Glycosylation of Wall Teichoic Acid in Staphylococcus aureus by TarM

Wall teichoic acid (WTA) glycopolymers are major constituents of cell envelopes in Staphylococcus aureus and related Gram-positive bacteria with important roles in cell wall maintenance, susceptibility to antimicrobial molecules, biofilm formation, and host interaction. Most S. aureus strains express polyribitol phosphate WTA substituted with d-alanine and N-acetylglucosamine (GlcNAc). WTA sugar modifications are highly variable and have been implicated in bacteriophage susceptibility and immunogenicity, but the pathway and enzymes of staphylococcal WTA glycosylation have remained unknown. Revisiting the structure of S. aureus RN4220 WTA by NMR analysis revealed the presence of canonical polyribitol phosphate WTA bearing only α-linked GlcNAc substituents. A RN4220 transposon mutant resistant to WTA-dependent phages was identified and shown to produce altered WTA, which exhibited faster electrophoretic migration and lacked completely the WTA α-GlcNAc residues. Disruption of a gene of unknown function, renamed tarM, was responsible for this phenotype. Recombinant TarM was capable of glycosylating WTA in vitro in a UDP-GlcNAc-dependent manner, thereby confirming its WTA GlcNAc-transferase activity. Deletion of the last seven amino acids from the C terminus abolished the activity of TarM. tarM-related genes were found in the genomes of several WTA-producing bacteria, suggesting that TarM-mediated WTA glycosylation is a general pathway in Gram-positive bacteria. Our study represents a basis for dissecting the biosynthesis and function of glycosylated WTA in S. aureus and other bacteria. Wall teichoic acid (WTA) glycopolymers are major constituents of cell envelopes in Staphylococcus aureus and related Gram-positive bacteria with important roles in cell wall maintenance, susceptibility to antimicrobial molecules, biofilm formation, and host interaction. Most S. aureus strains express polyribitol phosphate WTA substituted with d-alanine and N-acetylglucosamine (GlcNAc). WTA sugar modifications are highly variable and have been implicated in bacteriophage susceptibility and immunogenicity, but the pathway and enzymes of staphylococcal WTA glycosylation have remained unknown. Revisiting the structure of S. aureus RN4220 WTA by NMR analysis revealed the presence of canonical polyribitol phosphate WTA bearing only α-linked GlcNAc substituents. A RN4220 transposon mutant resistant to WTA-dependent phages was identified and shown to produce altered WTA, which exhibited faster electrophoretic migration and lacked completely the WTA α-GlcNAc residues. Disruption of a gene of unknown function, renamed tarM, was responsible for this phenotype. Recombinant TarM was capable of glycosylating WTA in vitro in a UDP-GlcNAc-dependent manner, thereby confirming its WTA GlcNAc-transferase activity. Deletion of the last seven amino acids from the C terminus abolished the activity of TarM. tarM-related genes were found in the genomes of several WTA-producing bacteria, suggesting that TarM-mediated WTA glycosylation is a general pathway in Gram-positive bacteria. Our study represents a basis for dissecting the biosynthesis and function of glycosylated WTA in S. aureus and other bacteria. IntroductionThe bacterial cell envelope represents an interface for the interaction of bacterial cells with host molecules, bacteriophages, other bacteria, and inanimate molecules or surfaces. The properties of bacterial cell surfaces are largely governed by proteins and glycopolymers, both of which have highly variable and often strain-specific compositions and roles (1Clarke S.R. Foster S.J. Adv. Microb. Physiol. 