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Journal of Bacteriology, November 2007, p. 7531-7538, Vol. 189, No. 21
0021-9193/07/$08.00+0     doi:10.1128/JB.00767-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Epsilon-Toxin Plasmids of Clostridium perfringens Type D Are Conjugative{triangledown} ,{dagger}

Meredith L. Hughes,1,{ddagger} Rachael Poon,1,{ddagger} Vicki Adams,1 Sameera Sayeed,2 Juliann Saputo,3 Francisco A. Uzal,3 Bruce A. McClane,1,2 and Julian I. Rood1*

Australian Research Council Centre of Excellence in Structural and Functional Microbial Genomics, Department of Microbiology, Monash University, Victoria 3800, Australia,1 Department of Molecular Genetics and Biochemistry, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261,2 California Animal Health and Food Safety Laboratory, San Bernardino Branch, School of Veterinary Medicine, University of California, Davis, San Bernardino, California 924083

Received 16 May 2007/ Accepted 3 August 2007

Isolates of Clostridium perfringens type D produce the potent epsilon-toxin (a CDC/U.S. Department of Agriculture overlap class B select agent) and are responsible for several economically significant enterotoxemias of domestic livestock. It is well established that the epsilon-toxin structural gene, etx, occurs on large plasmids. We show here that at least two of these plasmids are conjugative. The etx gene on these plasmids was insertionally inactivated using a chloramphenicol resistance cassette to phenotypically tag the plasmid. High-frequency conjugative transfer of the tagged plasmids into the C. perfringens type A strain JIR325 was demonstrated, and the resultant transconjugants were shown to act as donors in subsequent mating experiments. We also demonstrated the transfer of "unmarked" native {varepsilon}-toxin plasmids into strain JIR325 by exploiting the high transfer frequency. The transconjugants isolated in these experiments expressed functional {varepsilon}-toxin since their supernatants had cytopathic effects on MDCK cells and were toxic in mice. Using the widely accepted multiplex PCR approach for toxin genotyping, these type A-derived transconjugants were genotypically type D. These findings have significant implications for the C. perfringens typing system since it is based on the toxin profile of each strain. Our study demonstrated the fluid nature of the toxinotypes and their dependence upon the presence or absence of toxin plasmids, some of which have for the first time been shown to be conjugative.

* Corresponding author. Mailing address: Department of Microbiology, Monash University, Victoria 3800, Australia. Phone: (613) 9905 4825. Fax: (613) 9905 4811. E-mail: julian.rood{at}med.monash.edu.au

{triangledown} Published ahead of print on 24 August 2007.

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

{ddagger} M.L.H. and R.P. contributed equally to this paper and are joint first authors.

Journal of Bacteriology, November 2007, p. 7531-7538, Vol. 189, No. 21
0021-9193/07/$08.00+0     doi:10.1128/JB.00767-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



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  • Miyamoto, K., Li, J., Sayeed, S., Akimoto, S., McClane, B. A. (2008). Sequencing and Diversity Analyses Reveal Extensive Similarities between Some Epsilon-Toxin-Encoding Plasmids and the pCPF5603 Clostridium perfringens Enterotoxin Plasmid. J. Bacteriol. 190: 7178-7188 [Abstract] [Full Text]  
  • Teng, W. L., Bannam, T. L., Parsons, J. A., Rood, J. I. (2008). Functional Characterization and Localization of the TcpH Conjugation Protein from Clostridium perfringens. J. Bacteriol. 190: 5075-5086 [Abstract] [Full Text]  


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