ABSTRACTThe anaerobic pathogenClostridioides difficile, which is a primary cause of antibiotic-associated diarrhea, faces a variety of stresses in the environment and in the mammalian gut. To cope with these stresses, alternative sigma factor B (σB) is employed to modulate gene transcription, and σBis regulated by an anti-sigma factor, RsbW. To understand the role of RsbW inC. difficilephysiology, arsbWmutant (ΔrsbW), in which σBis assumed to be “always on,” was generated. ΔrsbWdid not show fitness defects in the absence of stress but tolerated acidic environments and detoxified reactive oxygen and nitrogen species better compared to the parental strain. ΔrsbWwas defective in spore and biofilm formation, but it displayed increased adhesion to human gut epithelia and was less virulent in aGalleria mellonellainfection model. A transcriptomic analysis to understand the unique phenotype of ΔrsbWshowed changes in expression of genes associated with stress responses, virulence, sporulation, phage, and several σB-controlled regulators, including the pleiotropic regulator sinRR′. While these profiles were distinct to ΔrsbW, changes in some σB-controlled stress-associated genes were similar to those reported in the absence of σB. Further analysis of ΔrsbWshowed unexpected lower intracellular levels of σB, suggesting an additional post-translational control mechanism for σBin the absence of stress. Our study provides insight into the regulatory role of RsbW and the complexity of regulatory networks mediating stress responses inC. difficile.IMPORTANCEPathogens likeClostridioides difficileface a range of stresses in the environment and within the host. Alternative transcriptional factors like sigma factor B (σB) enable the bacterium to respond quickly to different stresses. Anti-sigma factors like RsbW control sigma factors and therefore the activation of genes via these pathways. Some of these transcriptional control systems provideC. difficilewith the ability to tolerate and detoxify harmful compounds. Here, we investigate the role of RsbW inC. difficilephysiology. We demonstrate distinctive phenotypes for arsbWmutant in growth, persistence, and virulence and suggest alternate σBcontrol mechanisms inC. difficile. UnderstandingC. difficileresponses to external stress is key to designing better strategies to combat this highly resilient bacterial pathogen.