SummaryThe evolution of bacterial regulatory circuits often involves duplication of genes encoding transcription factors that may suffer both modifications in their detected signals, as well as, rewiring of their target operators. This, and subsequent horizontal gene transfer events contribute to generate a diverse array of regulatory pathways. In Salmonella, two homologous transcription factors CueR and GolS are responsible for Cu and Au sensing and resistance respectively. They share similarities not only in their sequence but also in their target binding sites, although they cluster separately among MerRmonovalent metal sensors. Here, we demonstrate that CueR and GolS can selectively distinguish their target binding sites by recognizing bases at positions 3Ј and 3 of their cognate operators. Swap of these bases results in switching regulator dependency. The differences in promoter architecture plus the environmentally controlled regulator's cytoplasmic availability warrant intra-regulon regulator-operator selectivity, and the proper response to metal injury. Furthermore, the presence of the distinctive operators' bases is widely extended among the two groups of MerRmonovalent metal sensors, providing evidence of the co-evolution of these factors and their target operators. This approach allows the prediction of regulator's dependency and the identification of transcription modules among groups of homologous transcription factors.

MerR metalloregulators alleviate toxicity caused by an excess of metal ions, such as copper, zinc, mercury, lead, cadmium, silver, or gold, by triggering the expression of specific efflux or detoxification systems upon metal detection. The sensor protein binds the inducer metal ion by using two conserved cysteine residues at the C-terminal metal-binding loop (MBL). Divalent metal ion sensors, such as MerR and ZntR, require a third cysteine residue, located at the beginning of the dimerization (␣5) helix, for metal coordination, while monovalent metal ion sensors, such as CueR and GolS, have a serine residue at this position. This serine residue was proposed to provide hydrophobic and steric restrictions to privilege the binding of monovalent metal ions. Here we show that the presence of alanine at this position does not modify the activation pattern of monovalent metal sensors. In contrast, GolS or CueR mutant sensors with a substitution of cysteine for the serine residue respond to monovalent metal ions or Hg(II) with high sensitivities. Furthermore, in a mutant deleted of the Zn(II) exporter ZntA, they also trigger the expression of their target genes in response to either Zn(II), Cd(II), Pb(II), or Co(II). IMPORTANCESpecificity in a stressor's recognition is essential for mounting an appropriate response. MerR metalloregulators trigger the expression of specific resistance systems upon detection of heavy metal ions. Two groups of these metalloregulators can be distinguished, recognizing either ؉1 or ؉2 metal ions, depending on the presence of a conserved serine in the former or a cysteine in the latter. Here we demonstrate that the serine residue in monovalent metal ion sensors excludes divalent metal ion detection, as its replacement by cysteine renders a pan-metal ion sensor. Our results indicate that the spectrum of signals detected by these sensors is determined not only by the metal-binding ligand availability but also by the metal-binding cavity flexibility.A set of bacterial metal ion sensors of the MerR family of transcriptional regulators are essential for controlling toxicity caused by an excess of essential heavy metal ions, such as copper (Cu) or zinc (Zn), or the presence of toxic-only heavy metal ions, such as mercury (Hg), lead (Pb), cadmium (Cd), silver (Ag), or gold (Au) ions (1). Detection of free metal ions in the cytoplasm by these sensors rapidly induces the transcription of sets of genes mostly coding for efflux or detoxification systems (2-5). Two functional regions composed of residues from both monomers can easily be distinguished in the structures of these dimeric regulators, namely, the DNA-binding and metal-binding regions. These regions are connected by two 34-residue antiparallel coiledcoil helices, one from each monomer, which cross over at the dimer interface. According to the current model, the signal detected at the metal-binding site is transmitted through allosteric changes to the DNA-binding domain, resulting in transcriptional activation of the target genes via a ...

Two homologous transcription factors, CueR and GolS, that belong to the MerR metalloregulatory family are responsible for Salmonella Cu and Au sensing and resistance, respectively. They share similarities not only in their sequences, but also in their target transcription binding sites. While CueR responds similarly to Au, Ag, or Cu to induce the expression of its target genes, GolS shows higher activation by Au than by Ag or Cu. We showed that the ability of GolS to distinguish Au from Cu resides in the metal-binding loop motif. Here, we identify the amino acids within the motif that determine in vivo metal selectivity. We show that residues at positions 113 and 118 within the metal-binding loop are the main contributors to metal selectivity. The presence of a Pro residue at position 113 favors the detection of Cu, while the presence of Pro at position 118 disfavors it. Our results highlight the molecular bases that allow these regulators to coordinate the correct metal ion directing the response to a particular metal injury.T ransition metal homeostasis influences many fundamental aspects of bacterial cell physiology and pathogenesis (1-3). The intracellular concentration of essential metals or the presence of harmful elements is monitored by a set of transcriptional regulators that modulate the expression of factors that rapidly restore metal homeostasis (4, 5). A large class of these metalloregulators belongs to the MerR family, a group of proteins that share similarity at the N-terminal DNA-binding domain (6-9). According to the current model, dimeric metal-sensing MerR regulators control gene transcription via a DNA distortion mechanism. Both the apo-and the metal-bound regulator recognize and interact with their target operators (a dyad-symmetric DNA sequence at the promoter region of their target genes). Binding of the metal ion at the C-terminal inductor-binding site would provoke an allosteric change at the N-terminal DNA binding region of the protein, which in turn transduces changes in the promoter structure resulting in transcription activation of the expression of genes coding mostly for efflux or detoxification systems (10-12).Most of the metal ion sensors of the MerR family are poorly selective because they cannot distinguish between cognate metals with similar physicochemical properties, including charge and coordination chemistry. For example, the Cu sensor CueR can discriminate between metal ions with ϩ1 and ϩ2 charges, but it cannot distinguish between monovalent metal ions of group 1B-i.e., Cu(I), Ag(I), and Au(I) (13). Structural studies indicate that CueR has only two coordinating ligands, the S-atoms of the conserved C112 and C120 residues which are appropriate for the interaction with the ϩ1 metal ion in a linear array but not to coordinate metal ions with a ϩ2 charge, which requires higher number of ligands (14). The recent identification of two Au-selective MerR sensors, first in the bacterial pathogen Salmonella enterica serovar Typhimurium (S. Typhimurium) and then in Cupriavidus met...