Abstract
Conjugation is fundamental for the acquisition of new genetic traits and the development of antibiotic resistance in pathogenic organisms. Here, we show that a hypothetical Clostridium perfringens protein, TcpK, which is encoded by the tetracycline resistance plasmid pCW3, is essential for efficient conjugative DNA transfer. Our studies reveal that TcpK is a member of the winged helix-turn-helix (wHTH) transcription factor superfamily and that it forms a dimer in solution. Furthermore, TcpK specifically binds to a nine-nucleotide sequence that is present as tandem repeats within the pCW3 origin of transfer (oriT). The X-ray crystal structure of the TcpK–TcpK box complex reveals a binding mode centered on and around the β-wing, which is different from what has been previously shown for other wHTH proteins. Structure-guided mutagenesis experiments validate the specific interaction between TcpK and the DNA molecule. Additional studies highlight that the TcpK dimer is important for specific DNA binding.
Highlights
Conjugation is fundamental for the acquisition of new genetic traits and the development of antibiotic resistance in pathogenic organisms
In our previous work, we demonstrated that the unique TcpM protein functions as the pCW3 DNA relaxase[6]
We have identified TcpK, a protein encoded by a gene upstream of the oriT site and tcpM, as being a major factor required for the efficient conjugation of the pCW3 plasmid
Summary
Conjugation is fundamental for the acquisition of new genetic traits and the development of antibiotic resistance in pathogenic organisms. In the Gram-positive pathogenic bacterium Clostridium perfringens the conjugative tetracycline resistance plasmid pCW3 is representative of a large class of closely related toxin and antibiotic resistance plasmids[1] Conjugative transfer of this plasmid is mediated by the highly conserved tcp locus, which encodes at least eight proteins that are known to be essential for efficient DNA transfer[2]. In vitro the nicking activity of TcpM does not appear to be sequence specific Given this finding, we hypothesized that conserved genes located upstream or downstream of the oriT site may encode accessory proteins that provide the specificity for relaxosome formation. We demonstrate that one of these genes, tcpK, which is located immediately upstream of the oriT site (Fig. 1a) and encodes a conserved protein of unknown structure and function, is important for DNA transfer and represents a new component of the conjugative machinery
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