Susana K. Checa

Bacterial sensing of and resistance to gold salts

The MerR family is a group of bacterial transcriptional regulators that respond to different environmental stimuli, such as heavy metals, oxidative stress or antibiotics. Here we characterize a new member of this family that is highly selective for Au ions. We show that this Salmonella regulator, named GolS, directly controls the expression of at least two transcriptional units specifically required for Au resistance. By chromosomal mutagenesis, we demonstrated that Au‐selectivity is accomplished by a metal‐binding motif in GolS. Among the monovalent metal‐ion sensing MerR regulators GolS clusters in a branch distant from enterobacterial CueR orthologues. We propose that GolS and its homologues evolved to cope with toxic concentration of Au ion, allowing microorganisms to withstand contaminated environments.

GolS controls the response to gold by the hierarchical induction of Salmonella-specific genes that include a CBA efflux-coding operon.

Salmonella employs a specific set of proteins that allows it to detect the presence of gold salts in the environment and to mount the appropriate resistance response. This includes a P‐type ATPase, GolT, and a small cytoplasmic metal binding protein, GolB. Their expression is controlled by a MerR‐like sensor, GolS, which is highly selective for Au ions. Here, we identify a new GolS‐controlled operon named gesABC which codes for a CBA efflux system, and establish its role in Au resistance. GesABC can also mediate drug resistance when induced by Au in a GolS‐dependent manner, in a strain deleted in the main drug transporter acrAB. The GolS‐controlled transcription of gesABC differs from the other GolS‐regulated loci. It is activated by gold, but not induced by copper, even in a strain deleted of the main Cu transporter gene copA, which triggers a substantial GolS‐dependent induction of golTS and golB. We demonstrate that the Au‐dependent induction of gesABC transcription requires higher GolS levels than for the other members of the gol regulon. This correlates with a divergent GolS operator in the gesABC promoter. We propose that the hierarchical induction within the gol regulon allows Salmonella to cope with Au‐contaminated environments.

Bacterial signaling systems as platforms for rational design of new generations of biosensors

Bacterial signal-responsive regulatory circuits have been employed as platform to design and construct whole-cell bacterial biosensors for reporting toxicity. A new generation of biosensors with improved performance and a wide application range has emerged after the application of synthetic biology concepts to biosensor design. Site-directed mutagenesis, directed evolution and domain swapping were applied to upgrade signal detection or to create novel sensor modules. Rewiring of the genetic circuits allows improving the determinations and reduces the heterogeneity of the response between individual reporter cells. Moreover, the assembly of natural or engineered modules to biosensor platforms provides innovative outputs, expanding the range of application of these devises, from monitoring toxics and bioremediation to killing targeted cells.

Bacterial gold sensing and resistance

Gold ions are mobilized and disseminated through the environment and enter into the cells by non-specific intake. To avoid deleterious effect that occurs even at very low concentrations, bacteria such as Salmonella enterica and Cupriavidus metallidurans use Au-specific MerR-type transcriptional regulators to detect the presence of these toxic ions, and control the expression of specific resistance factors. In contrast to the related copper sensor CueR, the Au-selective metalloregulatory proteins are able to distinguish Au(I) from Cu(I) or Ag(I). This is achieved by finely tuning a single dithiolate metal coordination with conserved cysteine residues at the metal binding site of the proteins to lower the affinity for Cu(I) in comparison to the Cu-sensors, while maintaining or even increasing the affinity for Au(I). In Salmonella, GolS not only privileges the binding of Au(I) over Cu(I) or Ag(I), but also distinguishes its target recognition sites in its regulated promoters minimizing cross-activation of CueR-controlled operators. In this sense, the presence of a selective Au sensory devise would allow species harbouring resident Cu-homeostasis systems to eliminate the toxic ion without affecting Cu acquisition in Au rich environments.

Downregulation of RpoN‐controlled genes protects Salmonella cells from killing by the cationic antimicrobial peptide polymyxin B

Salmonella enterica polymyxin B (PM) resistance is modulated mainly by substitutions of the acyl chains and the phosphate groups on the lipid A moiety of lipopolysaccharide. These modifications are mediated by genes under the control of the PmrA/PmrB and PhoP/PhoQ two-component regulatory systems. In this study, a deletion in the gene encoding the alternative sigma(54) factor, rpoN, was shown to increase PM resistance without affecting protamine sensitivity. The results presented here showed that the increased polymyxin resistance observed in the DeltarpoN mutant occurs through a PmrA/PhoP-independent pathway. Downregulation of one or more genes belonging to the RpoN regulon may provide an additional mechanism of defence against membrane-permeabilizing antimicrobial peptides that helps the pathogen to survive in different environments.

