. Microbiol. 2001; 42: 573-585Crossref PubMed Scopus (52) Google Scholar, 23Karatan E. Saulmon M.M. Bunn M.W. Ordal G.W. J. Biol. Chem. 2001; 276: 43618-43626Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar), although their mechanism of action remains elusive; less is known about the function of CheD. cheD mutant cells exhibit poorly methylated receptors, clockwise flagellar rotational bias (cells are tumbly), and decreased sensitivity to attractants (21Kirby J.R. Kristich C.J. Saulmon M.M. Zimmer M.A. Garrity L.F. Zhulin I.B. Ordal G.W. Mol. Microbiol. 2001; 42: 573-585Crossref PubMed Scopus (52) Google Scholar, 24Rosario M.M.R. Kirby J.R. Bochar D.A. Ordal G.W. Biochemistry. 1995; 34: 3823-3831Crossref PubMed Scopus (53) Google Scholar). Additionally, a recent report noted the unexpected observation that oneB. subtilis chemoreceptor, McpA, appeared to be absent in acheD mutant (21Kirby J.R. Kristich C.J. Saulmon M.M. Zimmer M.A. Garrity L.F. Zhulin I.B. Ordal G.W. Mol. Microbiol. 2001; 42: 573-585Crossref PubMed Scopus (52) Google Scholar). No link connecting these disparate phenotypes has been established. The apparent nonspecific nature of the chemotactic defects of the cheD mutant has precluded assignment of a specific biochemical function to CheD. Furthermore, its function cannot be inferred from sequence comparisons because all CheD homologs in the publicly available data base are putative proteins for which no function has been described. Although CheD function has remained a mystery, the discovery of CheD homologs within chemotaxis-like operons of a large number of bacterial and archaeal species (21Kirby J.R. Kristich C.J. Saulmon M.M. Zimmer M.A. Garrity L.F. Zhulin I.B. Ordal G.W. Mol. Microbiol. 2001; 42: 573-585Crossref PubMed Scopus (52) Google Scholar) suggests that CheD plays an important role in chemoreceptor-mediated sensory transduction for many organisms. This report presents the first evidence identifying a specific molecular function for the CheD protein. We demonstrate that B. subtilis CheD catalyzes amide hydrolysis of specific glutaminyl side chains of the B. subtilis chemoreceptor McpA. CheD can thus be classified as a chemoreceptor glutamine deamidase (E.C. 3.5.1.44). Because the cheD mutant exhibits a considerable clockwise prestimulus flagellar rotational bias (similar to a B. subtilis CheA null mutant) and cheD cells do not respond to most chemoattractants, we conclude that CheD-mediated receptor deamidation is required for productive communication of the conformational signals of the receptors to the CheA kinase. Bacterial strains and plasmids used in this study are listed in TableI. DNA manipulations were performed according to standard protocols. Plasmids pAIN750mcpA and pAIN750mcpC were constructed by first amplifying the corresponding mcp gene and its promoter from B. subtilis chromosomal DNA via PCR using Pfu polymerase (Stratagene). The amplification products were cloned into pAIN750 using primer-encoded EcoRI and BamHI restriction sites for mcpA orEcoRI and NotI restriction sites formcpC. Site-specific mutations in mcpA were created as described (13Zimmer M.A. Tiu J. Collins M.A. Ordal G.W. J. Biol. Chem. 2000; 275: 24264-24272Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar) using pAIN750mcpA as template. For use in an independent line of investigation, both pAIN750mcpA and pAIN750mcpC had aBglII restriction site incorporated into the region coding for the receptor c-terminal domain using a previously described method (13Zimmer M.A. Tiu J. Collins M.A. Ordal G.W. J. Biol. Chem. 2000; 275: 24264-24272Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). These nucleotide sequence alterations were phenotypically “silent”; that is, they did not result in any change of the amino acid sequence of the receptors.Table IStrains and plasmids used in this studyStrain or plasmidRelevant genotype or descriptionReferenceOI2836 cheB8∷cat16Kirsch M.L. Peters P.D. Hanlon D.W. Kirby J.R. Ordal G.W. J. Biol. Chem. 1993; 268: 18610-18616Abstract Full Text PDF PubMed Google ScholarOI2934 cheD1∷cat24Rosario M.M.R. Kirby J.R. Bochar D.A. Ordal G.W. Biochemistry. 