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Iron sulfur (Fe/S) proteins are ubiquitous and participate in multiple biological processes, from photosynthesis to DNA repair. Iron and sulfur are highly reactive chemical species, and the mechanisms allowing the multiprotein systems ISC and SUF to assist Fe/S cluster formation in vivo have attracted considerable attention. Here, A-Type components of these systems (ATCs for A-Type Carriers) are studied by phylogenomic and genetic analyses. ATCs that have emerged in the last common ancestor of bacteria were conserved in most bacteria and were acquired by eukaryotes and few archaea via horizontal gene transfers. Many bacteria contain multiple ATCs, as a result of gene duplication and/or horizontal gene transfer events. Based on evolutionary considerations, we could define three subfamilies: ATC-I, -II and -III. Escherichia coli, which has one ATC-I (ErpA) and two ATC-IIs (IscA and SufA), was used as a model to investigate functional redundancy between ATCs in vivo. Genetic analyses revealed that, under aerobiosis, E. coli IscA and SufA are functionally redundant carriers, as both are potentially able to receive an Fe/S cluster from IscU or the SufBCD complex and transfer it to ErpA. In contrast, under anaerobiosis, redundancy occurs between ErpA and IscA, which are both potentially able to receive Fe/S clusters from IscU and transfer them to an apotarget. Our combined phylogenomic and genetic study indicates that ATCs play a crucial role in conveying ready-made Fe/S clusters from components of the biogenesis systems to apotargets. We propose a model wherein the conserved biochemical function of ATCs provides multiple paths for supplying Fe/S clusters to apotargets. This model predicts the occurrence of a dynamic network, the structure and composition of which vary with the growth conditions. As an illustration, we depict three ways for a given protein to be matured, which appears to be dependent on the demand for Fe/S biogenesis.

Control of protein folding is a conserved function in all domains of life. The ubiquitous peptidyl-prolyl cis-trans isomerases (PPIases) catalyze the cis / trans -isomerization of peptidyl-prolyl bonds, often a rate-limiting step for protein folding. PPIases are involved in central processes in eucaryotes but their functions in bacteria are poorly understood. We applied a large-scale two-hybrid screen to identify novel targets of SlyD, a metallochaperone PPIase, from the model organism Escherichia coli , and the important gastric pathogen Helicobacter pylori . SlyD from both organisms interacts with metal-containing proteins including TCA cycle enzymes and with enzymes driving the biogenesis of iron-sulfur (Fe-S) clusters. In H. pylori , a ∆ slyD mutant contains strongly diminished amounts of MisSU, its sole Fe-S biogenesis proteins as well as diminished activity of the Fe-S aconitase. E. coli and H. pylori ∆slyD mutants are less susceptible to ampicillin and gentamicin antibiotics and under microaerobic conditions to the ROS-producing molecule hypochlorite. In addition, the intracellular ATP content and proton motive force are reduced in the H. pylori ∆ slyD mutant, and its gentamicin and hypochlorite susceptibility phenotypes are rescued upon restoration of MisSU expression. Measurement of malate and fumarate in the H. pylori ∆slyD mutant suggests that the two enzymes before and after fumarase in the TCA cycle are affected. We conclude that SlyD regulates the activity of TCA cycle enzymes by controlling the efficiency of their Fe-S cluster formation. This is the first report of the role of a bacterial PPIase in central metabolism and susceptibility to stress and antibiotics. Correct folding of proteins represents a crucial step for their functions. Among the chaperones that control protein folding, the ubiquitous PPIases catalyze the cis / trans -isomerization of peptidyl-prolyl bonds. Only few protein targets of PPIases have been reported in bacteria. To fill this knowledge gap, we performed a large-scale two-hybrid screen to search for targets of the Escherichia coli and Helicobacter pylori SlyD PPIase-metallochaperone. SlyD from both organisms interacts with enzymes (i) containing metal cofactors, (ii) from the central metabolism tricarboxylic acid (TCA) cycle, and (iii) involved in the formation of the essential and ancestral Fe-S cluster cofactor. E. coli and H. pylori ∆slyD mutants present similar phenotypes of diminished susceptibility to antibiotics and to oxidative stress. In H. pylori , measurements of the intracellular ATP content, proton motive force, and activity of TCA cycle proteins suggest that SlyD regulates TCA cycle enzymes by controlling the formation of their indispensable Fe-S clusters.

