Identification of the replication region in pBCNF5603, a bacteriocin-encoding plasmid, in the enterotoxigenic Clostridium perfringens strain F5603

Kazuaki Miyamoto, Soshi Seike, Teruhisa Takagishi, Kensuke Okui, Masataka Oda, Masaya Takehara, Masahiro Nagahama

Most recent studies of Clostridium perfringens plasmids have focused on toxin-encoding or antibiotic resistance plasmids. To cause intestinal disease, a toxigenic strain must grow in the intestines to levels allowing for sufficient toxin production and this in vivo growth often involves overcoming the normal intestinal microbial population. For this purpose, bacteriocin production might be important. In this study, as the first step in the genetic analysis of a co-existing plasmid with an enterotoxin gene (cpe)-encoding plasmid, the bacteriocin gene-encoding plasmid, pBCNF5603, was completely sequenced. This plasmid has some homology with two previously sequenced C. perfringens plasmids, namely, pCP13 carrying a cpb2 gene and pIP404 carrying a bcn gene. Using recombinant plasmids, the rep gene homologous to the PCP63 gene on pCP13 appeared to be functional. Comparative genomics indicated that the identified rep gene homologs were found on two additional toxin plasmids, pCP-OS1 and pCP-TS1. While functional analysis using recombinant plasmids indicated that pBCNF5603 and pCP13 are likely to be incompatible, the plasmid replication and partitioning region of pBCNF5603 alone was insufficient for stable maintenance of this plasmid. These findings suggest that pBCNF5603 evolved from recombination events between C. perfringens plasmids and inter-species mobile genetic element(s). In addition, the bcn-encoding plasmid, pBCNF5603, is likely to be included in the Inc family, which includes pCP13 and two variant iota-encoding plasmids. Furthermore, the bcn gene on pBCNF5603 could contribute to gastrointestinal disease induced by enterotoxigenic C. perfringens.