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BACKGROUND: Metabolomics plays a critical role in deciphering metabolic alterations within individuals, demanding the use of sophisticated analytical methodologies to navigate its intricate complexity. While many studies focus on single biofluid types, simultaneous analysis of multiple matrices enhances understanding of complex biological mechanisms. Consequently, the development of data fusion methods enabling multiblock analysis becomes essential for comprehensive insights into metabolic dynamics. RESULTS: This study introduces a novel guideline for jointly analyzing diverse metabolomic datasets (serum, urine, metadata) with a focus on metabolic differences between groups within a healthy cohort. The guideline presents two fusion strategies, 'Low-Level data fusion' (LLDF) and 'Mid-Level data fusion' (MLDF), employing a sequential application of Multivariate Curve Resolution with Alternating Least Squares (MCR-ALS), linking the outcomes of successive analyses. MCR-ALS is a versatile method for analyzing mixed data, adaptable at various stages of data processing-encompassing resonance integration, data compression, and exploratory analysis. The LLDF and MLDF strategies were applied to 1H NMR spectral data extracted from urine and serum samples, coupled with biochemical metadata sourced from 145 healthy volunteers. SIGNIFICANCE: Both methodologies effectively integrated and analysed multiblock datasets, unveiling the inherent data structure and variables associated with discernible factors among healthy cohorts. While both approaches successfully detected sex-related differences, the MLDF strategy uniquely revealed components linked to age. By applying this analysis, we aim to enhance the interpretation of intricate biological mechanisms and uncover variations that may not be easily discernible through individual data analysis.
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Metabolómica , Humanos , Metabolómica/métodos , Masculino , Femenino , Análisis Multivariante , Voluntarios Sanos , Adulto , Espectroscopía de Protones por Resonancia Magnética , Estudios de Cohortes , Persona de Mediana Edad , Análisis de los Mínimos Cuadrados , Adulto JovenRESUMEN
Proteins' extraordinary performance in recognition and catalysis has led to their use in a range of applications. However, proteins obtained from natural sources are oftentimes not suitable for direct use in industrial or diagnostic setups. Natural proteins, evolved to optimally perform a task in physiological conditions, usually lack the stability required to be used in harsher conditions. Therefore, the alteration of the stability of proteins is commonly pursued in protein engineering studies. Here, we achieved a substantial thermal stabilization of a bacterial Zn(II)-dependent phospholipase C by consensus sequence design. We retrieved and analyzed sequenced homologues from different sources, selecting a subset of examples for expression and characterization. A non-natural consensus sequence showed the highest stability and activity among those tested. Comparison of the stability parameters of this stabilized mutant and other natural variants bearing similar mutations allows us to pinpoint the sites most likely to be responsible for the enhancement. Point mutations in these sites alter the unfolding process of the consensus sequence. We show that the stabilized version of the protein retains full activity even in harsh oil degumming conditions, making it suitable for industrial applications.
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Proteínas , Zinc , Secuencia de Aminoácidos , Proteínas/metabolismo , Mutación , Secuencia de ConsensoRESUMEN
The increasing demand pressures the vegetable oil industry to develop novel refining methods. Degumming with type C phospholipases (PLCs) is a green technology and provides extra oil. However, natural PLCs are not active under the harsh conditions used in oil refining plants, requiring additional unit operations. These upfront capital expenditures and the associated operational costs hinder the adoption of this method. Here, we present a process based on ChPLC, a synthetic PLC obtained by consensus sequence design, possessing superior thermal stability and catalytic properties. Using ChPLC, crude soybean oil degumming was completed at 80 °C in 30 min, the temperature and residence time imposed by the design of existing oil refining plants. Remarkably, an extra yield of oil of 2% was obtained using 60% of the dose recommended for PLCs marketed today, saving upfront investments and reducing the operational cost of degumming. A techno-economic analysis indicates that, for medium size plants, ChPLC reduces the overall cost of soybean oil enzymatic degumming by 58%. The process presented here facilitates the implementation of enzymatic technologies to oil producers, regardless of their processing capacity, bringing potential annual benefits in the billion-dollar range for the global economy.
