The predicted acetoin dehydrogenase pathway represses sporulation ofClostridioides difficile

Abstract

ABSTRACTClostridioides difficileis a major gastrointestinal pathogen that is transmitted as a dormant spore. As an intestinal pathogen,C. difficilemust 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 onC. difficilesporulation. Specifically, we examined the impacts of pH and the metabolite acetoin onC. difficilegrowth and sporulation. We found that expression of the predicted acetoin dehydrogenase operon,acoRABCL, was pH-dependent and regulated by acetoin. Regulation of theC. difficile acolocus is distinct from other characterized systems and appears to involve a co-transcribed DeoR-family regulator rather than the sigma54-dependent activator. In addition, anacoAnull mutant produced significantly more spores and initiated sporulation earlier than the parent strain. However, unlike other Firmicutes, growth and culture density ofC. difficilewas not increased by acetoin availability or disruption of theacopathway. Together, these results indicate that acetoin, pH, and theacopathway play important roles in nutritional repression of sporulation inC. difficile, but acetoin metabolism does not support cell growth as a stationary phase energy source.IMPORTANCEClostridioides difficile,orC. diff, is an anaerobic bacterium that lives within the gut of many mammals and causes infectious diarrhea.C. difficileis 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 ofC. 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 ofC. difficileto 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 thatC. difficileuses acetoin differently than many other species and that acetoin has an important role as an environmental metabolite that influences spore formation.