A σ B -dependent sRNA provides a repressor function to the general stress response in Clostridioides difficile

Clostridioides difficile is an anaerobic, gram-positive gastrointestinal pathogen that poses a significant health threat worldwide. A recent RIL-seq characterization of the Hfq-mediated RNA-RNA interactome revealed that small regulatory RNAs (sRNAs) are key post-transcriptional regulators of virulence-related processes in C. difficile , including the initiation of sporulation. In the model Proteobacteria species E. coli and S. enterica , sRNAs often function as non-coding elements in vital stress responses mediated by alternative sigma factors, acting as repressors within these regulatory networks. However, whether this principle is broadly conserved across bacterial phyla remains largely unexplored. Here, we demonstrate that the Hfq-dependent sRNA SrsA is part of the regulatory network of the alternative sigma factor σ B , which governs the general stress response in Firmicutes. Consequently, SrsA is highly expressed during the transition from exponential to stationary phase and under infection-related stress conditions, such as oxidative stress. We show that SrsA represses a highly conserved small protein family that regulates sporulation and three genes in the methyl-fixing branch of the Wood-Ljungdahl pathway (WLP). Infection of mice further revealed that SrsA contributes to disease severity and bacterial recovery from antibiotic treatment in an acute and relapse mouse model of C. difficile infection. In summary, our data suggest that integrating SrsA into the σ B regulon helps coordinate sporulation and vital metabolic pathways of C. difficile in response to infection-relevant stress conditions. IMPORTANCE The central role of sRNAs as non-coding arms of stress responses represents a paradigm of sRNA function in Gammaproteobacteria. However, it remains unclear how broadly this regulatory principle is conserved across the bacterial tree of life. In our study, we identified the sRNA SrsA as a non-coding regulator involved in the sigma B-mediated stress response to nutrient limitation and radical stress in the intestinal pathogen Clostridioides difficile . We demonstrate that SrsA suppresses the production of a conserved family of spore regulators and of enzymes of the Wood-Ljungdahl pathway, establishing a role in regulating sporulation and gut colonization. Our work provides novel insights into the regulation of the Wood-Ljungdahl pathway, which is usually associated with autotrophic bacteria, and highlights the metabolic flexibility this anaerobic gut pathogen has evolved to survive in a nutrient-limited and inflamed gut environment.