TcpA, an FtsK/SpoIIIE homolog, is essential for transfer of the conjugative plasmid pCW3 in Clostridium perfringens

Abstract

The conjugative tetracycline resistance plasmid pCW3 is the paradigm conjugative plasmid in the anaerobic gram-positive pathogen Clostridium perfringens. Two closely related FtsK/SpoIIIE homologs, TcpA and TcpB, are encoded on pCW3, which is significant since FtsK domains are found in coupling proteins of gram-negative conjugation systems. To develop an understanding of the mechanism of conjugative transfer in C. perfringens, we determined the role of these proteins in the conjugation process. Mutation and complementation analysis was used to show that the tcpA gene was essential for the conjugative transfer of pCW3 and that the tcpB gene was not required for transfer. Furthermore, complementation of a pCW3DeltatcpA mutant with divergent tcpA homologs provided experimental evidence that all of the known conjugative plasmids from C. perfringens use a similar transfer mechanism. Functional genetic analysis of the TcpA protein established the essential role in conjugative transfer of its Walker A and Walker B ATP-binding motifs and its FtsK-like RAAG motif. It is postulated that TcpA is the essential DNA translocase or coupling protein encoded by pCW3 and as such represents a key component of the unique conjugation process in C. perfringens.

FIG. 1.

Bioinformatic analysis of TcpA and TcpB. (A) Domain structure analysis of TcpA and TcpB. The analysis was performed using Sosui (24) and Conserved Domain Database (37) searches, and the results were compared with related FtsK-HerA ATPase proteins. TMDs are represented by solid bars; coiled-coil domains are represented by the box with diagonal stripes; the AAD region is represented by a box with vertical stripes; and the Walker A, Walker B, and RAAG motifs are represented by dotted bars. Residues of the TcpA motifs targeted by amino acid substitutions are in bold type, and the conserved arginine residue of the RAAG motif is underlined. (B) Dendrogram constructed using ClustalW alignments of TcpA, TcpB, and FtsK-HerA members and the PHYLIP algorithm (17).

FIG. 2.

Model of TcpA structure. Swiss Model (54) was used to construct a putative model of the central region of TcpA (aa 212 to 420). The template structure was a C-terminal region of FtsK (aa 818 to 1329; PDB: 2iusA) (38).

FIG. 3.

Complementation of pCW3ΔtcpAB and pCW3ΔtcpA mutants. (A) Conjugation-deficient pCW3ΔtcpAB and pCW3ΔtcpA mutants were complemented with functional copies of tcpAB, tcpA, and tcpB and tested for the ability to transfer in mixed plate matings. (B) tcpA orthologs from pJIR26, pCPF4969, and pJGS1495 were tested for conjugative proficiency using mixed plate mating and compared to the wild-type pCW3 derivative. pCW3, wild-type positive control; pJIR1909, nontransferable negative control; M1, pCW3ΔtcpAB1; M2, pCW3ΔtcpAB2; M3, pCW3 ΔtcpA; V, pJIR750 vector control. The transfer efficiencies were expressed as the number of transconjugants per donor cell.

FIG. 4.

Phylogenetic tree of TcpA homologs. The amino acid sequences of seven TcpA orthologs from a range of C. perfringens conjugative plasmids were aligned using ClustalW. The level of identity of each protein to TcpA from pCW3 is indicated in parentheses. The accession numbers are as follows: pCW3, DQ366035; pCPF4969, NC007772; pJIR2774, DQ338473; pCPF5603, NC007773; and pJIR26, DQ338471. The sequences of pJGS1495 and pJGS1721 were obtained from G. Myers, I. Paulsen, J. Songer, B. McClane, R. Titball, J. Rood, and S. Melville (personal communication).

FIG. 5.

Effect of deletion and site-directed mutations on TcpA activity. Isogenic shuttle vector derivatives were used to complement the pCW3ΔtcpA mutant (M), and the conjugation frequencies of the resultant derivatives were determined. The positive control carried the wild-type tcpA gene (WT TcpA_1-530), and the negative control carried the shuttle vector [M(V)]. The mutated tcpA derivatives encoded TcpA_1-469, TcpA_1-365, TcpA_1-316, TcpA_Δ46-69, TcpA_Δ79-104, TcpA_Δ46-104, TcpA _K242A, TcpA _DE334/5AA, and TcpA _Q379A. The solid bars represent TMDs, and the Walker A box, the Walker B box, and the RAAG motif are represented by dotted bars, bars with horizontal stripes, and checked bars, respectively. The crosses indicate the motifs targeted by site-directed mutagenesis.