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Journal of Virology, December 2009, p. 12037-12045, Vol. 83, No. 23
0022-538X/09/$08.00+0     doi:10.1128/JVI.01256-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Bacteriophage-Mediated Toxin Gene Regulation in Clostridium difficile{triangledown}

Revathi Govind,1,2* Govindsamy Vediyappan,1 Rial D. Rolfe,1 Bruno Dupuy,2 and Joe A. Fralick1

Department of Microbiology and Immunology, Texas Tech University Health Sciences Center, Lubbock, Texas 79430,1 Unite Génétique Moléculaire Bactérienne, Institute Pasteur, 28 Rue du Docteur Roux, Paris, France 750152

Received 17 June 2009/ Accepted 14 September 2009

Clostridium difficile has been identified as the most important single identifiable cause of nosocomial antibiotic-associated diarrhea and colitis. Virulent strains of C. difficile produce two large protein toxins, toxin A and toxin B, which are involved in pathogenesis. In this study, we examined the effect of lysogeny by {Phi}CD119 on C. difficile toxin production. Transcriptional analysis demonstrated a decrease in the expression of pathogenicity locus (PaLoc) genes tcdA, tcdB, tcdR, tcdE, and tcdC in {Phi}CD119 lysogens. During this study we found that repR, a putative repressor gene of {Phi}CD119, was expressed in C. difficile lysogens and that its product, RepR, could downregulate tcdA::gusA and tcdR::gusA reporter fusions in Escherichia coli. We cloned and purified a recombinant RepR containing a C-terminal six-His tag and documented its binding to the upstream regions of tcdR in C. difficile PaLoc and in repR upstream region in {Phi}CD119 by gel shift assays. DNA footprinting experiments revealed similarities between the RepR binding sites in tcdR and repR upstream regions. These findings suggest that presence of a CD119-like temperate phage can influence toxin gene regulation in this nosocomially important pathogen.

* Corresponding author. Mailing address: Department of Microbiology and Immunology, Texas Tech University Health Sciences Center, Lubbock, TX 79430. Phone: (806) 743-4493. Fax: (806) 743-2334. E-mail: revathi.govind{at}ttuhsc.edu

{triangledown} Published ahead of print on 23 September 2009.

Journal of Virology, December 2009, p. 12037-12045, Vol. 83, No. 23
0022-538X/09/$08.00+0     doi:10.1128/JVI.01256-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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