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Clostridioides difficile is an anaerobic enteric pathogen that disseminates in the environment as a dormant spore. For C. difficile and other sporulating bacteria, the initiation of sporulation is a regulated process that prevents spore formation under favorable growth conditions. In Bacillus subtilis , one such mechanism for preventing sporulation is the Kinase Inhibitory Protein, KipI, which impedes activation of the main sporulation kinase. In addition, KipI functions as part of a complex that detoxifies the intermediate metabolite, 5-oxoproline (OP), a harmful by-product of glutamic acid. In this study, we investigate the orthologous Kip proteins in C. difficile to determine their roles in the regulation of sporulation and metabolism. Using deletion mutants in kipIA and the full kipOTIA operon, we show that unlike in B. subtilis, the Kip proteins have no significant impact on sporulation. However, we found that the kip operon encodes a functional oxoprolinase that facilitates detoxification of OP. Further, our data demonstrate that KipOTIA not only detoxifies OP, but also allows OP to be used as a nutrient source that supports the robust growth of C. difficile , thereby facilitating the conversion of a toxic byproduct of metabolism into an effective energy source.

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. Specifically, we examined the impacts of pH and the metabolite acetoin on C. difficile growth and sporulation. We found that expression of the predicted acetoin dehydrogenase operon, acoRABCL , was pH-dependent and regulated by acetoin. Regulation of the C. difficile aco locus is distinct from other characterized systems and appears to involve a co-transcribed DeoR-family regulator rather than the sigma 54 -dependent activator. In addition, an acoA 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 aco pathway. Together, these results indicate that acetoin, pH, and the aco pathway play important roles in nutritional repression of sporulation in C. difficile , but acetoin metabolism does not support cell growth as a stationary phase energy source., Importance: Clostridioides difficile, or C. diff , is an anaerobic bacterium that lives within the gut of many mammals and causes infectious diarrhea. C. difficile is able to survive outside of the gut and transmit to new hosts by forming dormant spores. It is known that the pH of the intestine and the nutrients available both affect the growth and sporulation of C. diffiicile, but the specific conditions that result in sporulation in the host are not clear. In this study, we investigated how pH and the metabolite acetoin affect the ability of C. difficile to grow, proliferate, and form spores. We found that a mutant lacking the predicted acetoin metabolism pathway form more spores, but their growth is not impacted. These results show that C. difficile uses acetoin differently than many other species and that acetoin has an important role as an environmental metabolite that influences spore formation.