1995; 34: 3823-3831Crossref PubMed Scopus (53) Google ScholarOI3545Δ10mcp che +26Hou S. Larsen R.W. Boudko D. Riley C.W. Karatan E. Zimmer M. Ordal G.W. Alam M. Nature. 2000; 403: 540-544Crossref PubMed Scopus (235) Google ScholarOI3920Δ10mcp cheR3∷catThis workOI3628Δ10mcp cheD1∷catThis workOI3922Δ10mcp cheR3∷cat cheD1∷catThis workOI3635Δ10mcp cheR3∷cat cheB8∷catThis workOI3921OI3545 amyE5720∷mcpAThis workOI3923OI3628 amyE5720∷mcpAThis workOI3924OI3922 amyE5720∷mcpAThis workOI3925OI3920 amyE5720∷mcpAThis workOI3926OI3635 amyE5720∷mcpAThis workOI3927OI3920 amyE5720∷mcpA Q593E Q594EThis workOI3928OI3922amyE5720∷mcpA Q593E Q594EThis workOI3929OI3920 amyE5720∷mcpA Q593A Q594AThis workOI3930OI3922amyE5720∷mcpA Q593A Q594AThis workOI3664OI3920 amyE5720∷mcpBThis workOI3932OI3922 amyE5720∷mcpBThis workOI3663OI3920 amyE5720∷mcpCThis workOI3931OI3922 amyE5720∷mcpCThis workRP3098 E. coli Δ(flhD-flhB)4,cheJ. S. ParkinsonTG-1 E. colicloning hostAmershampSE380 E. coli expression vectorInvitrogenpSKCloning vector; AmpRStratagenepEB112 B. subtilis-E. colishuttle vector; AmpR, KanR30Leonhardt H. Alonso J.C. J. Gen. Microbiol. 1988; 134: 605-609PubMed Google ScholarpWN5pEB112 containing cheD; AmpR, KanR24Rosario M.M.R. Kirby J.R. Bochar D.A. Ordal G.W. Biochemistry. 1995; 34: 3823-3831Crossref PubMed Scopus (53) Google ScholarpGXOBGST-CheD fusion expression vector; AmpR31Rosario M.M.R. Ordal G.W. Mol. Microbiol. 1996; 21: 511-518Crossref PubMed Scopus (46) Google ScholarpUSH1 B. subtilis-E. coli shuttle vector for constructing His6-tagged fusion proteins; CmR, KanR32Schön U. Schumann W. Gene (Amst.). 1994; 147: 91-94Crossref PubMed Scopus (30) Google ScholarpAIN620pUSH1 expressing His6-tagged-McpA cytoplasmic domainThis workpAIN150pWN5 containing consensus RBS upstream ofcheDThis workpAIN1510.8-kbHindIII/EcoRI fragment of pAIN150 cloned in pSKThis workpAIN1521.2-kbEcoRI/NotI fragment of pAIN620 cloned in pAIN151This workpAIN153vector for co-expression ofcheD and mcpA cytoplasmic fragment; AmpR, KanRThis workpDG1730 B. subtilis amyEintegration vector; AmpR, SpcR33Guerout-Fleury A.M. Frandsen N. Stragier P. Gene (Amst.). 1996; 180: 57-61Crossref PubMed Scopus (423) Google ScholarpAIN750253-bp BamHI/HindIII fragment of pSE380 cloned into BamHI/HindIII sites of pDG1730; AmpR, SpcRThis workpAIN750mcpA amyE integration vector containing mcpAThis workpAIN750mcpB amyE integration vector containing mcpB13Zimmer M.A. Tiu J. Collins M.A. Ordal G.W. J. Biol. Chem. 2000; 275: 24264-24272Abstract Full Text Full Text PDF PubMed Scopus (42) Google ScholarpAIN750mcpC amyE integration vector containing mcpCThis workpAIN750mcpAQQ-EE amyE integration vector containing mcpA Q593E Q594EThis workpAIN750mcpAQQ-AA amyE integration vector containing mcpA Q593A Q594AThis work Open table in a new tab A plasmid (pAIN620) designed to express a His6-tagged McpA cytoplasmic fragment (c-fragment)1 was constructed by amplifying the C-terminal domain of McpA from pAIN750mcpAusing Pfu polymerase and cloning the amplification product into the BamHI site of pUSH1 to create pAIN620. The amplification primers encoded BamHI sites and were designed to generate an in-frame N-terminal His-tagged fusion of the entire McpA cytoplasmic sequence beginning with McpA Arg-303. A second plasmid designed to co-express cheD with the c-fragment (pAIN153) was constructed via a multistep process as follows. Because the wild-type ribosome binding site (RBS) for cheD is a poor match for the B. subtilis consensus RBS (25Mountain A. Harwood C.R. Bacillus. Plenum Publishing Corp., New York1989: 73-114Crossref Google Scholar), resulting in low expression levels, site-specific mutations were introduced into pWN5 as described (13Zimmer M.A. Tiu J. Collins M.A. Ordal G.W. J. Biol. Chem. 2000; 275: 24264-24272Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar) to create a consensus RBS upstream of thecheD coding sequence (pAIN150). This procedure concomitantly introduced an adjacent HindIII site. The 0.8-kbHindIII/EcoRI fragment of pAIN150 containingcheD, and its RBS was subcloned into theHindIII/EcoRI sites of pSK, generating pAIN151. A 1.2-kb EcoRI/NotI fragment of pAIN620 containing the gene for the c-fragment, and its RBS was subcloned into theEcoRI/NotI sites of pAIN151, generating pAIN152. Finally, a 2.1-kb SalI fragment of pAIN152 containingcheD and the c-fragment was subcloned into theSalI site of pEB112, creating pAIN153. Strains carrying cheD1::cat orcheB8::cat alleles in combination with the 10 mcp mutations were constructed as described for OI3545 (26Hou S. Larsen R.W. Boudko D. Riley C.W. Karatan E. Zimmer M. Ordal G.W. Alam M. Nature. 