2006; 51: 187-224Crossref PubMed Scopus (207) Google Scholar, 2Weidenmaier C. Peschel A. Nat. Rev. Microbiol. 2008; 6: 276-287Crossref PubMed Scopus (514) Google Scholar). Although many bacterial surface proteins have been studied in detail, our knowledge on structures, biosynthetic pathways, and functions of cell wall glycopolymers is still incomplete. Nevertheless, it has become clear that many bacterial cell wall glycopolymers are crucial for bacterial cell wall maintenance, fitness, or virulence and represent promising targets for vaccines or antimicrobial compounds (2Weidenmaier C. Peschel A. Nat. Rev. Microbiol. 2008; 6: 276-287Crossref PubMed Scopus (514) Google Scholar).Staphylococcus aureus and most other Gram-positive bacteria express teichoic acid polymers at their surfaces that are either linked to membrane lipids (lipoteichoic acid) or to peptidoglycan (wall teichoic acid (WTA) 2The abbreviations used are: WTAwall teichoic acidTagteichoic acid glycerol phosphateTarteichoic acid ribitol phosphate typeRbo-Ppolyribitol phosphateWGAwheat germ agglutininMES2-(4-morpholino)-ethane-sulfonic acidCOSY1H,1H correlation spectroscopyTOCSYtotal correlation spectroscopyROESYrotating-frame nuclear Overhauser enhancement spectroscopyHSQC-DEPT13C,1H single quantum correlation distortionless enhancement by polarization transferHMBCheteronuclear multiple bond correlation experiment1H,31P HMQC-TOCSY1H,31P multiple quantum correlation-total correlation spectroscopyHRPhorseradish peroxidaseGLCgas-liquid chromatographyTricineN-2-hydroxy-1,1-bis(hydroxymethyl)ethylglycine.) (3Xia G. Kohler T. Peschel A. Int. J. Med. Microbiol. 2010; 300: 148-154Crossref PubMed Scopus (163) Google Scholar). WTA is composed of phosphate-containing repeating units with a very variable and often species-specific composition. Most S. aureus strains express polyribitol phosphate (Rbo-P)-type WTA, which is composed of repetitive d-ribitol units connected by 1,5-phosphodiester bonds (4Endl J. Seidl H.P. Fiedler F. Schleifer K.H. Arch. Microbiol. 1983; 135: 215-223Crossref PubMed Scopus (91) Google Scholar). The repeating units can be further substituted with d-alanine at C2-OH and/or with 2-acetamido-2-deoxy-d-glucopyranose (GlcNAc) at C4-OH via α- or β-glycosidic linkages whose relative abundance varies between individual strains (5Sanderson A.R. Strominger J.L. Nathenson S.G. J. Biol. Chem. 1962; 237: 3603-3613Abstract Full Text PDF PubMed Google Scholar, 6Vinogradov E. Sadovskaya I. Li J. Jabbouri S. Carbohydr. Res. 2006; 341: 738-743Crossref PubMed Scopus (49) Google Scholar). WTA accounts for a major portion of the total dry weight of the staphylococcal cell wall (7Neuhaus F.C. Baddiley J. Microbiol. Mol. Biol. Rev. 2003; 67: 686-723Crossref PubMed Scopus (739) Google Scholar) but does not play an essential role for S. aureus viability under laboratory conditions (8Weidenmaier 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 (417) Google Scholar, 9D'Elia M.A. Pereira M.P. Chung Y.S. Zhao W. Chau A. Kenney T.J. Sulavik M.C. Black T.A. Brown E.D. J. Bacteriol. 2006; 188: 4183-4189Crossref PubMed Scopus (156) Google Scholar), although important roles in biofilm formation and autolysin control have been documented (10Vergara-Irigaray M. Maira-Litrán T. Merino N. Pier G.B. Penadés J.R. Lasa I. Microbiology. 2008; 154: 865-877Crossref PubMed Scopus (75) Google Scholar, 11Schlag M. Biswas R. Krismer B. Kohler T. Zoll S. Schwarz H. Yu W. Peschel A. Gotz F. Mol. Microbiol. 