Structure and functional analysis of glycosyltransferases which are involved in the biosynthesis of bacterial polysaccharides

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Following results were obtained in the past three years.1.The rfe-rff region of Escherichia coli chromosome subcloned into a plasmid was analyzed. We found that the proteins encoded by rfe and orf2 genes were membrane proteins. We also analyzed the topology of these proteins with TnphoA method.2.We determined the entire nucleotide sequence of the K2-cps region of Klebsiella which encoded enzymes involved in the biosynthesis of K2 capsular polysaccharide. A sigma^<56> promoter and 19 ORFs were identified in the cps region and the functions of several ORFs were speculated from the results of homology search with the data base.3.We also determined the entire nucleotide sequence of the O9-rfb region of E.coli which was involved in the biosynthesis of the O polysaccharide of O9 LPS.GDPmannose synthetase, and three mannosyltransferases were identified in the region.4.We subloned 4 kinds of glycosyltransferase such as rfaG,rfaB,rfaI and rfaJ from the rfa region of E.coli K12 into a expression vector and expressed these enzymes. We also constructed deficient mutants of these genes, respectively, with a gene targeting method. By using these mutants, the expression and enzymatic activities of above glycosyltransferases were examined. Construction of site-directed mutant genes were currently proceeded for the identification of active sites.