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• W2783955862 abstract "The bacterial cell wall is an important and highly complex structure that is essential for bacterial growth because it protects bacteria from cell lysis and environmental insults. A typical Gram-positive bacterial cell wall is composed of peptidoglycan and the secondary cell wall polymers, wall teichoic acid (WTA) and lipoteichoic acid (LTA). In many Gram-positive bacteria, LTA is a polyglycerol-phosphate chain that is decorated with d-alanine and sugar residues. However, the function of and proteins responsible for the glycosylation of LTA are either unknown or not well-characterized. Here, using bioinformatics, genetic, and NMR spectroscopy approaches, we found that the Bacillus subtilis csbB and yfhO genes are essential for LTA glycosylation. Interestingly, the Listeria monocytogenes gene lmo1079, which encodes a YfhO homolog, was not required for LTA glycosylation, but instead was essential for WTA glycosylation. LTA is polymerized on the outside of the cell and hence can only be glycosylated extracellularly. Based on the similarity of the genes coding for YfhO homologs that are required in B. subtilis for LTA glycosylation or in L. monocytogenes for WTA glycosylation, we hypothesize that WTA glycosylation might also occur extracellularly in Listeria species. Finally, we discovered that in L. monocytogenes, lmo0626 (gtlB) was required for LTA glycosylation, indicating that the encoded protein has a function similar to that of YfhO, although the proteins are not homologous. Together, our results enable us to propose an updated model for LTA glycosylation and also indicate that glycosylation of WTA might occur through two different mechanisms in Gram-positive bacteria. The bacterial cell wall is an important and highly complex structure that is essential for bacterial growth because it protects bacteria from cell lysis and environmental insults. A typical Gram-positive bacterial cell wall is composed of peptidoglycan and the secondary cell wall polymers, wall teichoic acid (WTA) and lipoteichoic acid (LTA). In many Gram-positive bacteria, LTA is a polyglycerol-phosphate chain that is decorated with d-alanine and sugar residues. However, the function of and proteins responsible for the glycosylation of LTA are either unknown or not well-characterized. Here, using bioinformatics, genetic, and NMR spectroscopy approaches, we found that the Bacillus subtilis csbB and yfhO genes are essential for LTA glycosylation. Interestingly, the Listeria monocytogenes gene lmo1079, which encodes a YfhO homolog, was not required for LTA glycosylation, but instead was essential for WTA glycosylation. LTA is polymerized on the outside of the cell and hence can only be glycosylated extracellularly. Based on the similarity of the genes coding for YfhO homologs that are required in B. subtilis for LTA glycosylation or in L. monocytogenes for WTA glycosylation, we hypothesize that WTA glycosylation might also occur extracellularly in Listeria species. Finally, we discovered that in L. monocytogenes, lmo0626 (gtlB) was required for LTA glycosylation, indicating that the encoded protein has a function similar to that of YfhO, although the proteins are not homologous. Together, our results enable us to propose an updated model for LTA glycosylation and also indicate that glycosylation of WTA might occur through two different mechanisms in Gram-positive bacteria. The bacterial cell wall is a highly complex and very important structure; it maintains the cell shape and protects bacteria from cell lysis and environmental insults. The main cell wall components present in Gram-positive bacteria, such as Bacillus subtilis, Listeria monocytogenes, and Staphylococcus aureus, are peptidoglycan and teichoic acids. Teichoic acids are anionic carbohydrate-containing polymers that are present in two forms: wall teichoic acid (WTA), 4The abbreviations used are: WTAwall teichoic acidLTAlipoteichoic acidGTglycosyltransferaseIPTGisopropyl 1-thio-β-d-galactopyranosideLBLysogeny BrothBHIbrain heart infusionCamchloramphenicolErmerythromycinKankanamycinHRPhorseradish peroxidase. which is covalently linked to the N-acetylmuramic acid residues of the peptidoglycan polymer, and lipoteichoic acid (LTA), which is embedded in the cytoplasmic membrane via a lipid anchor (1Araki Y. Ito E. Linkage units in cell walls of Gram-positive bacteria.Crit. Rev. Microbiol. 