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Aceites de Plantas , Aceite de Soja , Fosfolipasas de Tipo C , TemperaturaRESUMEN
U-Omp19 is a bacterial protease inhibitor from Brucella abortus that inhibits gastrointestinal and lysosomal proteases, enhancing the half-life and immunogenicity of co-delivered antigens. U-Omp19 is a novel adjuvant that is in preclinical development with various vaccine candidates. However, the molecular mechanisms by which it exerts these functions and the structural elements responsible for these activities remain unknown. In this work, a structural, biochemical, and functional characterization of U-Omp19 is presented. Dynamic features of U-Omp19 in solution by NMR and the crystal structure of its C-terminal domain are described. The protein consists of a compact C-terminal beta-barrel domain and a flexible N-terminal domain. The latter domain behaves as an intrinsically disordered protein and retains the full protease inhibitor activity against pancreatic elastase, papain and pepsin. This domain also retains the capacity to induce CD8+ T cells in vivo of U-Omp19. This information may lead to future rationale vaccine designs using U-Omp19 as an adjuvant to deliver other proteins or peptides in oral formulations against infectious diseases, as well as to design strategies to incorporate modifications in its structure that may improve its adjuvanticity.
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In a joint DFT and chemometrics study applied to NMR spectra, we disclose the structure of the main decomposition products of hexamethylenetetramine. The combination of these techniques enabled us to propose the structures of near-identical intermediates of the process and to unveil the structure of the main decomposition product of this priviliged structure.
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MicroRNAs (miRNAs) are endogenous small RNAs of â¼21 nt that regulate multiple biological pathways in multicellular organisms. They derive from longer transcripts that harbor an imperfect stem-loop structure. In plants, the ribonuclease type III DICER-LIKE1 assisted by accessory proteins cleaves the precursor to release the mature miRNA. Numerous studies highlight the role of the precursor secondary structure during plant miRNA biogenesis; however, little is known about the relevance of the precursor sequence. Here, we analyzed the sequence composition of plant miRNA primary transcripts and found specifically located sequence biases. We show that changes in the identity of specific nucleotides can increase or abolish miRNA biogenesis. Most conspicuously, our analysis revealed that the identity of the nucleotides at unpaired positions of the precursor plays a crucial role during miRNA biogenesis in Arabidopsis.
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Arabidopsis/genética , Arabidopsis/metabolismo , MicroARNs/biosíntesis , MicroARNs/genética , ARN de Planta/biosíntesis , ARN de Planta/genética , Proteínas de Arabidopsis/metabolismo , Disparidad de Par Base , Proteínas de Ciclo Celular/metabolismo , Magnoliopsida/genética , Magnoliopsida/metabolismo , MicroARNs/química , MicroARNs/metabolismo , Simulación de Dinámica Molecular , Conformación de Ácido Nucleico , Polimorfismo de Nucleótido Simple , Procesamiento Postranscripcional del ARN , ARN de Planta/química , Ribonucleasa III/metabolismoRESUMEN
The Salmonella enterica PhoP/PhoQ two-component signaling system coordinates the spatiotemporal expression of key virulence factors that confer pathogenic traits. Through biochemical and structural analyses, we found that the sensor histidine kinase PhoQ acted as a receptor for long-chain unsaturated fatty acids (LCUFAs), which induced a conformational change in the periplasmic domain of the PhoQ protein. This resulted in the repression of PhoQ autokinase activity, leading to inhibition of the expression of PhoP/PhoQ-dependent genes. Recognition of the LCUFA linoleic acid (LA) by PhoQ was not stereospecific because positional and geometrical isomers of LA equally inhibited PhoQ autophosphorylation, which was conserved in multiple S. enterica serovars. Because orally acquired Salmonella encounters conjugated LA (CLA), a product of the metabolic conversion of LA by microbiota, in the human intestine, we tested how short-term oral administration of CLA affected gut colonization and systemic dissemination in a mouse model of Salmonella-induced colitis. Compared to untreated mice, CLA-treated mice showed increased gut colonization by wild-type Salmonella, as well as increased dissemination to the spleen. In contrast, the inability of the phoP strain to disseminate systemically remained unchanged by CLA treatment. Together, our results reveal that, by inhibiting PhoQ, environmental LCUFAs fine-tune the fate of Salmonella during infection. These findings may aid in the design of new anti-Salmonella therapies.
