Sequence Of E.coli O-antigen Gene Clusters And Identification Of The Sugar Biosynthesis Genes

Lipopolysaccharide (LPS), an important component of the outer membrane of Gram-negative bacteria, usually consists of three distinct regions: lipid A, core oligosaccharide, and O-antigen.The O-antigen is an important component of the outer membrane of Gram-negative bacteria, it consists of a number of repeats of an oligosaccharide attached to the core polysaccharide and extended into the extra cellular environment. It acts as a receptor for bacteriophages and is also important in the host immune response. The O-antigen is extremely variable, the variation in pathogenesis play an important role. Several studies have indicated that the composition and size of the O-antigen might be a reliable indicator of potential virulence.many forms of o-antigen in a species constitute a polymorphism in which the morphs vary in the set of genes present in the O-antigen gene cluster, the O-antigen diversity of E. coli can be achieved by two means: by obtaining new clusters from other species and by modifying the existing O-antigen gene cluster. The former is an important means of expansion of O-antigen diversity. Compared with glycosyltransferase genes and processing genes , which are commonly heterogeneous due to the different linkages involved, pathway gene are in general homologous regardless of species, for this reason they can be particularly important in effect of lateral gene transfer by homologous recombination involving the DNA flanking the locus in O antigen gene clusters, and pathway gene is also important in studies on evolutionary relationships of polysaccharide gene clusters from different species.O-antigen gene clusters located between galF and gnd from 20 E. coli and gene clusters between gnd and hisI from 3 E. coli were sequenced. Based on the similarity, we purposed the functions of gene and names of genes:1. The sequences located between galF and gnd are O-antigen gene clusters of E. coli strainsO-antigen of 20 E. coli are located between galF and gnd and transcribed from galF to gnd gene, and contain three classes genes: sugar biosynthesis genes, glycosyltransferase genes, and processing genes.The GC content of O-antigen is low: O-antigen gene clusters we sequenced are all of atypical low GC content for the species, Suggesting an origin from outside the species, Moreover, the GC content within the O-antigen clusters greatly differs from gene to gene, indicating that the gene clusters were assembled from several sources.2. Gene recombination done by sugar biosynthesis genes is an important way to create new O-antigen(1). The fucose-related genes of E. coli O156, E. coli O159 and S. dysenteriae 4 share high level identities, and this indicates that they were from the same ancestor. (2). O-antigen clusters of 14 E. coli strains contained rml gene that synthesize the rhamnose in20 strains. And rml genes were analyzed carefully and found it was important in gene recombination and gene transfer from one strain toe another strain.(3). The Fuc2NAc-related genes of E. coli O151, E. coli O29 and O26 share high level identities, and this indicates that they were from the same ancestor.3. The sequences located between gnd and hisI were part of O-antigen cluster of E. coli O155, and contained wzx gene that was processing gene, it was atypical feature.4. Identification of sugar biosynthesis genes: The colitose biosynthesis genes were identified by enzymes methods, the Qui3NAc pathway is proposed by bioinformatics and pathway genes were cloned and ligated to the expression plasmids, the relative proteins were isolated by affinity chromatogram. And the FucNAc biosynthesis genes were identified by indirect method. And mutation and complement experiments was a important way to identify the gene functions.5. We identified wzx and wzy genes and special primers that are highly specific to E. coli. This work provides the basis for a sensitive test for the rapid detection of E. coli.