1989; 17 (2692601): 121-135https://doi.org/10.1074/jbc.RA117.001614Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar, 2Neuhaus F.C. Baddiley J. A continuum of anionic charge: structures and functions of d-alanyl-teichoic acids in Gram-positive bacteria.Microbiol. Mol. Biol. Rev. 2003; 67 (14665680): 686-723https://doi.org/10.1074/jbc.RA117.001614Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar, 3Percy M.G. Gründling A. Lipoteichoic acid synthesis and function in Gram-positive bacteria.Annu. Rev. Microbiol. 2014; 68 (24819367): 81-100https://doi.org/10.1074/jbc.RA117.001614Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar). Recent studies have shown that WTA is crucial for the virulence and β-lactam resistance of S. aureus and L. monocytogenes (4Brown S. Xia G. Luhachack L.G. Campbell J. Meredith T.C. Chen C. Winstel V. Gekeler C. Irazoqui J.E. Peschel A. Walker S. Methicillin resistance in Staphylococcus aureus requires glycosylated wall teichoic acids.Proc. Natl. Acad. Sci. U.S.A. 2012; 109 (23027967): 18909-18914https://doi.org/10.1074/jbc.RA117.001614Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar, 5Krawczyk-Balska A. Lipiak M. Critical role of a ferritin-like protein in the control of Listeria monocytogenes cell envelope structure and stability under β-lactam pressure.PLoS One. 2013; 8 (24204978): e77808https://doi.org/10.1074/jbc.RA117.001614Abstract Full Text Full Text PDF PubMed Google Scholar, 6Spears P.A. Havell E.A. Hamrick T.S. Goforth J.B. Levine A.L. Thomas Abraham S.T. Heiss C. Azadi P. Orndorff P.E. Listeria monocytogenes wall teichoic acid decoration in virulence and cell-to-cell spread.Mol. Microbiol. 2016; 101 (26871418): 714-730https://doi.org/10.1074/jbc.RA117.001614Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar), whereas LTA is important for cell viability and cell division in these human pathogens (7Gründling A. Schneewind O. Synthesis of glycerol phosphate lipoteichoic acid in Staphylococcus aureus.Proc. Natl. Acad. Sci. U.S.A. 2007; 104 (17483484): 8478-8483https://doi.org/10.1074/jbc.RA117.001614Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar, 8Webb A.J. Karatsa-Dodgson M. Gründling A. Two-enzyme systems for glycolipid and polyglycerolphosphate lipoteichoic acid synthesis in Listeria monocytogenes.Mol. Microbiol. 2009; 74 (19682249): 299-314https://doi.org/10.1074/jbc.RA117.001614Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar). In the soil bacterium B. subtilis, the absence of LTA affects divalent cation homeostasis and leads to increased sensitivity to diverse antibiotics and lysozyme, and the absence of WTA leads to drastic morphological alterations (9Schirner K. Marles-Wright J. Lewis R.J. Errington J. Distinct and essential morphogenic functions for wall- and lipo-teichoic acids in Bacillus subtilis.EMBO J. 2009; 28 (19229300): 830-842https://doi.org/10.1074/jbc.RA117.001614Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar). It has also been shown that the deficiency in LTA synthesis leads to smaller colony sizes due to a failure in the execution of the colony developmental program in B. subtilis (10Mamou G. Fiyaksel O. Sinai L. Ben-Yehuda S. Deficiency in lipoteichoic acid synthesis causes a failure in executing the colony developmental program in Bacillus subtilis.Front. Microbiol. 2017; 8 (29114240): 1991https://doi.org/10.1074/jbc.RA117.001614Abstract Full Text Full Text PDF PubMed Google Scholar). Additionally, B. subtilis cells lacking both WTA and LTA are not viable, reflecting the importance of these cell polymers (9Schirner K. Marles-Wright J. Lewis R.J. Errington J. Distinct and essential morphogenic functions for wall- and lipo-teichoic acids in Bacillus subtilis.EMBO J. 2009; 28 (19229300): 830-842https://doi.org/10.1074/jbc.RA117.001614Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar). Due to the impact of WTA and LTA on cell viability and virulence, the enzymes required for their synthesis are considered suitable targets for the development of new antimicrobial compounds (11Xia G. Peschel A. Toward the pathway of S. aureus WTA biosynthesis.Chem. Biol. 2008; 15 (18291312): 95-96https://doi.org/10.1074/jbc.RA117.001614Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar, 12Richter S.G. Elli D. Kim H.K. Hendrickx A.P. Sorg J.A. Schneewind O. Missiakas D. Small molecule inhibitor of lipoteichoic acid synthesis is an antibiotic for Gram-positive bacteria.Proc. Natl. Acad. Sci. U.S.A. 2013; 110 (23401520): 3531-3536https://doi.org/10.1074/jbc.RA117.001614Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar, 13Pasquina L.W. Santa Maria J.P. Walker S. Teichoic acid biosynthesis as an antibiotic target.Curr. Opin. Microbiol. 