The gene cluster directing O-antigen biosynthesis in Yersinia enterocolitica serotype O:8: identification of the genes for mannose and galactose biosynthesis and the gene for the O-antigen polymerase

Abstract

Summary: Therfbgene cluster ofYersinia enterocoliticaserotype 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 inEscherichia coli.Deletion analysis generated a derivative that expressed semirough LPS, a phenotype typical of anrfcmutant lacking the O-antigen polymerase. The deletions and transcomplementation experiments allowed localization of therfcgene to the 3'-end of therfbgene 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 therfcgene is similar to otherrfcgenes. Nucleotide sequence analysis identified three other genes upstream ofrfc.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 therfbgene cluster. A gene similar to thegskgene downstream ofrfc,and genes similar toadkandhemHupstream of therfbgene cluster, were recognized. Thus therfbgene cluster of YeO8 is located between theadk-hemHandgskloci, and the order isadk-hemH-rfb-rfc-gskin the chromosome. Also in otherYersiniaspp., the locus downstream of thehemHgene is occupied by gene clusters associated with LPS biosynthesis.