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• W2110557005 abstract "The biosynthetic enzymes involved in wall teichoic acid biogenesis in Gram-positive bacteria have been the subject of renewed investigation in recent years with the benefit of modern tools of biochemistry and genetics. Nevertheless, there have been only limited investigations into the enzymes that glycosylate wall teichoic acid. Decades-old experiments in the model Gram-positive bacterium, Bacillus subtilis 168, using phage-resistant mutants implicated tagE (also called gtaA and rodD) as the gene coding for the wall teichoic acid glycosyltransferase. This study and others have provided only indirect evidence to support a role for TagE in wall teichoic acid glycosylation. In this work, we showed that deletion of tagE resulted in the loss of α-glucose at the C-2 position of glycerol in the poly(glycerol phosphate) polymer backbone. We also reported the first kinetic characterization of pure, recombinant wall teichoic acid glycosyltransferase using clean synthetic substrates. We investigated the substrate specificity of TagE using a wide variety of acceptor substrates and found that the enzyme had a strong kinetic preference for the transfer of glucose from UDP-glucose to glycerol phosphate in polymeric form. Further, we showed that the enzyme recognized its polymeric (and repetitive) substrate with a sequential kinetic mechanism. This work provides direct evidence that TagE is the wall teichoic acid glycosyltransferase in B. subtilis 168 and provides a strong basis for further studies of the mechanism of wall teichoic acid glycosylation, a largely uncharted aspect of wall teichoic acid biogenesis. The biosynthetic enzymes involved in wall teichoic acid biogenesis in Gram-positive bacteria have been the subject of renewed investigation in recent years with the benefit of modern tools of biochemistry and genetics. Nevertheless, there have been only limited investigations into the enzymes that glycosylate wall teichoic acid. Decades-old experiments in the model Gram-positive bacterium, Bacillus subtilis 168, using phage-resistant mutants implicated tagE (also called gtaA and rodD) as the gene coding for the wall teichoic acid glycosyltransferase. This study and others have provided only indirect evidence to support a role for TagE in wall teichoic acid glycosylation. In this work, we showed that deletion of tagE resulted in the loss of α-glucose at the C-2 position of glycerol in the poly(glycerol phosphate) polymer backbone. We also reported the first kinetic characterization of pure, recombinant wall teichoic acid glycosyltransferase using clean synthetic substrates. We investigated the substrate specificity of TagE using a wide variety of acceptor substrates and found that the enzyme had a strong kinetic preference for the transfer of glucose from UDP-glucose to glycerol phosphate in polymeric form. Further, we showed that the enzyme recognized its polymeric (and repetitive) substrate with a sequential kinetic mechanism. This work provides direct evidence that TagE is the wall teichoic acid glycosyltransferase in B. subtilis 168 and provides a strong basis for further studies of the mechanism of wall teichoic acid glycosylation, a largely uncharted aspect of wall teichoic acid biogenesis. Wall teichoic acids are anionic, phosphate-rich polymers that constitute a substantial portion of the cell wall of Gram-positive bacteria. Although the precise function of these polymers is unknown, they have been shown to play a role in critical cellular processes, namely cell shape determination in Bacillus subtilis (1D'Elia M.A. Millar K.E. Beveridge T.J. Brown E.D. J. Bacteriol. 