Scs system links copper and redox homeostasis in bacterial pathogens.

The bacterial envelope is an essential compartment involved in metabolism and metabolites transport, virulence, and stress defense. Its roles become more evident when homeostasis is challenged during host-pathogen interactions. In particular, the presence of free radical groups and excess copper in the periplasm causes noxious reactions, such as sulfhydryl group oxidation leading to enzymatic inactivation and protein denaturation. In response to this, canonical and accessory oxidoreductase systems are induced, performing quality control of thiol groups, and therefore contributing to restoring homeostasis and preserving survival under these conditions. Here, we examine recent advances in the characterization of the Dsb-like, Salmonella-specific Scs system. This system includes the ScsC/ScsB pair of Cu+-binding proteins with thiol-oxidoreductase activity, an alternative ScsB-partner, the membrane-linked ScsD, and a likely associated protein, ScsA, with a role in peroxide resistance. We discuss the acquisition of the scsABCD locus and its integration into a global regulatory pathway directing envelope response to Cu stress during the evolution of pathogens that also harbor the canonical Dsb systems. The evidence suggests that the canonical Dsb systems cannot satisfy the extra demands that the host-pathogen interface imposes to preserve functional thiol groups. This resulted in the acquisition of the Scs system by Salmonella. We propose that the ScsABCD complex evolved to connect Cu and redox stress responses in this pathogen as well as in other bacterial pathogens.

Unique underlying principles shaping copper homeostasis networks.

Copper is essential in cells as a cofactor for key redox enzymes. Bacteria have acquired molecular components that sense, uptake, distribute, and expel copper ensuring that cuproenzymes are metallated and steady-state metal levels are maintained. Toward preventing deleterious reactions, proteins bind copper ions with high affinities and transfer the metal via ligand exchange, warranting that copper ions are always complexed. Consequently, the directional copper distribution within cell compartments and across cell membranes requires specific dynamic interactions and metal exchange between cognate holo-apo protein partners. These metal exchange reactions are determined by thermodynamic and kinetics parameters and influenced by mass action. Then, copper distribution can be conceptualized as a molecular system of singular interacting elements that maintain a physiological copper homeostasis. This review focuses on the impact of copper high-affinity binding and exchange reactions on the homeostatic mechanisms, the conceptual models to describe the cell as a homeostatic system, the various molecule functions that contribute to copper homeostasis, and the alternative system architectures responsible for copper homeostasis in model bacteria.

The interplay of the metallosensor CueR with two distinct CopZ chaperones defines copper homeostasis in Pseudomonas aeruginosa.

Copper homeostasis in pathogenic bacteria is critical for cuproprotein assembly and virulence. However, in vivo biochemical analyses of these processes are challenging, which has prevented defining and quantifying the homeostatic interplay between Cu+-sensing transcriptional regulators, chaperones, and sequestering molecules. The cytoplasm of Pseudomonas aeruginosa contains a Cu+-sensing transcriptional regulator, CueR, and two homologous metal chaperones, CopZ1 and CopZ2, forming a unique system for studying Cu+ homeostasis. We found here that both chaperones exchange Cu+, albeit at a slow rate, reaching equilibrium after 3 h, a time much longer than P. aeruginosa duplication time. Therefore, they appeared as two separate cellular Cu+ pools. Although both chaperones transferred Cu+ to CueR in vitro, experiments in vivo indicated that CopZ1 metallates CueR, eliciting the translation of Cu+ efflux transporters involved in metal tolerance. Although this observation was consistent with the relative Cu+ affinities of the three proteins (CopZ1 < CueR < CopZ2), in vitro and in silico analyses also indicated a stronger interaction between CopZ1 and CueR that was independent of Cu+ In contrast, CopZ2 function was defined by its distinctly high abundance during Cu2+ stress. Under resting conditions, CopZ2 remained largely in its apo form. Metal stress quickly induced CopZ2 expression, and its holo form predominated, reaching levels commensurate with the cytoplasmic Cu+ levels. In summary, these results show that CopZ1 acts as chaperone delivering Cu+ to the CueR sensor, whereas CopZ2 functions as a fast-response Cu+-sequestering storage protein. We propose that equivalent proteins likely play similar roles in most bacterial systems.

