Summary: The gene cluster of serotype O:8 (YeO8) strain 8081-c was cloned by cosmid cloning. Restriction mapping, deletion analysis and transposon mutagenesis showed that about 19 kb of the cloned DNA is essential for the synthesis and expression of the YeO8 O-side-chain in Deletion analysis generated a derivative that expressed semirough LPS, a phenotype typical of an mutant lacking the O-antigen polymerase. The deletions and transcomplementation experiments allowed localization of the gene to the 3'-end of the gene cluster. The deduced YeO8 Rfc did not share significant amino acid sequence similarity with any other protein, but its amino acid composition and hydrophobicity profile are similar to those of identified Rfc proteins. In addition, the codon usage of the gene is similar to other genes. Nucleotide sequence analysis identified three other genes upstream of Two of the gene products showed 60-70% identity to the RfbM and RfbK proteins that are biosynthetic enzymes for the GDPmannose pathway of enterobacteria. The third gene product was about 50-80% identical to the bacterial GalE protein, UDPglucose 4-epimerase, which catalyses the epimerization of UDPglucose to UDPgalactose. Since mannose and galactose are both present in the YeO8 O-antigen repeat unit, the above three genes are likely to belong to the gene cluster. A gene similar to the gene downstream of and genes similar to and upstream of the gene cluster, were recognized. Thus the gene cluster of YeO8 is located between the and loci, and the order is in the chromosome. Also in other spp., the locus downstream of the gene is occupied by gene clusters associated with LPS biosynthesis.


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