2010; 75: 864-873Crossref PubMed Scopus (191) Google Scholar). Of note, WTA is of pivotal importance during host colonization and infection inasmuch as it facilitates attachment of the bacteria to WTA binding epithelial or endothelial cell receptors (8Weidenmaier 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 (417) Google Scholar, 12Weidenmaier C. Peschel A. Xiong Y.Q. Kristian S.A. Dietz K. Yeaman M.R. Bayer A.S. J. Infect. Dis. 2005; 191: 1771-1777Crossref PubMed Scopus (178) Google Scholar) and protects the bacterial cells from bactericidal agents such as skin antimicrobial fatty acids (13Kohler T. Weidenmaier C. Peschel A. J. Bacteriol. 2009; 191: 4482-4484Crossref PubMed Scopus (75) Google Scholar). Moreover, alanyl residues in teichoic acid play important roles in resistance to cationic antimicrobial host defense factors, antibiotics, and bacteriocins (14Peschel 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 (793) Google Scholar, 15Peschel A. Vuong C. Otto M. Götz F. Antimicrob. Agents Chemother. 2000; 44: 2845-2847Crossref PubMed Scopus (201) Google Scholar). In contrast, WTA sugar modifications have been implicated in the ability of WTA to elicit specific antibody responses (16Juergens W.G. Sanderson A.R. Strominger J.L. J. Exp. Med. 1963; 117: 925-935Crossref PubMed Scopus (14) Google Scholar, 17Nathenson S.G. Ishimoto N. Anderson J.S. Strominger J.L. J. Biol. Chem. 1966; 241: 651-658Abstract Full Text PDF PubMed Google Scholar, 18Torii M. Kabat E.A. Bezer A.E. J. Exp. Med. 1964; 120: 13-29Crossref PubMed Scopus (31) Google Scholar) and binding of bacteriophages to S. aureus (19Chatterjee A.N. Mirelman D. Singer H.J. Park J.T. J. Bacteriol. 1969; 100: 846-853Crossref PubMed Google Scholar, 20Shaw D.R. Mirelman D. Chatterjee A.N. Park J.T. J. Biol. Chem. 1970; 245: 5101-5106Abstract Full Text PDF PubMed Google Scholar), Bacillus subtilis (21Young F.E. Proc. Natl. Acad. Sci. U.S.A. 1967; 58: 2377-2384Crossref PubMed Scopus (86) Google Scholar, 22Glaser L. Ionesco H. Schaeffer P. Biochim. Biophys. Acta. 1966; 124: 415-417Crossref PubMed Scopus (28) Google Scholar), or Listeria species (23Wendlinger G. Loessner M.J. Scherer S. Microbiology. 1996; 142: 985-992Crossref PubMed Scopus (93) Google Scholar).Most steps of WTA backbone biosynthesis, which occurs on the universal lipid carrier undecaprenyl phosphate (C55-P), have recently been elucidated in B. subtilis 168 (24Schertzer J.W. Brown E.D. J. Biol. Chem. 2003; 278: 18002-18007Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar, 25Bhavsar A.P. Truant R. Brown E.D. J. Biol. Chem. 2005; 280: 36691-36700Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar, 26Ginsberg C. Zhang Y.H. Yuan Y. Walker S. ACS Chem. Biol. 2006; 1: 25-28Crossref PubMed Scopus (59) Google Scholar) and S. aureus (27Brown S. Zhang Y.H. Walker S. Chem. Biol. 2008; 15: 12-21Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar, 28Meredith T.C. Swoboda J.G. Walker S. J. Bacteriol. 2008; 190: 3046-3056Crossref PubMed Scopus (73) Google Scholar), and many of the involved enzymes have been characterized in vitro. The same holds true for the pathway of WTA and lipoteichoic acid alanylation, which consists of four proteins that activate d-alanine by ATP hydrolysis, link it to the dedicated carrier protein DltC, translocate it across the cytoplasmic membrane, and connect it with WTA or lipoteichoic acid repeating units (29Perego 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 (212) Google Scholar, 30Debabov D.V. Heaton M.P. Zhang Q. Stewart K.D. Lambalot R.H. Neuhaus F.C. J. Bacteriol. 