Genetic Basis Of Bacterial Surface Antigens In Salmonella And E. Coli

Bacterial surface antigens, such as 0 antigen, H antigen and K antigen, are essential component of bacteria.O-antigen (O-polysaccharide), consisting of a number of repeats of an oligosaccharide unit (O-unit), is an essential part of lipopolysaccharide (LPS) present on the cell surface of Gram-negative bacteria. It is the most variable cell wall constituent due to a high diversity of sugars present, their arrangement, and the linkages between them. The O-antigen variation is the basis for bacterial serotyping, and at least 180 O-serogroups in.Escherichia coli and 46 in Salmonella enterica have been recognized. The genes involved in the biosynthesis of O antigen are generally found on the chromosome as an O antigen gene cluster, and genetic variation in the gene cluster is the major basis for the diversity of O antigen forms. Genes involved in O antigen synthesis are classified into 3 main classes:(1) nucleotide sugar synthesis genes; (2) genes for transfer of sugars; (3) O unit processing genes encoding flippase (Wzx) and polymerase (Wzy). The biosynthesis of disaccharides, oligosaccharides and polysaccharides involves the action of hundreds of different glycosyltransferases. the enzymes that catalyse the transfer of sugar moieties from activated donor molecules to specific acceptor molecules, forming glycosidic bonds.classification of glycosyltransferases using nucleotide diphospho-sugar, nucleotide monophospho-sugars and sugar phosphates and related proteins into distinct sequence-based families. About 90 glycosyltransferase (GT) families have been identified.It is believed that E. coli and Salmonella diverged from a common ancestor about 140 million years ago. However, previous studies shown that there are only three cases in which the O antigen are identical in the two species. In this study,22 Salmonella O antigen gene clusters were sequenced and analyzed. Together with the data in our lab (some are unpublished data) and Genbank, now the DNA sequence of all 46 Salmonella O antigen gene clusters were obtained. The characteristic of Salmonella O antigen gene clusters are similar to those of E. coli. Anomalies are found in some Salmonella O antigen gene clusters, indicating some of them may be formed recently. The comparison shows that there are 18 identical or closely related O antigen gene clusters between Salmonella and E. coli, indicating a more close relationship than expected. The molecular evolution analysis show that the identical O antigen gene cluster of Salmonella and E. coli originated from a common ancestor. The genes in the 0 antigen gene cluster were found to diverge at a higher rate than the housekeeping genes, indicating they are under ongoing selection pressure for better adaptation to the situation. The glycosyltransferase and 0 unit processing gene are found to diverge at a higher rate than nucleotide sugar synthesis genes, which may be caused by the difference in the pressure exerted by natural selection.We have cloned and identified the genes (wfaP in 056 and wfgD in 0152) within the two 0 antigen gene clusters that encode glucosyltransferases involved in the synthesis of this linkage. The two genes share 59% and 46% identity at DNA and amino acid level, respectively. A synthetic substrate analog of the natural acceptor substrate undecaprenol-pyrophosphate-lipid (GlcNAc-PP-PhU) was used as an acceptor and UDP-Glc as a donor substrate to demonstrate that both wfgD and wfaP encode -1,3-Glc transferases. The wfeD gene in the O-antigen gene cluster of S. boydii type 14 was proposed to encode a galactosyltransferase (GalT) involved in O-antigen synthesis. We show here that the wfeD gene product is a 4GalT that synthesizes the Gal l-4GlcNAc-R linkage. The enzyme was shown to be specific for the UDP-Gal donor substrate and required pyrophosphate in the acceptor substrate. Divalent metal ions such as Mn2+, Ni2+, and surprisingly also Pb2+, were able to enhance enzyme activity.Flagella confer bacterial motility and contribute to bacterial pathogenesis. Flagellin, also known as H antigen, is one of major surface antigens in Gram-negative bacteria. Flagellar phase variation is an important mechanism for bacterial survival against host defense systems. Although E. coli has been generally considered monophasic, occurrence of flagellar phase variation has been demonstrated in some strains. In E. coli H3 and H47, the flagellar phase variation is mediated by the integration and excision of the flk GI controlled by a site-specific recombinase, which provides a switch for alternating expression of fliC and flkA flagellin gene. This is the first time that a molecular mechanism for flagellar phase variation has been reported in E. coli.But until now, only the H17 flagellin-specifying gene, flnA, has not been found. In this study, we identified the flnA gene and investagated the mechanism of flagellar pahse variation involved in E. coli H17 serotype strain. By generating fliC-expressing variants of H17 strains, unilateral flagellar phase variation in this strain was shown to be mediated by excision of the flnA region and the excised region existed as extrachromosomal circular form, indicating that there is a genomic island. The function of the proposed integrase gene was confirmed by deletion and a complementation test. A general model for flagellar phase variation in E. coli H17 serotype strain can be expressed as fliCoff+flnAon→fliCon+flnAnone.