A Single Serine Residue Determines Selectivity to Monovalent Metal Ions in Metalloregulators of the MerR Family

UNLABELLED 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 (α5) 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). IMPORTANCE Specificity in a stressors 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.

Identification of a Salmonella ancillary copper detoxification mechanism by a comparative analysis of the genome-wide transcriptional response to copper and zinc excess

Copper and zinc are essential metal ions, but toxic in excess. Bacteria have evolved different strategies to control their intracellular concentrations, ensuring proper supply while avoiding toxicity, including the induction of metal-specific as well as non-specific mechanisms. We compared the transcriptional profiles of Salmonella Typhimurium after exposure to either copper or zinc ions in both rich and minimal media. Besides metal-specific regulatory networks many global stress-response pathways react to an excess of either of these metal ions. Copper excess affects both zinc and iron homeostasis by inducing transcription of these metal-specific regulons. In addition to the control of zinc-specific regulons, zinc excess affects the Cpx regulon and the σ(E) envelope-stress responses. Finally, novel metal-specific upregulated genes were detected including a new copper-detoxification pathway that involves the siderophore enterobactin and the outer-membrane protein TolC. This work sheds light onto the transcriptional landscape of Salmonella after copper or zinc overload, and discloses a new mechanism of copper detoxification.

Protein Signatures That Promote Operator Selectivity among Paralog MerR Monovalent Metal Ion Regulators

Background: Two nucleotide bases distinguish promoters controlled by paralog MerR monovalent metalloregulators, avoiding cross-activation. Results: Specific residues within the DNA-binding region of the regulators were identified as responsible for the selectivity in the operator recognition. Conclusion: Co-evolution of both the regulator and its target operator sequences prevents cross-activation of paralog regulatory circuits. Significance: The basis for regulator/operator specificity among MerR monovalent metalloregulators is described. Two paralog transcriptional regulators of the MerR family, CueR and GolS, are responsible for monovalent metal ion sensing and resistance in Salmonella enterica. Although similar in sequence and also in their target binding sites, these proteins differ in signal detection and in the set of target genes they control. Recently, we demonstrated that selective promoter recognition depends on the presence of specific bases located at positions 3′ and 3 within the operators they interact with. Here, we identify the amino acid residues within the N-terminal DNA-binding domain of these sensor proteins that are directly involved in operator discrimination. We demonstrate that a methionine residue at position 16 of GolS, absolutely conserved among GolS-like proteins but absent in all CueR-like xenologs, is the key to selectively recognize operators that harbor the distinctive GolS-operator signature, whereas the residue at position 19 finely tunes the regulator/operator interaction. Furthermore, swapping these residues switches the set of genes recognized by these transcription factors. These results indicate that co-evolution of a regulator and its cognate operators within the bacterial cell provides the conditions to avoid cross-recognition and guarantees the proper response to metal injury.

Dissecting the Metal Selectivity of MerR Monovalent Metal Ion Sensors in Salmonella

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.

Selective detection of gold using genetically engineered bacterial reporters

Salmonella typhimurium harbours a Au‐resistance system whose expression is controlled by GolS, a transcriptional regulator of the MerR family that selectively detects Au with high sensitivity. We developed both Salmonella and genetically engineered Escherichia coli strains as Au‐selective whole‐cell biosensors by coupling the strictly regulated GolS‐dependent golB promoter to the gfp reporter gene. The bio‐reporters were evaluated under different laboratory conditions and calibrated for their use as selective Au detectors. Due to the intrinsic characteristics of the regulatory protein, the transgenic E. coli sensor exhibits low background, high signal‐to‐noise ratio, and improved sensitivity for detection of Au ions in a wide range of concentrations (up to 470 nM) with a calculated detection limit of ∼33 nM (6 µg L−1 or parts per billion) Au(I). The fluorescent Au‐sensing bacteria exhibit also minimal interference by chemically related metals such as Cu or Ag that are commonly found in Au deposits. These highly specific and sensitive Au detectors might allow the development of rapid and robust screening tools to improve discovery and extraction procedures. Biotechnol. Bioeng. 2011;108: 2553–2560.