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Proteínas Bacterianas/metabolismo , Histidina Quinasa/metabolismo , Ácido Linoleico/metabolismo , Infecciones por Salmonella/metabolismo , Salmonella typhimurium/metabolismo , Transducción de Señal , Animales , Proteínas Bacterianas/genética , Femenino , Histidina Quinasa/genética , Ácido Linoleico/genética , Ratones , Fosforilación , Infecciones por Salmonella/genética , Salmonella typhimurium/genética , Salmonella typhimurium/patogenicidadRESUMEN
The ribonuclease III (RNase III) cleaves dsRNA in specific positions generating mature RNAs. RNase III enzymes play important roles in RNA processing, post-transcriptional gene expression, and defense against viral infection. The enzyme's active site contains Mg2+ ions bound by a network of acidic residues and water molecules, but there is a lack of information about their specific roles. In this work, multiple steered molecular dynamics simulations at QM/MM level were performed to explore the hydrolysis reaction carried out by the enzyme. Free energy profiles modifying the features of the active site are obtained and the role of Mg2+ ions, the solvent molecules and the residues of the active site are discussed in detail. Our results show that Mg2+ ions carry out different roles in the hydrolysis process positioning the substrate for the attack from a coordinated nucleophile and activating it to perform hydrolysis reaction, cleaving the dsRNA backbone in a SN2 substitution. In addition, water molecules present in the active site lower the energy barrier of the process. RNase III hydrolyzes dsRNA to generate mature RNAs. For this purpose, its active site contains Mg2+ which has an important role during the reaction. Results show that the Mg2+ activates the solvent molecule that produces the nucleophilic attack and the surrounding waters contribute significantly to the hydrolysis process.
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Bacterias/enzimología , Magnesio/metabolismo , Teoría Cuántica , ARN Bicatenario/metabolismo , Ribonucleasa III/metabolismo , Hidrólisis , Simulación de Dinámica Molecular , Conformación Proteica , Procesamiento Postranscripcional del ARN , Ribonucleasa III/químicaRESUMEN
Many evolutionarily conserved microRNAs (miRNAs) in plants regulate transcription factors with key functions in development. Hence, mutations in the core components of the miRNA biogenesis machinery cause strong growth defects. An essential aspect of miRNA biogenesis is the precise excision of the small RNA from its precursor. In plants, miRNA precursors are largely variable in size and shape and can be processed by different modes. Here, we optimized an approach to detect processing intermediates during miRNA biogenesis. We characterized a miRNA whose processing is triggered by a terminal branched loop. Plant miRNA processing can be initiated by internal bubbles, small terminal loops or branched loops followed by dsRNA segments of 15-17 bp. Interestingly, precision and efficiency vary with the processing modes. Despite the various potential structural determinants present in a single a miRNA precursor, DCL1 is mostly guided by a predominant structural region in each precursor in wild-type plants. However, our studies in fiery1, hyl1 and se mutants revealed the existence of cleavage signatures consistent with the recognition of alternative processing determinants. The results provide a general view of the mechanisms underlying the specificity of miRNA biogenesis in plants.
