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• W2220881036 abstract "Post-translational modification of proteins is a ubiquitous mechanism of signal transduction in all kingdoms of life. One such modification is addition of O-linked N-acetylglucosamine to serine or threonine residues, known as O-GlcNAcylation. This unusual type of glycosylation is thought to be restricted to nucleocytoplasmic proteins of eukaryotes and is mediated by a pair of O-GlcNAc-transferase and O-GlcNAc hydrolase enzymes operating on a large number of substrate proteins. Protein O-GlcNAcylation is responsive to glucose and flux through the hexosamine biosynthetic pathway. Thus, a close relationship is thought to exist between the level of O-GlcNAc proteins within and the general metabolic state of the cell. Although isolated apparent orthologues of these enzymes are present in bacterial genomes, their biological functions remain largely unexplored. It is possible that understanding the function of these proteins will allow development of reductionist models to uncover the principles of O-GlcNAc signaling. Here, we identify orthologues of both O-GlcNAc cycling enzymes in the genome of the thermophilic eubacterium Thermobaculum terrenum. The O-GlcNAcase and O-GlcNAc-transferase are co-expressed and, like their mammalian orthologues, localize to the cytoplasm. The O-GlcNAcase orthologue possesses activity against O-GlcNAc proteins and model substrates. We describe crystal structures of both enzymes, including an O-GlcNAcase·peptide complex, showing conservation of active sites with the human orthologues. Although in vitro activity of the O-GlcNAc-transferase could not be detected, treatment of T. terrenum with an O-GlcNAc-transferase inhibitor led to inhibition of growth. T. terrenum may be the first example of a bacterium possessing a functional O-GlcNAc system. Post-translational modification of proteins is a ubiquitous mechanism of signal transduction in all kingdoms of life. One such modification is addition of O-linked N-acetylglucosamine to serine or threonine residues, known as O-GlcNAcylation. This unusual type of glycosylation is thought to be restricted to nucleocytoplasmic proteins of eukaryotes and is mediated by a pair of O-GlcNAc-transferase and O-GlcNAc hydrolase enzymes operating on a large number of substrate proteins. Protein O-GlcNAcylation is responsive to glucose and flux through the hexosamine biosynthetic pathway. Thus, a close relationship is thought to exist between the level of O-GlcNAc proteins within and the general metabolic state of the cell. Although isolated apparent orthologues of these enzymes are present in bacterial genomes, their biological functions remain largely unexplored. It is possible that understanding the function of these proteins will allow development of reductionist models to uncover the principles of O-GlcNAc signaling. Here, we identify orthologues of both O-GlcNAc cycling enzymes in the genome of the thermophilic eubacterium Thermobaculum terrenum. The O-GlcNAcase and O-GlcNAc-transferase are co-expressed and, like their mammalian orthologues, localize to the cytoplasm. The O-GlcNAcase orthologue possesses activity against O-GlcNAc proteins and model substrates. We describe crystal structures of both enzymes, including an O-GlcNAcase·peptide complex, showing conservation of active sites with the human orthologues. Although in vitro activity of the O-GlcNAc-transferase could not be detected, treatment of T. terrenum with an O-GlcNAc-transferase inhibitor led to inhibition of growth. T. terrenum may be the first example of a bacterium possessing a functional O-GlcNAc system. Post-translational modifications of proteins are essential for cell signaling and regulation of cell biological processes. Probably the best understood type of such modification is protein phosphorylation, which can affect the conformation of the modified protein and as a result its activity, localization, or association with other proteins (for reviews, see Refs. 1.Johnson L.N. Barford D. The effects of phosphorylation on the structure and function of proteins.Annu. Rev. Biophys. Biomol. Struct. 