| 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, comprising repeats of an O unit of generally two to six sugars, is one of the most variable cell constituents and contributes major antigenic variability, O-antigen variation is thought to be a major factor in bacterial adaptation to different hosts or situations. Genes responsible for the synthesis of O-antigens are clustered in bacterial choromosome, and these gene clusters provides the best opportunity to study bacterial evolution because of their highly variable nature.S. boydii type 13 may have important status and may play a role in the evolution of Shigella and E. coli. We research the genetic, structure and evolution of its O-antigen gene cluster at first in the world. We find that its O polysaccharide is acid labile owing to the presence of a glycosyl phosphate linkage in the main chain. We conclude that at least part of the S. boydii type 13 O antigen gene cluster originated from V. cholerae or a related species.Both structural and genetic organizations of Escherichia coli O52 O-antigen were studied. O52 employs an ABC transporter dependent pathway for translocation and polymerization of the O units and its O antigen is a disaccharide heteropolymer containing two unusual sugars, furanoid dTDP-D-fucose and 6-deoxy -D- manno - heptose. We showed that the O antigen gene cluster is located between galF and gnd. It is the first time to report that ABC transporter is involved in translocation of a heteropolysacc- haride O-antigen in E. coli. Furthermore, we indentify wzm gene and orf16 gene by deletion and complement and confirm the involvement of wzm in the biosynthesis process of E. coli O52 O-antigen. While the result show that orf16 gene has lost the function. We also surppose the synthesis pathway of furanoid dTDP-D-fucose.The O-antigen gene cluster of Shigella bodyii 10 is sequenced and share the almost same sequence except for IS629, the identity is in the range from 99.8-100% which suggest that the gene cluster of S. bodyii 6 is assembled by intraspecies lateraltransfer with an insertion of IS629 which makes orf8 lose its function of ribosyltransferase, while the O-antigen of S. bodyii 6 lost ribose in the branch.We find an exemple that E. coli O164 origin from Shigella dysenteriae 3. The insertion of 2bp results in the movement of orf and made the gene unfunction.We also research the O-antigen gene cluster of E. coli O56 and O24 which have similar O-antigen structure. Their genes sequences share high identity which indicates that they havenear evolutional relationship. We delete its wzx, wzy and or/9 genes and find that all of them are founctional by complemention experiments.Four groups of Shigella and E.coli strains that have identical O-antigen structure are studied. All of the results show that Shigella, which still stands as a genus with four species today, in reality are clones of E.coli. We find that the site of homologous recombination in rmlBDAC change with different strain.We also make the NJ tree of manB and manC from all E.coli strains, and find that manB is colanic acid-like which let us understand more better the homologous recombination in manB and manC and lateral transfer.There is a very rare sugar in the O antigen gene cluster of E.coli O136 which is EEEC. We finish its sequence and identify its wzy gene by deletion and complemention experiments.We find that insert sequence is important for producing new O-antigen, IS makes the important genes lose their founction and results in new pathogenic bacteria. We also find that IS acts as an important role in DNA lateral transfer in O antigen gene cluster.By PCR testing against representative strains for the 166 E.coli and 43 Shigella O serogroups, we identified genes including glycosyltransferse genes and the O-antigen processing genes are highly specific to Shigella and E.coli. This work provides the basis for a sensit... |
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