Membrane association and multimerization of TcpT, the cognate ATPase ortholog of the Vibrio cholerae toxin-coregulated-pilus biogenesis apparatus.
The toxin-coregulated pilus (TCP) is one of the major virulence factors of Vibrio cholerae. Biogenesis of this type 4 pilus (Tfp) requires a number of structural components encoded by the tcp operon. TcpT, the cognate putative ATPase, is required for TCP biogenesis and all TCP-mediated functions. We studied the stability and localization of TcpT in cells containing in-frame deletions in each of the tcp genes. TcpT was detectable in each of the biogenesis mutants except the DeltatcpT strain. TcpT was localized to the inner membrane (IM) in a TcpR-dependent manner. TcpR is a predicted bitopic inner membrane protein of the TCP biogenesis apparatus. Using metal affinity pull-down experiments, we demonstrated interaction between TcpT and TcpR. Using Escherichia coli as a heterologous system, we investigated direct interaction between TcpR and TcpT. We report that TcpR is sufficient for TcpT IM localization per se; however, stable IM localization of TcpT requires an additional V. cholerae-specific factor(s). A LexA-based two-hybrid system was utilized to define interaction domains of the two proteins. We demonstrate a strong interaction between the cytoplasmic domain of TcpR and the N-terminal 100 amino acid residues of TcpT. We also demonstrated the ability of the C-terminal domain of TcpT to multimerize.
Tripathi
Shital A
SA
Department of Microbiology and Immunology, Dartmouth Medical School, HB7550, Hanover, NH 03755, USA.
Taylor
Ronald K
RK
D000251:Adenosine Triphosphatases / Q000235:genetics* / Q000378:metabolism*; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D002462:Cell Membrane / Q000737:chemistry / Q000201:enzymology*; D019281:Dimerization; D004926:Escherichia coli / Q000235:genetics; D010861:Fimbriae, Bacterial / Q000235:genetics*; D017353:Gene Deletion; D005798:Genes, Bacterial; D008565:Membrane Proteins / Q000235:genetics* / Q000378:metabolism*; D009876:Operon; D011485:Protein Binding; D025941:Protein Interaction Mapping; D020798:Two-Hybrid System Techniques; D014734:Vibrio cholerae / Q000737:chemistry / Q000201:enzymology* / Q000472:pathogenicity; D037521:Virulence Factors / Q000235:genetics*
D001426:Bacterial Proteins; D008565:Membrane Proteins; D037521:Virulence Factors; D000251:Adenosine Triphosphatases