title: Translocation failure in a type-4 pilin operon: rfb and tcpT mutants in Vibrio cholerae.
abstract: Defined chromosomal mutations that lead to assembly failure of the toxin 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-defective phenotypes acting at different locations. Mutants in the perosamine biosynthesis pathway of V. cholerae 569B result in an rfb phenotype with an LPS consisting only of core oligosaccharide and lipid A. Such strains are unable to assemble TCP, and TcpA subunits are found in the periplasm and membrane fractions. In both rfb and tcpT mutants, the export defect is specific and complete. TcpT is a member of a large family of cytoplasmic membrane-associated ATP-binding proteins which are essential in type-4 pilin systems and in many non-pilin outer membrane transporters in Gram-negative bacteria. The behaviour of translocation-arrested TcpA in rfb and tcpT mutants is indistinguishable from that within assembled pilus under a range of conditions including flotation in density gradients, chemical cross-linking, and detergent extraction experiments. From the data presently available, it would appear 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.
authors.affiliation: Department of Microbiology and Immunology, University of Adelaide, Australia.
authors.forename: J R
authors.initials: JR
authors.lastname: Iredell
authors.forename: P A
authors.initials: PA
authors.lastname: Manning
mesh_terms: D001425:Bacterial Outer Membrane Proteins / Q000235:genetics* / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D001692:Biological Transport; D002462:Cell Membrane / Q000378:metabolism; D036781:Fimbriae Proteins; D010861:Fimbriae, Bacterial / Q000235:genetics*; D005798:Genes, Bacterial; D008358:Mannose / Q000031:analogs & derivatives / Q000096:biosynthesis; D009154:Mutation; D019081:O Antigens / Q000096:biosynthesis; D009876:Operon; D014734:Vibrio cholerae / Q000235:genetics* / Q000378:metabolism / Q000648:ultrastructure
chemical_list: D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; D019081:O Antigens; C085807:TcpA protein, Vibrio cholerae; C079397:rfb protein, Bacteria; D036781:Fimbriae Proteins; C057033:4-amino-4,6-dideoxy-D-mannose; D008358:Mannose