1993; 22: 199-232Crossref PubMed Scopus (220) Google Scholar and 2.Nishi H. Hashimoto K. Panchenko A.R. Phosphorylation in protein-protein binding: effect on stability and function.Structure. 2011; 19: 1807-1815Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar). Conserved in all domains of life, protein phosphorylation is governed by a plethora of kinases and reciprocal phosphatases (3.Manning G. Whyte D.B. Martinez R. Hunter T. Sudarsanam S. The protein kinase complement of the human genome.Science. 2002; 298: 1912-1934Crossref PubMed Scopus (6224) Google Scholar). These enzyme pairs are characterized by a high specificity for a small number of target proteins that they modify. In contrast, modification of cytoplasmic proteins with a single O-linked N-acetylglucosamine (O-GlcNAc) 4The abbreviations used are:O-GlcNAcO-linked N-acetylglucosamineOGTO-GlcNAc-transferaseOGAO-GlcNAcaseTPRtetratricopeptide repeatCpC. perfringensTtT. terrenumDMSOdimethyl sulfoxide4MU-GlcNAc4-methylumbelliferyl-N-acetyl-β-d-glucosaminidehTab1human TAK1-binding protein 1GT41glycosyltransferase 41GH84glycoside hydrolase 84hOGThuman OGThOGAhuman OGAAc4-5S-GlcNAcperacetylated 5S-GlcNAcr.m.s.d.root mean square deviationTLRtetratricopeptide-like regionOgO. granulosus. is a regulatory post-translational modification that is dependent on a single O-GlcNAc-transferase (OGT), which transfers the O-GlcNAc moiety onto target proteins, and O-GlcNAcase (OGA), which can reverse this process (4.Vocadlo D.J. O-GlcNAc processing enzymes: catalytic mechanisms, substrate specificity, and enzyme regulation.Curr. Opin. Chem. Biol. 2012; 16: 488-497Crossref PubMed Scopus (104) Google Scholar). This unusual type of protein glycosylation occurs exclusively on nucleocytoplasmic proteins in metazoa (5.Holt G.D. Hart G.W. The subcellular distribution of terminal N-acetylglucosamine moieties. Localization of a novel protein-saccharide linkage, O-linked GlcNAc.J. Biol. Chem. 1986; 261: 8049-8057Abstract Full Text PDF PubMed Google Scholar, 6.Hart G.W. Haltiwanger R.S. Holt G.D. Kelly W.G. Glycosylation in the nucleus and cytoplasm.Annu. Rev. Biochem. 1989; 58: 841-874Crossref PubMed Scopus (322) Google Scholar). Since its discovery 30 years ago during a study of nucleoporins (7.Torres C.-R. Hart G.W. Topography and polypeptide distribution of terminal N-acetylglucosamine residues on the surfaces of intact lymphocytes. Evidence for O-linked GlcNAc.J. Biol. Chem. 1984; 259: 3308-3317Abstract Full Text PDF PubMed Google Scholar), over a thousand proteins have now been shown to be modified by O-GlcNAc (8.Love D.C. Hanover J.A. The hexosamine signaling pathway: deciphering the “O-GlcNAc code.”.Sci. STKE. 2005; 2005: re13Crossref PubMed Scopus (383) Google Scholar). O-GlcNAc is a small, uncharged moiety, and the molecular basis of its impact on protein function is not well understood. However, there is evidence that protein O-GlcNAcylation can affect protein localization (9.Park J. Han D. Kim K. Kang Y. Kim Y. O-GlcNAcylation disrupts glyceraldehyde-3-phosphate dehydrogenase homo-tetramer formation and mediates its nuclear translocation.Biochim. Biophys. Acta. 2009; 1794: 254-262Crossref PubMed Scopus (48) Google Scholar), activity (10.Yi W. Clark P.M. Mason D.E. Keenan M.C. Hill C. Goddard 3rd, W.A. Peters E.C. Driggers E.M. Hsieh-Wilson L.C. Phosphofructokinase 1 glycosylation regulates cell growth and metabolism.Science. 