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Journal of Bacteriology, November 2008, p. 7178-7188, Vol. 190, No. 21
0021-9193/08/$08.00+0     doi:10.1128/JB.00939-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Sequencing and Diversity Analyses Reveal Extensive Similarities between Some Epsilon-Toxin-Encoding Plasmids and the pCPF5603 Clostridium perfringens Enterotoxin Plasmid{triangledown} ,{dagger}

Kazuaki Miyamoto,1,{ddagger} Jihong Li,2,{ddagger} Sameera Sayeed,2,§ Shigeru Akimoto,1 and Bruce A. McClane2*

Department of Microbiology, Wakayama Medical University School of Medicine, 811-1 Kimiidera, Wakayama, 641-0012, Japan,1 Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 152612

Received 9 July 2008/ Accepted 22 August 2008

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, but etx genetics have been poorly studied in type B isolates. This study reports the first sequencing of any etx plasmid, i.e., pCP8533etx, from type B strain NCTC8533. This etx plasmid is 64.7 kb, carries tcp 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-like etx 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 different etx plasmids. Sequences upstream of the etx gene vary between type B isolates and some type D isolates that do not carry a pCP8533etx-like etx plasmid. However, nearly all type B and D isolates have an etx locus with an upstream IS1151, and those etx loci typically reside near a dcm ORF. These results suggest that pCPF5603 and pCP8533etx evolved from insertion of mobile genetic elements carrying enterotoxin or etx genes, respectively, onto a common progenitor plasmid.

* Corresponding author. Mailing address: Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261. Phone: (412) 648-9022. Fax: (412) 624-1401. E-mail: bamcc{at}pitt.edu

{triangledown} Published ahead of print on 5 September 2008.

{dagger} Supplemental material for this article may be found at http://jb.asm.org/.

{ddagger} Kazuaki Miyamoto and Jihong Li contributed equally to this work.

§ Present address: Science Department, Marywood University, 2300 Adams Ave., Scranton, PA 18509.

Journal of Bacteriology, November 2008, p. 7178-7188, Vol. 190, No. 21
0021-9193/08/$08.00+0     doi:10.1128/JB.00939-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.





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