Medicago truncatula Ferroportin2 mediates iron import into nodule symbiosomes.

Iron is an essential cofactor for symbiotic nitrogen fixation, required by many of the enzymes involved, including signal transduction proteins, O2 homeostasis systems, and nitrogenase itself. Consequently, host plants have developed a transport network to deliver essential iron to nitrogen-fixing nodule cells. Ferroportin family members in model legume Medicago truncatula were identified and their expression was determined. Yeast complementation assays, immunolocalization, characterization of a tnt1 insertional mutant line, and synchrotron-based X-ray fluorescence assays were carried out in the nodule-specific M. truncatula ferroportin Medicago truncatula nodule-specific gene Ferroportin2 (MtFPN2) is an iron-efflux protein. MtFPN2 is located in intracellular membranes in the nodule vasculature and in inner nodule tissues, as well as in the symbiosome membranes in the interzone and early-fixation zone of the nodules. Loss-of-function of MtFPN2 alters iron distribution and speciation in nodules, reducing nitrogenase activity and biomass production. Using promoters with different tissular activity to drive MtFPN2 expression in MtFPN2 mutants, we determined that expression in the inner nodule tissues is sufficient to restore the phenotype, while confining MtFPN2 expression to the vasculature did not improve the mutant phenotype. These data indicate that MtFPN2 plays a primary role in iron delivery to nitrogen-fixing bacteroids in M. truncatula nodules.

An important role for periplasmic storage in Pseudomonas aeruginosa copper homeostasis revealed by a combined experimental and computational modeling study.

Biological systems require precise copper homeostasis enabling metallation of cuproproteins while preventing metal toxicity. In bacteria, sensing, transport, and storage molecules act in coordination to fulfill these roles. However, there is not yet a kinetic schema explaining the system integration. Here, we report a model emerging from experimental and computational approaches that describes the dynamics of copper distribution in Pseudomonas aeruginosa. Based on copper uptake experiments, a minimal kinetic model describes well the copper distribution in the wild-type bacteria but is unable to explain the behavior of the mutant strain lacking CopA1, a key Cu+ efflux ATPase. The model was expanded through an iterative hypothesis-driven approach, arriving to a mechanism that considers the induction of compartmental pools and the parallel function of CopA and Cus efflux systems. Model simulations support the presence of a periplasmic copper storage with a crucial role under dyshomeostasis conditions in P. aeruginosa. Importantly, the model predicts not only the interplay of periplasmic and cytoplasmic pools but also the existence of a threshold in the concentration of external copper beyond which cells lose their ability to control copper levels.

Copper homeostasis networks in the bacterium Pseudomonas aeruginosa.

Bacterial copper (Cu+) homeostasis enables both precise metallation of diverse cuproproteins and control of variable metal levels. To this end, protein networks mobilize Cu+ to cellular targets with remarkable specificity. However, the understanding of these processes is rather fragmented. Here, we use genome-wide transcriptomic analysis by RNA-Seq to characterize the response of Pseudomonas aeruginosa to external 0.5 mm CuSO4, a condition that did not generate pleiotropic effects. Pre-steady-state (5-min) and steady-state (2-h) Cu+ fluxes resulted in distinct transcriptome landscapes. Cells quickly responded to Cu2+ stress by slowing down metabolism. This was restored once steady state was reached. Specific Cu+ homeostasis genes were strongly regulated in both conditions. Our system-wide analysis revealed induction of three Cu+ efflux systems (a P1B-ATPase, a porin, and a resistance-nodulation-division (RND) system) and of a putative Cu+-binding periplasmic chaperone and the unusual presence of two cytoplasmic CopZ proteins. Both CopZ chaperones could bind Cu+ with high affinity. Importantly, novel transmembrane transporters probably mediating Cu+ influx were among those largely repressed upon Cu+ stress. Compartmental Cu+ levels appear independently controlled; the cytoplasmic Cu+ sensor CueR controls cytoplasmic chaperones and plasma membrane transporters, whereas CopR/S responds to periplasmic Cu+ Analysis of ΔcopR and ΔcueR mutant strains revealed a CopR regulon composed of genes involved in periplasmic Cu+ homeostasis and its putative DNA recognition sequence. In conclusion, our study establishes a system-wide model of a network of sensors/regulators, soluble chaperones, and influx/efflux transporters that control the Cu+ levels in P. aeruginosa compartments.