2013; 16 (23916223): 531-537https://doi.org/10.1074/jbc.RA117.001614Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar). wall teichoic acid lipoteichoic acid glycosyltransferase isopropyl 1-thio-β-d-galactopyranoside Lysogeny Broth brain heart infusion chloramphenicol erythromycin kanamycin horseradish peroxidase. The biosynthesis of LTA has been extensively studied in B. subtilis, S. aureus, and L. monocytogenes (3Percy M.G. Gründling A. Lipoteichoic acid synthesis and function in Gram-positive bacteria.Annu. Rev. Microbiol. 2014; 68 (24819367): 81-100https://doi.org/10.1074/jbc.RA117.001614Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar, 8Webb A.J. Karatsa-Dodgson M. Gründling A. Two-enzyme systems for glycolipid and polyglycerolphosphate lipoteichoic acid synthesis in Listeria monocytogenes.Mol. Microbiol. 2009; 74 (19682249): 299-314https://doi.org/10.1074/jbc.RA117.001614Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar, 14Fischer W. Bacterial phosphoglycolipids and lipoteichoic acids.in: Kates M. Glycolipids, Phosphoglycolipids, and Sulfoglycolipids. Springer US, Boston, MA1990: 123-234Crossref Google Scholar, 15Kiriukhin M.Y. Debabov D.V. Shinabarger D.L. Neuhaus F.C. Biosynthesis of the glycolipid anchor in lipoteichoic acid of Staphylococcus aureus RN4220: role of YpfP, the diglucosyldiacylglycerol synthase.J. Bacteriol. 2001; 183 (11344159): 3506-3514https://doi.org/10.1074/jbc.RA117.001614Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar, 16Reichmann N.T. Gründling A. Location, synthesis and function of glycolipids and polyglycerolphosphate lipoteichoic acid in Gram-positive bacteria of the phylum Firmicutes.FEMS Microbiol. Lett. 2011; 319 (21388439): 97-105https://doi.org/10.1074/jbc.RA117.001614Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar, 17Wörmann M.E. Corrigan R.M. Simpson P.J. Matthews S.J. Gründling A. Enzymatic activities and functional interdependencies of Bacillus subtilis lipoteichoic acid synthesis enzymes.Mol. Microbiol. 2011; 79 (21255105): 566-583https://doi.org/10.1074/jbc.RA117.001614Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar). B. subtilis produces type I LTA, which is composed of an unbranched 1–3-linked polyglycerol-phosphate (GroP) backbone chain that is attached to the outer layer of the bacterial membrane via a glycolipid anchor (14Fischer W. Bacterial phosphoglycolipids and lipoteichoic acids.in: Kates M. Glycolipids, Phosphoglycolipids, and Sulfoglycolipids. Springer US, Boston, MA1990: 123-234Crossref Google Scholar, 16Reichmann N.T. Gründling A. Location, synthesis and function of glycolipids and polyglycerolphosphate lipoteichoic acid in Gram-positive bacteria of the phylum Firmicutes.FEMS Microbiol. Lett. 2011; 319 (21388439): 97-105https://doi.org/10.1074/jbc.RA117.001614Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar). Each GroP subunit can be modified with d-alanine or GlcNAc residues (18Fischer W. Rösel P. The alanine ester substitution of lipoteichoic acid (LTA) in Staphylococcus aureus.FEBS Lett. 1980; 119 (7428933): 224-226https://doi.org/10.1074/jbc.RA117.001614Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar, 19Iwasaki H. Shimada A. Ito E. Comparative studies of lipoteichoic acids from several Bacillus strains.J. Bacteriol. 1986; 167 (3733670): 508-516https://doi.org/10.1074/jbc.RA117.001614Abstract Full Text Full Text PDF PubMed Google Scholar, 20Fischer W. Physiology of lipoteichoic acids in bacteria.Adv. Microb. Physiol. 1988; 29 (3289326): 233-302https://doi.org/10.1074/jbc.RA117.001614Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar, 21Iwasaki H. Shimada A. Yokoyama K. Ito E. Structure and glycosylation of lipoteichoic acids in Bacillus strains.J. Bacteriol. 