2006; 188: 8313-8316Crossref PubMed Scopus (142) Google Scholar) and pathogenesis in Staphylococcus aureus (2Weidenmaier C. Kokai-Kun J.F. Kristian S.A. Chanturiya T. Kalbacher H. Gross M. Nicholson G. Neumeister B. Mond J.J. Peschel A. Nat. Med. 2004; 10: 243-245Crossref PubMed Scopus (430) Google Scholar, 3D'Elia M.A. Henderson J.A. Beveridge T.J. Heinrichs D.E. Brown E.D. J. Bacteriol. 2009; 191: 4030-4034Crossref PubMed Scopus (56) Google Scholar). Of the Gram-positive organisms studied to date, most produce either a poly(glycerol phosphate) or poly(ribitol phosphate) polymer as the major wall teichoic acid (4Neuhaus F.C. Baddiley J. Microbiol. Mol. Biol. Rev. 2003; 67: 686-723Crossref PubMed Scopus (774) Google Scholar). The main chain hydroxyl groups on both of these polymers are subject to modification with d-alanine and glycosyl residues. The d-alanylation modification of teichoic acids has been extensively studied and has been shown to play an important role in modulating the properties of the bacterial cell envelope (e.g. in regulating resistance to certain antimicrobial molecules) (4Neuhaus F.C. Baddiley J. Microbiol. Mol. Biol. Rev. 2003; 67: 686-723Crossref PubMed Scopus (774) Google Scholar, 5Peschel A. Otto M. Jack R.W. Kalbacher H. Jung G. Götz F. J. Biol. Chem. 1999; 274: 8405-8410Abstract Full Text Full Text PDF PubMed Scopus (807) Google Scholar, 6Peschel A. Vuong C. Otto M. Götz F. Antimicrob. Agents Chemother. 2000; 44: 2845-2847Crossref PubMed Scopus (206) Google Scholar). By contrast, there have been limited investigations into wall teichoic acid glycosylation, and its functional significance is unknown. The wall teichoic acid biosynthetic pathway has largely been elucidated in the Gram-positive model organism, B. subtilis 168. This organism produces a linear 1,3-linked poly(glycerol phosphate) polymer that is modified at position 2 of glycerol with d-alanine or glucose (4Neuhaus F.C. Baddiley J. Microbiol. Mol. Biol. Rev. 2003; 67: 686-723Crossref PubMed Scopus (774) Google Scholar). Classical genetic experiments in B. subtilis led to the isolation of the tag (teichoic acid glycerol phosphate) gene cluster for wall teichoic acid synthesis (7Mauël C. Young M. Margot P. Karamata D. Mol. Gen. Genet. 1989; 215: 388-394Crossref PubMed Scopus (59) Google Scholar, 8Mauël C. Young M. Karamata D. J. Gen. Microbiol. 1991; 137: 929-941Crossref PubMed Scopus (73) Google Scholar), and studies over the past decade using recombinant proteins have assigned biochemical functions to nearly all of the proteins involved in poly(glycerol phosphate) synthesis (9Ginsberg C. Zhang Y.H. Yuan Y. Walker S. ACS Chem. Biol. 2006; 1: 25-28Crossref PubMed Scopus (60) Google Scholar, 10Bhavsar A.P. Truant R. Brown E.D. J. Biol. Chem. 2005; 280: 36691-36700Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar, 11Park Y.S. Sweitzer T.D. Dixon J.E. Kent C. J. Biol. Chem. 1993; 268: 16648-16654Abstract Full Text PDF PubMed Google Scholar, 12Schertzer J.W. Brown E.D. J. Biol. Chem. 2003; 278: 18002-18007Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar, 13Swoboda J.G. Campbell J. Meredith T.C. Walker S. Chembiochem. 2010; 11: 35-45Crossref PubMed Scopus (270) Google Scholar). In addition, the pathway responsible for teichoic acid d-alanylation has been characterized in B. subtilis (14Perego M. Glaser P. Minutello A. Strauch M.A. Leopold K. Fischer W. J. Biol. Chem. 1995; 270: 15598-15606Abstract Full Text Full Text PDF PubMed Scopus (220) Google Scholar) as well as in other bacteria, such as S. aureus (5Peschel A. Otto M. Jack R.W. Kalbacher H. Jung G. Götz F. J. Biol. Chem. 1999; 274: 8405-8410Abstract Full Text Full Text PDF PubMed Scopus (807) Google Scholar). Together, these