2012; 337: 975-980Crossref PubMed Scopus (415) Google Scholar), and stability (11.Olivier-Van Stichelen S. Dehennaut V. Buzy A. Zachayus J.-L. Guinez C. Mir A.-M. El Yazidi-Belkoura I. Copin M.-C. Boureme D. Loyaux D. Ferrara P. Lefebvre T. O-GlcNAcylation stabilizes β-catenin through direct competition with phosphorylation at threonine 41.FASEB J. 2014; 28: 3325-3338Crossref PubMed Scopus (98) Google Scholar). Additionally, the genetic disruption of ogt is lethal in vertebrates (12.Zachara N.E. Hart G.W. O-GlcNAc a sensor of cellular state: the role of nucleocytoplasmic glycosylation in modulating cellular function in response to nutrition and stress.Biochim. Biophys. Acta. 2004; 1673: 13-28Crossref PubMed Scopus (338) Google Scholar, 13.Frank L.A. Sutton-McDowall M.L. Brown H.M. Russell D.L. Gilchrist R.B. Thompson J.G. Hyperglycaemic conditions perturb mouse oocyte in vitro developmental competence via β-O-linked glycosylation of heat shock protein 90.Hum. Reprod. 2014; 29: 1292-1303Crossref PubMed Scopus (24) Google Scholar, 14.O'Donnell N. Zachara N.E. Hart G.W. Marth J.D. OGT-dependent X-chromosome-linked protein glycosylation is a requisite modification in somatic cell function and embryo viability.Mol. Cell. Biol. 2004; 24: 1680-1690Crossref PubMed Scopus (339) Google Scholar, 15.Yang Y.R. Song M. Lee H. Jeon Y. Choi E.-J. Jang H.-J. Moon H.Y. Byun H.-Y. Kim E.-K. Kim D.H. Lee M.N. Koh A. Ghim J. Choi J.H. Lee-Kwon W. Kim K.T. Ryu S.H. Suh P.-G. O-GlcNAcase is essential for embryonic development and maintenance of genomic stability.Aging Cell. 2012; 11: 439-448Crossref PubMed Scopus (161) Google Scholar, 16.Webster D.M. Teo C.F. Sun Y. Wloga D. Gay S. Klonowski K.D. Wells L. Dougan S.T. O-GlcNAc modifications regulate cell survival and epiboly during zebrafish development.BMC Dev. Biol. 2009; 9: 28Crossref PubMed Scopus (77) Google Scholar, 17.Dehennaut V. Lefebvre T. Leroy Y. Vilain J.-P. Michalski J.-C. Bodart J.-F. Survey of O-GlcNAc level variations in Xenopus laevis from oogenesis to early development.Glycoconj. J. 2009; 26: 301-311Crossref PubMed Scopus (19) Google Scholar, 18.Dehennaut V. Lefebvre T. Sellier C. Leroy Y. Gross B. Walker S. Cacan R. Michalski J.-C. Vilain J.-P. Bodart J.-F. O-Linked N-acetylglucosaminyltransferase inhibition prevents G2/M transition in Xenopus laevis oocytes.J. Biol. Chem. 2007; 282: 12527-12536Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar) and Drosophila (19.Sinclair D.A. Syrzycka M. Macauley M.S. Rastgardani T. Komljenovic I. Vocadlo D.J. Brock H.W. Honda B.M. Drosophila O-GlcNAc transferase (OGT) is encoded by the Polycomb group (PcG) gene, super sex combs (sxc).Proc. Natl. Acad. Sci. U.S.A. 2009; 106: 13427-13432Crossref PubMed Scopus (184) Google Scholar). Given that protein O-GlcNAcylation occurs on serine and threonine residues, it has been suggested to show a degree of interplay with phosphorylation and that O-GlcNAcylation controls a number of signal transduction pathways (20.Hart G.W. Slawson C. Ramirez-Correa G. Lagerlof O. Cross talk between O-GlcNAcylation and phosphorylation: roles in signaling, transcription, and chronic disease.Annu. Rev. Biochem. 2011; 80: 825-858Crossref PubMed Scopus (919) Google Scholar). O-linked N-acetylglucosamine O-GlcNAc-transferase O-GlcNAcase tetratricopeptide repeat C. perfringens T. terrenum dimethyl sulfoxide 4-methylumbelliferyl-N-acetyl-β-d-glucosaminide human TAK1-binding protein 1 glycosyltransferase 41 glycoside hydrolase 84 human OGT human OGA peracetylated 5S-GlcNAc root mean square deviation tetratricopeptide-like region O. granulosus. A typical eukaryotic OGT enzyme comprises an N-terminal tetratricopeptide repeat domain, which is required for interaction with some of the substrate proteins, and a C-terminal catalytic domain formed by two lobes separated by an intervening domain of unknown function (Fig. 1A) (21.Clarke A.J. Hurtado-Guerrero R. Pathak S. Schüttelkopf A.W. Borodkin V. Shepherd S.M. Ibrahim A.F. van Aalten D.M. Structural insights into mechanism and specificity of O-GlcNAc transferase.EMBO J. 2008; 27: 2780-2788Crossref PubMed Scopus (91) Google Scholar, 22.Lazarus M.B. Nam Y. Jiang J. Sliz P. Walker S. Structure of human O-GlcNAc transferase and its complex with a peptide substrate.Nature. 