micromachines Article Potential Whole-Cell Biosensors for Detection of Metal Using MerR Family Proteins from Enterobacter sp. YSU and Stenotrophomonas maltophilia OR02 Georgina Baya 1, Stephen Muhindi 2, Valentine Ngendahimana 3 and Jonathan Caguiat 1,* 1 Department of Biological and Chemical Sciences, Youngstown State University, Youngstown, OH 44555, USA; [email protected] 2 Department of Biological Sciences, University of Toledo, Toledo, OH 43606, USA; [email protected] 3 Biology Department, Lone Star College-CyFair, 9191 Barker Cypress Rd, Cypress, TX 77433, USA; [email protected] * Correspondence: [email protected]; Tel.: +1-330-941-2063 Abstract: Cell-based biosensors harness a cell’s ability to respond to the environment by repurposing its sensing mechanisms. MerR family proteins are activator/repressor switches that regulate the expression of bacterial metal resistance genes and have been used in metal biosensors. Upon metal binding, a conformational change switches gene expression from off to on. The genomes of the multi- metal resistant bacterial strains, Stenotrophomonas maltophilia Oak Ridge strain 02 (S. maltophilia 02) and Enterobacter sp. YSU, were recently sequenced. Sequence analysis and gene cloning identified three mercury resistance operons and three MerR switches in these strains. Transposon mutagenesis and sequence analysis identified Enterobacter sp. YSU zinc and copper resistance operons, which ap- pear to be regulated by the protein switches, ZntR and CueR, respectively. Sequence analysis and Citation: Baya, G.; Muhindi, S.; reverse transcriptase polymerase chain reaction (RT-PCR) showed that a CueR switch appears to Ngendahimana, V.; Caguiat, J. activate a S. maltophilia 02 copper transport gene in the presence of CuSO4 and HAuCl4·3H2O. Potential Whole-Cell Biosensors for In previous studies, genetic engineering replaced metal resistance genes with the reporter genes for Detection of Metal Using MerR β-galactosidase, luciferase or the green fluorescence protein (GFP). These produce a color change of a Family Proteins from Enterobacter sp. reagent, produce light, or fluoresce in the presence of ultraviolet (UV) light, respectively. Coupling YSU and Stenotrophomonas maltophilia these discovered operons with reporter genes has the potential to create whole-cell biosensors for OR02. Micromachines 2021, 12, 142. HgCl , ZnCl , CuSO and HAuCl ·3H O. https://doi.org/10.3390/mi12020142 2 2 4 4 2 Academic Editor: Lawrence Kulinsky Keywords: whole-cell biosensor; MerR family protein; ZntR; CueR; bacterial metal resistance; HgCl2; Received: 7 January 2021 ZnCl2; CuSO4; HAuCl4·3H2O; Enterobacter; Stenotrophomonas maltophilia Accepted: 27 January 2021 Published: 29 January 2021 Publisher’s Note: MDPI stays neutral 1. Introduction with regard to jurisdictional claims in Whole-cell biosensors are highly anticipated in the field of on-site detection [1–3]. published maps and institutional affil- Metal-resistant bacteria control the expression of their metal resistant genes using protein iations. switches. They activate or turn them on in the presence of toxic metals and repress them or turn them off in the absence of metal to conserve energy. The Tn21 mercury resistance operon (mer) from Shigella flexneri has been well studied [4] and the protein switch, MerR, controls its expression [5,6]. The operon consists of the genes, merT, merP, merC, merA, merD, Copyright: © 2021 by the authors. merE (merTPCADE) and merR (Figure1a) [ 7,8]. The genes, merTPCADE, are transcribed Licensee MDPI, Basel, Switzerland. in the opposite direction in relation to merR. MerT, MerC and MerE are cytoplasmic This article is an open access article membrane proteins, while MerP is a periplasmic protein [9,10]. All four transport divalent distributed under the terms and mercury (Hg(II)) into the cells. Then, MerA, mercuric reductase, reduces it to elemental conditions of the Creative Commons mercury (Hg(0)), which volatilizes from the microorganisms environment [11]. MerR is Attribution (CC BY) license (https:// an activator/repressor protein switch [12], which binds to the operator region of the creativecommons.org/licenses/by/ merTPCADE and merR promoters and represses the expression of merTPCADE and merR in 4.0/). Micromachines 2021, 12, 142. https://doi.org/10.3390/mi12020142 https://www.mdpi.com/journal/micromachines Micromachines 2021, 12, 142 2 of 17 the absence of Hg(II). When Hg(II) is present, it activates the expression of merTPCADE, while still repressing the expression of merR. Even under this repressed state, enough merR is transcribed and translated to produce MerR protein that efficiently regulates merTPCADE Micromachines 2021, 12, x FOR PEER REVIEW 3 of 16 expression. When the concentration of mercury decreases below toxic levels, MerD restores the operon to the repressed state [13,14]. FigureFigure 1. 