2000; 403: 540-544Crossref PubMed Scopus (235) Google Scholar) using OI2934 (cheD) or OI2836 (cheB) as host strains. ThecheR3::cat allele was introduced into relevant strains via PBS1 co-transduction with the nearbytrpF + marker. Chemoreceptors were expressed inB. subtilis strains by integration into the amyElocus as described (13Zimmer M.A. Tiu J. Collins M.A. Ordal G.W. J. Biol. Chem. 2000; 275: 24264-24272Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). LB is 1% tryptone, 0.5% yeast extract, 0.5% NaCl. Bacteria were diluted 1:100 into LB from saturated overnight cultures and incubated at 37 °C with agitation (250 rpm) until they reached early stationary phase. Cells were pelleted, resuspended in protoplast buffer (20% sucrose, 25 mm potassium phosphate, 10 mmMgCl2, 30 mm sodium lactate, 1 mmEDTA, pH 7 (27Ullah A.H.J. Ordal G.W. J. Bacteriol. 1981; 145: 958-965Crossref PubMed Google Scholar)) plus 5 mg/ml lysozyme atA 600 of 1.0, and incubated at 37 °C as before for 35 m. 1-ml protoplast samples were pelleted and resuspended in 0.1 ml of 4× SDS sample-loading buffer (28Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning, A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1989Google Scholar). Electrophoresis and electroblotting were performed as described (21Kirby J.R. Kristich C.J. Saulmon M.M. Zimmer M.A. Garrity L.F. Zhulin I.B. Ordal G.W. Mol. Microbiol. 2001; 42: 573-585Crossref PubMed Scopus (52) Google Scholar) using 10-cm polyacrylamide gels run at 25 mA (constant current) for 3 h. Where indicated, 25-cm gels were used and run at 350 V (constant voltage) for 15 h. When analyzing c-fragments, 10-cm gels were run at 250 V (constant voltage) for 2 h. Anti-McpB antibody (to detect McpA, c-fragment, and McpB) and anti-McpC antibody (to detect McpC) were used as described (21Kirby J.R. Kristich C.J. Saulmon M.M. Zimmer M.A. Garrity L.F. Zhulin I.B. Ordal G.W. Mol. Microbiol. 2001; 42: 573-585Crossref PubMed Scopus (52) Google Scholar). C-fragments were purified under denaturing conditions using nickel-nitrilotriacetic acid agarose (Qiagen), essentially according to the manufacturer's instructions. A 1-liter culture of LB plus antibiotic (10 μg/ml chloramphenicol or 100 μg/ml ampicillin) was inoculated 1:100 with an overnight culture of RP3098 harboring pAIN620 (for the unmodified c-fragment) or pAIN153 (for the CheD-modified c-fragment) and grown at 37 °C with agitation (250 rpm) to an A 600 of 0.8. IPTG was added to 1 mm; incubation was continued as before, and cells were harvested by centrifugation (5000 ×g, 5 min) after 3 h. Cell pellets were frozen at −70 °C. Thawed cell pellets were resuspended in Buffer B (8m urea, 0.1 m NaH2PO4, 0.01 m Tris, pH 8) and incubated at room temperature for 1 h with mixing. The supernatants were clarified by two serial centrifugations (5000 × g, 5 min; 20,000 ×g, 40 min) and incubated with nickel-nitrilotriacetic acid agarose resin (pre-equilibrated in Buffer B) at room temperature for 1 h with mixing. The resin was batch-washed twice with Buffer B and twice with Buffer C (Buffer B with pH adjusted to 6.3) and then applied to a gravity flow column and washed with 10 column volumes of Buffer C. Elution was performed in 20 ml of Buffer E (Buffer B with pH adjusted to 4.5). The eluates were concentrated to ∼1 ml using a cellulose ultrafiltration membrane (Millipore) in an Amicon ultrafiltration cell. These samples were dialyzed against three changes of 25 mm NH4HCO3 at 4 °C, and