SEROTYPE CONVERSION IN VIBRIO-CHOLERAE O1

Vibrio cholerae O1 exists as two major serotypes, Inaba and Ogawa, whi ch are associated with the O antigen of the lipopolysaccharide and are capable of unequal reciprocal interconversion. The 20-kilobase rfb re gions encoding O-antigen biosynthesis in strains 569B (Inaba) and O17 (Ogawa) have been cloned in Escherichia coli K-12 and the nucleotide s equences have been determined. Besides several base substitutions and a small deletion in the 569B sequence relative to O17, there is a sing le nucleotide change resulting in a TGA stop codon within the gene for the 32-kDa RfbT protein. We have demonstrated that rfbT is responsibl e for serotype conversion (Inaba to Ogawa). The construction of a spec ific rfbT mutation in the Ogawa strain O17, and the ability of the gen e from O17 to complement Inaba strains to Ogawa, confirmed rfbT as the gene required for the Ogawa serotype. By Southern hybridization and s equencing of PCR products of a number of strains, we have shown that t he changes observed in one Inaba strain (569B) are not conserved in ot her Inaba strains. This may explain why some Inaba strains are able to convert to Ogawa whereas others are not. The protein encoded by rfbT has been identified and expressed in E. coli K-12 using a phage T7 exp ression system. Amino-terminal analysis of partially purified protein has identified the translational start of the protein. Primer extensio n studies have enabled the 5' end of the mRNA to be defined. It exists as a separate transcript from the rest of the rfb region, and the dis tinctive G + C content of rfbT suggests that it has been acquired from a non-Vibrio source.