The rfb gene cluster of Yersinia entemcolitica serotype 0:8 (Ye08) 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 Kiinamyllynkatu 13, 20520 Turku, Finland essential for the synthesis and expression of the Ye08 0-side-chain in Escherichia coli. Deletion analysis generated a derivative that expressed semirough LPS, a phenotype typical of an rfc mutant lacking the 0-antigen polymerase. The

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... eletions and transcomplementation experiments allowed localization of the rfc gene to the 3'-end of the rfb gene cluster. The deduced Ye08 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 rfc gene is similar to other rfc genes. Nucleotide sequence analysis identified three other genes upstream of rfc. Two of the gene products showed 60-70°/0 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 O/ o identical to the bacterial GalE protein, UDPglucose bepimerase, which catalyses the epimerization of UDPglucose to UDPgalactose. Since mannose and galactose are both present in the Ye08 0-antigen repeat unit, the above three genes are likely to belong to the rib gene cluster. A gene similar to the gsk gene downstream of rfc, and genes similar to adk and hemH upstream of the rfb gene cluster, were recognized. Thus the gene cluster of Ye08 is located between the ad&-hemH and gsk loci, and the order is adk-hemH-dbfc-gsk in the chromosome. Also in other Yersinia spp., the locus downstream of the hemH gene is occupied by gene clusters associated with LPS biosynthesis.