1989; 171 (2914853): 424-429https://doi.org/10.1074/jbc.RA117.001614Abstract Full Text Full Text PDF PubMed Google Scholar). L. monocytogenes also produces a type I LTA; however, in this organism, the GroP subunits are substituted with d-alanine and galactose residues (22Hether N.W. Jackson L.L. Lipoteichoic acid from Listeria monocytogenes.J. Bacteriol. 1983; 156 (6415040): 809-817Crossref PubMed Google Scholar, 23Uchikawa K. Sekikawa I. Azuma I. Structural studies on lipoteichoic acids from four Listeria strains.J. Bacteriol. 1986; 168 (3093460): 115-122https://doi.org/10.1074/jbc.RA117.001614Abstract Full Text Full Text PDF PubMed Google Scholar). The enzymes required for the d-alanylation of LTA are encoded by the dltABCD operon and have been characterized in a number of studies (2Neuhaus F.C. Baddiley J. A continuum of anionic charge: structures and functions of d-alanyl-teichoic acids in Gram-positive bacteria.Microbiol. Mol. Biol. Rev. 2003; 67 (14665680): 686-723https://doi.org/10.1074/jbc.RA117.001614Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar, 24Heaton M.P. Neuhaus F.C. Biosynthesis of d-alanyl-lipoteichoic acid: cloning, nucleotide sequence, and expression of the Lactobacillus casei gene for the d-alanine-activating enzyme.J. Bacteriol. 1992; 174 (1385594): 4707-4717https://doi.org/10.1074/jbc.RA117.001614Abstract Full Text Full Text PDF PubMed Google Scholar, 25Abachin E. Poyart C. Pellegrini E. Milohanic E. Fiedler F. Berche P. Trieu-Cuot P. Formation of d-alanyl-lipoteichoic acid is required for adhesion and virulence of Listeria monocytogenes.Mol. Microbiol. 2002; 43 (11849532): 1-14https://doi.org/10.1074/jbc.RA117.001614Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar). In contrast to the d-alanylation process, little is known about the enzymes responsible for LTA glycosylation. Fischer and others proposed a model for the addition of sugar residues to LTA based on biochemical studies performed three decades ago (21Iwasaki H. Shimada A. Yokoyama K. Ito E. Structure and glycosylation of lipoteichoic acids in Bacillus strains.J. Bacteriol. 1989; 171 (2914853): 424-429https://doi.org/10.1074/jbc.RA117.001614Abstract Full Text Full Text PDF PubMed Google Scholar, 26Mancuso D.J. Chiu T.H. Biosynthesis of glucosyl monophosphoryl undecaprenol and its role in lipoteichoic acid biosynthesis.J. Bacteriol. 1982; 152 (7130126): 616-625Crossref PubMed Google Scholar, 27Yokoyama K. Araki Y. Ito E. The function of galactosyl phosphorylpolyprenol in biosynthesis of lipoteichoic acid in Bacillus coagulans.Eur. J. Biochem. 1988; 173 (3360021): 453-458https://doi.org/10.1074/jbc.RA117.001614Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar, 28Fischer W. Lipoteichoic acid and lipids in the membrane of Staphylococcus aureus.Med. Microbiol. Immunol. 1994; 183 (7935161): 61-76https://doi.org/10.1074/jbc.RA117.001614Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar). According to this model, a cytoplasmic glycosyltransferase (GT) uses a nucleotide-activated sugar to form a C55-P sugar intermediate, which is subsequently transported across the membrane by a flippase enzyme. The sugar molecule is subsequently transferred onto LTA by the action of a second GT (27Yokoyama K. Araki Y. Ito E. The function of galactosyl phosphorylpolyprenol in biosynthesis of lipoteichoic acid in Bacillus coagulans.Eur. J. Biochem. 1988; 173 (3360021): 453-458https://doi.org/10.1074/jbc.RA117.001614Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar, 28Fischer W. Lipoteichoic acid and lipids in the membrane of Staphylococcus aureus.Med. Microbiol. Immunol. 1994; 183 (7935161): 61-76https://doi.org/10.1074/jbc.RA117.001614Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar). As the polyglycerol-phosphate backbone of LTA is polymerized on the outside of the cell, this final step needs to be catalyzed by a GT with an extracellular active site. Recently, GtlA (locus tag Lmo0933 in strain EGD-e) has been identified as the putative cytoplasmic GT involved in the glycosylation process of LTA in the L. monocytogenes strain 10403S (29Percy M.G. Karinou E. Webb A.J. Gründling A. Identification of a lipoteichoic acid glycosyltransferase enzyme reveals that GW-domain-containing proteins can be retained in the cell wall of Listeria monocytogenes in the absence of lipoteichoic acid or its modifications.J. Bacteriol. 