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Proteínas de Arabidopsis/genética , Arabidopsis/genética , MicroARNs/genética , Monoéster Fosfórico Hidrolasas/genética , Proteínas de Unión al ARN/genética , Sitios de Unión , Biología Computacional , Regulación de la Expresión Génica de las Plantas , Biblioteca de Genes , MicroARNs/biosíntesis , Mutación , Plantas Modificadas Genéticamente , Reacción en Cadena de la Polimerasa , Estructura Secundaria de Proteína , Procesamiento Postranscripcional del ARN , ARN Bicatenario/genética , Plantones , Transcripción Genética , TransgenesRESUMEN
DCL1 is the ribonuclease that carries out miRNA biogenesis in plants. Substrate pri-miRNA recognition by DCL1 requires two double stranded RNA binding domains located at the C-terminus of the protein. We have previously shown that the first of these domains, DCL1-A, is intrinsically disordered and folds upon binding pri-miRNA. Integrating NMR and SAXS data, we study here the conformational landscape of free DCL1-A through an ensemble description. Our results reveal that secondary structure elements, corresponding to the folded form of the protein, are transiently populated in the unbound state. The conformation of one of the dsRNA binding regions in the free protein shows that, at a local level, RNA recognition proceeds through a conformational selection mechanism. We further explored the stability of the preformed structural elements via temperature and urea destabilization. The C-terminal helix is halfway on the folding pathway in free DCL1-A, constituting a potential nucleation site for the final folding of the protein. In contrast, the N-terminal helix adopts stable non-native structures that could hinder the correct folding of the protein in the absence of RNA. This description of the unfolded form allows us to understand details of the mechanism of binding-induced folding of the protein.
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Proteínas de Arabidopsis/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas Intrínsecamente Desordenadas/química , MicroARNs/metabolismo , Ribonucleasa III/metabolismo , Arabidopsis , Proteínas de Arabidopsis/química , Proteínas de Ciclo Celular/química , Dicroismo Circular , Espectroscopía de Resonancia Magnética , MicroARNs/química , Modelos Químicos , Unión Proteica , Conformación Proteica , Dominios Proteicos , Pliegue de Proteína , Ribonucleasa III/química , Dispersión del Ángulo Pequeño , Temperatura , Difracción de Rayos XRESUMEN
The intrinsically disordered protein domain DCL1-A is the first report of a complete double stranded RNA binding domain folding upon binding. DCL1-A recognizes the dsRNA by acquiring a well-folded structure after engagement with its interaction partner. Despite the structural characterization of the interaction complex underlying the recognition of dsRNA has been established, the dynamics of disorder-to-order transitions in the binding process remains elusive. Here we have developed a coarse-grained structure-based model with consideration of electrostatic interactions to explore the mechanism of the coupled folding and binding. Our approach led to remarkable agreements with both experimental and theoretical results. We quantified the global binding-folding landscape, which indicates a synergistic binding induced folding mechanism. We further investigated the effect of electrostatic interactions in this coupled folding and binding process. It reveals that non-native electrostatic interactions dominate the initial stage of the recognition. Our results help improve our understanding of the induced folding of the IDP DCL1-A upon binding to dsRNA. Such methods developed here can be applied for further explorations of the dynamics of coupled folding and binding systems.
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Proteínas de Arabidopsis/química , Proteínas de Ciclo Celular/química , Pliegue de Proteína , ARN/química , Ribonucleasa III/química , Simulación por Computador , Cinética , Modelos Moleculares , Unión Proteica , Dominios Proteicos , Electricidad Estática , Relación Estructura-Actividad , TermodinámicaRESUMEN
Extremophilic microorganisms are a rich source of enzymes, the enzymes which can serve as industrial catalysts that can withstand harsh processing conditions. An example is thermostable ß-glucosidases that are addressing a challenging problem in the biodiesel industry: removing steryl glucosides (SGs) from biodiesel. Steryl glucosidases (SGases) must be tolerant to heat and solvents in order to function efficiently in biodiesel. The amphipathic nature of SGs also requires enzymes with an affinity for water/solvent interfaces in order to achieve efficient hydrolysis. Additionally, the development of an enzymatic process involving a commodity such as soybean biodiesel must be cost-effective, necessitating an efficient manufacturing process for SGases. This review summarizes the identification of microbial SGases and their applications, discusses biodiesel refining processes and the development of analytical methods for identifying and quantifying SGs in foods and biodiesel, and considers technologies for strain engineering and process optimization for the heterologous production of a SGase from Thermococcus litoralis. All of these technologies might be used for the production of other thermostable enzymes. Structural features of SGases and the feasibility of protein engineering for novel applications are explored.