studies have begun to describe a model for wall teichoic acid biosynthesis and modification in B. subtilis 168. Synthesis occurs through the sequential action of several enzymes on the cytoplasmic face of the cell membrane on an undecaprenyl phosphate molecule. TagO and TagA add N-acetylglucosamine 1-phosphate and N-acetylmannosamine residues, respectively. TagB primes the undecaprenyl-pyrophosphoryl disaccharide with a single unit of glycerol 3-phosphate to complete formation of the linkage unit. The polymerase, TagF, then catalyzes the addition of 30–50 units of glycerol 3-phosphate, a substrate that is provided by TagD in the activated form, CDP-glycerol. Once synthesis is complete, the polymer is exported by TagGH to the outside of the cell, where it is attached to the 6-hydroxyl of N-acetylmuramic acid of peptidoglycan by an unknown transferase and modified with cationic d-alanyl esters (Fig. 1) (4Neuhaus F.C. Baddiley J. Microbiol. Mol. Biol. Rev. 2003; 67: 686-723Crossref PubMed Scopus (774) Google Scholar, 13Swoboda J.G. Campbell J. Meredith T.C. Walker S. Chembiochem. 2010; 11: 35-45Crossref PubMed Scopus (270) Google Scholar). Significant gaps still remain, however, in our understanding of wall teichoic acid synthesis, most notably in relation to wall teichoic acid glycosylation. The putative gene coding for the wall teichoic acid glycosyltransferase in B. subtilis 168 was first identified using phage-resistant mutants. Mutations in tagE have been shown to be associated with resistance to bacteriophages ϕ25 and ϕ29, which recognize glucose residues on teichoic acid as a receptor (15Young F.E. Proc. Natl. Acad. Sci. U.S.A. 1967; 58: 2377-2384Crossref PubMed Scopus (87) Google Scholar, 16Young F.E. Smith C. Reilly B.E. J. Bacteriol. 1969; 98: 1087-1097Crossref PubMed Google Scholar, 17Honeyman A.L. Stewart G.C. Mol. Microbiol. 1989; 3: 1257-1268Crossref PubMed Scopus (36) Google Scholar). A similar approach involving phage-resistant mutants was recently used to identify the wall teichoic acid glycosyltransferase in S. aureus. An elegant transposon mutagenesis screen for resistance to phage 80 led to the isolation of tarM (18Xia G. Maier L. Sanchez-Carballo P. Li M. Otto M. Holst O. Peschel A. J. Biol. Chem. 2010; 285: 13405-13415Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar). Disruption of this gene led to wall teichoic acid that completely lacked N-acetylglucosamine. The wall teichoic acid glycosyltransferase activity of TarM was subsequently confirmed by demonstration that crude extracts containing recombinant enzyme catalyzed the transfer of N-acetylglucosamine onto an uncharacterized membrane acceptor in vitro (18Xia G. Maier L. Sanchez-Carballo P. Li M. Otto M. Holst O. Peschel A. J. Biol. Chem. 2010; 285: 13405-13415Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar). Glaser and Burger (19Glaser L. Burger M.M. J. Biol. Chem. 1964; 239: 3187-3191Abstract Full Text PDF PubMed Google Scholar) conducted the first and only in vitro study of poly(glycerol phosphate) polymer glycosylation in B. subtilis nearly 50 years ago. This was a traditional study of multistep purification of glycosyltransferase activity from wild-type cells where the poly(glycerol phosphate) acceptor was provided in the form of membrane vesicles derived from B. subtilis. Thus, neither the enzyme nor the acceptor substrate was homogenous or unambiguously identified. Nevertheless, the TagE protein was later ascribed this activity following work using phage-resistant mutants that linked mutations in the encoding gene to the loss of glucose associated with the poly(glycerol phosphate) polymer (16Young F.E. Smith C. Reilly B.E. J. Bacteriol. 