Genome-Scale Mapping Reveals Complex Regulatory Activities of RpoN in <named-content content-type="genus-species">Yersinia pseudotuberculosis</named-content>

ABSTRACT RpoN, an alternative sigma factor commonly known as σ54, is implicated in persistent stages of Yersinia pseudotuberculosis infections in which genes associated with this regulator are upregulated. We here combined phenotypic and genomic assays to provide insight into its role and function i...

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Autores principales:	A. K. M. Firoj Mahmud, Kristina Nilsson, Anna Fahlgren, Roberto Navais, Rajdeep Choudhury, Kemal Avican, Maria Fällman
Formato:	article
Lenguaje:	EN
Publicado:	American Society for Microbiology 2020
Materias:	ChIP-Seq RNA-seq RpoN Yersinia antisense binding genome mapping Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/a3a6c3d7038f4728b7349f8495a1f005
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spelling	oai:doaj.org-article:a3a6c3d7038f4728b7349f8495a1f0052021-12-02T18:44:44ZGenome-Scale Mapping Reveals Complex Regulatory Activities of RpoN in <named-content content-type="genus-species">Yersinia pseudotuberculosis</named-content>10.1128/mSystems.01006-202379-5077https://doaj.org/article/a3a6c3d7038f4728b7349f8495a1f0052020-12-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mSystems.01006-20https://doaj.org/toc/2379-5077ABSTRACT RpoN, an alternative sigma factor commonly known as σ54, is implicated in persistent stages of Yersinia pseudotuberculosis infections in which genes associated with this regulator are upregulated. We here combined phenotypic and genomic assays to provide insight into its role and function in this pathogen. RpoN was found essential for Y. pseudotuberculosis virulence in mice, and in vitro functional assays showed that it controls biofilm formation and motility. Mapping genome-wide associations of Y. pseudotuberculosis RpoN using chromatin immunoprecipitation coupled with next-generation sequencing identified an RpoN binding motif located at 103 inter- and intragenic sites on both sense and antisense strands. Deletion of rpoN had a large impact on gene expression, including downregulation of genes encoding proteins involved in flagellar assembly, chemotaxis, and quorum sensing. There were also clear indications of cross talk with other sigma factors, together with indirect effects due to altered expression of other regulators. Matching differential gene expression with locations of the binding sites implicated around 130 genes or operons potentially activated or repressed by RpoN. Mutagenesis of selected intergenic binding sites confirmed both positive and negative regulatory effects of RpoN binding. Corresponding mutations of intragenic sense sites had less impact on associated gene expression. Surprisingly, mutating intragenic sites on the antisense strand commonly reduced expression of genes carried by the corresponding sense strand. IMPORTANCE The alternative sigma factor RpoN (σ54), which is widely distributed in eubacteria, has been implicated in controlling gene expression of importance for numerous functions including virulence. Proper responses to host environments are crucial for bacteria to establish infection, and regulatory mechanisms involved are therefore of high interest for development of future therapeutics. Little is known about the function of RpoN in the intestinal pathogen Y. pseudotuberculosis, and we therefore investigated its regulatory role in this pathogen. This regulator was indeed found to be critical for establishment of infection in mice, likely involving its requirement for motility and biofilm formation. The RpoN regulon involved both activating and suppressive effects on gene expression which could be confirmed with mutagenesis of identified binding sites. This is the first study of its kind of RpoN in Y. pseudotuberculosis, revealing complex regulation of gene expression involving both productive and silent effects of its binding to DNA, providing important information about RpoN regulation in enterobacteria.A. K. M. Firoj MahmudKristina NilssonAnna FahlgrenRoberto NavaisRajdeep ChoudhuryKemal AvicanMaria FällmanAmerican Society for MicrobiologyarticleChIP-SeqRNA-seqRpoNYersiniaantisense bindinggenome mappingMicrobiologyQR1-502ENmSystems, Vol 5, Iss 6 (2020)
institution	DOAJ
collection	DOAJ
language	EN
topic	ChIP-Seq RNA-seq RpoN Yersinia antisense binding genome mapping Microbiology QR1-502
