[Molecular characterization of O-antigen gene cluster of Escherichia coli O23 reference strain and identification of UDP-N-acetylglucosamine 4-epimerase].

Lipopolysaccharide (LPS) is one of the major components of the outer membrane of gram-negative bacteria. It is an amphipathic molecule composing of lipid A, a core oligosaccharide and an O-specific antigen. O-antigen, which is a repeat unit polysaccharide, is a major contribution to the antigenic variability of the bacterial cell surface. Genes involved in O-antigen biosynthesis are generally found to be clustered between the housekeeping genes galF and gnd on the chromosome of E. coli. E. coli O23 is one of the enterotoxigenic E. coli causing pediatric diarrhea in the developing world. The O-antigen gene cluster of E. coli O23 type strain was amplified by long-range PCR using primers based on galF and gnd and then sequenced. Except for galF and gnd, seven open reading frames were identified and assigned functions on the basis of their similarity to those from available databases. The seven genes include a UDP-N-acetylglucosamine 4-epimerase gene (gne), the O-antigen polymerase gene (wzy), the O-antigen transferase gene (wzx) and four glycosyltransferase genes (orf2, orf4, orf5, orf6). The UDP-N-acetylglucosamine 4-epimerase (Gne) was identified by mutation and complementation complement tests. The structure of Gne was predicted by the homology modeling method, and the active sites were also analyzed. The phylogenetic and structural analysis showed that the Gne derived from the common ancestor with E. coli O23 Gne were UDP-GlcNAc/UDP-GalNAc epimerases. The specific DNA used for rapid molecular genotyping for E. coli O23 was also identified.

[Sequence of Escherichia coli O11 O-antigen gene cluster and identification of molecular markers specific to O11].

Escherichia coli O11 belongs to Shiga toxin-producing Escherichia coli (STEC), which can cause food-borne disease, hemorrhagic colitis, and hemolytic-uremic syndrome (HUS) in humans. Because of its character of specificity, the O-antigen gene cluster provides the best material for the selection of molecular markers which can be used for rapid genotyping of bacterial strain. In this study, the E.coli O11 O-antigen gene cluster was amplified by Long-range PCR and was sequenced using Shotgun-sequencing approach. Twelve open reading frames were assigned functions on the basis of homology in the E. coli O11 O-antigen gene cluster, including UDP-N-acetyl glucosamine-4-epimerase gene (gne), genes responsible for the biosynthesis of GDP-L-fucose (gmd, fcl, gmm, manC, manB), glycosyl transferase genes, O-unit flippase gene (wzx) and O-antigen polymerase gene (wzy). By polymerase chain reaction against representative stains for all the 166 E. coli and 43 Shigella O serotypes, two genes and four pairs of primers were identified to be specific to E. coli O11. Further PCR was done to detect E. coli O11 from the environmental specimens, and the sensitivities for detecting E.coli O11 from the pork and dejecta specimens were 0.25 cfu/g and 2.5 x 10(3) cfu/g, respectively. Moreover, eight probes were designed and proved to be unique to E. coli O11, which provides the basis for a sensitive test of the rapid detection of E. coli O11 by DNA microarray method.

[Sequence of Escherichia coli O138 O-antigen gene cluster and gne identification by bioinformatics].

E. coli O138 is one of the enterotoxigenic Escherichia coli, causing the postweaning diarrhea and edema disease of weaned pigs. The O-antigen gene cluster of E. coli O138 was sequenced and found to contain the genes rmlB-DAC and gne, gna for the biosynthesis of nucleotide sugars dTDP-rhamnose and UDP-GalNAcA, respectively, genes encoding for O unit flippase(wzx), O-antigen polymerase(wzy) and 3 potential transferase genes. The possible biosynthesis pathway for rare UDP-GalNAcA was proposed. Two genes specific to E. coli O138 were identified. This work provides the basis for a sensitive test by PCR for the rapid detection of E. coli O138. Phylogenetic tree for Gne and GalE proteins was generated and comparisons were made among different strains, and results revealed that these proteins are similar in the second structure, and Gne of E. coli O138 was identified by bioinformatics.

[Sequence of Escherichia coli O141 O-antigen gene cluster and analyses of its evolutionary history].

Lipopolysaccharide (LPS) is one of the major components of the outer membrane of gram-negative bacteria. It is an amphipathic molecule compose of lipid A, a core oligosaccharide and an O-specific antigen. O-antigen, which is a repeat-unit polysaccharide, is a major contribution to the antigenic variability of the bacterial cell surface. The genes of O-antigen gene cluster are responsible for the synthesis of the O-antigen. The O-antigen gene cluster of E. coli O141 was sequenced and found to contain the genes rmlBDAC and manBC for the biosynthesis of nucleotide sugars dTDP-rhamnose and GDP-mannose, respectively, encoding genes for Ounit flippase (wzx), O-antigen polymerase (wzy) and potential transferase genes. The possible biosynthesis pathway for O-antigen of E. coli O141 was proposed. Two genes specific to E. coli O141 were identified. This work provides the basis for a sensitive test by PCR for the rapid detection of E. coli O141. Phylogenetic trees for the rmlB, rmlD, rmlA, and rmlC genes and manB, manC genes were generated and the comparisons were made among different strains. We find that these genes are typical E. coli genes and might have been involved in recombination events between O-antigen gene clusters.