1996; 178: 3869-3876Crossref PubMed Google Scholar). In contrast, WTA glycosylation has hardly been investigated. Nevertheless, WTA β-GlcNAc-transferase activity (EC 2.4.1.70) has been detected in crude cell extracts of S. aureus strain Copenhagen in the 1960s (31Nathenson S.G. Strominger J.L. J. Biol. Chem. 1963; 238: 3161-3169Abstract Full Text PDF PubMed Google Scholar), and a S. aureus strain H mutant 52B2 deficient in WTA GlcNAc-transferase activity has been described (20Shaw D.R. Mirelman D. Chatterjee A.N. Park J.T. J. Biol. Chem. 1970; 245: 5101-5106Abstract Full Text PDF PubMed Google Scholar), the genetic basis of which has remained unknown.Here we on the and of a S. aureus protein TarM that is responsible for the glycosylation of WTA with α-GlcNAc and UDP-GlcNAc-dependent WTA GlcNAc-transferase activity in vitro. Moreover, we that TarM a in the mutant S. aureus strain 52B2 that to a and TarM. Our study the for the function of the sugar residues by WTA in S. and host has been in most Gram-positive bacteria produce WTA or related cell wall glycopolymers, the for the and strain-specific in WTA have remained largely The involved in biosynthesis of WTA with or glycerol phosphate repeating units and with d-alanine have been elucidated (7Neuhaus F.C. Baddiley J. Microbiol. Mol. Biol. Rev. 2003; 67: 686-723Crossref PubMed Scopus (739) Google Scholar, A.P. Brown E.D. Mol. Microbiol. 2006; PubMed Scopus Google Scholar, J.G. J. T.C. Walker S. 2010; PubMed Scopus Google Scholar) and represent a basis for of or the most promising enzymes for study a that further the of the WTA biosynthetic TarM is the described the glycosylation of the WTA and the WTA whose function has been in vitro via a Moreover, we for the a NMR of WTA with α-GlcNAc from S. with the NMR of WTA with from strain E. Sadovskaya I. Li J. Jabbouri S. Carbohydr. Res. 2006; 341: 738-743Crossref PubMed Scopus (49) Google Scholar) our study a basis for dissecting the structure and function of glycosylated WTA in S. WTA with d-alanine occurs of the across the cytoplasmic membrane, it has remained and WTA is with sugar residues. The that TarM a and is a protein that it in the Moreover, its on the sugar which is not in the cell the that WTA glycosylation by TarM the WTA is to the of the cytoplasmic was in vitro in the of other and it on WTA not connected to interaction with to be for TarM to the to be TarM of the of the WTA biosynthetic whose have been shown to a in B. subtilis A. R. P. J. J. Bacteriol. 2008; 190: PubMed Scopus (75) Google Scholar) or TarM with the WTA or (27Brown S. Zhang Y.H. Walker S. Chem. Biol. 2008; 15: 12-21Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar, 28Meredith T.C. Swoboda J.G. Walker S. J. Bacteriol. 2008; 190: 3046-3056Crossref PubMed Scopus (73) Google Scholar) in note, the that the and 52B2 and of WTA not that the WTA by and and the unknown that the to peptidoglycan are not in their by the presence or of GlcNAc on the repeating represents a to the teichoic acid of which has recently been shown to be to translocate polymers the M. A.S. A. A. W. J. Bacteriol. PubMed Scopus Google Scholar, W. A. Mol. Microbiol. PubMed Scopus Google is related to the of of which and C. T. B. Res. 2009; PubMed Scopus Google Scholar). TarM does not with or bacterial GlcNAc-transferase with activity and to represent a of Moreover, we proteins with clear to TarM in several other Gram-positive bacteria that TarM is of a used Of note, related proteins were from bacteria to produce glycosylated WTA such as Staphylococcus and Listeria that bacteria have to the protein with we found the most protein proteins from S. to be a with from Listeria whose WTA structure has been protein and with TarM its we not TarM in other Listeria that WTA glycosylation is a species-specific in the Nevertheless, several genes involved in WTA glycosylation have been identified in L. whose WTA is composed of repeating units of the GlcNAc can be further substituted with or at the or of K. I. I. J. PubMed Scopus Google Scholar). genes the Fiedler F. S. J. Bacteriol. PubMed Scopus Google Scholar), and J.R. E.A. Infect. 2008; PubMed Scopus Google Scholar) and a protein of unknown function N. Fiedler F. P. S. S. J. Bacteriol. 