Redefining the Clostridioides difficile σ(B) Regulon: σ(B) 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 (σ(B)). In C. difficile, σ(B) has been implicated in protection against stressors such as reactive oxygen species (ROS) and antimicrobial compounds. Here, we used an anti-σ(B) antibody to demonstrate time-limited overproduction of σ(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 σ(B) gene expression. Inducible σ(B) overproduction uncouples σ(B) expression from its native regulatory network and allows for the refinement of the previously proposed σ(B) regulon. At least 32% of the regulon was found to consist of genes involved in the response to reactive radicals. Direct gene activation by C. difficile σ(B) was demonstrated through in vitro runoff transcription of specific target genes (cd0350, cd3614, cd3605, and 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 σ(B)-dependent gene activation. IMPORTANCE Sigma 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 σ(B) by evaluating the transcriptional effects of σ(B) overproduction, provide biochemical evidence of direct transcriptional activation by σ(B), and show that σ(B)-dependent genes can be activated by antimicrobials. Together, our data suggest that σ(B) is a key player in dealing with toxic radicals.