1969; 98: 1087-1097Crossref PubMed Google Scholar). In this work, we have demonstrated that precise deletion of tagE results in the absence of α-glucose at the C-2 position along the poly(glycerol phosphate) polymer backbone. Furthermore, we have conducted the first biochemical study of purified, recombinant TagE with pure synthetic acceptor substrates and have shown that the enzyme catalyzes the transfer of glucose from UDP-glucose onto a poly(glycerol phosphate) polymer acceptor at an appreciable rate in vitro. Using a robust HPLC-based assay to monitor wall teichoic acid glycosyltransferase activity, we have explored the sugar donor and acceptor specificity of the enzyme and have investigated its steady state kinetic mechanism. TagE showed a strong kinetic preference for UDP-glucose as its sugar donor and utilized a sequential (ternary complex) kinetic mechanism to catalyze the addition of glucose onto acceptor substrates. This study unambiguously establishes TagE as the wall teichoic acid glycosyltransferase in B. subtilis 168. Strains, plasmids, and oligonucleotides used in this work are listed in Table 1. Escherichia coli and B. subtilis strains were grown in Luria-Bertani (LB) medium. Ampicillin was used at a concentration of 50 μg/ml (E. coli), whereas spectinomycin was used at a concentration of 150 μg/ml (B. subtilis). HotStar TaqPCR reagents, gel extraction, and plasmid miniprep kits were purchased from Qiagen (Mississauga, Canada). Vent polymerase was obtained from New England Biolabs (Beverly, MA), the Expand PCR system was purchased from Roche Applied Science, and the GatewayTM cloning system was from Invitrogen. Cloning was performed in the E. coli strain Novablue (Novagen, Madison, WI) according to established protocols (20Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989: 1.47-1.84Google Scholar). B. subtilis competent cells were prepared and transformed as described previously (21Cutting S.M. Vander Horn P.B. Harwood C.R. Cutting S.M. Molecular Biological Methods for Bacillus. John Wiley & Sons, Inc., New York1990: 175-209Google Scholar). SPO1 phage was obtained from the Bacillus Genetic Stock Center (Ohio State University, Columbus, OH). All chemicals were purchased from Sigma unless otherwise specified. UDP-[14C]glucose and scintillation fluid were purchased from PerkinElmer Life Sciences. CDP-glycerol was synthesized according to established methods (22Badurina D.S. Zolli-Juran M. Brown E.D. Biochim. Biophys. Acta. 2003; 1646: 196-206Crossref PubMed Scopus (35) Google Scholar). MnaA, TarA, TagB, TarD, and TagF were purified as described previously (10Bhavsar A.P. Truant R. Brown E.D. J. Biol. Chem. 2005; 280: 36691-36700Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar, 12Schertzer J.W. Brown E.D. J. Biol. Chem. 2003; 278: 18002-18007Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar, 22Badurina D.S. Zolli-Juran M. Brown E.D. Biochim. Biophys. Acta. 2003; 1646: 196-206Crossref PubMed Scopus (35) Google Scholar, 23Pereira M.P. Schertzer J.W. D'Elia M.A. Koteva K.P. Hughes D.W. Wright G.D. Brown E.D. Chembiochem. 2008; 9: 1385-1390Crossref PubMed Scopus (29) Google Scholar). Chromatography was performed on a Waters HPLC system (Mississauga, Canada).TABLE 1Strains, plasmids, and oligonucleotides used in this studyStrain, plasmid, or oligonucleotideDescriptionSourceStrainsNovablueGeneral E. coli cloning strain (endA1 hsdR17(rK12− mK12+)supE44 thi-1 recA1 gyrA96 relA1 lacF′ [proA+ B+ lacIqZΔM15::Tn10(TcR)])NovagenEB863E. coli strain used for protein overexpression (F− ompT hsdSB(rB− mB−) gal dcm araB::T7RNAP-tetA)InvitrogenEB6Wild-type B. subtilis (hisA1 argC2 metC3)Ref. 34Bhavsar A.P. Beveridge T.J. Brown E.D. J. Bacteriol. 