Functional characterization and localization of the TcpH conjugation protein from Clostridium perfringens

In Clostridium perfringens, conjugative plasmids encode important virulence factors, such as toxins and resistance determinants. All of these plasmids carry a conjugation locus that consists of 11 genes: intP and tcpA to tcpJ. Three proteins, TcpA, a potential coupling protein, TcpF, a putative ATPase that is similar to ORF15 from Tn916, and TcpH, which contains VirB6-like domains, are essential for conjugation in the prototype conjugative plasmid pCW3. To analyze the functional domains of TcpH, a putative structural component of the mating-pair formation complex and deletion and site-directed mutants were constructed and analyzed. The results showed that the N-terminal 581 residues and the conserved 242VQQPW246 motif were required for conjugative transfer. Bacterial two-hybrid and biochemical studies showed that TcpH interacted with itself and with TcpC. An analysis of the tcpH mutants demonstrated that the region required for these interactions also was localized to the N-terminal 581 residues and that the function of the C-terminal region of TcpH was independent of protein-protein interactions. Finally, immunofluorescence studies showed that TcpH and TcpF were located at both cell poles of donor C. perfringens cells. The results provide evidence that TcpH is located in the cell membrane, where it oligomerizes and interacts with TcpC to form part of the mating-pair formation complex, which is located at the cell poles and is closely associated with TcpF.