Barbara Jann

Structural studies of the O-specific side chain of the lipopolysaccharide from Escherichia coli O:7.

The structure of the O-specific side-chain of the lipopolysaccharide from Escherichia coli O:7 has been investigated, using n.m.r. spectroscopy, methylation analysis, partial hydrolysis, and Smith degradation as the principal methods. It is concluded that the polysaccharide is constructed of repeating pentasaccharide units having the structure (formula; see text) where D-QuipNAc stands for 4-acetamido-4,6-dideoxy-D-glucopyranose. The 13C-n.m.r. spectrum of the polysaccharide has been interpreted completely.

On a bacteriophage T3 and T4 receptor region within the cell wall lipopolysaccharide of escherichia coli B

Abstract † Mutants of Escherichia coli B with increasing structural defects in the cell wall lipopolysaccharide (above) were tested for adsorption resistance to bacteriophages T3 and T4, and the lipopolysaccharide extracted from these mutants was assayed for loss of ability to inactivate the viruses. Absence of terminal glucose, or of any of the substituents (branch heptose, phosphate and pyrophosphorylethanolamine) on the two chain heptoses was found to have little effect on T3 and T4 susceptibility. However, all mutants lacking both glucoses were resistant to both phages.

Genetic and biochemical analysis of Shigella dysenteriae 1 O antigen polysaccharide biosynthesis in Escherichia coli K-12: structure and functions of the rfb gene cluster

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.

Structure of the amino acid-containing capsular polysaccharide (K54 antigen) from escherichia coli O6:K54:H10

The structure of the K54-antigenic polysaccharide (K54 antigen) of Escherichia coli O6:K54:H10 was elucidated by determination of the composition, 1H- and 13C-n.m.r. spectroscopy, periodate oxidation, and a study of the oligosaccharides obtained by partial hydrolysis with acid. The K54 polysaccharide consists of----3)-beta-D-glucosyluronic acid-(1----3)-alpha-L-rhamnosyl-(1----repeating-units. Of the glucuronic acid residues, approximately 85% are substituted in the ratio 9:1 with L-threonine and L-serine amidically linked to the carboxyl group. The K54 polysaccharide has a molecular weight of approximately 160,000, corresponding to approximately 380 repeating-units.

Structure of the 3-deoxy-D-manno-octulosonic acid-(KDO)-containing capsular polysaccharide (K14 antigen) from Escherichia coli 06:K14:H31.

The chemical structure of the K14-antigenic polysaccharide (K14 antigen) of Escherichia coli 06:K14:H31 was elucidated by determination of the composition, 1H- and 13C-n.m.r. spectroscopy, periodate oxidation, and study of the oligosaccharides obtained by partial hydrolysis. The polysaccharide consists of [O-(2-acetamido-2-deoxy-beta-D-galactopyranosyl)-(1 leads to 5)-O-(3-deoxy-beta-D-manno-octulopyranosylonic acid)-(2 leads to 6)] repeating units, approximately 60% of the octonic acid units being O-acetylated and approximately 10% O-propionylated at O-8. The sequence of acetylated and propionylated residues is not known. The serologically-specific part of the K14 antigen residues in the polysaccharide part.

Genetic analysis of Escherichia coli 09 rfb: identification and DNA sequence of phosphomannomutase and GDP-mannose pyrophosphorylase genes

Subcloning, transposon insertion, and deletion analysis revealed that the Escherichia coli O9 rfb region is about 12 kb in size. The region encodes at least seven polypeptides of 89, 74, 55, 50, 44, 41 and 39.5 kDa. Southern hybridization analysis of rfb regions of E. coli O8 and O9, and Klebsiella O3 and O5 serotypes (all of these O polysaccharides are mannose homopolymers and the structures of the repeating unit of E. coli O9 and Klebsiella O3 are identical) showed that a central region specific for E. coli O9 and Klebsiella O3 is flanked by two regions common to all four. Complementation experiments using strains with known defects and specific tests for the enzymic activity showed that the 50 and 55 kDa polypeptides, encoded by the common region, are phosphomannomutase (PMM) and GDP-mannose pyrophosphorylase (GMP), respectively. Nucleotide sequencing of the region revealed the presence of two genes, rfbK and rfbM, analogous to the corresponding genes of Salmonella typhimurium. In E. coli O9, rfbK and rfbM encode proteins of 460 amino acids (50,809 Da) and 471 amino acids (52,789 Da). The amino acid sequence of GMP was conserved in RfbMs of E. coli O7 and Salmonella groups B, C1 and C2, CpsB of S. typhimurium, AlgA of Pseudomonas aeruginosa, and XanB of Xanthomonas campestris. The phylogenetic trees of PMM and GMP were different in topology and in the evolutionary distances from ancestors.

Structure of the serine-containing capsular poly-saccharide K40 antigen from Escherichia coli O8:K40:H9

The structure of the K40 antigenic capsular polysaccharide (K40 antigen) of E. coli O8:K40:H9 was elucidated by determination of the composition, 1H- and 13C-n.m.r. spectroscopy, periodate oxidation and Smith degradation, and methylation analysis. The K40 polysaccharide consists of [(O-beta-D-glucopyranosyluronic acid)-(1----4)-O-(2-acetamido-2-deoxy-alpha-D-glucopyranosyl)-(1----6)-O -(2-acetamido-2-deoxy-alpha-D-glucopyranosyl)-(1----4)] repeating units. All of the glucuronic acid residues are substituted amidically with L-serine.

NMR investigation of the 6-deoxy-l-talose-containing O45, O45-related (O45rel), and O66 polysaccharides of Escherichia coli

The structures of the 6-deoxytalose-containing O-specific polysaccharides from the O45 antigen, an O45-related antigen (O45rel), and the O66 antigen (lipopolysaccharides, LPSs) of Escherichia coli were elucidated by chemical characterization and by one- and two-dimensional 1H and 13C NMR spectroscopy. The O45 and O45-related polysaccharides have the following general structure: [formula: see text] For the O45 antigen, X is alpha-D-FucpNAc and for the O45-related antigen, X is beta-D-GlcpNAc. The structure of the O66 polysaccharide is [formula: see text]

Structure of the K16 antigen from Escherichia coli O7:K16:H-, a Kdo-containing capsular polysaccharide.

The K16-antigen from E. coli Rk 21510 (O7:K16:H-) is shown to consist of the repeating unit ----2)-beta-D-Ribf-(1----3)-beta-D-Ribf-(1----5)-alpha-Kd op-(2---- of which approximately 33% is O-acetylated at position 3 of the 2-linked ribose.

Structure of the K95 antigen from Escherichia coli O75:K95:H5, a capsular polysaccharide containing furanosidic KDO-residues

Abstract The structure of the K95 antigenic capsular polysaccharide (K95 antigen) of Escherichia coli O75:K95:H5 was elucidated by determination of the composition, 1 H- and 13 C-n.m.r. spectroscopy, periodate oxidation, and methylation analysis. The K95 polysaccharide, which contains furanosidic 3-deoxy- d - manno -2-octulosonic acid (KDO f ) residues, consists of →3)-β- d -Rib-(1→8)-KDO f -(2→ repeating units, has a molecular weight of ∼25,000 (∼65 repeating units), and is randomly O -acetylated (1 acetyl group per repeating unit at unknown positions).