2016; 198 (27185829): 2029-2042https://doi.org/10.1074/jbc.RA117.001614Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar), although biochemical evidence for such an activity is still lacking. This protein is anchored by two C-terminal transmembrane helices to the membrane and contains a large N-terminal cytoplasmic enzymatic domain. NMR analysis of LTA produced by a gtlA mutant strain confirmed the absence of galactose modifications. Additionally, cell extracts obtained from the gtlA mutant strain showed a stronger LTA signal on western blots using a polyglycerol phosphate–specific antibody as compared with a WT strain, suggesting that the LTA backbone structure is better recognized by the antibody in the absence of sugar modifications (29Percy M.G. Karinou E. Webb A.J. Gründling A. Identification of a lipoteichoic acid glycosyltransferase enzyme reveals that GW-domain-containing proteins can be retained in the cell wall of Listeria monocytogenes in the absence of lipoteichoic acid or its modifications.J. Bacteriol. 2016; 198 (27185829): 2029-2042https://doi.org/10.1074/jbc.RA117.001614Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar). GtlA belongs to the GT2 family of glycosyltransferases and is characterized by a GT-A fold. GT-A fold glycosyltransferases assume a Rossmann fold with seven or more β-sheets, which is typical for proteins that bind nucleotides (30Rao S.T. Rossmann M.G. Comparison of super-secondary structures in proteins.J. Mol. Biol. 1973; 76 (4737475): 241-256https://doi.org/10.1074/jbc.RA117.001614Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar, 31Breton C. Snajdrová L. Jeanneau C. Koca J. Imberty A. Structures and mechanisms of glycosyltransferases.Glycobiology. 2006; 16 (16037492): 29R-37Rhttps://doi.org/10.1074/jbc.RA117.001614Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar); in L. monocytogenes, the likely substrate of GtlA is UDP-galactose. A second feature of enzymes with a GT-A fold is the presence of a conserved DXD motif, which interacts with the phosphate group of the nucleotide-activated sugar. This interaction requires a divalent cation, which in many cases is a Mn2+ ion (31Breton C. Snajdrová L. Jeanneau C. Koca J. Imberty A. Structures and mechanisms of glycosyltransferases.Glycobiology. 2006; 16 (16037492): 29R-37Rhttps://doi.org/10.1074/jbc.RA117.001614Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar, 32Breton C. Bettler E. Joziasse D.H. Geremia R.A. Imberty A. Sequence-function relationships of prokaryotic and eukaryotic galactosyltransferases.J. Biochem. 1998; 123 (9603985): 1000-1009https://doi.org/10.1074/jbc.RA117.001614Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar, 33Breton C. Imberty A. Structure/function studies of glycosyltransferases.Curr. Opin. Struct. Biol. 1999; 9 (10508766): 563-571https://doi.org/10.1074/jbc.RA117.001614Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar). However, the remaining proteins required for the glycosylation of LTA in L. monocytogenes are still unknown. Also, none of the enzymes involved in the LTA glycosylation process of B. subtilis, including the enzyme required for the production of the C55-P sugar intermediate, the enzyme required for the transfer of this intermediate from the inner to the outer leaflet of the membrane, or the enzyme that transfers the sugar residue to the polyglycerol backbone, have been identified. Here, we show that deletion of the csbB and yfhO genes in B. subtilis led to a loss of sugar modifications on LTA. Interestingly, the L. monocytogenes YfhO homolog, Lmo1079, is not involved in LTA but WTA glycosylation. Instead, we found that the absence of Lmo0626 (here renamed GtlB) has an impact on LTA glycosylation, and we hypothesize that this protein performs the extracellular LTA glycosylation step in L. monocytogenes. With this, not only did we discover additional genes required for LTA glycosylation, but the work also allowed us to propose an alternative, extracellular, glycosylation mechanism for wall teichoic acid. We have recently reported that the annotated glycosyltransferase GtlA probably catalyzes the first step of the LTA glycosylation process in L. monocytogenes (29Percy M.G. Karinou E. Webb A.J. Gründling A. Identification of a lipoteichoic acid glycosyltransferase enzyme reveals that GW-domain-containing proteins can be retained in the cell wall of Listeria monocytogenes in the absence of lipoteichoic acid or its modifications.J. Bacteriol. 