Fine-tuning of Substrate Affinity Leads to Alternative Roles of Mycobacterium tuberculosis Fe2+-ATPases.

Little is known about iron efflux transporters within bacterial systems. Recently, the participation of Bacillus subtilis PfeT, a P1B4-ATPase, in cytoplasmic Fe(2+) efflux has been proposed. We report here the distinct roles of mycobacterial P1B4-ATPases in the homeostasis of Co(2+) and Fe(2+) Mutation of Mycobacterium smegmatis ctpJ affects the homeostasis of both ions. Alternatively, an M. tuberculosis ctpJ mutant is more sensitive to Co(2+) than Fe(2+), whereas mutation of the homologous M. tuberculosis ctpD leads to Fe(2+) sensitivity but no alterations in Co(2+) homeostasis. In vitro, the three enzymes are activated by both Fe(2+) and Co(2+) and bind 1 eq of either ion at their transport site. However, equilibrium binding affinities and activity kinetics show that M. tuberculosis CtpD has higher affinity for Fe(2+) and twice the Fe(2+)-stimulated activity than the CtpJs. These parameters are paralleled by a lower activation and affinity for Co(2+) Analysis of Fe(2+) and Co(2+) binding to CtpD by x-ray absorption spectroscopy shows that both ions are five- to six-coordinate, constrained within oxygen/nitrogen environments with similar geometries. Mutagenesis studies suggest the involvement of invariant Ser, His, and Glu residues in metal coordination. Interestingly, replacement of the conserved Cys at the metal binding pocket leads to a large reduction in Fe(2+) but not Co(2+) binding affinity. We propose that CtpJ ATPases participate in the control of steady state Fe(2+) levels. CtpD, required for M. tuberculosis virulence, is a high affinity Fe(2+) transporter involved in the rapid response to iron dyshomeostasis generated upon redox stress.

The Listeria monocytogenes Fur-regulated virulence protein FrvA is an Fe(II) efflux P1B4 -type ATPase.

Listeria monocytogenes FrvA (Lmo0641) is critical for virulence in the mouse model and is an ortholog of the Bacillus subtilis Fur- and PerR-regulated Fe(II) efflux P1B4 -type ATPase PfeT. Previously, FrvA was suggested to protect against heme toxicity. Here, we demonstrate that an frvA mutant is sensitive to iron intoxication, but not to other metals. Expression of frvA is induced by high iron and this induction requires Fur. FrvA functions in vitro as a divalent cation specific ATPase most strongly activated by ferrous iron. When expressed in B. subtilis, FrvA increases resistance to iron both in wild-type and in a pfeT null strain. FrvA is a high affinity Fe(II) exporter and its induction imposes severe iron limitation in B. subtilis resulting in derepression of both Fur- and PerR-regulated genes. FrvA also recognizes Co(II) and Zn(II) as substrates and can complement B. subtilis strains defective in the endogenous export systems for these cations. Building on these results, we conclude that FrvA functions in the efflux of Fe(II), and not heme during listerial infection.

Evolution of a plant-specific copper chaperone family for chloroplast copper homeostasis.

Metallochaperones traffic copper (Cu(+)) from its point of entry at the plasma membrane to its destination. In plants, one destination is the chloroplast, which houses plastocyanin, a Cu-dependent electron transfer protein involved in photosynthesis. We present a previously unidentified Cu(+) chaperone that evolved early in the plant lineage by an alternative-splicing event of the pre-mRNA encoding the chloroplast P-type ATPase in Arabidopsis 1 (PAA1). In several land plants, recent duplication events created a separate chaperone-encoding gene coincident with loss of alternative splicing. The plant-specific Cu(+) chaperone delivers Cu(+) with specificity for PAA1, which is flipped in the envelope relative to prototypical bacterial ATPases, compatible with a role in Cu(+) import into the stroma and consistent with the canonical catalytic mechanism of these enzymes. The ubiquity of the chaperone suggests conservation of this Cu(+)-delivery mechanism and provides a unique snapshot into the evolution of a Cu(+) distribution pathway. We also provide evidence for an interaction between PAA2, the Cu(+)-ATPase in thylakoids, and the Cu(+)-chaperone for Cu/Zn superoxide dismutase (CCS), uncovering a Cu(+) network that has evolved to fine-tune Cu(+) distribution.