1999; PubMed Google of which with WTA from B. subtilis strain is a L. Ionesco H. Schaeffer P. Biochim. Biophys. Acta. 1966; 124: 415-417Crossref PubMed Scopus (28) Google Scholar) with a structure as the described in this study for S. aureus strain RN4220 for the sugar identified a TarM with protein in B. subtilis that and with RN4220 TarM. its gene is in the of the WTA and acid gene of B. subtilis it is to be a involved in the of residues to the WTA The protein and of B. subtilis 168 has been implicated in the of residues to glycerol phosphate WTA by genetic C. M. P. D. Mol. PubMed Scopus (59) Google Scholar, Stewart Mol. Microbiol. PubMed Scopus (36) Google Scholar). The of is and with of TarM were found in Staphylococcus Staphylococcus Staphylococcus and Bacillus whose WTA molecules are composed of either glycerol of other repeating units (4Endl J. Seidl H.P. Fiedler F. Schleifer K.H. Arch. Microbiol. 1983; 135: 215-223Crossref PubMed Scopus (91) Google Scholar), or have not been of TarM proteins the of the proteins suggesting that the for the is governed by only in or structure of of the structure of TarM to the basis of and of have that S. aureus strains produce WTA with either only α-GlcNAc or or with both of linkages S.G. Ishimoto N. Anderson J.S. Strominger J.L. J. Biol. Chem. 1966; 241: 651-658Abstract Full Text PDF PubMed Google Scholar, S.G. Strominger J.L. J. Biol. Chem. 1962; 237: Full Text PDF PubMed Google Scholar). Of note, have for the of WTA to elicit specific (16Juergens W.G. Sanderson A.R. Strominger J.L. J. Exp. Med. 1963; 117: 925-935Crossref PubMed Scopus (14) Google Scholar) found that WTA of S. aureus Copenhagen in that were only to but not to the is to that the of WTA with GlcNAc in to the of S. aureus that the ability of host to a In it that the WTA GlcNAc residues to staphylococcal to epithelial and endothelial cells via interaction with host in a (8Weidenmaier 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 (417) Google Scholar, 12Weidenmaier C. Peschel A. Xiong Y.Q. Kristian S.A. Dietz K. Yeaman M.R. Bayer A.S. J. Infect. Dis. 2005; 191: 1771-1777Crossref PubMed Scopus (178) Google Scholar, C. Kokai-Kun J.F. E. Kohler T. G. H. Götz F. Peschel A. Int. J. Med. Microbiol. 2008; PubMed Scopus Google Scholar), which is of the of binding to S. aureus to the binding and roles of the WTA GlcNAc in our study a in the knowledge on WTA biosynthesis and represents a basis for on the role of WTA glycosylation in interaction. the of phages as antimicrobial agents such as S. it and important to the basis of and In this our be of importance for the of proteins and enzymes from staphylococcal phages that be used as antimicrobial IntroductionThe bacterial cell envelope represents an interface for the interaction of bacterial cells with host molecules, bacteriophages, other bacteria, and inanimate molecules or surfaces. The properties of bacterial cell surfaces are largely governed by proteins and glycopolymers, both of which have highly variable and often strain-specific compositions and roles (1Clarke S.R. Foster S.J. Adv. Microb. Physiol. 2006; 51: 187-224Crossref PubMed Scopus (207) Google Scholar, 2Weidenmaier C. Peschel A. Nat. Rev. Microbiol. 2008; 6: 276-287Crossref PubMed Scopus (514) Google Scholar). Although many bacterial surface proteins have been studied in detail, our knowledge on structures, biosynthetic pathways, and functions of cell wall glycopolymers is still incomplete. Nevertheless, it has become clear that many bacterial cell wall glycopolymers are crucial for bacterial cell wall maintenance, fitness, or virulence and represent promising targets for vaccines or antimicrobial compounds (2Weidenmaier C. Peschel A. Nat. Rev. Microbiol. 