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Role of the rfe gene in the synthesis of the O8 antigen in Escherichia coli K-12.

By P D Rick, G L Hubbard and K Barr


The Escherichia coli O8 antigen is a mannan composed of the trisaccharide repeat unit -->3)-alpha-Man-(1-->2)-alpha-Man-(1-->2)-alpha-Man-(1--> (K. Reske and K. Jann, Eur. J. Biochem. 67:53-56, 1972), and synthesis of the O8 antigen is rfe dependent (G. Schmidt, H. Mayer, and P. H. Mäkelä, J. Bacteriol. 127:755-762, 1976). The rfe gene has recently been identified as encoding a tunicamycin-sensitive UDP-GlcNAc:undecaprenylphosphate GlcNAc-1-phosphate transferase (U. Meier-Dieter, K. Barr, R. Starman, L. Hatch, and P. D. Rick, J. Biol. Chem. 267:746-753, 1992). However, the role of rfe in O8 side chain synthesis is not understood. Thus, the role of the rfe gene in the synthesis of the O8 antigen was investigated in an rfbO8+ (rfb genes encoding O8 antigen) derivative of E. coli K-12 mutant possessing a defective phosphoglucose isomerase (pgi). The in vivo synthesis of O8 side chains was inhibited by the antibiotic tunicamycin. In addition, putative lipid carrier-linked O8 side chains accumulated in vivo when lipopolysaccharide outer core synthesis was precluded by growing cells in the absence of exogenously supplied glucose. The lipid carrier-linked O8 antigen was extracted from cells and treated with mild acid in order to release free O8 side chains. The water-soluble O8 side chains were then purified by affinity chromatography using Sepharose-bound concanavalin A. Characterization of the affinity-purified O8 side chains revealed the occurrence of glucosamine in the reducing terminal position of the polysaccharide chains. The data presented suggest that GlcNAc-pyrophosphorylundecaprenol functions as the acceptor of mannose residues for the in vivo synthesis of O8 side chains in E. coli K-12

Topics: Research Article
Year: 1994
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Provided by: PubMed Central
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