2011; 469: 564-567Crossref PubMed Scopus (327) Google Scholar, 23.Martinez-Fleites C. He Y. Davies G.J. Structural analyses of enzymes involved in the O-GlcNAc modification.Biochim. Biophys. Acta. 2010; 1800: 122-133Crossref PubMed Scopus (34) Google Scholar). OGT uses a form of substrate-assisted catalysis to transfer N-acetylglucosamine from the sugar-nucleotide donor UDP-GlcNAc onto specific serine or threonine residues of the substrate (24.Schimpl M. Zheng X. Borodkin V.S. Blair D.E. Ferenbach A.T. Schüttelkopf A.W. Navratilova I. Aristotelous T. Albarbarawi O. Robinson D.A. Macnaughtan M.A. van Aalten D.M. O-GlcNAc transferase invokes nucleotide sugar pyrophosphate participation in catalysis.Nat. Chem. Biol. 2012; 8: 969-974Crossref PubMed Scopus (105) Google Scholar). O-GlcNAcase is a glycoside hydrolase responsible for hydrolysis of the link between the modified protein and the O-GlcNAc moiety (25.Dong D.L. Hart G.W. Purification and characterization of an O-GlcNAc selective N-acetyl-β-d-glucosaminidase from rat spleen cytosol.J. Biol. Chem. 1994; 269: 19321-19330Abstract Full Text PDF PubMed Google Scholar). In metazoa, OGA consists of a glycoside hydrolase catalytic domain and a putative acetyltransferase domain(Fig. 1B) (26.Dennis R.J. Taylor E.J. Macauley M.S. Stubbs K.A. Turkenburg J.P. Hart S.J. Black G.N. Vocadlo D.J. Davies G.J. Structure and mechanism of a bacterial β-glucosaminidase having O-GlcNAcase activity.Nat. Struct. Mol. Biol. 2006; 13: 365-371Crossref PubMed Scopus (164) Google Scholar, 27.Rao F.V. Dorfmueller H.C. Villa F. Allwood M. Eggleston I.M. van Aalten D.M. Structural insights into the mechanism and inhibition of eukaryotic O-GlcNAc hydrolysis.EMBO J. 2006; 25: 1569-1578Crossref PubMed Scopus (167) Google Scholar, 28.Schimpl M. Schüttelkopf A.W. Borodkin V.S. van Aalten D.M. Human OGA binds substrates in a conserved peptide recognition groove.Biochem. J. 2010; 432: 1-7Crossref PubMed Scopus (54) Google Scholar, 29.Gao Y. Wells L. Comer F.I. Parker G.J. Hart G.W. Dynamic O-glycosylation of nuclear and cytosolic proteins: cloning and characterization of a neutral, cytosolic β-N-acetylglucosaminidase from human brain.J. Biol. Chem. 2001; 276: 9838-9845Abstract Full Text Full Text PDF PubMed Scopus (516) Google Scholar, 30.Toleman C. Paterson A.J. Whisenhunt T.R. Kudlow J.E. Characterization of the histone acetyltransferase (HAT) domain of a bifunctional protein with activable O-GlcNAcase and HAT activities.J. Biol. Chem. 2004; 279: 53665-53673Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar, 31.Forsythe M.E. Love D.C. Lazarus B.D. Kim E.J. Prinz W.A. Ashwell G. Krause M.W. Hanover J.A. Caenorhabditis elegans ortholog of a diabetes susceptibility locus: oga-1 (O-GlcNAcase) knockout impacts O-GlcNAc cycling, metabolism, and dauer.Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 11952-11957Crossref PubMed Scopus (129) Google Scholar), although the relationship between the O-GlcNAcase and acetyltransferase activities of this enzyme is not understood. One of the key questions in the O-GlcNAc signaling field is how two single enzymes, OGA and OGT, can together build a dynamic and inducible O-GlcNAc proteome of over a thousand O-GlcNAc proteins, whereas over 600 kinases/phosphatases are needed to carefully regulate site-specific protein phosphorylation in response to extracellular cues. Elucidating this essential mechanism is challenging in the model organisms where the greatest progress in understanding this modification has been achieved to date (i.e. mouse and Drosophila) and where OGT knock-outs are unfortunately lethal (19.Sinclair D.A. Syrzycka M. Macauley M.S. Rastgardani T. Komljenovic I. Vocadlo D.J. Brock H.W. Honda B.M. Drosophila O-GlcNAc transferase (OGT) is encoded by the Polycomb group (PcG) gene, super sex combs (sxc).Proc. Natl. Acad. Sci. U.S.A. 2009; 106: 13427-13432Crossref PubMed Scopus (184) Google Scholar, 32.Shafi R. Iyer S.P. Ellies L.G. O'Donnell N. Marek K.W. Chui D. Hart G.W. Marth J.D. The O-GlcNAc transferase gene resides on the X chromosome and is essential for embryonic stem cell viability and mouse ontogeny.Proc. Natl. Acad. Sci. U.S.A. 2000; 97: 5735-5739Crossref PubMed Scopus (594) Google Scholar). Thus, discovery of a much simpler, reductionist model system to study the basic mechanisms of O-GlcNAc signaling would be of considerable benefit. O-GlcNAcylation is predominantly thought of as restricted to metazoa as OGT and OGA were initially identified across the Animalia kingdom (20.Hart G.W. Slawson C. Ramirez-Correa G. Lagerlof O. Cross talk between O-GlcNAcylation and phosphorylation: roles in signaling, transcription, and chronic disease.Annu. Rev. Biochem. 2011; 80: 825-858Crossref PubMed Scopus (919) Google Scholar). Two OGT orthologues, SPINDLY and SECRET AGENT, were subsequently identified in plants and are implicated in the gibberellin signaling pathway (33.Thornton T.M. Swain S.M. Olszewski N.E. Gibberellin signal transduction presents … the SPY who O-GlcNAc'd me.Trends Plant Sci. 1999; 4: 424-428Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar, 34.Hartweck L.M. Scott C.L. Olszewski N.E. Two O-linked N-acetylglucosamine transferase genes of Arabidopsis thaliana L. Heynh. have overlapping functions necessary for gamete and seed development.Genetics. 2002; 161: 1279-1291Crossref PubMed Google Scholar). These two OGTs show a level of functional redundancy, and disruption of both genes is lethal (34.Hartweck L.M. Scott C.L. Olszewski N.E. Two O-linked N-acetylglucosamine transferase genes of Arabidopsis thaliana L. Heynh. have overlapping functions necessary for gamete and seed development.Genetics. 2002; 161: 1279-1291Crossref PubMed Google Scholar). Furthermore, SECRET AGENT was shown to self-O-GlcNAcylate in vitro when expressed in Escherichia coli (34.Hartweck L.M. Scott C.L. Olszewski N.E. Two O-linked N-acetylglucosamine transferase genes of Arabidopsis thaliana L. Heynh. have overlapping functions necessary for gamete and seed development.Genetics. 2002; 161: 1279-1291Crossref PubMed Google Scholar). However, other modified plant proteins remain to be identified, and there is currently no evidence of a functional OGA homologue in plant genomes. Strikingly, many prokaryotic genomes of various genera appear to encode orthologues of both OGT and OGA. Some of these orthologues have been widely used in structural and enzymatic approaches to understand the molecular mechanism of O-GlcNAc transfer and hydrolysis (21.Clarke A.J. Hurtado-Guerrero R. Pathak S. Schüttelkopf A.W. Borodkin V. Shepherd S.M. Ibrahim A.F. van Aalten D.M. Structural insights into mechanism and specificity of O-GlcNAc transferase.EMBO J. 2008; 27: 2780-2788Crossref PubMed Scopus (91) Google Scholar, 26.Dennis R.J. Taylor E.J. Macauley M.S. Stubbs K.A. Turkenburg J.P. Hart S.J. Black G.N. Vocadlo D.J. Davies G.J. Structure and mechanism of a bacterial β-glucosaminidase having O-GlcNAcase activity.Nat. Struct. Mol. Biol. 2006; 13: 365-371Crossref PubMed Scopus (164) Google