1.The The TnTn2121 mercury resistance ( mer) operon operon and and MerR MerR protein protein activator/repressor activator/repressor switch. switch. (a) ( aThe) The genes genes merTPCADmerTPCAD areare transcribed transcribed toto thethe rightright andand merRmerR is transcribed to to the the left. left. The The MerR MerR protein protein binds binds as as a adimer dimer toto the the DNA DNA operator operator regionregion (underlined) (underlined) between between the the− −10 and −−3535 RNA RNA polymerase polymerase binding binding regions regions (high (highlightedlighted in inblack). black). The The non-optimal non-optimal 19 19 base pair (bp) spacer between the -10 and -35 regions prevents RNA polymerase from initiating transcription efficiently. base pair (bp) spacer between the −10 and −35 regions prevents RNA polymerase from initiating transcription efficiently. In the absence of Hg(II), MerR binding to the DNA acts as an off switch to repress transcription. In the presence of Hg(II), In the absence of Hg(II), MerR binding to the DNA acts as an off switch to repress transcription. In the presence of MerR acts as an on switch by shortening the distance between and aligning the −10 and −35 regions so that RNA polymer- Hg(II),ase can MerR initiate acts transcription as an on switch of the by merTPCAD shortening genes the distance efficiently. between Transcription and aligning begins the at the−10 nucleotide and −35 regionsbase (bold) so that below RNA polymerase+ in +1. The can first initiate three base transcription pairs, CAT, of theon themerTPCAD far left marksgenes the efficiently. beginning Transcription of MerR translation. begins at The the last nucleotide three base base pairs, (bold) belowATG, +on in the +1 far. The right first marks three the base be pairs,ginning CAT, of MerT on the translation. far left marks (b) The the Tn beginning21 MerR amino of MerR acid translation. residue primary The last sequence. three base pairs,The helix-turn-helix ATG, on the far domain right marksbinds to the the beginning operator region of MerT of the translation. promoter in (b the) The DNA. Tn21 TheMerR coupling amino domain acid residuelinks the primaryDNA sequence.binding domain The helix-turn-helix to the metal binding domain domain. binds The to the metal operator bindin regiong domain of theconsists promoter of the indime therization DNA. Thehelix, coupling metal binding domain loop and 2-turn α-helix. The dimerization helix links two identical MerR polypeptides to form an antiparallel coiled coil. links the DNA binding domain to the metal binding domain. The metal binding domain consists of the dimerization helix, The # below the sequences designates conserved cysteine amino acid residues which bind to Hg(II). The * and numbers metal binding loop and 2-turn α-helix. The dimerization helix links two identical MerR polypeptides to form an antiparallel above the MerR primary sequence denote the position of every 10 amino acid residues. coiled coil. The # below the sequences designates conserved cysteine amino acid residues which bind to Hg(II). The * and numbers above the MerR primary2. Materials sequence and denote Methods the position of every 10 amino acid residues. 2.1. BacterialThe MerR Strains, protein Plasmids has three and Media domains: a DNA binding domain, a mercury binding do- mainStenotrophomonas and a coupling domain maltophilia (Figure Oak1 Ridgeb) [ 12 strain,15–17 02]. It(ATCC binds #53510) as a homodimer, was purchased two identical from polypeptides,the American Type to the Culture operator Collection in the DNA (Manassas, using a helix-turn-helixVA, USA), Enterobacter motif. Thesp. YSU binding was or operatordescribed region previously in the [29] DNA and is locatedStrataClone between SoloPack the − 10Competent and −35 RNACells polymeraseand the plasmid, binding sitespSC-A-amp/kan, of the promoter were andpurchased consists from of anAgilent inverted (Santa repeat Clara, (Figure CA, USA)1a). as Each components polypeptide of inthe the StrataClone homodimer PCR binds (Polymerase to one ofChain the repeats.Reaction) The Cloning mercury Kit. TransforMax™ binding domain EC100D™ contains a dimerizationpir-116 Escherichia helix, coli a metal (E. coli binding) was purchased loop and afrom 2-turn Lucigen alpha-helix. (Middleton, The dimerization WI, USA) [30]. helices link theLennox dimer LB together medium in was an antiparallel purchased orientationfrom Amresco and (Solon, form a coiledOH, USA), coil [ 18and]. TheR3A-tris protein usesmedium the was sulfhydryl described groups previously from three[29]. When cysteine required, amino LB acid or R3A-tris residues, medium cys82,