2008; 6: 276-287Crossref PubMed Scopus (514) Google Scholar).Staphylococcus aureus and most other Gram-positive bacteria express teichoic acid polymers at their surfaces that are either linked to membrane lipids (lipoteichoic acid) or to peptidoglycan (wall teichoic acid (WTA) 2The abbreviations used are: WTAwall teichoic acidTagteichoic acid glycerol phosphateTarteichoic acid ribitol phosphate typeRbo-Ppolyribitol phosphateWGAwheat germ agglutininMES2-(4-morpholino)-ethane-sulfonic acidCOSY1H,1H correlation spectroscopyTOCSYtotal correlation spectroscopyROESYrotating-frame nuclear Overhauser enhancement spectroscopyHSQC-DEPT13C,1H single quantum correlation distortionless enhancement by polarization transferHMBCheteronuclear multiple bond correlation experiment1H,31P HMQC-TOCSY1H,31P multiple quantum correlation-total correlation spectroscopyHRPhorseradish peroxidaseGLCgas-liquid chromatographyTricineN-2-hydroxy-1,1-bis(hydroxymethyl)ethylglycine.) (3Xia G. Kohler T. Peschel A. Int. J. Med. Microbiol. 2010; 300: 148-154Crossref PubMed Scopus (163) Google Scholar). WTA is composed of phosphate-containing repeating units with a very variable and often species-specific composition. Most S. aureus strains express polyribitol phosphate (Rbo-P)-type WTA, which is composed of repetitive d-ribitol units connected by 1,5-phosphodiester bonds (4Endl J. Seidl H.P. Fiedler F. Schleifer K.H. Arch. Microbiol. 1983; 135: 215-223Crossref PubMed Scopus (91) Google Scholar). The repeating units can be further substituted with d-alanine at C2-OH and/or with 2-acetamido-2-deoxy-d-glucopyranose (GlcNAc) at C4-OH via α- or β-glycosidic linkages whose relative abundance varies between individual strains (5Sanderson A.R. Strominger J.L. Nathenson S.G. J. Biol. Chem. 1962; 237: 3603-3613Abstract Full Text PDF PubMed Google Scholar, 6Vinogradov E. Sadovskaya I. Li J. Jabbouri S. Carbohydr. Res. 2006; 341: 738-743Crossref PubMed Scopus (49) Google Scholar). WTA accounts for a major portion of the total dry weight of the staphylococcal cell wall (7Neuhaus F.C. Baddiley J. Microbiol. Mol. Biol. Rev. 2003; 67: 686-723Crossref PubMed Scopus (739) Google Scholar) but does not play an essential role for S. aureus viability under laboratory conditions (8Weidenmaier 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 (417) Google Scholar, 9D'Elia M.A. Pereira M.P. Chung Y.S. Zhao W. Chau A. Kenney T.J. Sulavik M.C. Black T.A. Brown E.D. J. Bacteriol. 2006; 188: 4183-4189Crossref PubMed Scopus (156) Google Scholar), although important roles in biofilm formation and autolysin control have been documented (10Vergara-Irigaray M. Maira-Litrán T. Merino N. Pier G.B. Penadés J.R. Lasa I. Microbiology. 2008; 154: 865-877Crossref PubMed Scopus (75) Google Scholar, 11Schlag M. Biswas R. Krismer B. Kohler T. Zoll S. Schwarz H. Yu W. Peschel A. Gotz F. Mol. Microbiol. 2010; 75: 864-873Crossref PubMed Scopus (191) Google Scholar). Of note, WTA is of pivotal importance during host colonization and infection inasmuch as it facilitates attachment of the bacteria to WTA binding epithelial or endothelial cell receptors (8Weidenmaier 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 (417) Google Scholar, 12Weidenmaier C. Peschel A. Xiong Y.Q. Kristian S.A. Dietz K. Yeaman M.R. Bayer A.S. J. Infect. Dis. 2005; 191: 1771-1777Crossref PubMed Scopus (178) Google Scholar) and protects the bacterial cells from bactericidal agents such as skin antimicrobial fatty acids (13Kohler T. Weidenmaier C. Peschel A. J. Bacteriol. 2009; 191: 4482-4484Crossref PubMed Scopus (75) Google Scholar). Moreover, alanyl residues in teichoic acid play important roles in resistance to cationic antimicrobial host defense factors, antibiotics, and bacteriocins (14Peschel 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 (793) Google Scholar, 15Peschel A. Vuong C. Otto M. Götz F. Antimicrob. Agents Chemother. 