title: A single amino acid substitution in a mannosyltransferase, WbdA, converts the Escherichia coli O9 polysaccharide into O9a: generation of a new O-serotype group.
abstract: wbdA is a mannosyltransferase gene that is involved in synthesis of the Escherichia coli O9a polysaccharide, a mannose homopolymer with a repeating unit of 2-alphaMan-1,2-alphaMan-1,3-alphaMan-1, 3-alphaMan-1. The equivalent structural O polysaccharide in the E. coli O9 and Klebsiella O3 strains is 2-alphaMan-1,2-alphaMan-1, 2-alphaMan-1,3-alphaMan-1,3-alphaMan-1, with an excess of one mannose in the 1,2 linkage. We have cloned wbdA genes from these O9 and O3 strains and shown by genetic and functional studies that wbdA is the only gene determining the O-polysaccharide structure of O9 or O9a. Based on functional analysis of chimeric genes and site-directed mutagenesis, we showed that a single amino acid substitution, C55R, in WbdA of E. coli O9 converts the O9 polysaccharide into O9a. DNA sequencing revealed the substitution to be conserved in other E. coli O9a strains. The reverse substitution, R55C, in WbdA of E. coli O9a resulted in lipopolysaccharide synthesis showing no ladder profile instead of the conversion of O9a to O9. This suggests that more than one amino acid substitution in WbdA is required for conversion from O9a to O9.
authors.affiliation: Unit of Biosystems, School of Informatics and Sciences, Nagoya University, Nagoya, Aichi 464-8601, Japan. j45811a@nucc.cc.nagoya-u.ac.jp
authors.forename: N
authors.initials: N
authors.lastname: Kido
authors.forename: H
authors.initials: H
authors.lastname: Kobayashi
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001483:Base Sequence; D002236:Carbohydrate Conformation; D003001:Cloning, Molecular; D004269:DNA, Bacterial; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D005798:Genes, Bacterial; D007709:Klebsiella / Q000235:genetics; D008358:Mannose / Q000096:biosynthesis*; D008364:Mannosyltransferases / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D016296:Mutagenesis; D019906:Nuclear Magnetic Resonance, Biomolecular; D011108:Polymers; D011134:Polysaccharides / Q000096:biosynthesis*; D012703:Serotyping
chemical_list: D004269:DNA, Bacterial; D011108:Polymers; D011134:Polysaccharides; D008364:Mannosyltransferases; D008358:Mannose