title: Electroporation of DNA sequences from the pathogenicity locus (PaLoc) of toxigenic Clostridium difficile into a non-toxigenic strain.
abstract: Toxigenic Clostridium difficile is the etiologic agent of C. difficile-associated diarrhoea (CDAD), the most common cause of hospital-acquired infectious 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. difficile. Genetic and virulence studies at the molecular level in C. difficile have been hindered by the lack of techniques for DNA manipulation in this species. We describe the electroporation of DNA fragments from a toxigenic isolate into a non-toxigenic strain of C. difficile. Using previously described 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 resulting transformed clones were screened for the introduced gene fragments by PCR, which confirmed their presence. This is the first description of introduction of DNA into C. difficile by electroporation.
authors.affiliation: Department of Internal Medicine, Division of Infectious and Immunologic Diseases, University of California-Davis, PSSB Suite 500, Sacramento, CA 95817, USA. grit.ackermann@ucdmc.ucdavis.edu
authors.forename: G
authors.initials: G
authors.lastname: Ackermann
authors.forename: Y J
authors.initials: YJ
authors.lastname: Tang
authors.forename: J P
authors.initials: JP
authors.lastname: Henderson
authors.forename: A C
authors.initials: AC
authors.lastname: Rodloff
authors.forename: J
authors.initials: J
authors.lastname: Silva
authors.forename: S H
authors.initials: SH
authors.lastname: Cohen
mesh_terms: D001426:Bacterial Proteins; D001427:Bacterial Toxins / Q000235:genetics*; D003001:Cloning, Molecular; D016360:Clostridioides difficile / Q000235:genetics* / Q000472:pathogenicity*; D003015:Clostridium Infections / Q000209:etiology*; D004269:DNA, Bacterial / Q000235:genetics; D018274:Electroporation / Q000379:methods*; D006801:Humans; D016133:Polymerase Chain Reaction; D014774:Virulence
chemical_list: D001426:Bacterial Proteins; D001427:Bacterial Toxins; D004269:DNA, Bacterial; C031815:toxB protein, Clostridium difficile