A Recombination Directionality Factor Controls the Cell Type-Specific Activation of σ(K) and the Fidelity of Spore Development in Clostridium difficile

The strict anaerobe Clostridium difficile is the most common cause of nosocomial diarrhea, and the oxygen-resistant spores that it forms have a central role in the infectious cycle. The late stages of sporulation require the mother cell regulatory protein σ(K). In Bacillus subtilis, the onset of σ(K) activity requires both excision of a prophage-like element (skin(Bs)) inserted in the sigK gene and proteolytical removal of an inhibitory pro-sequence. Importantly, the rearrangement is restricted to the mother cell because the skin(Bs) recombinase is produced specifically in this cell. In C. difficile, σ(K) lacks a pro-sequence but a skin(Cd) element is present. The product of the skin(Cd) gene CD1231 shares similarity with large serine recombinases. We show that CD1231 is necessary for sporulation and skin(Cd) excision. However, contrary to B. subtilis, expression of CD1231 is observed in vegetative cells and in both sporangial compartments. Nevertheless, we show that skin(Cd) excision is under the control of mother cell regulatory proteins σ(E) and SpoIIID. We then demonstrate that σ(E) and SpoIIID control the expression of the skin(Cd) gene CD1234, and that this gene is required for sporulation and skin(Cd) excision. CD1231 and CD1234 appear to interact and both proteins are required for skin(Cd) excision while only CD1231 is necessary for skin(Cd) integration. Thus, CD1234 is a recombination directionality factor that delays and restricts skin(Cd) excision to the terminal mother cell. Finally, while the skin(Cd) element is not essential for sporulation, deletion of skin(Cd) results in premature activity of σ(K) and in spores with altered surface layers. Thus, skin(Cd) excision is a key element controlling the onset of σ(K) activity and the fidelity of spore development.