| Popis: |
Bacterial conjugation is a major contributor to horizontal gene transfer, and is the predominant means by which plasmid-borne antibiotic resistance and toxin genes are dispersed. In the anaerobic pathogen Clostridium perfringens, the mechanism of plasmid transfer is currently being elucidated. The 11 gene transfer clostridial plasmid (tcp) locus is required for conjugative plasmid transfer and includes the intP and tcpA-tcpJ genes, variants of which are present in all conjugative resistance and toxin plasmids from C. perfringens. In this laboratory previous studies on the prototype conjugative resistance plasmid pCW3 have shown that the tcpA, tcpF and tcpH genes are essential for the conjugative transfer of pCW3. TcpC is a conserved hypothetical protein consisting of 359 amino acid residues with low-level (24%) amino acid sequence identity to Orf13 from Tn916. It has a potential coiled-coil domain and a putative transmembrane domain (TMD) within the N-terminal region. By carrying out mutational and complementation studies this study showed that TcpC is involved in pCW3 conjugation. tcpC mutants were reduced in their ability to transfer by a 100, 000 fold, which was subsequently complemented to wild-type levels. These results provided evidence that TcpC is required for the efficient conjugative transfer of pCW3. In collaboration with Dr. Corrine Porter, the X-ray crystal structure of the 261 amino acid truncated TcpC99-359 protein was determined to 1.8A, which revealed the presence of a homotrimer in a single crystallographic asymmetric unit. Each monomer had two structurally identical globular domains separated by a large poly-linker of seven amino acids and each of these globular domains had structural similarity to that of the predicted periplasmic domain of VirB8 from both Agrobacterium tumefaciens and Brucella suis and also to nuclear transport factor 2 (NTF2) of eukaryotic cells. In addition, this study also showed that TcpC localized to the membrane independently of the other Tcp proteins. A novel interaction between TcpC and the putative peptidoglycan hydrolase TcpG was identified by bacterial two-hybrid analysis. Functional genetic analysis together with bacterial two-hybrid analysis showed that the N-terminal region of TcpC, especially the region spanning residues 57-79, which forms a putative transmembrane domain TMD is essential for interactions with itself, TcpA, TcpH and TcpG and therefore for TcpC function. The results showed that both of the C-terminal globular domains were functional, with the C-terminal domain having a major role in TcpC protein-protein interactions. The conserved residues FFK, which are located within the C-terminal domain, were shown to be essential for its proper folding and for interactions with the putative coupling protein TcpA. Finally, based on these data it was concluded that by forming homo- and heterooligomeric protein complexes, TcpC contributes to the stability and integrity of the pCW3 mating pair formation complex facilitating its efficient conjugative transfer. Peptidoglycan hydrolases specifically associated with conjugation systems are proposed to facilitate the correct assembly of the transfer apparatus by creating a temporally and spatially controlled local opening in the peptidoglycan layer. At the start of this study no reports were available investigating the role of such enzymes in gram positive conjugation systems. Unusually, two putative peptidoglycan hydrolases TcpG and TcpI are encoded by the tcp locus. Mutation and complementation analysis was used to demonstrate that the tcpG gene was required for efficient conjugative transfer of pCW3, but not the tcpI gene. Furthermore, it was also shown that the two predicted catalytic domains of TcpG were active in C. perfringens and that the conserved predicted catalytic site residues E-111, D-136 and C-238 present within these functional domains were essential for TcpG function. In addition, it was shown that an autoaggregation phenotype was associated with E. coli cells producing TcpG, and that a purified recombinant TcpG had peptidoglycan hydrolyzing activity on cognate peptidoglycan. Based on these results it was concluded that TcpG, but not TcpI is required for efficient conjugative transfer of pCW3 and it was postulated that TcpG is a putative peptidoglycan hydrolase that is likely to facilitate the proper assembly of the pCW3 translocation complex. Based on the results obtained in this study and other pCW3 conjugation related studies carried out in this laboratory a model for the transfer of pCW3 is described. |
