The disruption of the tcdC gene resulted in an on average 1.7 fold higher transcription level of tcdC in time compared to the wild type strain, although this difference was not found to be statistically significant. It should be noted that we detect these differences because the real time PCR probe detects a region of the gene upstream of the ClosTron insertion site ( Figure 1A ). This finding might indicate some kind of feedback mechanism on TcdC expression. Similar to tcdC gene expression, the disruption of tcdC resulted in a slightly higher transcription level of the other PaLoc genes, although this was generally not significant. Moreover, the increased transcription level of the toxin genes did not result in generated a PCR product of 302 bp for the CT::tcdC strains. Primers tcdC1 and tcdC2 generated a 699 bp PCR product for the wild type and for the CT::tcdC strain a PCR product of circa 2800 bp. (C) Southern blot analysis of EcoRV digested genomic DNA of wild type and CT::tcdC strains with a Group II intron, ermB gene and tcdC specific probes. Note that probing with the ermB probe results in 2 bands for the CT::tcdC strains, since wild type already carries a copy of the ermB gene in the genome [35] . (D) Western blot analysis of TcdC production in wild type and CT::tcdC strain 8 hours post inoculation. The arrow indicates the location of TcdC protein based on MW and absence of the protein in the PaLoc negative Type 035 strain. Note that cross-reaction of TcdC antibody with a protein of similar MW was also observed in Carter et al. [30] . (E) Growth curves of C. difficile 630DErm and C. difficile CT::tcdC mutant strains. The absorbance (OD 600 ) was measured over 48 hrs of growth in TY medium. The error bars indicate the standard error of the mean of six experiments. doi:10.1371/journal.pone.0043247.g001 a detectable increase in toxin levels as measured with two independent assays.