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J. Bacteriol. doi:10.1128/JB.00008-07
Copyright (c) 2007, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

Membrane Association and Multimerization of TcpT, the Cognate ATPase Ortholog of the Vibrio cholerae TCP Biogenesis Apparatus

Department of Microbiology and Immunology, Dartmouth Medical School, Hanover, New Hampshire 03755

^* To whom correspondence should be addressed. Email: ronald.k.taylor{at}dartmouth.edu.

	Abstract

TCP, the toxin co-regulated pilus, 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 tcpT 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 E. 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 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.