Structures and gene clusters of the O-specific polysaccharides of the lipopolysaccharides of Escherichia coli O69 and O146 containing glycolactilic acids: ether conjugates of D-GlcNAc and D-Glc with (R)- and (S)-lactic acid.

Based on the O-specific polysaccharides of the lipopolysaccharides (O-polysaccharides, O-antigens), strains of a clonal species Escherichia coli are classified into 184 O serogroups. In this work, structures of the O-polysaccharides of E. coli O69 and O146 were elucidated and gene clusters for their biosynthesis were characterized. The O-polysaccharides were released from the lipopolysaccharides by mild acid hydrolysis and studied by sugar analysis and one- and two-dimensional 1H and 13C NMR spectroscopy before and after O-deacetylation. The O146 polysaccharide was also studied by Smith degradation. The O69 and O146 polysaccharides were found to contain ether conjugates of monosaccharides with lactic acid called glycolactilic acids: 2-acetamido-2-deoxy-4-O-[(R)-1-carboxyethyl]-D-glucose (D-GlcNAc4Rlac) and 3-O-[(S)-1-carboxyethyl]-D-glucose (D-Glc3Slac), respectively. Structures of the pentasaccharide repeats of the O-polysaccharides were established, and that of E. coli O69 was found to differ in the presence of D-GlcNAc4Rlac from the structure reported for this bacterium earlier (Erbing C, Kenne L, Lindberg B. 1977. Carbohydr Res. 56:371-376). The O-antigen gene clusters of E. coli O69 and O146 between conserved genes galF and gnd were analyzed taking into account the O-polysaccharide structures established, and functions of putative genes for synthesis of D-Glc3Slac and D-GlcNAc4Rlac and for glycosyltransferases were assigned based on homology with O-antigen biosynthesis genes of other enteric bacteria. It was found that in E. coli and Shigella spp. predicted enolpyruvate reductases of the biosynthesis pathway of glycolactilic acids, LarR and LarS, which catalyze formation of conjugates with (R)- or (S)-lactic acid, respectively, are distinguished by sequence homology and size.

Characterization of the dTDP-D-fucofuranose biosynthetic pathway in Escherichia coli O52.

D-fucofuranose (D-Fucf) is a component of Escherichia coli O52 O antigen. This uncommon sugar is also the sugar moiety of the anticancer drug--gilvocarcin V produced by many streptomycetes. In E. coli O52, rmlA, rmlB, fcf1 and fcf2 were proposed in a previous study by our group to encode the enzymes of the dTDP-D-Fucf (the nucleotide-activated form of D-Fucf) biosynthetic pathway. In this study, Fcf1 and Fcf2 from E. coli O52 were expressed, purified and assayed for their respective activities. Novel product peaks from enzyme-substrate reactions were detected by capillary electrophoresis and the structures of the product compounds were elucidated by electro-spray ionization mass spectrometry and nuclear magnetic resonance spectroscopy. Fcf1 was confirmed to be a dTDP-6-deoxy-D-xylo-hex-4-ulopyranose reductase for the conversion of dTDP-6-deoxy-D-xylo-hex-4-ulopyranose to dTDP-D-fucopyranose (dTDP-D-Fucp), and Fcf2 a dTDP-D-Fucp mutase for the conversion of dTDP-D-Fucp to dTDP-D-Fucf. The K(m) of Fcf1 for dTDP-6-deoxy-D-xylo-hex-4-ulopyranose was determined to be 0.38 mM, and of Fcf2 for dTDP-D-Fucp to be 3.43 mM. The functional role of fcf1 and fcf2 in the biosynthesis of E. coli O52 O antigen were confirmed by mutation and complementation tests. This is the first time that the biosynthetic pathway of dTDP-D-Fucf has been fully characterized.