Redefining the Clostridioides difficile σ Regulon: σ 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). In , σ has been implicated in protection against stressors such as reactive oxygen species (ROS) and antimicrobial compounds. Here, we used an anti-σ antibody to demonstrate time-limited overproduction of σ in despite its toxicity at higher cellular concentrations. This toxicity eventually led to the loss of the plasmid used for anhydrotetracycline-induced σ gene expression. Inducible σ overproduction uncouples σ expression from its native regulatory network and allows for the refinement of the previously proposed σ regulon. At least 32% of the regulon was found to consist of genes involved in the response to reactive radicals. Direct gene activation by σ was demonstrated through runoff transcription of specific target genes (, , , and ). 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-dependent gene activation. Sigma B is the alternative sigma factor governing stress response in many Gram-positive bacteria. In , a mutant shows pleiotropic transcriptional effects. Here, we determine genes that are likely direct targets of σ by evaluating the transcriptional effects of σ overproduction, provide biochemical evidence of direct transcriptional activation by σ, and show that σ-dependent genes can be activated by antimicrobials. Together, our data suggest that σ is a key player in dealing with toxic radicals.