Redefining the Clostridioides difficile σB regulon: σB activates genes involved in detoxifying radicals that can result from the exposure to antimicrobials and hydrogen peroxide

In Gram-positive organisms the general stress response is regulated by the alternative sigma factor sigma B (σB). In C. difficile this sigma factor has been implicated in protection against stressors such as reactive oxygen species and antimicrobial compounds. Here, we show that an anti-σB antibody can be used to demonstrate overexpression of σB in C. difficile and that this overexpression is tolerated for short periods of time, despite its toxicity. Toxicity associated with σB-overexpression leads to loss of the plasmid used for anhydrotetracycline-induced σB expression. By uncoupling σB from its native regulatory network, we refined the previously proposed σB regulon and confirmed a direct role for σB in the induction of selected genes using in vitro run-off transcription reactions. Finally, using a plate-based luciferase reporter assay we demonstrated that different antibiotics and hydrogen peroxide induce genes positively regulated by σB in a manner dependent on this sigma factor. Together, our work suggests that lethal exposure to antibiotics may result in the formation of toxic radicals that lead to σB-dependent gene activation. Clostridioides difficile 630∆erm∆sigB (Clostron mutant) containing either Ptet-slucopt (control) or Ptet-sigB was grown until OD≈0.3 (exponential phase) and induced for one hour with 100 ng/ul anhydrotetracyclin (ATc). Biological quadruplicates were used for RNA isolation and the DNA array