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

• Published in: mSphere 2020-09, Vol.5 (5)
• Main Authors: Boekhoud, Ilse M., Michel, Annika-Marisa, Corver, Jeroen, Jahn, Dieter, Smits, Wiep Klaas
• Format: Article
• Language: English
• Subjects: Antibodies; Antimicrobial agents; Clostridium difficile; Gene expression; Gram-positive bacteria; Hydrogen peroxide; in vitro transcription; Investigations; luciferase; Molecular Biology and Physiology; Molecular weight; Plasmids; Radicals; Reactive oxygen species; regulon; RNA polymerase; Sigma factor; sigma factors; Toxicity; Transcription activation
• ISSN: 2379-5042; 2379-5042
• DOI: 10.1128/mSphere.00728-20

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. 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.