2001; 183: 6688-6693Crossref PubMed Scopus (51) Google ScholarEB2252tagE deletion strain derived from EB6 (hisA1 argC2 metC3 tagE::spec)This studyPlasmidspUS19pUC19 derivative used as a source for a specR cassetteRef. 35Benson A.K. Haldenwang W.G. J. Bacteriol. 1993; 175: 2347-2356Crossref PubMed Google ScholarpDEST17-tagEExpression plasmid for N-terminal His6-tagged TagEThis studyOligonucleotidestagE-F5′-ggggacaagtttctacaaaaaagcaggcttcttgtctttacatgcggtgagtgaatc-3′tagE-R5′-ggggaccactttgtacaagaaagctgggtcttaactctcttttatttccgtgaccctc-3′tagE-a5′-ggctatagtcgtttactctgatac-3′tagE-b5′-ctataaactatttaaataacagatttaaaaaattataaacagttaaaggcaatttctcttgg-3′tagE-c5′-attaatttgttcgtatgtattcaaatatatcctcctcactttttttactccctttcggcatcta-3′tagE-d5′-gttaagttactgttaacataaggaata-3′spec-F5′-agtgaggaggatatatttgaatac-3′spec-R5′-ttataatttttttaatctgttat-3′ Open table in a new tab To create a clean ΔtagE strain, primers tagE-a and tagE-b, tagE-c and tagE-d, and spec-F and spec-R were used with Vent polymerase to amplify chromosomal DNA or plasmid DNA in the latter case. The PCR products were purified and used as templates in a final reaction with primers tagE-a and tagE-d to create a product wherein a spectinomycin resistance cassette beginning at its translational start site and lacking a terminator was flanked by 1-kb regions surrounding the tagE locus. The 3-kb PCR product was transformed into EB6 to create a tagE deletion strain (EB2252). The resulting strain was confirmed by PCR with spectinomycin cassette-specific primers and primers designed to anneal to sequences outside the region of recombination. The ΔtagE strain was also examined for resistance to bacteriophage SPO1. A liquid culture of wild-type B. subtilis 168 and the ΔtagE strain was grown overnight at 30 °C in LB medium. An aliquot of both cultures was streaked onto an LB-agar plate to form a lawn using a sterile cotton swab, and then 10 μl of SPO1 bacteriophage was spotted onto the plate. Plates were incubated overnight at 37 °C and then examined for a clear zone of lysis. Strains were grown overnight in 100 ml of LB medium at 30 °C. Cell wall isolation and phosphate content determination were carried out as described previously (24Bhavsar A.P. Erdman L.K. Schertzer J.W. Brown E.D. J. Bacteriol. 2004; 186: 7865-7873Crossref PubMed Scopus (83) Google Scholar). Teichoic acid was released from peptidoglycan by treatment with 1% acetic acid (95 °C, 1 h). Subsequent purifications were carried out by size exclusion chromatography using a Bio-Gel P-6 column calibrated with blue dextran. The detection of carbohydrate material was accomplished using a phenol-sulfuric acid assay (25Westphal O. Jann K. Methods Carbohydr. Chem. 1965; 5: 83-91Google Scholar). One- and two-dimensional 1H and 31P NMR spectra were recorded on a Bruker NSC 600 spectrometer. The temperature was kept at 300 K in all experiments. Prior to performing the NMR experiments, the samples were lyophilized three times with D2O (99.9%). Trimethylsilyl propionate (δH 0.00, δC 0.0) in D2O was used as a reference for both 1H and 13C experiments. Orthophosphoric acid (δP 0.0) was used as the external reference for the 31P NMR experiments. The GatewayTM recombination-based cloning system and primers tagE-F and tagE-R were used to create a pDEST17-tagE vector for the expression of N-terminal hexahistidine-tagged TagE. The plasmid was transformed into E. coli BL21(AI) cells (Invitrogen). The sequence of tagE inserted into pDEST17-tagE was confirmed by sequencing. E. coli BL21(AI) cells harboring pDEST17-tagE were grown at 37 °C in LB medium supplemented with 50 μg/ml ampicillin to an A600 of 0.8. The culture was then induced with 0.2% (w/v) arabinose and grown for 20 h at 16 °C. The cells were harvested by