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 Vol. 5; no. 5
• Main Authors: Boekhoud, Ilse M., Michel, Annika-Marisa, Corver, Jeroen, Jahn, Dieter, Smits, Wiep Klaas
• Format: Journal Article
• Language: English
• Published: Washington American Society for Microbiology 16-09-2020
• 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.