A metabolite dehydrogenase pathway represses sporulation of Clostridioides difficile

Clostridioides difficile is a major gastrointestinal pathogen that is transmitted as a dormant spore. As an intestinal pathogen, C. difficile must contend with variable environmental conditions, including fluctuations in pH and nutrient availability. Nutrition and pH both influence growth and spore formation, but how pH and nutrition jointly influence sporulation are not known. In this study, we investigated the dual impact of pH and pH-dependent metabolism on C. difficile sporulation.

We examined the impacts of pH and the metabolite acetoin on C. difficile growth, gene expression, and sporulation.

We found that expression of the predicted acetoin dehydrogenase operon, CD0035-CD0039, was pH-dependent and repressed by acetoin and pyruvate. Regulation of the C. difficile CD0035-CD0039 locus is distinct from characterized orthologous systems and appears to involve a co-transcribed DeoR-family regulator, rather than a sigma⁵⁴-dependent activator. In addition, an CD0036 null mutant produced significantly more spores and initiated sporulation earlier than the parent strain. However, unlike other Firmicutes, growth and culture density of C. difficile was not increased by acetoin availability or disruption of the dehydrogenase pathway.

Together, these results indicate that acetoin, pH, and the CD0036-CD0039 dehydrogenase pathway play important roles in nutritional repression of sporulation in C. difficile. However, the data do not support the involvement of the CD0036-CD0039 pathway in acetoin metabolism and acetoin is not a significant stationary phase energy source for C. difficile.