centrifugation (8000 × g for 15 min) and then washed with 0.85% NaCl. Cells were resuspended in purification buffer (20 mm sodium phosphate, pH 7.2, 500 mm NaCl, and 5% glycerol) containing 0.1 mg/ml DNase I, 0.1 mg/ml RNase A, and Calbiochem Protease Inhibitor Mixture Set III (Roche Applied Science). Cells were lysed by passage through a cell disruptor, and then the lysate was spun at 20,000 × g for 15 min. The pellet was resuspended in purification buffer, and CHAPS was added to a final concentration of 2% (w/v). The resuspended pellet was then incubated for 1 h at 4 °C with gentle rocking. Following centrifugation at 20,000 × g for 15 min, the supernatant was filtered through a 0.45-μm filter and applied to a 5-ml Hi-Trap His column (Amersham Biosciences) pre-equilibrated in purification buffer containing 15 mm imidazole. TagE was eluted in a stepwise manner in purification buffer containing 25, 50, and 400 mm imidazole. Fractions were visualized by Coomassie-stained SDS-PAGE, and pure fractions of TagE were pooled and dialyzed overnight in dialysis buffer containing 20 mm Tris, pH 7.5, 150 mm NaCl, 1 mm DTT, and 10% glycerol. Following dialysis, the purified protein was separated into aliquots and stored at −80 °C. Soluble lipid α, β, and ϕ.1 analogues were synthesized according to established methods (9Ginsberg C. Zhang Y.H. Yuan Y. Walker S. ACS Chem. Biol. 2006; 1: 25-28Crossref PubMed Scopus (60) Google Scholar, 23Pereira M.P. Schertzer J.W. D'Elia M.A. Koteva K.P. Hughes D.W. Wright G.D. Brown E.D. Chembiochem. 2008; 9: 1385-1390Crossref PubMed Scopus (29) Google Scholar). Reactions for the synthesis of the lipid ϕ.40 analog contained 50 mm Tris, pH 8.0, 30 mm MgCl2, 100 nm TagF, and 39 equivalents of CDP-glycerol per lipid ϕ.1 analog. Lipid ϕ.n analogues ranging from 5 to 80 glycerol phosphate units were synthesized by varying the ratio of CDP-glycerol molecules to the lipid ϕ.1 analog. Reaction progress was determined by monitoring the conversion of CDP-glycerol to CMP at 271 nm by previously described methods (23Pereira M.P. Schertzer J.W. D'Elia M.A. Koteva K.P. Hughes D.W. Wright G.D. Brown E.D. Chembiochem. 2008; 9: 1385-1390Crossref PubMed Scopus (29) Google Scholar). All reactions were allowed to proceed to near completion before being filtered through a 30,000 molecular weight cut-off centrifugal filter (Millipore, Billerica, MA). A polymer containing only glycerol phosphate residues was synthesized as reported previously by incubating 4 mm CDP-glycerol with 100 nm TagF in a buffer containing 50 mm Tris, pH 8.0, and 30 mm MgCl2 at room temperature overnight (26Schertzer J.W. Brown E.D. J. Bacteriol. 2008; 190: 6940-6947Crossref PubMed Scopus (17) Google Scholar). Reactions were conducted at room temperature in buffer containing 50 mm Tris, pH 7.4, 30 mm MgCl2, and TagE (1–50 nm) with UDP-glucose as the sugar donor and the lipid ϕ.40 analog as the acceptor unless otherwise specified. Reactions were quenched by the addition of urea to a final concentration of 6 m. Substrates and products of the TagE reaction were separated by reversed phase chromatography on an Inertsil ODS-3 column (Canadian Life Sciences, Peterborough, Canada) with the ion pairing agent tetrabutylammonium hydrogen sulfate. Each sample was eluted at a flow rate of 1 ml/min using a linear gradient of buffer PicA (15 mm potassium phosphate, pH 7.0, 10 mm tetrabutylammonium hydrogen sulfate) to PicB (15 mm potassium phosphate, pH 7.0, 10 mm tetrabutylammonium hydrogen sulfate, 30% (v/v) acetonitrile). UDP-glucose and UDP were detected by absorbance at 262 nm, and turnover was calculated on the basis of the ratio of the integrated peaks. For reactions containing UDP-[14C]glucose, substrates