Metal acquisition is crucial for all cells and for the virulence of many bacterial pathogens. In particular, nickel is a virulence determinant for the human gastric pathogen Helicobacter pylori as it is the cofactor of two enzymes essential for in vivo colonization, urease and a [NiFe] hydrogenase. To import nickel despite its scarcity in the human body, H. pylori requires efficient uptake mechanisms that are only partially defined. Indeed, alternative ways of nickel entry were predicted to exist in addition to the well-described NixA permease. Using a genetic screen, we identified an ABC transporter, that we designated NiuBDE, as a novel H. pylori nickel transport system. Unmarked mutants carrying deletions of nixA, niuD and/or niuB, were constructed and used to measure (i) tolerance to toxic nickel exposure, (ii) intracellular nickel content by ICP-OES, (iii) transport of radioactive nickel and (iv) expression of a reporter gene controlled by nickel concentration. We demonstrated that NiuBDE and NixA function separately and are the sole nickel transporters in H. pylori. NiuBDE, but not NixA, also transports cobalt and bismuth, a metal currently used in H. pylori eradication therapy. Both NiuBDE and NixA participate in nickel-dependent urease activation at pH 5 and survival under acidic conditions mimicking those encountered in the stomach. However, only NiuBDE is able to carry out this activity at neutral pH and is essential for colonization of the mouse stomach. Phylogenomic analyses indicated that both nixA and niuBDE genes have been acquired via horizontal gene transfer by the last common ancestor of the gastric Helicobacter species. Our work highlights the importance of this evolutionary event for the emergence of Helicobacter gastric species that are adapted to the hostile environment of the stomach where the capacity of Helicobacter to import nickel and thereby activate urease needs to be optimized.

Cellular metal homeostasis is a critical process for all organisms, requiring tight regulation. In the major pathogen Helicobacter pylori , the acquisition of nickel is an essential virulence determinant as this metal is a cofactor for the acid-resistance enzyme, urease. Nickel uptake relies on the NixA permease and the NiuBDE ABC transporter. Till now, bacterial metal transporters were reported to be controlled at their transcriptional level. Here we uncovered post-translational regulation of the essential Niu transporter in H . pylori . Indeed, we demonstrate that SlyD, a protein combining peptidyl-prolyl isomerase (PPIase), chaperone, and metal-binding properties, is required for the activity of the Niu transporter. Using two-hybrid assays, we found that SlyD directly interacts with the NiuD permease subunit and identified a motif critical for this contact. Mutants of the different SlyD functional domains were constructed and used to perform in vitro PPIase activity assays and four different in vivo tests measuring nickel intracellular accumulation or transport in H . pylori . In vitro , SlyD PPIase activity is down-regulated by nickel, independently of its C-terminal region reported to bind metals. In vivo , a role of SlyD PPIase function was only revealed upon exposure to high nickel concentrations. Most importantly, the IF chaperone domain of SlyD was shown to be mandatory for Niu activation under all in vivo conditions. These data suggest that SlyD is required for the active functional conformation of the Niu permease and regulates its activity through a novel mechanism implying direct protein interaction, thereby acting as a gatekeeper of nickel uptake. Finally, in agreement with a central role of SlyD, this protein is essential for the colonization of the mouse model by H . pylori .

