title: Biosynthesis of the polymannose lipopolysaccharide O-antigens from Escherichia coli serotypes O8 and O9a requires a unique combination of single- and multiple-active site mannosyltransferases.
abstract: The Escherichia coli O9a and O8 O-antigen serotypes represent model systems for the ABC transporter-dependent synthesis of bacterial polysaccharides. The O9a and O8 antigens are linear mannose homopolymers containing conserved reducing termini (the primer-adaptor), a serotype-specific repeat unit domain, and a terminator. Synthesis of these glycans occurs on the polyisoprenoid lipid-linked primer, undecaprenol pyrophosphoryl-GlcpNAc, by two conserved mannosyltransferases, WbdC and WbdB, and a serotype-specific mannosyltransferase, WbdA. The glycan structure and pattern of conservation in the O9a and O8 mannosyltransferases are not consistent with the existing model of O9a biosynthesis. Here we establish a revised pathway using a combination of in vivo (mutant complementation) experiments and in vitro strategies with purified enzymes and synthetic acceptors. WbdC and WbdB synthesize the adaptor region, where they transfer one and two α-(1→3)-linked mannose residues, respectively. The WbdA enzymes are solely responsible for forming the repeat unit domains of these O-antigens. WbdA(O9a) has two predicted active sites and polymerizes a tetrasaccharide repeat unit containing two α-(1→3)- and two α-(1→2)-linked mannopyranose residues. In contrast, WbdA(O8) polymerizes trisaccharide repeat units containing single α-(1→3)-, α-(1→2)-, and β-(1→2)-mannopyranoses. These studies illustrate assembly systems exploiting several mannosyltransferases with flexible active sites, arranged in single- and multiple-domain formats.
authors.affiliation: Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario N1G 2W1, Canada.
authors.forename: Laura K
authors.initials: LK
authors.lastname: Greenfield
authors.affiliation: Alberta Glycomics Centre and Department of Chemistry, University of Alberta, Edmonton, Alberta T6G 2G2, Canada.
authors.forename: Michele R
authors.initials: MR
authors.lastname: Richards
authors.affiliation: Institute for Biological Sciences, National Research Council of Canada, Ottawa, Ontario K1A 0R6, Canada.
authors.forename: Jianjun
authors.initials: J
authors.lastname: Li
authors.affiliation: Institute for Biological Sciences, National Research Council of Canada, Ottawa, Ontario K1A 0R6, Canada.
authors.forename: Warren W
authors.initials: WW
authors.lastname: Wakarchuk
authors.affiliation: Alberta Glycomics Centre and Department of Chemistry, University of Alberta, Edmonton, Alberta T6G 2G2, Canada.
authors.forename: Todd L
authors.initials: TL
authors.lastname: Lowary
authors.affiliation: Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario N1G 2W1, Canada. Electronic address: cwhitfie@uoguelph.ca.
authors.forename: Chris
authors.initials: C
authors.lastname: Whitfield
mesh_terms: D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D008351:Mannans / Q000096:biosynthesis* / Q000235:genetics; D008364:Mannosyltransferases / Q000235:genetics / Q000378:metabolism*; D019081:O Antigens / Q000096:biosynthesis* / Q000235:genetics; D011106:Polyisoprenyl Phosphates / Q000378:metabolism
chemical_list: D029968:Escherichia coli Proteins; D008351:Mannans; D019081:O Antigens; D011106:Polyisoprenyl Phosphates; C097323:polymannose; C088139:undecaprenyl pyrophosphate; D008364:Mannosyltransferases