and products were separated on a Waters Shodex KW-803 column in buffer containing 0.1% ammonium hydrogen carbonate and 10% acetonitrile and detected by inline scintillation counting. All initial rate data were fitted by non-linear least squares regression to the equations in either SigmaPlot 8.0 or the Enzyme Kinetics Module 1.1 (SPSS Inc., Chicago, IL). The Michaelis-Menten equation (Equation 1) and equations that describe sequential (Equation 2) and ping-pong (Equation 3) mechanisms are given below. V=Vmax[S]Km+[S]Eq. 1 V=Vmax[A][B]KjaKmb+Kmb[A]+Kma[B]+[A][B]Eq. 2 V=Vmax[A][B]Kmb[A]+Kma[B]+[A][B]Eq. 3 A and B are the reactants, Kma and Kmb are the Michaelis constants for A and B, and Kia is the dissociation constant for A from the enzyme complex EA (27Cleland W.W. Boyer P.D. The Enzymes. Academic Press, Inc., New York1970: 1-65Google Scholar). A 14C-lipid ϕ.5 analog was synthesized by incubating a lipid ϕ.1 analog (100 μm) with a mixture of CDP-glycerol (300 μm) and [14C]CDP-glycerol (100 μm at 0.01 μCi/μl) in a reaction buffer containing 50 mm Tris, pH 8.0, 30 mm MgCl2 and 100 nm TagF. Reactions were allowed to proceed to completion, and the 14C-lipid ϕ.5 analog (30 μm) was subsequently incubated with 4 mm UDP-glucose and 50 nm TagE for 3 h. Reaction progress was determined by paired ion chromatography-HPLC at 262 nm. The non-glycosylated and glycosylated 14C-lipid ϕ.5 analogues were filtered through a 30,000 molecular weight cut-off centrifugal filter (Millipore, Billerica, MA) and then incubated with 4 mm unlabeled CDP-glycerol and 100 nm TagF for 5 h at room temperature. Reaction substrates and products were separated by size exclusion chromatography using a Waters Shodex KW-803 column in buffer containing 0.1% ammonium hydrogen carbonate and 10% acetonitrile at 0.5 ml/min. All injections contained 0.1 μCi of radiolabeled substrate, and reaction products were visualized by inline scintillation counting. To determine whether tagE codes for the wall teichoic acid glycosyltransferase, we created a ΔtagE strain by allelic replacement of tagE with a spectinomycin resistance cassette. Given that tagDEF are encoded in an operon, we left the last 26 bp of the tagE coding sequence intact to ensure that the native ribosome binding site of tagF located in the 3′-end of tagE was not disrupted. Phosphate analysis showed that the ΔtagE strain contained wild-type levels of phosphate in its cell wall (Fig. 2A), confirming that there were no polar effects on tagF. Furthermore, the ΔtagE strain exhibited no major changes in morphology or growth kinetics compared with the wild-type parental strain (data not shown). We then tested both strains for resistance to bacteriophage SPO1, which recognizes glycosylated teichoic acid as a receptor (28Yasbin R.E. Maino V.C. Young F.E. J. Bacteriol. 1976; 125: 1120-1126Crossref PubMed Google Scholar). B. subtilis 168 was susceptible to SPO1 phage, whereas the ΔtagE strain was resistant (Fig. 2B). This strongly suggests that deletion of tagE leads to the loss of glucose along the wall teichoic acid polymer. To confirm this, we isolated wall teichoic acid from B. subtilis 168 and the ΔtagE strain and analyzed the polymers by 1H NMR. As shown in Fig. 2C, the 1H NMR spectrum revealed an anomeric proton signal at δ5.07 (J1,2 2.1 Hz) that could be assigned to α-glucose at the C-2 position in wall teichoic acid isolated from wild-type B. subtilis 168. This finding is consistent with the previous stereochemical assignment of the glucose linkage (19Glaser L. Burger M.M. J. Biol. Chem. 1964; 239: 3187-3191Abstract Full Text PDF PubMed Google Scholar). By contrast, this signal was absent in the 1H NMR spectrum of wall teichoic acid from the