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Biotecnología/métodos , Glucosidasas/biosíntesis , Glucosidasas/química , Biocombustibles , Celulasas/biosíntesis , Celulasas/química , Celulasas/genética , Estabilidad de Enzimas , Glucosidasas/genética , Calor , Hidrólisis , Ingeniería de Proteínas , Solventes/química , Glycine maxRESUMEN
Thioredoxin is a ubiquitous small protein that catalyzes redox reactions of protein thiols. Additionally, thioredoxin from E. coli (EcTRX) is a widely-used model for structure-function studies. In a previous paper, we characterized several single-point mutants of the C-terminal helix (CTH) that alter global stability of EcTRX. However, spectroscopic signatures and enzymatic activity for some of these mutants were found essentially unaffected. A comprehensive structural characterization at the atomic level of these near-invariant mutants can provide detailed information about structural variability of EcTRX. We address this point through the determination of the crystal structures of four point-mutants, whose mutations occurs within or near the CTH, namely L94A, E101G, N106A and L107A. These structures are mostly unaffected compared with the wild-type variant. Notably, the E101G mutant presents a large region with two alternative traces for the backbone of the same chain. It represents a significant shift in backbone positions. Enzymatic activity measurements and conformational dynamics studies monitored by NMR and molecular dynamic simulations show that E101G mutation results in a small effect in the structural features of the protein. We hypothesize that these alternative conformations represent samples of the native-state ensemble of EcTRX, specifically the magnitude and location of conformational heterogeneity.
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Escherichia coli/metabolismo , Mutación/genética , Tiorredoxinas/química , Tiorredoxinas/genética , Alquilación , Cristalografía por Rayos X , Cisteína/genética , Espectroscopía de Resonancia Magnética , Simulación de Dinámica Molecular , Proteínas Mutantes/química , Concentración Osmolar , Oxidación-Reducción , Conformación ProteicaRESUMEN
Double electron-electron resonance (DEER) can be used to study the structure of a protein in its native cellular environment. Until now, this has required isolation, inâ vitro labeling, and reintroduction of the protein back into the cells. We describe a completely biosynthetic approach that avoids these steps. It exploits genetically encodable lanthanide-binding tags (LBT) to form self-assembling Gd(III) metal-based spin labels and enables direct in-cell measurements. This approach is demonstrated using a pair of LBTs encoded one at each end of a 3-helix bundle expressed in E.â coli grown on Gd(III) -supplemented medium. DEER measurements directly on these cells produced readily detectable time traces from which the distance between the Gd(III) labels could be determined. This work is the first to use biosynthetically produced self-assembling metal-containing spin labels for non-disruptive in-cell structural measurements.
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Escherichia coli/citología , Gadolinio/química , Marcadores de Spin , Espectroscopía de Resonancia por Spin del Electrón , Escherichia coli/crecimiento & desarrollo , Tamaño de la PartículaRESUMEN
Double stranded RNA (dsRNA) participates in several biological processes, where RNA molecules acquire secondary structure inside the cell through base complementarity. The double stranded RNA binding domain (dsRBD) is one of the main protein folds that is able to recognize and bind to dsRNA regions. The N-terminal dsRBD of DCL1 in Arabidopsis thaliana (DCL1-1), in contrast to other studied dsRBDs, lacks a stable structure, behaving as an intrinsically disordered protein. DCL1-1 does however recognize dsRNA by acquiring a canonical fold in the presence of its substrate. Here we present a detailed modeling and molecular dynamics study of dsRNA recognition by DCL1-1. We found that DCL1-1 forms stable complexes with different RNAs and we characterized the residues involved in binding. Although the domain shows a binding loop substantially shorter than other homologs, it can still interact with the dsRNA and results in bending of the dsRNA A-type helix. Furthermore, we found that R8, a non-conserved residue located in the first dsRNA binding region, recognizes preferentially mismatched base pairs. We discuss our findings in the context of the function of DCL1-1 within the microRNA processing complex.