2016; 198 (27185829): 2029-2042https://doi.org/10.1074/jbc.RA117.001614Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar). However, the enzymes involved in this process in other bacteria, including B. subtilis, remain unknown. To identify proteins required for LTA glycosylation in B. subtilis, the L. monocytogenes GtlA protein sequence was used as a query sequence in a BLASTP search against the B. subtilis 168 genome. This identified three homologs, YkcC (e-value: 1e−158), CsbB (e-value: 5e−68), and YkoT (e-value: 1e−60), all of which are encoded in a two-gene operon (Fig. 1A). Analysis using the Pfam database (http://pfam.xfam.org/) 5Please note that the JBC is not responsible for the long-term archiving and maintenance of this site or any other third party hosted site. indicated that YkcC, CsbB, and YkoT encode GT-A fold family 2 glycosyltransferases, which are known to transfer sugar moieties from nucleotide-activated sugars, such as UDP-glucose, UDP-GlcNAc, or UDP-galactose to a variety of substrates, including the lipid carrier C55-P (34Liu J. Mushegian A. Three monophyletic superfamilies account for the majority of the known glycosyltransferases.Protein Sci. 2003; 12 (12824488): 1418-1431https://doi.org/10.1074/jbc.RA117.001614Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar, 35Breazeale S.D. Ribeiro A.A. McClerren A.L. Raetz C.R. A formyltransferase required for polymyxin resistance in Escherichia coli and the modification of lipid A with 4-amino-4-deoxy-l-arabinose: identification and function of UDP-4-deoxy-4-formamido-L-arabinose.J. Biol. Chem. 2005; 280 (15695810): 14154-14167https://doi.org/10.1074/jbc.RA117.001614Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar, 36Lairson L.L. Henrissat B. Davies G.J. Withers S.G. Glycosyltransferases: structures, functions, and mechanisms.Annu. Rev. Biochem. 2008; 77 (18518825): 521-555https://doi.org/10.1074/jbc.RA117.001614Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar). To assess whether YkcC, CsbB, and YkoT are involved in the LTA glycosylation process in B. subtilis, ykcC, csbB, and ykoT deletion strains were constructed by replacing the respective gene in B. subtilis 168 with an antibiotic resistance marker. To determine whether deletion of one of these genes impacts LTA synthesis, anti-LTA western blot analysis was performed on cell extracts derived from the WT and mutant B. subtilis strains. The LTA isolated from strain 168ΔcsbB yielded a stronger signal as compared with the WT strain (Fig. 1B), indicating that the structure or the amount of the LTA polymer is changed in the absence of CsbB. In contrast, the anti-LTA signal for strains with a ykcC or ykoT deletion was indistinguishable from that seen for the WT (Fig. 1B). To determine the chemical structure of LTA in the WT and mutant strains, the polymer was isolated and analyzed by 1D 1H NMR. LTA purified from WT B. subtilis 168 showed the expected spectrum; peaks derived from the CH2 groups of the GroP backbone (colored green in Fig. 2A), the CH2 and CH3 groups of the fatty acid chain (colored orange in Fig. 2A), the non-exchangeable protons from the d-Ala substitutions (colored blue in Fig. 2A), and the GlcNAc modification (colored yellow in Fig. 2A) could be assigned, as described previously (29Percy M.G. Karinou E. Webb A.J. Gründling A. Identification of a lipoteichoic acid glycosyltransferase enzyme reveals that GW-domain-containing proteins can be retained in the cell wall of Listeria monocytogenes in the absence of lipoteichoic acid or its modifications.J. Bacteriol. 2016; 198 (27185829): 2029-2042https://doi.org/10.1074/jbc.RA117.001614Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar, 37Morath S. Geyer A. Hartung T. Structure-function relationship of cytokine induction by lipoteichoic acid from Staphylococcus aureus.J. Exp. Med. 2001; 193 (11157059): 393-397https://doi.org/10.1074/jbc.RA117.001614Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar, 38Morath S. Geyer A. Spreitzer I. Hermann C. Hartung T. Structural decomposition and heterogeneity of commercial lipoteichoic acid preparations.Infect. Immun. 