| Budget Amount *help |
¥3,300,000 (Direct Cost: ¥3,300,000)
Fiscal Year 1999: ¥800,000 (Direct Cost: ¥800,000)
Fiscal Year 1998: ¥2,500,000 (Direct Cost: ¥2,500,000)
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| Research Abstract |
O polysaccharides of Gram-negative bacteria are structurally polymorphic, and consequently, they are utilized as the O-antigen for serological typing. The O polysaccharides and covalently binds to lipid A through the core-oligosaccharide portion. Each O polysaccharide consists of many repeating units of an oligosaccaride composed of several sugars with various linkages. The wbィイD1*ィエD1 gene cluster is responsible for the O polysaccharide synthesis. The wbィイD1*ィエD1 cluster of E. coli O9a strain F719 is constituted of eight genes, two for GDP-mannose synthesis, two for the putative ABC-transporter, three for mannosyltransferases, and one whose function is unknown. wbdA is a mannosyltransferase gene that is involved is synthesis of the Escherichia coli O9a polysaccharide, a mannose homopolymer with a repeating unit of 2-αman-1,2-αman-1,3-αman-1,3-αman-1. The equivalent structural O polysaccharide in the polysaccharide in the E. coli O9 and Klebsiella O3 strains is 2-αman-1,2-αman-1,2-αman-1,3-αman-1,3-αman-1, with an excess of one mannose in the 1,2-linkage. We have cloned wbdA genes from the latter strains and shown by genetic and functional studies that wbdA is one only gene determining the O polysaccharide structure of O9 or O9a. Based on functional analysis of chimeric genes and site-directed mutagenesis, we could show that a single amino acid substitution, C55R, in the WbdA of E. coli O9 converts the O9 polysaccharide into O9a. DNA sequencing revealed the substitution to conserved in other E. coli O9a strains. The reverse substitution, R55C, in the WbdA of E. coli O9a results in LPS synthesis showing no ladder profile instead of the conversion of O9a to O9. This suggests that more than one amino acid substitution in the WbdA is required for conversion from O9a to O9.
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