Scholar, 27.Rao F.V. Dorfmueller H.C. Villa F. Allwood M. Eggleston I.M. van Aalten D.M. Structural insights into the mechanism and inhibition of eukaryotic O-GlcNAc hydrolysis.EMBO J. 2006; 25: 1569-1578Crossref PubMed Scopus (167) Google Scholar, 35.Martinez-Fleites C. Macauley M.S. He Y. Shen D.L. Vocadlo D.J. Davies G.J. Structure of an O-GlcNAc transferase homolog provides insight into intracellular glycosylation.Nat. Struct. Mol. Biol. 2008; 15: 764-765Crossref PubMed Scopus (85) Google Scholar, 36.Sousa P.R. de Alencar N.A. Lima A.H. Lameira J. Alves C.N. Protein-ligand interaction study of CpOGA in complex with GlcNAcstatin.Chem. Biol. Drug Des. 2013; 81: 284-290Crossref PubMed Scopus (4) Google Scholar, 37.Schimpl M. Borodkin V.S. Gray L.J. van Aalten D.M. Synergy of peptide and sugar in O-GlcNAcase substrate recognition.Chem. Biol. 2012; 19: 173-178Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar, 38.Dorfmueller H.C. Borodkin V.S. Schimpl M. Zheng X. Kime R. Read K.D. van Aalten D.M. Cell-penetrant, nanomolar O-GlcNAcase inhibitors selective against lysosomal hexosaminidases.Chem. Biol. 2010; 17: 1250-1255Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar) despite the lack of any functional insight into their physiological roles. Several are secreted pathogenicity factors, like the NagJ from Clostridium perfringens (39.Shimizu T. Ohtani K. Hirakawa H. Ohshima K. Yamashita A. Shiba T. Ogasawara N. Hattori M. Kuhara S. Hayashi H. Complete genome sequence of Clostridium perfringens, an anaerobic flesh-eater.Proc. Natl. Acad. Sci. U.S.A. 2002; 99: 996-1001Crossref PubMed Scopus (588) Google Scholar) (hereafter CpOGA), precluding a role in modulating intracellular O-GlcNAc signaling. A noteworthy exception is the recently identified OGT homologue found in the cyanobacterium Synechococcus elongates that appears to be involved in phosphorus retention within the cell, and genetic disruption causes the cells to aggregate (40.Sokol K.A. Olszewski N. The putative eukaryotic-like O-GlcNAc transferase of the cyanobacterium Synechococcus elongatus PCC7942 hydrolyzes UDP-GlcNAc and is involved in multiple cellular processes.J. Bacteriol. 2015; 197: 354-361Crossref PubMed Scopus (7) Google Scholar). The underlying biological mechanisms of these phenotypes are not understood, and the organism lacks a predicted OGA homologue, precluding the existence of a dynamic O-GlcNAc proteome in this organism. It is possible that the single S. elongates OGT resembles the O-GlcNAcylation system found in plants. In this report, we describe the identification of the first complete putative bacterial protein O-GlcNAcylation system, found in the soil thermophile Thermobaculum terrenum (41.Botero L.M. Brown K.B. Brumefield S. Burr M. Castenholz R.W. Young M. McDermott T.R. Thermobaculum terrenum gen. nov., sp. nov.: a non-phototrophic gram-positive thermophile representing an environmental clone group related to the Chloroflexi (green non-sulfur bacteria) and Thermomicrobia.Arch. Microbiol. 2004; 181: 269-277Crossref PubMed Scopus (31) Google Scholar). By means of protein sequence searches, we identified orthologues of both OGT and OGA in this organism. We show that both proteins are expressed in T. terrenum under laboratory conditions and that both proteins are retained in the cytoplasm. The OGA orthologue is active in vitro on both a synthetic substrate and O-GlcNAc proteins, and treatment of T. terrenum with an OGT-specific inhibitor leads to growth inhibition. Unfortunately, throughout our experimental procedures, we were unable to identify proteins modified by the OGT homologue or detect in vitro activity of the recombinant protein. Finally, we use crystal structures of both enzymes to demonstrate conservation of the catalytic