2002; 70 (11796629): 938-944https://doi.org/10.1074/jbc.RA117.001614Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar, 39Morath S. Stadelmaier A. Geyer A. Schmidt R.R. Hartung T. Synthetic lipoteichoic acid from Staphylococcus aureus is a potent stimulus of cytokine release.J. Exp. Med. 2002; 195 (12070290): 1635-1640https://doi.org/10.1074/jbc.RA117.001614Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar). The spectra, including the peaks for the nonexchangeable protons derived from the GlcNAc residues, were essentially identical for the LTA isolated from the ykcC and ykoT deletion strains to that of the WT strain, indicating that the proteins encoded by these two genes are not involved in the LTA glycosylation process in B. subtilis under the conditions used (Fig. S1). In contrast, the GlcNAc-specific peaks were absent in the NMR spectra obtained from the LTA isolated from the csbB mutant (Fig. 2B). To confirm that the phenotype is due to inactivation of csbB, a copy of csbB expressed from its native promoter was introduced into the amyE locus of the csbB mutant. The LTA western blot signal (Fig. 1B) and NMR peaks corresponding to GlcNAc (Fig. 2C) were restored to WT levels in the complementation strain. These results highlight that CsbB is required for the decoration of LTA with GlcNAc residues in B. subtilis during vegetative growth. Although biochemical evidence is still lacking, we presume that CsbB functions as the cytoplasmic LTA glycosyltransferase.Figure 2NMR analysis of LTA isolated from WT B. subtilis 168, csbB mutant, and complementation strains. Shown are NMR spectra of LTA derived from B. subtilis strains 168 (WT) (A), csbB mutant (B), and the csbB+csbB complementation strain (C). Colored boxes and labels indicate nonexchangeable protons derived from the different LTA components. Peaks were assigned as described previously (17Wörmann M.E. Corrigan R.M. Simpson P.J. Matthews S.J. Gründling A. Enzymatic activities and functional interdependencies of Bacillus subtilis lipoteichoic acid synthesis enzymes.Mol. Microbiol. 2011; 79 (21255105): 566-583https://doi.org/10.1074/jbc.RA117.001614Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar, 37Morath S. Geyer A. Hartung T. Structure-function relationship of cytokine induction by lipoteichoic acid from Staphylococcus aureus.J. Exp. Med. 2001; 193 (11157059): 393-397https://doi.org/10.1074/jbc.RA117.001614Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar, 38Morath S. Geyer A. Spreitzer I. Hermann C. Hartung T. Structural decomposition and heterogeneity of commercial lipoteichoic acid preparations.Infect. Immun. 2002; 70 (11796629): 938-944https://doi.org/10.1074/jbc.RA117.001614Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar, 39Morath S. Stadelmaier A. Geyer A. Schmidt R.R. Hartung T. Synthetic lipoteichoic acid from Staphylococcus aureus is a potent stimulus of cytokine release.J. Exp. Med. 2002; 195 (12070290): 1635-1640https://doi.org/10.1074/jbc.RA117.001614Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar). The different peaks for the protons and acetyl group of GlcNAc are labeled with 1H, 4H, and Ac, respectively. Gray boxes highlight peaks resulting from residual citrate, a buffer component used during the LTA purification procedure. The spectra are representative of three independent experiments.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Analysis of the genomic ykcC, csbB, and ykoT re" @default.
• W2783955862 created "2018-01-26" @default.
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• W2783955862 creator A5081883664 @default.
• W2783955862 date "2018-03-01" @default.
• W2783955862 modified "2023-10-14" @default.
• W2783955862 title "Discovery of genes required for lipoteichoic acid glycosylation predicts two distinct mechanisms for wall teichoic acid glycosylation" @default.
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• W2783955862 doi "https://doi.org/10.1074/jbc.ra117.001614" @default.
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