DNA-Binding and Transcription Activation by Unphosphorylated Response Regulator AgrR From Cupriavidus metallidurans Involved in Silver Resistance.

Md Muntasir Ali,Kristel Mijnendonckx,Ann Provoost,Daniel Charlier,Rob Van Houdt

doi:10.3389/fmicb.2020.01635

Md Muntasir Ali, Kristel Mijnendonckx + Show 3 more

Open Access

https://doi.org/10.3389/fmicb.2020.01635

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Abstract

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.

Highlights

The numerous applications of silver, a precious metal with recognized antimicrobial and antiviral activity, in industry, commerce, hygiene and healthcare have greatly favored its release and spread in the environment (Silver et al, 2006; EdwardsJones, 2009; Mijnendonckx et al, 2013a; Huy et al, 2017)
The genome-wide expression analysis of the silverresistant mutant NA4S was previously performed via the microarray platform of the C. metallidurans type strain CH34 (Mijnendonckx et al, 2019)
The NA4 systems homologous to known silver detoxification systems were not differentially expressed in NA4S (Table 1)

Summary

Introduction

The numerous applications of silver, a precious metal with recognized antimicrobial and antiviral activity, in industry, commerce, hygiene and healthcare have greatly favored its release and spread in the environment (Silver et al, 2006; EdwardsJones, 2009; Mijnendonckx et al, 2013a; Huy et al, 2017). Increased exposure of microorganisms to enhanced silver concentrations may favor the development of resistance and, silverresistant bacteria have already been isolated from environmental, industrial and even clinical environments (Finley et al, 2015). The emergence of such organisms may impede the utilization of silver as antimicrobial agent, especially for medical applications. It has been shown that in some instances, increased metal resistance may be accompanied by an increase in antibiotic resistance (McHugh et al, 1975; Choudhury and Kumar, 1998; Perron et al, 2004; Dieppois et al, 2012; Pal et al, 2017; Wang et al, 2017; Chen et al, 2019)

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