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Proteínas de Arabidopsis/química , Arabidopsis/enzimología , Proteínas de Ciclo Celular/química , MicroARNs/química , Modelos Químicos , Simulación de Dinámica Molecular , ARN Bicatenario/química , ARN de Planta/química , Ribonucleasa III/química , Proteínas de Arabidopsis/metabolismo , Proteínas de Ciclo Celular/metabolismo , MicroARNs/metabolismo , ARN Bicatenario/metabolismo , ARN de Planta/metabolismo , Ribonucleasa III/metabolismoRESUMEN
A genetically encodable paramagnetic spin-label capable of self-assembly from naturally available components would offer a means for studying the in-cell structure and interactions of a protein by electron paramagnetic resonance (EPR). Here, we demonstrate pulse electron-electron double resonance (DEER) measurements on spin-labels consisting of Mn(II) ions coordinated to a sequence of histidines, so-called His-tags, that are ubiquitously added by genetic engineering to facilitate protein purification. Although the affinity of His-tags for Mn(II) was low (800 µM), Mn(II)-bound His-tags yielded readily detectable DEER time traces even at concentrations expected in cells. We were able to determine accurately the distance between two His-tag Mn(II) spin-labels at the ends of a rigid helical polyproline peptide of known structure, as well as at the ends of a completely cell-synthesized 3-helix bundle. This approach not only greatly simplifies the labeling procedure but also represents a first step towards using self-assembling metal spin-labels for in-cell distance measurements.
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Histidina/química , Manganeso/química , Proteínas/química , Marcadores de Spin , Espectroscopía de Resonancia por Spin del Electrón , Simulación de Dinámica Molecular , Péptidos/química , Estructura Terciaria de ProteínaRESUMEN
DCL1 is the ribonuclease that carries out miRNA biogenesis in plants. The enzyme has two tandem double stranded RNA binding domains (dsRBDs) in its C-terminus. Here we show that the first of these domains binds precursor RNA fragments when isolated and cooperates with the second domain in the recognition of substrate RNA. Remarkably, despite showing RNA binding activity, this domain is intrinsically disordered. We found that it acquires a folded conformation when bound to its substrate, being the first report of a complete dsRBD folding upon binding. The free unfolded form shows tendency to adopt folded conformations, and goes through an unfolded bound state prior to the folding event. The significance of these results is discussed by comparison with the behavior of other dsRBDs.
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Proteínas de Arabidopsis/química , Proteínas de Ciclo Celular/química , Proteínas de Unión al ARN/química , Ribonucleasa III/química , Proteínas de Arabidopsis/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas Intrínsecamente Desordenadas/química , Modelos Moleculares , Unión Proteica , Estabilidad Proteica , Estructura Terciaria de Proteína , ARN/química , ARN/metabolismo , Pliegue del ARN , Proteínas de Unión al ARN/metabolismo , Ribonucleasa III/metabolismoRESUMEN
MicroRNAs have turned out to be important regulators of gene expression. These molecules originate from longer transcripts that are processed by ribonuclease III (RNAse III) enzymes. Dicer proteins are essential RNAse III enzymes that are involved in the generation of microRNAs (miRNAs) and other small RNAs. The correct function of Dicer relies on the participation of accessory dsRNA binding proteins, the exact function of which is not well-understood so far. In plants, the double stranded RNA binding protein Hyponastic Leaves 1 (HYL1) helps Dicer Like protein (DCL1) to achieve an efficient and precise excision of the miRNAs from their primary precursors. Here we dissected the regions of HYL1 that are essential for its function in Arabidopsis thaliana plant model. We generated mutant forms of the protein that retain their structure but affect its RNA-binding properties. The mutant versions of HYL1 were studied both in vitro and in vivo, and we were able to identify essential aminoacids/residues for its activity. Remarkably, mutation and even ablation of one of the purportedly main RNA binding determinants does not give rise to any major disturbances in the function of the protein. We studied the function of the mutant forms in vivo, establishing a direct correlation between affinity for the pri-miRNA precursors and protein activity.
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Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Arabidopsis/crecimiento & desarrollo , MicroARNs/metabolismo , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/genética , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Sitios de Unión , Proteínas de Ciclo Celular/metabolismo , Dicroismo Circular , Regulación de la Expresión Génica de las Plantas , Modelos Moleculares , Mutación , Estructura Secundaria de Proteína , ARN de Planta/metabolismo , Proteínas de Unión al ARN/metabolismo , Ribonucleasa III/metabolismoRESUMEN
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.