Sequencing and Diversity Analyses Reveal Extensive Similarities between Some Epsilon-Toxin-Encoding Plasmids and the pCPF5603Clostridium perfringensEnterotoxin Plasmid

Clostridium perfringens type B and D isolates produce epsilon-toxin, the third most potent clostridial toxin. The epsilon-toxin gene (etx ) is plasmid borne in type D isolates, butetx genetics have been poorly studied in type B isolates. This study reports the first sequencing of anyetx plasmid, i.e., pCP8533etx, from type B strain NCTC8533. Thisetx plasmid is 64.7 kb, carriestcp conjugative transfer genes, and encodes additional potential virulence factors including beta2-toxin, sortase, and collagen adhesin but not beta-toxin. Interestingly, nearly 80% of pCP8533etx open reading frames (ORFs) are also present on pCPF5603, an enterotoxin-encoding plasmid from type A isolate F5603. Pulsed-field gel electrophoresis and overlapping PCR indicated that a pCP8533etx-likeetx plasmid is also present in most, if not all, other type B isolates and some beta2-toxin-positive,cpe -negative type D isolates, while other type D isolates carry differentetx plasmids. Sequences upstream of theetx gene vary between type B isolates and some type D isolates that do not carry a pCP8533etx-likeetx plasmid. However, nearly all type B and D isolates have anetx locus with an upstream IS1151 , and thoseetx loci typically reside near adcm ORF. These results suggest that pCPF5603 and pCP8533etx evolved from insertion of mobile genetic elements carrying enterotoxin oretx genes, respectively, onto a common progenitor plasmid.