Investigating transfer of large chromosomal regions containing the pathogenicity locus between clostridium difficile strains

The genomes of all sequenced Clostridium difficile isolates contain multiple mobile genetic elements. The chromosomally located pathogenicity locus (PaLoc), encoding the cytotoxins TcdA and TcdB, was previously hypothesized to be a mobile genetic element; however, mobility was not demonstrated. Here we describe the methods used to facilitate and detect the transfer of the PaLoc from a toxigenic strain into non-toxigenic strains of C. difficile. Although the precise mechanism of transfer has not yet been elucidated, a number of controls are described which indicate transfer occurs via a cell-to-cell-mediated conjugation-like transfer mechanism. Importantly, transfer of the PaLoc was shown to occur on large chromosomal fragments of variable sizes, indicating that homologous recombination is likely to be responsible for the insertion events.