2000; 44: 2845-2847Crossref PubMed Scopus (201) Google Scholar). In contrast, WTA sugar modifications have been implicated in the ability of WTA to elicit specific antibody responses (16Juergens W.G. Sanderson A.R. Strominger J.L. J. Exp. Med. 1963; 117: 925-935Crossref PubMed Scopus (14) Google Scholar, 17Nathenson S.G. Ishimoto N. Anderson J.S. Strominger J.L. J. Biol. Chem. 1966; 241: 651-658Abstract Full Text PDF PubMed Google Scholar, 18Torii M. Kabat E.A. Bezer A.E. J. Exp. Med. 1964; 120: 13-29Crossref PubMed Scopus (31) Google Scholar) and binding of bacteriophages to S. aureus (19Chatterjee A.N. Mirelman D. Singer H.J. Park J.T. J. Bacteriol. 1969; 100: 846-853Crossref PubMed Google Scholar, 20Shaw D.R. Mirelman D. Chatterjee A.N. Park J.T. J. Biol. Chem. 1970; 245: 5101-5106Abstract Full Text PDF PubMed Google Scholar), Bacillus subtilis (21Young F.E. Proc. Natl. Acad. Sci. U.S.A. 1967; 58: 2377-2384Crossref PubMed Scopus (86) Google Scholar, 22Glaser L. Ionesco H. Schaeffer P. Biochim. Biophys. Acta. 1966; 124: 415-417Crossref PubMed Scopus (28) Google Scholar), or Listeria species (23Wendlinger G. Loessner M.J. Scherer S. Microbiology. 1996; 142: 985-992Crossref PubMed Scopus (93) Google Scholar).Most steps of WTA backbone biosynthesis, which occurs on the universal lipid carrier undecaprenyl phosphate (C55-P), have recently been elucidated in B. subtilis 168 (24Schertzer J.W. Brown E.D. J. Biol. Chem. 2003; 278: 18002-18007Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar, 25Bhavsar A.P. Truant R. Brown E.D. J. Biol. Chem. 2005; 280: 36691-36700Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar, 26Ginsberg C. Zhang Y.H. Yuan Y. Walker S. ACS Chem. Biol. 2006; 1: 25-28Crossref PubMed Scopus (59) Google Scholar) and S. aureus (27Brown S. Zhang Y.H. Walker S. Chem. Biol. 2008; 15: 12-21Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar, 28Meredith T.C. Swoboda J.G. Walker S. J. Bacteriol. 2008; 190: 3046-3056Crossref PubMed Scopus (73) Google Scholar), and many of the involved enzymes have been characterized in vitro. The same holds true for the pathway of WTA and lipoteichoic acid alanylation, which consists of four proteins that activate d-alanine by ATP hydrolysis, link it to the dedicated carrier protein DltC, translocate it across the cytoplasmic membrane, and connect it with WTA or lipoteichoic acid repeating units (29Perego 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 (212) Google Scholar, 30Debabov D.V. Heaton M.P. Zhang Q. Stewart K.D. Lambalot R.H. Neuhaus F.C. J. Bacteriol. 1996; 178: 3869-3876Crossref PubMed Google Scholar). In contrast, WTA glycosylation has hardly been investigated. Nevertheless, WTA β-GlcNAc-transferase activity (EC 2.4.1.70) has been detected in crude cell extracts of S. aureus strain Copenhagen in the 1960s (31Nathenson S.G. Strominger J.L. J. Biol. Chem. 1963; 238: 3161-3169Abstract Full Text PDF PubMed Google Scholar), and a S. aureus strain H mutant 52B2 deficient in WTA GlcNAc-transferase activity has been described (20Shaw D.R. Mirelman D. Chatterjee A.N. Park J.T. J. Biol. Chem. 1970; 245: 5101-5106Abstract Full Text PDF PubMed Google Scholar), the genetic basis of which has remained unknown.Here we on the and of a S. aureus protein TarM that is responsible for the glycosylation of WTA with α-GlcNAc and UDP-GlcNAc-dependent WTA GlcNAc-transferase activity in vitro. Moreover, we that TarM a in the mutant S. aureus strain 52B2 that to a and TarM. Our study the for the function of the sugar residues by WTA in S. and host interaction.