Electroporation of DNA sequences from the pathogenicity locus (PaLoc) of toxigenic Clostridium difficile into a non-toxigenic strain

Toxigenic Clostridium difficile is the etiologic agent of C. difficile-asso ciated diarrhoea (CDAD), the most common cause of hospital-acquired infecti ous diarrhoea. The genes tcdA and tcdB, which encode for the toxin A and B proteins, are part of the pathogenicity locus (PaLoc) of toxigenic C. diffi cile. Genetic and virulence studies at the molecular level in C. difficile have been hindered by the lack of techniques for DNA manipulation in this s pecies. We describe the electroporation of DNA fragments from a toxigenic i solate into a non-toxigenic strain of C. difficile. Using previously descri bed methods of electroporation into Clostridium spp., the complete toxin B gene and polymerase chain reaction (PCR) fragments of the PaLoc were cloned and electroporated into a non-toxigenic strain of C. difficile. The result ing transformed clones were screened for the introduced gene fragments by P CR, which confirmed their presence. This is the first description of introd uction of DNA into C. difficile by electroporation. (C) 2001 Academic Press .