TcpM: a novel relaxase that mediates transfer of large conjugative plasmids from Clostridium perfringens

Summary Conjugative transfer of toxin and antibiotic resistance plasmids in Clostridium perfringens is mediated by the tcp conjugation locus. Surprisingly, neither a relaxase gene nor an origin of transfer (oriT) has been identified on these plasmids, which are typified by the 47 kb tetracycline resistance plasmid pCW3. The tcpM gene (previously called intP) encodes a potential tyrosine recombinase that was postulated to be an atypical relaxase. Mutagenesis and complementation studies showed that TcpM was required for wild‐type transfer of pCW3 and that a tyrosine residue, Y259, was essential for TcpM activity, which was consistent with the need for a relaxase‐mediated hydrophilic attack at the oriT site. Other catalytic residues conserved in tyrosine recombinases were not required for TcpM activity, suggesting that TcpM was not a site‐specific recombinase. Mobilization studies led to the identification of the oriT site, which was located in the 391 bp intergenic region upstream of tcpM. The oriT site was localized to a 150 bp region, and gel mobility shift studies showed that TcpM could bind to this region. Based on these studies we postulate that conjugative transfer of pCW3 involves the atypical relaxase TcpM binding to and processing the oriT site to initiate plasmid transfer. No relaxase gene or oriT site had been recognized on conjugative plasmids from Clostridium perfringens. We have now identified a novel pCW3‐encoded relaxase, TcpM, that is required for wild‐type conjugative transfer. TcpM binds to an oriT site that we define here. TcpM was originally identified as a potential tyrosine recombinase, but we show that only the conserved Y259 residue is required for transfer, indicating that TcpM does not act as a tyrosine recombinase in conjugation.