The cell wall protein 66 gene ( cwp66 ) encodes a cell wall protein, which is the second major cell surface antigen of C. difficile . Although immunological approaches, such as antibodies and purified recombinant proteins, have been implemented to study the role of Cwp66 in cell adhesion, no deletion mutant of the cwp66 gene has yet been characterized.

The alternative σ factor TcdR controls the synthesis of two major enterotoxins: TcdA and TcdB in Clostridioides difficile. Four potential TcdR-dependent promoters in the pathogenicity locus of C. difficile showed different activities. In this study, we constructed a heterologous system in Bacillus subtilis to investigate the molecular basis of TcdR-dependent promoter activity. The promoters of the two major enterotoxins showed strong TcdR-dependent activity, while the two putative TcdR-dependent promoters in the upstream region of the tcdR gene did not show detectable activity, suggesting that the autoregulation of TcdR may need other unknown factors involved. Mutation analysis indicated that the divergent -10 region is the key determinant for different activities of the TcdR-dependent promoters. Analysis of the TcdR model predicted by AlphaFold2 suggested that TcdR should be classified into group 4, i.e., extracytoplasmic function, σ70 factors. The results of this study provide the molecular basis of the TcdR-dependent promoter recognition for toxin production. This study also suggests the feasibility of the heterologous system in analyzing σ factor functions and possibly in drug development targeting these factors.