Metal acquisition and intracellular trafficking are crucial for all cells and metal ions have been recognized as virulence determinants in bacterial pathogens. Virulence of the human gastric pathogen Helicobacter pylori is dependent on nickel, cofactor of two enzymes essential for in vivo colonization, urease and [NiFe] hydrogenase. We found that two small paralogous nickel-binding proteins with high content in Histidine (Hpn and Hpn-2) play a central role in maintaining non-toxic intracellular nickel content and in controlling its intracellular trafficking. Measurements of metal resistance, intracellular nickel contents, urease activities and interactomic analysis were performed. We observed that Hpn acts as a nickel-sequestration protein, while Hpn-2 is not. In vivo, Hpn and Hpn-2 form homo-multimers, interact with each other, Hpn interacts with the UreA urease subunit while Hpn and Hpn-2 interact with the HypAB hydrogenase maturation proteins. In addition, Hpn-2 is directly or indirectly restricting urease activity while Hpn is required for full urease activation. Based on these data, we present a model where Hpn and Hpn-2 participate in a common pathway of controlled nickel transfer to urease. Using bioinformatics and top-down proteomics to identify the predicted proteins, we established that Hpn-2 is only expressed by H. pylori and its closely related species Helicobacter acinonychis. Hpn was detected in every gastric Helicobacter species tested and is absent from the enterohepatic Helicobacter species. Our phylogenomic analysis revealed that Hpn acquisition was concomitant with the specialization of Helicobacter to colonization of the gastric environment and the duplication at the origin of hpn-2 occurred in the common ancestor of H. pylori and H. acinonychis. Finally, Hpn and Hpn-2 were found to be required for colonization of the mouse model by H. pylori. Our data show that during evolution of the Helicobacter genus, acquisition of Hpn and Hpn-2 by gastric Helicobacter species constituted a decisive evolutionary event to allow Helicobacter to colonize the hostile gastric environment, in which no other bacteria persistently thrives. This acquisition was key for the emergence of one of the most successful bacterial pathogens, H. pylori.

Iron/sulfur cluster (ISC)-containing proteins are essential components of cells. In most eukaryotes, Fe/S clusters are synthesized by the mitochondrial ISC machinery, the cytosolic iron/sulfur assembly system, and, in photosynthetic species, a plastid sulfur-mobilization (SUF) system. Here we show that the anaerobic human protozoan parasite Blastocystis, in addition to possessing ISC and iron/sulfur assembly systems, expresses a fused version of the SufC and SufB proteins of prokaryotes that it has acquired by lateral transfer from an archaeon related to the Methanomicrobiales, an important lineage represented in the human gastrointestinal tract microbiome. Although components of the Blastocystis ISC system function within its anaerobic mitochondrion-related organelles and can functionally replace homologues in Trypanosoma brucei , its SufCB protein has similar biochemical properties to its prokaryotic homologues, functions within the parasite’s cytosol, and is up-regulated under oxygen stress. Blastocystis is unique among eukaryotic pathogens in having adapted to its parasitic lifestyle by acquiring a SUF system from nonpathogenic Archaea to synthesize Fe/S clusters under oxygen stress.

Nickel is a virulence determinant for the human gastric pathogen Helicobacter pylori. Indeed, H. pylori possesses two nickel-enzymes that are essential for in vivo colonization, [NiFe] hydrogenase and urease, an abundant virulence factor that contains 24 nickel ions per active complex. Because of these two enzymes, survival of H. pylori relies on an important supply of nickel, implying a tight control of its distribution and storage. In this review, we will present the pathways of activation of the nickel enzymes as well as original mechanisms found in H. pylori for the uptake, trafficking and distribution of nickel between the two enzymes. These include (i) an outer-membrane nickel uptake system, the FrpB4 TonB-dependent transporter, (ii) overlapping protein complexes and interaction networks involved in nickel trafficking and distribution between urease and hydrogenase and, (iii) Helicobacter specific nickel-binding proteins that are involved in nickel storage and can play the role of metallo-chaperones. Finally, we will discuss the implication of the nickel trafficking partners in virulence and propose them as novel therapeutic targets for treatments against H. pylori infection.

Recent structural and biochemical studies have identified a novel control mechanism of gene expression mediated through the secondary channel of RNA Polymerase (RNAP) during transcription initiation. Specifically, the small nucleotide ppGpp, along with DksA, a RNAP secondary channel interacting factor, modifies the kinetics of transcription initiation, resulting in, among other events, down-regulation of ribosomal RNA synthesis and up-regulation of several amino acid biosynthetic and transport genes during nutritional stress. Until now, this mode of regulation of RNAP was primarily associated with ppGpp. Here, we identify TraR, a DksA homolog that mimics ppGpp/DksA effects on RNAP. First, expression of TraR compensates for dksA transcriptional repression and activation activities in vivo. Second, mutagenesis of a conserved amino acid of TraR known to be critical for DksA function abolishes its activity, implying both structural and functional similarity to DksA. Third, unlike DksA, TraR does not require ppGpp for repression of the rrnB P1 promoter in vivo and in vitro or activation of amino acid biosynthesis/transport genes in vivo. Implications for DksA/ppGpp mechanism and roles of TraR in horizontal gene transfer and virulence are discussed.