machinery, suggesting that this may represent a bona fide O-GlcNAc system orthologous to that found in metazoa. T. terrenum strain YNP1 was obtained from ATCC. T. terrenum was routinely maintained at 65 °C with agitation in NYZ broth (10 g of casamino acids (Thermo Fisher), 5 g of yeast extract (Merck), 5 g of NaCl/liter) solidified with 0.8% Gelzan CM Gelrite (Sigma-Aldrich) when necessary. T. terrenum cells were streaked from a glycerol stock onto an NYZ plate and incubated at 65 °C for 5 days. A single colony was inoculated into 5 ml of NYZ broth supplemented with 0.2% glucose, and the starter culture was incubated at 65 °C for 2 days with vigorous agitation and used to inoculate experimental cultures. Bacillus subtilis strain 168 (Marburg) was routinely maintained and propagated in LB medium (10 g of Bacto tryptone (BD Biosciences), 5 g of yeast extract (Merck), 10 g of NaCl/liter). E. coli was routinely maintained in LB broth supplemented with 100 μg/ml ampicillin as required at 37 °C. Primers and plasmids used in this work are listed in Table 1. The coding frames of Tter_2822 and Tter_0116 genes were amplified using appropriate primer pairs from the genomic DNA of T. terrenum prepared using phenol/chloroform extraction. The amplified fragments were cloned into pGEX-6P-1 vector (GE Healthcare) using a restriction-free approach (42.van den Ent F. Löwe J. RF cloning: a restriction-free method for inserting target genes into plasmids.J. Biochem. Biophys. Methods. 2006; 67: 67-74Crossref PubMed Scopus (416) Google Scholar). Point mutations were introduced by site-directed mutagenesis using primers listed in Table 1 and verified by sequencing. All plasmids were cloned and maintained in E. coli DH5α.TABLE 1Plasmids and primers1 Restriction-free cloning primer fragments homologous to the vector are underlined. The bases for site-directed mutagenesis substitutions are in bold. Open table in a new tab 1 Restriction-free cloning primer fragments homologous to the vector are underlined. The bases for site-directed mutagenesis substitutions are in bold. Full-length recombinant TtOGT and TtOGA proteins were expressed as N-terminally GST-tagged fusions in E. coli BL21. Transformed strains were grown in autoinduction medium at 37 °C with agitation until A600 0.3 at which point the temperature was lowered to 18 °C and incubation was continued overnight. Cells were harvested by centrifugation at 4 °C (35 min 4,500 × g). The cell pellets were resuspended in lysis buffer (50 mm HEPES, pH 7.5, 250 mm NaCl, 0.5 mm tris(2-carboxyethyl)phosphine) supplemented with 0.1 mg/ml DNase I and protease inhibitor mixture (1 mm benzamidine, 0.2 mm PMSF, 5 mm leupeptin) and disrupted using a continuous flow cell disruptor (three passes, 15,000 p.s.i.). After removing the cell debris (45 min, 30,000 × g), the supernatant was subjected to glutathione affinity chromatography using GSH-Sepharose beads (GE Healthcare) according to the manufacturer's instructions, and the desired product protein was liberated using PreScission protease (GE Healthcare). The cleaved protein was concentrated using centrifugal concentrators (Sartorius) and loaded onto a 300-ml prepacked SuperdexTM 75 column (GE Healthcare) equilibrated with lysis buffer. The protein peak was pooled and concentrated to 10 mg/ml for TtOGT and 60 mg/ml for TtOGA and used fresh in further experiments. For the purpose of raising polyclonal antibodies, samples of the purified proteins were submitted to Dundee Cell Products for antibody production in rabbits. The antibodies were affinity-purified against the full-length purified proteins as described previously (43.Ostrowski A. Mehert A. Prescott A. Kiley T.B. Stanley-Wall N.R. YuaB functions synergistically with the exopolysaccharide and TasA amyloid fibers to allow biofilm formation by Bacillus subtilis.J. Bacteriol. 