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 many gram-positive bacteria the general stress response is regulated at the transcriptional level by the alternative sigma factor sigma B ({sigma}B). In C. difficile {sigma}B has been implicated in protection against stressors such as reactive oxygen species and antimicrobial compounds. Here, we used an anti-{sigma}B antibody to demonstrate time-limited overproduction of {sigma}B in C. difficile despite its toxicity at higher cellular concentrations. This toxicity eventually led to the loss of the plasmid used for anhydrotetracycline-induced {sigma}B gene expression. Inducible {sigma}B overproduction uncouples {sigma}B expression from its native regulatory network and allowed for the refinement of the previously proposed {sigma}B regulon. At least 32% the regulon was found to consist of genes involved in the response to reactive radicals. Direct gene activation by C. difficile {sigma}B was demonstrated through in vitro run-off transcription of specific target genes (cd0350, cd3614, cd3605, cd2963). Finally, we demonstrated that different antimicrobials and hydrogen peroxide induce these genes in a manner dependent on this sigma factor, using a plate-based luciferase reporter assay. Together, our work suggests that lethal exposure to antimicrobials may result in the formation of toxic radicals that lead to {sigma}B-dependent gene activation. ImportanceSigma B is the alternative sigma factor governing stress response in many gram-positive bacteria. In C. difficile, a sigB mutant shows pleiotropic transcriptional effects. Here, we determine genes that are likely direct targets of {sigma}B by evaluating the transcriptional effects of {sigma}B overproduction, provide biochemical evidence of direct transcriptional activation by {sigma}B, and show that {sigma}B-dependent genes can be activated by antimicrobials. Together our data suggest that {sigma}B is a key player in dealing with toxic radicals.