TRANSLOCATION FAILURE IN A TYPE-4 PILIN OPERON - RFB AND TCPT MUTANTSIN VIBRIO-CHOLERAE

Defined chromosomal mutations that lead to assembly failure of the tox in coregulated pilus (TCP) of Vibrio cholerae provide useful insights into the biogenesis of a type-4 pilus. Mutants in rfb affecting LPS O- antigen biosynthesis, and strains depleted of the cytoplasmic membrane -associated ATP-binding protein TcpT, provide contrasting TCP export-d efective phenotypes acting at different locations. Mutants in the pero samine biosynthesis pathway of V. cholerae 569B result in an rfb pheno type with an LPS consisting only of core oligosaccharide and lipid A. Such strains are unable to assemble TCP, and TcpA subunits are found i n the periplasm and membrane fractions. In both rfb and tcpT mutants, the export defect is specific and complete. TcpT is a member of a larg e family of cytoplasmic membrane-associated ATP-binding proteins which are essential in type-4 pilin systems and in many non-pilin outer mem brane transporters in Gram-negative bacteria. The behaviour of translo cation-arrested TcpA in rfb and tcpT mutants is indistinguishable from that within assembled pilus under a range of conditions including flo tation in density gradients, chemical cross-linking, and detergent ext raction experiments. From the data presently available, it would appea r that TcpA requires TcpT-mediated translocation from the cytoplasmic membrane and that TcpT stabilizes the subunit at or immediately beyond this stage, before crossing the outer membrane. (C) 1997 Elsevier Sci ence B.V.