2011; 193: 4821-4831Crossref PubMed Scopus (95) Google Scholar). To establish T. terrenum growth kinetics, 50-ml cultures were inoculated to an A600 of 0.025 from the starter cultures. The A600 of cultures was measured twice a day. To identify localization of TtOGT and TtOGA, 10-ml samples were removed from each culture, and the cell pellet was separated from the medium supernatant by centrifugation for 10 min at 4,000 × g. The decanted supernatant was filtered through a 0.2-μm syringe filter to remove any unpelleted cells. The cells were lysed by sonication in 500 μl of PBS, and the medium fraction was concentrated using VivaSpin 20 10-kDa-molecular mass cutoff spin concentrators (Sartorius) to 200 μl. To remove medium contaminants, the concentrated fraction was precipitated with methanol/chloroform and dissolved in 100 μl of PBS with 1% SDS, yielding a 400-fold concentration factor. The concentration of total protein in each of the samples was estimated by Coomassie staining of the SDS-PAGE gel. Standardized samples were resolved by10% SDS-PAGE. The gel was blotted onto nitrocellulose membrane using a Novex Semi-Dry Blotter, and the membranes were blocked in 3% milk in TBS, 0.1% Tween 20. The primary purified antibodies against TtOGT (dilution, 1:100), TtOGA (dilution, 1:1,000), and B. subtilis RpoD (44.Cairns L.S. Marlow V.L. Kiley T.B. Birchall C. Ostrowski A. Aldridge P.D. Stanley-Wall N.R. FlgN is required for flagellum-based motility by Bacillus subtilis.J. Bacteriol. 2014; 196: 2216-2226Crossref PubMed Scopus (15) Google Scholar) (dilution, 1:1000) were incubated with the membranes overnight at 4 °C and detected with HRP-conjugated anti-rabbit secondary antibodies. To assess the effects of peracetylated 5S-GlcNAc (Ac4-5S-GlcNAc) on growth of T. terrenum, the cells were inoculated to A600 0.025 in 5 ml of NYZ broth supplemented with 0.2% glucose and 15–1,000 μm Ac4-5S-GlcNAc prepared in 100 μl of DMSO. Controls of cells treated with 100 μl of pure DMSO and untreated cells were included. The growth of cells was monitored by removing 100-μl samples and A600 measurement over the course of 92 h with sampling every 24 h from the moment of inoculation. Steady-state kinetics of wild type and mutant TtOGA were determined using the fluorogenic substrate 4-methylumbelliferyl-N-acetyl-β-d-glucosaminide (4MU-GlcNAc; Sigma). 50-μl reaction mixtures contained 0.2 nm enzyme in TBS buffer supplemented with 0.1 mg/ml BSA and 56–1,600 μm substrate in 2% DMSO. The fluorescence of the product, 4-methylumbelliferone (4MU), was quantified using an FLX 800 microplate fluorescence reader (Bio-Tek) with excitation and emission wavelengths of 360 and 460 nm, respectively. Experiments were performed in triplicate. Results were corrected for the background emission from the BSA, buffer, and the 4MU-GlcNAc, and the background-corrected data were fitted to the Michaelis-Menten equation using GraphPad Prism 5.0. To determine the IC50 of GlcNAcstatin G, 4 pm wild type TtOGA enzyme was incubated with the inhibitor (0.04–2,470 nm) for 1 min prior to starting the reaction by addition of the substrate. The substrate concentration was constant and equivalent to the Km determined from steady-state kinetics (90 μm). IC50 values were obtained by fitting the background-corrected fluorescence intensity data to a four-paramete" @default.
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• W2220881036 date "2015-12-01" @default.
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• W2220881036 title "Evidence for a Functional O-Linked N-Acetylglucosamine (O-GlcNAc) System in the Thermophilic Bacterium Thermobaculum terrenum" @default.
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