Oral Presentation Australian Society for Microbiology Annual Scientific Meeting 2018

Identification and characterisation of a new family of conjugative plasmids in Clostridium perfringens (#147)

Thomas D Watts 1 , Callum J Vidor 1 , Milena M Awad 1 , Dena Lyras 1 , Julian I Rood 1 , Vicki Adams 1
  1. Infection and Immunity Program, Biomedicine Discovery Institute and Department of Microbiology, Monash University, Clayton, Vic, Australia

Clostridium perfringens produces an extensive arsenal of toxins that are involved in human diseases ranging from mild enterotoxaemia and food poisoning to potentially fatal clostridial myonecrosis. Many C. perfringens toxins are encoded by genes found on members of the pCW3-family of conjugative plasmids. These plasmids have a high level of sequence similarity, including 11 genes that constitute the unique tcp conjugation locus. Recently, another series of plasmids, the pCP13-like family, have been shown to harbour important toxin genes, including genes that encode the novel binary clostridial enterotoxin, BEC. This family of plasmids was thought to be non-conjugative, but recent studies have shown that an approximately 25 kb region encoded by pCP13 has similarity to the cst conjugation locus of the Paeniclostridium (formerly Clostridium) sordellii toxin plasmid pCS1-1 and a putative conjugation locus on the Clostridium botulinum toxin plasmid pCLL. To determine if pCP13 was conjugative, a genetically marked derivative was constructed by insertionally inactivating the cpb2 toxin gene with an erythromycin resistance determinant. The marked pCP13 derivative transferred at high frequency to a plasmid-free recipient. One presumed product (PcpB4) of the putative pCP13 conjugation locus has a conserved type 4 secretion system (T4SS)-like VirB4 ATPase domain. To determine if the pcpB4 gene was required for conjugation it was insertionally inactivated and the resultant mutant assessed in a conjugation assay. The results showed that it had a greatly reduced transfer frequency (seven orders of magnitude lower) compared to wild-type pCP13. The mutant was complemented in trans with the wild-type pcpB4 gene, which restored the transfer frequency to wild-type levels. Cross-complementation of the pcpB4 mutant with its cstB4 homologue (51.9% amino acid sequence identity) from P. sordellii partially restored its ability to transfer. In conclusion, we have identified a novel conjugation locus that has now been shown to be functional in C. perfringens. This study has significant implications for our understanding of the movement of toxin genes in this important pathogen.