aliquots were frozen at −70 °C. For purification of glutathione S-transferase-CheD fusion protein (GST-CheD), a saturated overnight culture of pGXOB was diluted 1:100 into 1 liter of LB plus 100 μg/ml ampicillin and grown at 37 °C (250 rpm) to A 600 of 0.8. IPTG was added to 1 mm, and the culture was incubated (200 rpm) overnight at room temperature. Cells were pelleted and frozen as above. Thawed cell pellets were resuspended in phosphate-buffered saline (150 mm NaCl, 16 mm Na2HPO4, 4 mm NaH2PO4, pH 7.3) and disrupted by sonication. The supernatant was clarified as above and applied to a 2-ml gravity flow glutathione-Sepharose 4B column (AmershamBiosciences). The column was washed with 15 column volumes of phosphate-buffered saline, and elution was performed using 10 ml of elution buffer (50 mm Tris, 5 mm glutathione, pH 8). The eluate was concentrated as above. The sample was dialyzed against three changes of 50 mm Tris (pH 8), 10% glycerol at 4 °C, and aliquots were frozen at −70 °C. For in vitro reactions, 1 μm c-fragment was incubated with the indicated amounts of GST-CheD at room temperature in reaction buffer (50 mm Tris, pH 7.5, 0.1 mm dithiothreitol, 1 mm MgCl2, 0.5 mm EDTA). At the indicated times, samples (10 μl) were removed and mixed with 10 μl of 2× SDS loading buffer. Immunoblotting was performed as described above. CNBr digestion was performed essentially as described (29Sun Y. Bauer M.D. Keough T.W. Lacey M.P. Chapman J.R. Protein and Peptide Analysis by Mass Spectrometry. Humana Press, New Jersey1996: 185-210Google Scholar). Aliquots of purified c-fragments (0.5–1 nmol) were dried under vacuum using a SpeedVac and resuspended in 0.1m HCl. A small, unweighed crystal of CNBr was added; the reaction was vortexed to dissolve the crystal and incubated overnight at room temperature in the dark. 4 volumes of water were added, and the reaction was dried as before. Cleavage products were resuspended in 0.1% trifluoroacetic acid. Peptide separation and automated Edman degradation were performed at the UIUC Protein Sciences Facility. Electrophoretic mobility of a B. subtilis chemoreceptor is dependent on the cheD gene product—Previous work to characterize several B. subtilis chemoreceptors demonstrated that themcpB gene product fractionated near its expectedM r (71,800) during SDS-PAGE (14Hanlon D.W. Ordal G.W. J. Biol. Chem. 1994; 269: 14038-14046Abstract Full Text PDF PubMed Google Scholar). Despite being encoded with a nearly identical M r as McpB, themcpA gene product (Mr-72,400) fractionated as several distinct bands at a significantly higher apparentM r (∼95,000). Recently, however, immunoblot analysis revealed the unexpected absence of McpA-specific cross-reacting material in the lysate of a cheD mutant (21Kirby J.R. Kristich C.J. Saulmon M.M. Zimmer M.A. Garrity L.F. Zhulin I.B. Ordal G.W. Mol. Microbiol. 2001; 42: 573-585Crossref PubMed Scopus (52) Google Scholar). In an attempt to elucidate the biochemical role of the cheDgene product, we investigated the apparent absence of McpA. One possibility to explain the absence of McpA is that the synthesis or stability of McpA is impaired in the cheD mutant. We found this possibility unlikely since two other B. subtilisreceptors (McpB and McpC) were detected at wild-type levels in thecheD lysate (21Kirby J.R. Kristich C.J. Saulmon M.M. Zimmer M.A. Garrity L.F. Zhulin I.B. Ordal G.W. Mol. Microbiol. 2001; 42: 573-585Crossref PubMed Scopus (52) Google Scholar). However, the antibody used for detection of McpA also cross-reacts with McpB (21Kirby J.R. Kristich C.J. Saulmon M.M. Zimmer M.A. Garrity L.F. Zhulin I.B. Ordal G.W. Mol. Microbiol. 