Redefining the <named-content content-type="genus-species">Clostridioides difficile</named-content> σ<sup>B</sup> Regulon: σ<sup>B</sup> Activates Genes Involved in Detoxifying Radicals That Can Result from the Exposure to Antimicrobials and Hydrogen Peroxide

ABSTRACT In many Gram-positive bacteria, the general stress response is regulated at the transcriptional level by the alternative sigma factor sigma B (σB). In C. difficile, σB has been implicated in protection against stressors such as reactive oxygen species (ROS) and antimicrobial compounds. Here...

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Autores principales:	Ilse M. Boekhoud, Annika-Marisa Michel, Jeroen Corver, Dieter Jahn, Wiep Klaas Smits
Formato:	article
Lenguaje:	EN
Publicado:	American Society for Microbiology 2020
Materias:	Clostridium difficile antimicrobial agents in vitro transcription luciferase regulon sigma factors Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/0b85a566a1b94306951aa24ede8f8e69
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spelling	oai:doaj.org-article:0b85a566a1b94306951aa24ede8f8e692021-11-15T15:30:59ZRedefining the <named-content content-type="genus-species">Clostridioides difficile</named-content> σ<sup>B</sup> Regulon: σ<sup>B</sup> Activates Genes Involved in Detoxifying Radicals That Can Result from the Exposure to Antimicrobials and Hydrogen Peroxide10.1128/mSphere.00728-202379-5042https://doaj.org/article/0b85a566a1b94306951aa24ede8f8e692020-10-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mSphere.00728-20https://doaj.org/toc/2379-5042ABSTRACT In many Gram-positive bacteria, the general stress response is regulated at the transcriptional level by the alternative sigma factor sigma B (σB). In C. difficile, σB has been implicated in protection against stressors such as reactive oxygen species (ROS) and antimicrobial compounds. Here, we used an anti-σB antibody to demonstrate time-limited overproduction of σB in C. difficile despite its toxicity at higher cellular concentrations. This toxicity eventually led to the loss of the plasmid used for anhydrotetracycline-induced σB gene expression. Inducible σB overproduction uncouples σB expression from its native regulatory network and allows for the refinement of the previously proposed σB 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 σB 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 σB-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 σB by evaluating the transcriptional effects of σB overproduction, provide biochemical evidence of direct transcriptional activation by σB, and show that σB-dependent genes can be activated by antimicrobials. Together, our data suggest that σB is a key player in dealing with toxic radicals.Ilse M. BoekhoudAnnika-Marisa MichelJeroen CorverDieter JahnWiep Klaas SmitsAmerican Society for MicrobiologyarticleClostridium difficileantimicrobial agentsin vitro transcriptionluciferaseregulonsigma factorsMicrobiologyQR1-502ENmSphere, Vol 5, Iss 5 (2020)
institution	DOAJ
collection	DOAJ
language	EN
topic	Clostridium difficile antimicrobial agents in vitro transcription luciferase regulon sigma factors Microbiology QR1-502
spellingShingle	Clostridium difficile antimicrobial agents in vitro transcription luciferase regulon sigma factors Microbiology QR1-502 Ilse M. Boekhoud Annika-Marisa Michel Jeroen Corver Dieter Jahn Wiep Klaas Smits Redefining the <named-content content-type="genus-species">Clostridioides difficile</named-content> σ<sup>B</sup> Regulon: σ<sup>B</sup> Activates Genes Involved in Detoxifying Radicals That Can Result from the Exposure to Antimicrobials and Hydrogen Peroxide
description	ABSTRACT In many Gram-positive bacteria, the general stress response is regulated at the transcriptional level by the alternative sigma factor sigma B (σB). In C. difficile, σB has been implicated in protection against stressors such as reactive oxygen species (ROS) and antimicrobial compounds. Here, we used an anti-σB antibody to demonstrate time-limited overproduction of σB in C. difficile despite its toxicity at higher cellular concentrations. This toxicity eventually led to the loss of the plasmid used for anhydrotetracycline-induced σB gene expression. Inducible σB overproduction uncouples σB expression from its native regulatory network and allows for the refinement of the previously proposed σB 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 σB 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 σB-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 σB by evaluating the transcriptional effects of σB overproduction, provide biochemical evidence of direct transcriptional activation by σB, and show that σB-dependent genes can be activated by antimicrobials. Together, our data suggest that σB is a key player in dealing with toxic radicals.
format	article
author	Ilse M. Boekhoud Annika-Marisa Michel Jeroen Corver Dieter Jahn Wiep Klaas Smits
author_facet	Ilse M. Boekhoud Annika-Marisa Michel Jeroen Corver Dieter Jahn Wiep Klaas Smits
author_sort	Ilse M. Boekhoud
title	Redefining the <named-content content-type="genus-species">Clostridioides difficile</named-content> σ<sup>B</sup> Regulon: σ<sup>B</sup> Activates Genes Involved in Detoxifying Radicals That Can Result from the Exposure to Antimicrobials and Hydrogen Peroxide
title_short	Redefining the <named-content content-type="genus-species">Clostridioides difficile</named-content> σ<sup>B</sup> Regulon: σ<sup>B</sup> Activates Genes Involved in Detoxifying Radicals That Can Result from the Exposure to Antimicrobials and Hydrogen Peroxide
title_full	Redefining the <named-content content-type="genus-species">Clostridioides difficile</named-content> σ<sup>B</sup> Regulon: σ<sup>B</sup> Activates Genes Involved in Detoxifying Radicals That Can Result from the Exposure to Antimicrobials and Hydrogen Peroxide
title_fullStr	Redefining the <named-content content-type="genus-species">Clostridioides difficile</named-content> σ<sup>B</sup> Regulon: σ<sup>B</sup> Activates Genes Involved in Detoxifying Radicals That Can Result from the Exposure to Antimicrobials and Hydrogen Peroxide
title_full_unstemmed	Redefining the <named-content content-type="genus-species">Clostridioides difficile</named-content> σ<sup>B</sup> Regulon: σ<sup>B</sup> Activates Genes Involved in Detoxifying Radicals That Can Result from the Exposure to Antimicrobials and Hydrogen Peroxide
title_sort	redefining the <named-content content-type="genus-species">clostridioides difficile</named-content> σ<sup>b</sup> regulon: σ<sup>b</sup> activates genes involved in detoxifying radicals that can result from the exposure to antimicrobials and hydrogen peroxide
publisher	American Society for Microbiology
publishDate	2020
url	https://doaj.org/article/0b85a566a1b94306951aa24ede8f8e69
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Redefining the <named-content content-type="genus-species">Clostridioides difficile</named-content> σ<sup>B</sup> Regulon: σ<sup>B</sup> Activates Genes Involved in Detoxifying Radicals That Can Result from the Exposure to Antimicrobials and Hydrogen Peroxide

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