ΔtagE strain (Fig. 2C). Taken together, these results demonstrate that tagE is involved in wall teichoic acid glycosylation in B. subtilis 168. Having confirmed a role for TagE in wall teichoic acid glycosylation, we sought to investigate the activity of the enzyme in vitro. The reaction catalyzed by TagE in our in vitro assay is depicted in Scheme 1. Recombinant TagE that had been purified to homogeneity (supplemental Fig. S1) was incubated with the activated sugar donor UDP-glucose and a soluble analog of lipid ϕ.40, the product of the TagF reaction. The nomenclature of lipid-linked teichoic acid intermediates is summarized in Table 2 (29Pereira M.P. Brown E.D. Moran A.B. Hoist P. Itzstein O. Microbial Glycobiology: Structures, Relevance and Applications. Elsevier Science Publishing Co., Inc., New York2008: 337-350Google Scholar). The lipid ϕ.40 analog consists of 40 glycerol phosphate units that are in a 1,3-linkage and attached to a lipid ϕ.1 analog. We chose to synthesize a poly(glycerol phosphate) polymer of this length given that wall teichoic acid polymers in the cell wall of B. subtilis 168 typically contain 30–50 units of glycerol phosphate (12Schertzer J.W. Brown E.D. J. Biol. Chem. 2003; 278: 18002-18007Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar). The transfer of glucose from UDP-glucose onto the lipid ϕ.40 analog was monitored using an HPLC-based assay that measures UDP production. Using radiolabeled UDP-[14C]glucose, we confirmed that the production of UDP in this assay was stoichiometric with the transfer of glucose from UDP-glucose onto the acceptor (data not shown). We took great care to ensure that, under our assay conditions, the lipid ϕ.40 analog-dependent production of UDP was linear with both time and the amount of enzyme added (Fig. 3). By analyzing the dependence of the reaction velocity on enzyme concentration, we estimated a turnover of 16 s−1 for TagE under conditions where both substrates were saturating.TABLE 2Nomenclature for wall teichoic acid intermediates (29Pereira M.P. Brown E.D. Moran A.B. Hoist P. Itzstein O. Microbial Glycobiology: Structures, Relevance and Applications. Elsevier Science Publishing Co., Inc., New York2008: 337-350Google Scholar)EnzymeSubstrate or substrate analogChemical compositionaLipid ϕ.1 is the product of the TagB reaction and contains a single glycerol phosphate unit. Lipid ϕ.1 serves as a substrate for TagF, which catalyzes the addition of n glycerol phosphate units. und, undecaprenyl; P, phosphate; GlcNAc, N-acetylglucosamine; ManNAc, N-acetylmannosamine; GroP, glycerol phosphate.TagALipid αGlcNAc-1-P-P-undTagBLipid βManNAc-β-(1–4)-GlcNAc-1-P-P-undTagFLipid ϕ.n(GroP)n-ManNAc-β-(1–4)-GlcNAc-1-P-P-undTagALipid α analogGlcNAc-1-P-P-tridecaneTagBLipid β analogManNAc-β-(1–4)-GlcNAc-1-P-P-tridecaneTagFLipid ϕ.n analog(GroP)n-ManNAc-β-(1–4)-GlcNAc-1-P-P-tridecanea Lipid ϕ.1 is the product of the TagB reaction and contains a single glycerol phosphate unit. Lipid ϕ.1 serves as a substrate for TagF, which catalyzes the addition of n glycerol phosphate units. und, undecaprenyl; P, phosphate; GlcNAc, N-acetylglucosamine; ManNAc, N-acetylmannosamine; GroP, glycerol phosphate. Open table in a new tab FIGURE 3Dependence of TagE activity on time and enzyme concentration. Reactions contained 3 mm UDP-glucose, 15 μm lipid ϕ.40 analog, and 1 (●), 2.5 (○), 5 (▴), or 10 nm (△) TagE. Reactions were quenched with urea to a final concentration of 6 m following 1-, 3-, 6-, and 12-min incubations. The conversion of UDP-glucose to UDP was monitored at 262 nm following separation by paired ion HPLC. Inset, plot of initial velocity versus TagE conce" @default.
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