In an alternative approach that addresses many of the issues above, the role of tcdC in toxin expression could be addressed by removing it from a background in which it is normally functional. To this end, we generated two independent isogenic ClosTronbased tcdC mutants strain that could be directly compared to its wild type counterpart, in which the TcdC protein was expected to be functional. Our data obtained with these mutant strains show that TcdC does not exert a major or even significant effect on the transcription of the PaLoc genes or the expression levels of the toxins under the conditions tested.