We generated two independent isogenic tcdC mutants by insertional inactivation of the tcdC gene in the wild type strain 630Derm using ClosTron technology [24, 25] . Briefly, the Perutka algorithm on the ClosTron website (http://www.clostron.com) was used to design primers ( Table 2) for retargeting the Group II intron (Sigma; Targetron). The retargeted intron was cloned using the restriction enzymes BsrGI and HindIII into plasmids pMTL007C-E2 and the constructs were verified by sequencing [25] . The verified plasmid (pDB001) was transformed to E. coli CA434 and transferred to the wild type strain 630Derm via conjugation [34, 41] . The selection of C. difficile transconjugants was done by subculturing on pre-reduced BHIS agar supplemented with thiamphenicol (Sigma; 10 mg/ml) and C. difficile selective supplement (Oxoid). This was followed by several rounds of subculturing on pre-reduced BHIS agar supplemented with lincomycin (Sigma; 20 mg/ml) and C. difficile selective supplement to promote integration of the GroupII intron into the gene of interest. Chromosomal DNA isolated from the transconjugants using a QIAamp blood kit (Qiagen) was used in conventional PCRs and sequence runs to confirm the disruption of tcdC and the nucleotide position of the insertion in the tcdC gene. Primers used for cloning and sequencing are listed in Table 2 .