spellingShingle	ChIP-Seq RNA-seq RpoN Yersinia antisense binding genome mapping Microbiology QR1-502 A. K. M. Firoj Mahmud Kristina Nilsson Anna Fahlgren Roberto Navais Rajdeep Choudhury Kemal Avican Maria Fällman Genome-Scale Mapping Reveals Complex Regulatory Activities of RpoN in <named-content content-type="genus-species">Yersinia pseudotuberculosis</named-content>
description	ABSTRACT RpoN, an alternative sigma factor commonly known as σ54, is implicated in persistent stages of Yersinia pseudotuberculosis infections in which genes associated with this regulator are upregulated. We here combined phenotypic and genomic assays to provide insight into its role and function in this pathogen. RpoN was found essential for Y. pseudotuberculosis virulence in mice, and in vitro functional assays showed that it controls biofilm formation and motility. Mapping genome-wide associations of Y. pseudotuberculosis RpoN using chromatin immunoprecipitation coupled with next-generation sequencing identified an RpoN binding motif located at 103 inter- and intragenic sites on both sense and antisense strands. Deletion of rpoN had a large impact on gene expression, including downregulation of genes encoding proteins involved in flagellar assembly, chemotaxis, and quorum sensing. There were also clear indications of cross talk with other sigma factors, together with indirect effects due to altered expression of other regulators. Matching differential gene expression with locations of the binding sites implicated around 130 genes or operons potentially activated or repressed by RpoN. Mutagenesis of selected intergenic binding sites confirmed both positive and negative regulatory effects of RpoN binding. Corresponding mutations of intragenic sense sites had less impact on associated gene expression. Surprisingly, mutating intragenic sites on the antisense strand commonly reduced expression of genes carried by the corresponding sense strand. IMPORTANCE The alternative sigma factor RpoN (σ54), which is widely distributed in eubacteria, has been implicated in controlling gene expression of importance for numerous functions including virulence. Proper responses to host environments are crucial for bacteria to establish infection, and regulatory mechanisms involved are therefore of high interest for development of future therapeutics. Little is known about the function of RpoN in the intestinal pathogen Y. pseudotuberculosis, and we therefore investigated its regulatory role in this pathogen. This regulator was indeed found to be critical for establishment of infection in mice, likely involving its requirement for motility and biofilm formation. The RpoN regulon involved both activating and suppressive effects on gene expression which could be confirmed with mutagenesis of identified binding sites. This is the first study of its kind of RpoN in Y. pseudotuberculosis, revealing complex regulation of gene expression involving both productive and silent effects of its binding to DNA, providing important information about RpoN regulation in enterobacteria.
format	article
author	A. K. M. Firoj Mahmud Kristina Nilsson Anna Fahlgren Roberto Navais Rajdeep Choudhury Kemal Avican Maria Fällman
author_facet	A. K. M. Firoj Mahmud Kristina Nilsson Anna Fahlgren Roberto Navais Rajdeep Choudhury Kemal Avican Maria Fällman
author_sort	A. K. M. Firoj Mahmud
title	Genome-Scale Mapping Reveals Complex Regulatory Activities of RpoN in <named-content content-type="genus-species">Yersinia pseudotuberculosis</named-content>
title_short	Genome-Scale Mapping Reveals Complex Regulatory Activities of RpoN in <named-content content-type="genus-species">Yersinia pseudotuberculosis</named-content>
title_full	Genome-Scale Mapping Reveals Complex Regulatory Activities of RpoN in <named-content content-type="genus-species">Yersinia pseudotuberculosis</named-content>
title_fullStr	Genome-Scale Mapping Reveals Complex Regulatory Activities of RpoN in <named-content content-type="genus-species">Yersinia pseudotuberculosis</named-content>
title_full_unstemmed	Genome-Scale Mapping Reveals Complex Regulatory Activities of RpoN in <named-content content-type="genus-species">Yersinia pseudotuberculosis</named-content>
title_sort	genome-scale mapping reveals complex regulatory activities of rpon in <named-content content-type="genus-species">yersinia pseudotuberculosis</named-content>
publisher	American Society for Microbiology
publishDate	2020
url	https://doaj.org/article/a3a6c3d7038f4728b7349f8495a1f005
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Genome-Scale Mapping Reveals Complex Regulatory Activities of RpoN in <named-content content-type="genus-species">Yersinia pseudotuberculosis</named-content>

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