2001; 42: 573-585Crossref PubMed Scopus (52) Google Scholar); therefore, if McpA and McpB co-fractionated, they would be indistinguishable. We hypothesized that the cheD mutation affected the electrophoretic mobility of McpA such that it co-fractionated with McpB. To test this hypothesis, we expressed a single receptor gene (mcpA ormcpB) in two backgrounds containing mutations in the 10 known receptor genes (Δ10mcp); one wasche +, and the other was a cheD mutant derivative. We assessed the electrophoretic mobility of each receptor during SDS-PAGE (Fig. 1). McpB exhibited no mobility changes due to the cheD mutation under these conditions (not shown, but see below). In contrast, McpA displayed a substantial difference in mobility due to the cheD mutation (Fig. 1, compare lanes 1 and 2). ThecheD mutation resulted in an McpA protein that did not fractionate as several bands at the previously described relatively high apparent M r; instead, it fractionated as a single band closer to its expected M r. Thus, CheD is required for an electrophoretic mobility shift of McpA to a higher apparent M r. Chemoreceptors of E. coli are known to undergo electrophoretic mobility shifts due to changes in the extent of their modification (methylation, demethylation, or deamidation) catalyzed by the CheR methyltransferase and the CheB methylesterase (17Boyd A. Simon M.I. J. Bacteriol. 1980; 143: 809-815Crossref PubMed Google Scholar, 18Chelsky D. Dahlquist F.W. Proc. Natl. Acad. Sci. U. S. A. 1980; 77: 2434-2438Crossref PubMed Scopus (35) Google Scholar). To test the possibility that CheD-dependent mobility shifts of McpA were due to analogous changes in the level of its modification catalyzed by the B. subtilis methyltransferase or methylesterase, we expressed mcpA in Δ10mcpstrains harboring cheR (methyltransferase) or cheR cheB (methyltransferase and methylesterase) mutations. Immunoblot analysis of McpA reveals that the mobility shift to a higher apparentM r occurs normally in both backgrounds and is dependent on CheD (Fig. 1, compare lanes 3–5). Thus, neither of the two enzymes shown previously to modify bacterial chemoreceptors is required for the CheD-dependent mobility shift of McpA to a higher apparent M r, implying that CheD is itself involved in chemoreceptor modification. Although neither of the two previously known chemoreceptor modification enzymes is required for the CheD-dependent mobility shift of McpA, it is apparent from Fig. 1 that methylation of McpA substantially affects its mobility independently of the CheD-dependent modification. Unmethylated McpA (derived from the cheR host) fractionates as a single band of a relatively high apparent M r (lanes 3and 5), whereas the presence of CheR results in several forms of McpA that fractionate at a lower apparentM r (lane 1), presumably as a result of methylation at one or more methylatable sites. Under these conditions, we were able to resolve a total of four bands in the wild-type background (lane 1); the two bands of intermediateM r appear to “smear” together, but shorter exposures of the same blot clearly reveal distinct bands (not shown). In addition, the presence of CheR also causes McpA to fractionate at a lower apparent M r in the absence of CheD, again presumably as a result of methylation (compare lanes 2 and4). This effect of methylation on chemoreceptor mobility has been well documented for chemoreceptors of E. coli (17Boyd A. Simon M.I. J. Bacteriol. 1980; 143: 809-815Crossref PubMed Google Scholar, 18Chelsky D. Dahlquist F.W. Proc. Natl. Acad. Sci. U. S. A. 1980; 77: 2434-2438Crossref PubMed Scopus (35) Google Scholar) and is consistent with the observation of Hanlon and Ordal (14Hanlon D.W. Ordal G.W. J. Biol. Chem. 1994; 269: 14038-14046Abstract Full Text PDF PubMed Google Scholar) that the mcpA gene product accounts for several distinct methylated bands in extracts of wild-type B. subtilis. In contrast to the effect of methylation on chemoreceptor mobility, E. colireceptors that are deamidated migrate more slowly through acrylamide gels (19Rollins C. Dahlquist F.W. Cell. 1981; 25: 333-340Abstract Full Text PDF PubMed Scopus (23) Google Scholar, 20Kehry M.R. Dahlquist F.W. Cell. 1982; 29: 761-772Abstract Full Text PDF PubMed Scopus (34) Google Scholar). The DNA-encoded amino acid" @default.
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• W2057989817 title "Bacillus subtilis CheD Is a Chemoreceptor Modification Enzyme Required for Chemotaxis" @default.
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