Metal acquisition and intracellular trafficking are crucial for all cells and metal ions have been recognized as virulence determinants in bacterial pathogens. Virulence of the human gastric pathogen Helicobacter pylori is dependent on nickel, cofactor of two enzymes essential for in vivo colonization, urease and [NiFe] hydrogenase. We found that two small paralogous nickel-binding proteins with high content in Histidine (Hpn and Hpn-2) play a central role in maintaining non-toxic intracellular nickel content and in controlling its intracellular trafficking. Measurements of metal resistance, intracellular nickel contents, urease activities and interactomic analysis were performed. We observed that Hpn acts as a nickel-sequestration protein, while Hpn-2 is not. In vivo, Hpn and Hpn-2 form homo-multimers, interact with each other, Hpn interacts with the UreA urease subunit while Hpn and Hpn-2 interact with the HypAB hydrogenase maturation proteins. In addition, Hpn-2 is directly or indirectly restricting urease activity while Hpn is required for full urease activation. Based on these data, we present a model where Hpn and Hpn-2 participate in a common pathway of controlled nickel transfer to urease. Using bioinformatics and top-down proteomics to identify the predicted proteins, we established that Hpn-2 is only expressed by H. pylori and its closely related species Helicobacter acinonychis. Hpn was detected in every gastric Helicobacter species tested and is absent from the enterohepatic Helicobacter species. Our phylogenomic analysis revealed that Hpn acquisition was concomitant with the specialization of Helicobacter to colonization of the gastric environment and the duplication at the origin of hpn-2 occurred in the common ancestor of H. pylori and H. acinonychis. Finally, Hpn and Hpn-2 were found to be required for colonization of the mouse model by H. pylori. Our data show that during evolution of the Helicobacter genus, acquisition of Hpn and Hpn-2 by gastric Helicobacter species constituted a decisive evolutionary event to allow Helicobacter to colonize the hostile gastric environment, in which no other bacteria persistently thrives. This acquisition was key for the emergence of one of the most successful bacterial pathogens, H. pylori.

Iron sulfur (Fe/S) proteins are ubiquitous and participate in multiple biological processes, from photosynthesis to DNA repair. Iron and sulfur are highly reactive chemical species, and the mechanisms allowing the multiprotein systems ISC and SUF to assist Fe/S cluster formation in vivo have attracted considerable attention. Here, A-Type components of these systems (ATCs for A-Type Carriers) are studied by phylogenomic and genetic analyses. ATCs that have emerged in the last common ancestor of bacteria were conserved in most bacteria and were acquired by eukaryotes and few archaea via horizontal gene transfers. Many bacteria contain multiple ATCs, as a result of gene duplication and/or horizontal gene transfer events. Based on evolutionary considerations, we could define three subfamilies: ATC-I, -II and -III. Escherichia coli, which has one ATC-I (ErpA) and two ATC-IIs (IscA and SufA), was used as a model to investigate functional redundancy between ATCs in vivo. Genetic analyses revealed that, under aerobiosis, E. coli IscA and SufA are functionally redundant carriers, as both are potentially able to receive an Fe/S cluster from IscU or the SufBCD complex and transfer it to ErpA. In contrast, under anaerobiosis, redundancy occurs between ErpA and IscA, which are both potentially able to receive Fe/S clusters from IscU and transfer them to an apotarget. Our combined phylogenomic and genetic study indicates that ATCs play a crucial role in conveying ready-made Fe/S clusters from components of the biogenesis systems to apotargets. We propose a model wherein the conserved biochemical function of ATCs provides multiple paths for supplying Fe/S clusters to apotargets. This model predicts the occurrence of a dynamic network, the structure and composition of which vary with the growth conditions. As an illustration, we depict three ways for a given protein to be matured, which appears to be dependent on the demand for Fe/S biogenesis.