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DNA-Binding and Transcription Activation by Unphosphorylated Response Regulator AgrR From Cupriavidus metallidurans Involved in Silver Resistance
Even though silver and silver nanoparticles at low concentrations are considered safe for human health, their steadily increasing use and associated release in nature is not without risk since it may result in the selection of silver-resistant microorganisms, thus impeding the utilization of silver...
Autores principales: | , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Frontiers Media S.A.
2020
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7380067/ https://www.ncbi.nlm.nih.gov/pubmed/32765465 http://dx.doi.org/10.3389/fmicb.2020.01635 |
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author | Ali, Md Muntasir Provoost, Ann Mijnendonckx, Kristel Van Houdt, Rob Charlier, Daniel |
author_facet | Ali, Md Muntasir Provoost, Ann Mijnendonckx, Kristel Van Houdt, Rob Charlier, Daniel |
author_sort | Ali, Md Muntasir |
collection | PubMed |
description | Even though silver and silver nanoparticles at low concentrations are considered safe for human health, their steadily increasing use and associated release in nature is not without risk since it may result in the selection of silver-resistant microorganisms, thus impeding the utilization of silver as antimicrobial agent. Furthermore, increased resistance to metals may be accompanied by increased antibiotic resistance. Inactivation of the histidine kinase and concomitant upregulation of the cognate response regulator (RR) of the AgrRS two-component system was previously shown to play an important role in the increased silver resistance of laboratory adapted mutants of Cupriavidus metallidurans. However, binding of AgrR, a member of the OmpR/PhoP family of RRs with a conserved phosphoreceiver aspartate residue, to potential target promoters has never been demonstrated. Here we identify differentially expressed genes in the silver-resistant mutant NA4S in non-selective conditions by RNA-seq and demonstrate sequence-specific binding of AgrR to six selected promoter regions of upregulated genes and divergent operons. We delimit binding sites by DNase I and in gel copper-phenanthroline footprinting of AgrR-DNA complexes, and establish a high resolution base-specific contact map of AgrR-DNA interactions using premodification binding interference techniques. We identified a 16-bp core AgrR binding site (AgrR box) arranged as an imperfect inverted repeat of 6 bp (ATTACA) separated by 4 bp variable in sequence (6-4-6). AgrR interacts with two major groove segments and the intervening minor groove, all aligned on one face of the helix. Furthermore, an additional in phase imperfect direct repeat of the half-site may be observed slightly up and/or downstream of the inverted repeat at some operators. Mutant studies indicated that both inverted and direct repeats contribute to AgrR binding in vitro and AgrR-mediated activation in vivo. From the position of the AgrR box it appears that AgrR may act as a Type II activator for most investigated promoters, including positive autoregulation. Furthermore, we show in vitro binding and in vivo activation with dephosphomimetic AgrR mutant D51A, indicating that unphosphorylated AgrR is the active form of the RR in mutant NA4S. |
format | Online Article Text |
id | pubmed-7380067 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2020 |
publisher | Frontiers Media S.A. |
record_format | MEDLINE/PubMed |
spelling | pubmed-73800672020-08-05 DNA-Binding and Transcription Activation by Unphosphorylated Response Regulator AgrR From Cupriavidus metallidurans Involved in Silver Resistance Ali, Md Muntasir Provoost, Ann Mijnendonckx, Kristel Van Houdt, Rob Charlier, Daniel Front Microbiol Microbiology Even though silver and silver nanoparticles at low concentrations are considered safe for human health, their steadily increasing use and associated release in nature is not without risk since it may result in the selection of silver-resistant microorganisms, thus impeding the utilization of silver as antimicrobial agent. Furthermore, increased resistance to metals may be accompanied by increased antibiotic resistance. Inactivation of the histidine kinase and concomitant upregulation of the cognate response regulator (RR) of the AgrRS two-component system was previously shown to play an important role in the increased silver resistance of laboratory adapted mutants of Cupriavidus metallidurans. However, binding of AgrR, a member of the OmpR/PhoP family of RRs with a conserved phosphoreceiver aspartate residue, to potential target promoters has never been demonstrated. Here we identify differentially expressed genes in the silver-resistant mutant NA4S in non-selective conditions by RNA-seq and demonstrate sequence-specific binding of AgrR to six selected promoter regions of upregulated genes and divergent operons. We delimit binding sites by DNase I and in gel copper-phenanthroline footprinting of AgrR-DNA complexes, and establish a high resolution base-specific contact map of AgrR-DNA interactions using premodification binding interference techniques. We identified a 16-bp core AgrR binding site (AgrR box) arranged as an imperfect inverted repeat of 6 bp (ATTACA) separated by 4 bp variable in sequence (6-4-6). AgrR interacts with two major groove segments and the intervening minor groove, all aligned on one face of the helix. Furthermore, an additional in phase imperfect direct repeat of the half-site may be observed slightly up and/or downstream of the inverted repeat at some operators. Mutant studies indicated that both inverted and direct repeats contribute to AgrR binding in vitro and AgrR-mediated activation in vivo. From the position of the AgrR box it appears that AgrR may act as a Type II activator for most investigated promoters, including positive autoregulation. Furthermore, we show in vitro binding and in vivo activation with dephosphomimetic AgrR mutant D51A, indicating that unphosphorylated AgrR is the active form of the RR in mutant NA4S. Frontiers Media S.A. 2020-07-17 /pmc/articles/PMC7380067/ /pubmed/32765465 http://dx.doi.org/10.3389/fmicb.2020.01635 Text en Copyright © 2020 Ali, Provoost, Mijnendonckx, Van Houdt and Charlier. http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. |
spellingShingle | Microbiology Ali, Md Muntasir Provoost, Ann Mijnendonckx, Kristel Van Houdt, Rob Charlier, Daniel DNA-Binding and Transcription Activation by Unphosphorylated Response Regulator AgrR From Cupriavidus metallidurans Involved in Silver Resistance |
title | DNA-Binding and Transcription Activation by Unphosphorylated Response Regulator AgrR From Cupriavidus metallidurans Involved in Silver Resistance |
title_full | DNA-Binding and Transcription Activation by Unphosphorylated Response Regulator AgrR From Cupriavidus metallidurans Involved in Silver Resistance |
title_fullStr | DNA-Binding and Transcription Activation by Unphosphorylated Response Regulator AgrR From Cupriavidus metallidurans Involved in Silver Resistance |
title_full_unstemmed | DNA-Binding and Transcription Activation by Unphosphorylated Response Regulator AgrR From Cupriavidus metallidurans Involved in Silver Resistance |
title_short | DNA-Binding and Transcription Activation by Unphosphorylated Response Regulator AgrR From Cupriavidus metallidurans Involved in Silver Resistance |
title_sort | dna-binding and transcription activation by unphosphorylated response regulator agrr from cupriavidus metallidurans involved in silver resistance |
topic | Microbiology |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7380067/ https://www.ncbi.nlm.nih.gov/pubmed/32765465 http://dx.doi.org/10.3389/fmicb.2020.01635 |
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