The Clostridium difficile and Escherichia coli (E. coli) strains and plasmids used in this study are described in Table 1 . E. coli strains were grown in Luria Bertani (LB, USB cooperation) medium supplemented with appropriate antibiotics when required. C. difficile strains were grown anaerobically in a microaerobic cabinet (Don Whitley VA1000) at 37uC in pre-reduced 3% Bacto Tryptose (Difco), 2% Yeast extract (Difco) and 0.1% thioglycolate (pH 7.4) medium (TY) or Brain Heart Infusion broth (Oxoid) supplemented with 0.5% yeast extract and 0.01% L-cysteine (Sigma) (BHIS) [40, 41] . When required, the broths were supplemented with appropriated antibiotics. For RNA extraction and toxin quantification, C. difficile 630DErm (wild type) and two independent isogenic tcdC mutant strains (CT::tcdC) were serially diluted and pre-cultured (overnight) in pre-reduced TY broth. Mid-logarithmic growth phase pre-cultures (OD 600 0.4-0.8) were used to inoculate pre-reduced TY broth to a starting OD 600 of 0.05 (60.01). Optical density readings and samples for total toxin quantification were taken at 2, 3, 4, 5, 6 and 8 hours post inoculation in the exponential growth phase and at 12, 24 and 48 hours post inoculation in the stationary phase. Samples for RNA extraction were taken at 6, 8, 12, and 24 hours post inoculation. Samples for Western blot detection of TcdC were taken at 8 hours post inoculation. We routinely monitored the purity of the 