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ABSTRACT: Production of the somatic antigen, O-specific polysaccharide of Shigella dysenteriae 1 is determined by the chromosomal rfb gene cluster and the rfp gene located on the 9 kb plasmid pHW400 carried by this organism. When transferred to Escherichia coli K-12, which produces lipopolysaccharide consisting only of core oligosaccharide linked to lipid A, rfp gene-containing plasmids caused modification of the core oligosaccharide leading to the appearance of core molecules with new electrophoretic mobilities. Chemical analysis of the modified core has shown that it is substituted with a galactose residue which is the first sugar of the O-polysaccharide repeat unit.
Microbial Pathogenesis 07/1986; 1(3):299-306. · 1.94 Impact Factor
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ABSTRACT: The genetic organization and functions of the Shigella dysenteriae 1 rfb gene cluster, which specifies the somatic O antigen in this organism, have been studied in Escherichia coli K-12 by insertion and deletion mutagenesis of pSS9, a pBR322 hybrid containing the Shigella rfb genes. On the basis of the sensitivity/resistance to rough-specific bacteriophage T3 of E. coli K-12 derivatives containing mutant pSS9 plasmids, of the banding patterns and immunoreactivity of LPS isolated from such derivatives and electrophoresed on SDS-polyacrylamide gels, and of the sugar composition of the polysaccharide portion of the LPS determined by chemical analysis, six determinants for O antigen production were identified and localized. At least two determinants are involved in synthesis of TDP-rhamnose and the transfer of a rhamnose residue to the galactose-substituted core. One of these functions is probably TDP-rhamnose synthetase. A third function effects the transfer of a second rhamnose residue to the rha----gal-substituted core. A fourth function, for which evidence was obtained for two determinants (cistrons), is N-acetylglucosamine transferase, whereas a sixth determinant is necessary for extension of the first completed side chain repeat unit to the full O antigen polymer. These results confirmed the previously-determined chemical composition of the S. dysenteriae 1 O antigen and demonstrated that the order of the sugars is glcNAc----rha----rha----gal with gal as the first sugar linked to the core. Evidence was obtained for at least two transcriptional units in the rfb gene cluster and the approximate locations of two promoters are suggested. The detection of new electrophoretic species of LPS that may correspond to LPS biosynthetic intermediates, and the finding on the cell surfaces of structures corresponding to LPS core substituted with one or more O-specific sugars, appear to be novel findings.
Microbial Pathogenesis 07/1986; 1(3):307-24. · 1.94 Impact Factor
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ABSTRACT: A procedure is described for the efficient transfer of fractionated bacterial lipopolysaccharide (LPS) from SDS-polyacrylamide gels to nitrocellulose filters, and its subsequent display by a peroxidase-linked antibody. The method is sensitive, and reveals and resolves high molecular weight LPS molecules having side chain lengths of up to and greater than 30 repeat units. It is useful for the rapid analysis of LPS in bacterial outer membrane preparations.
Archives of Microbiology 01/1985; 140(2-3):198-201. · 1.43 Impact Factor
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ABSTRACT: The genetic organization and functions of the Shigella dysenteriae 1 rfb gene cluster, which specifies the somatic O antigen in this organism, have been studied in Escherichia coli K-12 by insertion and deletion mutagenesis of pSS9, a pBR322 hybrid containing the Shigellarfb genes. On the basis of the sensitivity/resistance to rough-specific bacteriophage T3 of E. coli K-12 derivatives containing mutant pSS9 plasmids, of the banding patterns and immunoreactivity of LPS isolated from such derivatives and electrophoresed on SDS-polyacrylamide gels, and of the sugar composition of the polysaccharide portion of the LPS determined by chemical analysis, six determinants for O antigen production were identified and localized. At least two determinants are involved in synthesis of TDP-rhamnose and the transfer of a rhamnose residue to the galactose-substituted core. One of these functions is probably TDP-rhamnose synthetase. A third function effects the transfer of a second rhamnose residue to the rha → gal-substituted core. A fourth function, for which evidence was obtained for two determinants (cistrons), is n-acetylglucosamine transferase, whereas a sixth determinant is necessary for extension of the first completed side chain repeat unit to the full O antigen polymer. These results confirmed the previously-determined chemical composition of the S. dysenteriae 1 O antigen and demonstrated that the order of the sugars is glcNAc → rha → rha → gal with gal as the first sugar linked to the core. Evidence was obtained for at least two transcriptional units in the rfb gene cluster and the approximate locations of two promoters are suggested. The detection of new electrophoretic species of LPS that may correspond to LPS biosynthetic intermediates, and the finding on the cell surfaces of structures corresponding to LPS core substituted with one or more O-specific sugars, appear to be novel findings.
Microbial Pathogenesis.
