The recombinant plasmid used for complementation of the cdtA mutant was pJIR3107, described previously 47 (link). C. difficile strains were grown in TY broth (3.0% tryptone, 2.0% yeast extract and 0.1% sodium thioglycollate) at 37°C in a Don Whitley A35 Anaerobic Workstation in an atmosphere of 10% (v/v) H2, 10% (v/v) CO2 and 80% (v/v) N2. Toxins were partially purified and concentrated eight-fold from 72 hour C. difficile culture supernatants by methanol-chloroform precipitation 48 (link). Protein concentrations were determined using the BCA protein assay kit (Pierce) as per the manufacturer’s instructions. Concentrated supernatant proteins (10 μg) were separated by 10% (w/v) SDS-PAGE and transferred onto a nitrocellulose membrane (Whatman). Membranes were detected as previously described 29 (link). CDTa and CDTb were detected using a CDTa-specific antibody and C. perfringens Ib-specific antibody that is cross reactive with CDTb 49 (link), respectively. CDTa and CDTb antibodies were detected using horseradish peroxidase conjugated anti-rabbit goat antibodies (Millipore) The Western Lightning Chemiluminescence reagent kit (Perkin-Elmer) was used to detect the blots, following the manufacturer’s instructions and blots were recorded by exposure to X-ray film (Fujifilm).