Translocation failure in a type-4 pilin operon: rfb and tcpT mutants in Vibrio cholerae

• Published in: Gene Vol. 192; no. 1; pp. 71 - 77
• Main Authors: Iredell, Jon R, Manning, Paul A
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 11.06.1997
• Subjects: ATPase; Bacterial Outer Membrane Proteins - genetics; Bacterial Outer Membrane Proteins - metabolism; Bacterial Proteins - genetics; Bacterial Proteins - metabolism; Biological Transport; Cell Membrane - metabolism; Fimbriae Proteins; Fimbriae, Bacterial - genetics; General secretory pathway; Genes, Bacterial; Mannose - analogs & derivatives; Mannose - biosynthesis; Mutation; O Antigens - biosynthesis; Operon; Protein export; rfb; Subunit translocation; TcpA; TcpT; Toxin coregulated pilus (TCP); Type-4 pilus assembly; Vibrio cholerae; Vibrio cholerae - genetics; Vibrio cholerae - metabolism; Vibrio cholerae - ultrastructure; General secretory pathway; CM, cytoplasmic membrane; EDTA, ethylenediaminetetraacetic acid; IEM, immuno-electron microscopy; TcpA; Ab, antibody(ies); Subunit translocation; PBS, phosphate-buffered saline (0.14 M NaCl, 27 mM KCl, 1.5 mM K 2HPO 4, 8 mM Na 2HPO 4); Vc, V. cholerae; SDS, sodium dodecyl sulfate; Type-4 pilus assembly; EM, electron microscopy; wt, wild type; Toxin coregulated pilus (TCP); TcpT; PAGE, polyacrylamide gel electrophoresis; Protein export; ATP, adenosine triphosphate; OM, outer membrane; P., Pseudomonas; ATPase; Km, kanamycin; TEM, transmission electron microscopy; rfb; ECL, enhanced chemiluminescence; K., Klebsiella; nt, nucleotide(s); TCP, toxin-coregulated pilus; E., Escherichia; LPS, lipopolysaccharide; IF, immunofluorescence; V., Vibrio; tcp, gene(s) encoding Tcp protein(s)
• ISSN: 0378-1119; 1879-0038
• DOI: 10.1016/S0378-1119(97)00040-1

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.