PfeT, a P1B4 -type ATPase, effluxes ferrous iron and protects Bacillus subtilis against iron intoxication.

Iron is an essential element for nearly all cells and limited iron availability often restricts growth. However, excess iron can also be deleterious, particularly when cells expressing high affinity iron uptake systems transition to iron rich environments. Bacillus subtilis expresses numerous iron importers, but iron efflux has not been reported. Here, we describe the B. subtilis PfeT protein (formerly YkvW/ZosA) as a P1B4 -type ATPase in the PerR regulon that serves as an Fe(II) efflux pump and protects cells against iron intoxication. Iron and manganese homeostasis in B. subtilis are closely intertwined: a pfeT mutant is iron sensitive, and this sensitivity can be suppressed by low levels of Mn(II). Conversely, a pfeT mutant is more resistant to Mn(II) overload. In vitro, the PfeT ATPase is activated by both Fe(II) and Co(II), although only Fe(II) efflux is physiologically relevant in wild-type cells, and null mutants accumulate elevated levels of intracellular iron. Genetic studies indicate that PfeT together with the ferric uptake repressor (Fur) cooperate to prevent iron intoxication, with iron sequestration by the MrgA mini-ferritin playing a secondary role. Protection against iron toxicity may also be a key role for related P1B4 -type ATPases previously implicated in bacterial pathogenesis.

Mechanism of ATPase-mediated Cu+ export and delivery to periplasmic chaperones: the interaction of Escherichia coli CopA and CusF.

Cellular copper homeostasis requires transmembrane transport and compartmental trafficking while maintaining the cell essentially free of uncomplexed Cu(2+/+). In bacteria, soluble cytoplasmic and periplasmic chaperones bind and deliver Cu(+) to target transporters or metalloenzymes. Transmembrane Cu(+)-ATPases couple the hydrolysis of ATP to the efflux of cytoplasmic Cu(+). Cytosolic Cu(+) chaperones (CopZ) interact with a structural platform in Cu(+)-ATPases (CopA) and deliver copper into the ion permeation path. CusF is a periplasmic Cu(+) chaperone that supplies Cu(+) to the CusCBA system for efflux to the extracellular milieu. In this report, using Escherichia coli CopA and CusF, direct Cu(+) transfer from the ATPase to the periplasmic chaperone was observed. This required the specific interaction of the Cu(+)-bound form of CopA with apo-CusF for subsequent metal transfer upon ATP hydrolysis. As expected, the reverse Cu(+) transfer from CusF to CopA was not observed. Mutation of CopA extracellular loops or the electropositive surface of CusF led to a decrease in Cu(+) transfer efficiency. On the other hand, mutation of Met and Glu residues proposed to be part of the metal exit site in the ATPase yielded enzymes with lower turnover rates, although Cu(+) transfer was minimally affected. These results show how soluble chaperones obtain Cu(+) from transmembrane transporters. Furthermore, by explaining the movement of Cu(+) from the cytoplasmic pool to the extracellular milieu, these data support a mechanism by which cytoplasmic Cu(+) can be precisely directed to periplasmic targets via specific transporter-chaperone interactions.

Differential roles for the Co(2+) /Ni(2+) transporting ATPases, CtpD and CtpJ, in Mycobacterium tuberculosis virulence.

The genome of Mycobacterium tuberculosis encodes two paralogous P1 B 4 -ATPases, CtpD (Rv1469) and CtpJ (Rv3743). Both proteins showed ATPase activation by Co(2+) and Ni(2+) , and both appear to be required for metal efflux from the cell. However, using a combination of biochemical and genetic studies we found that these proteins play non-redundant roles in virulence and metal efflux. CtpJ expression is induced by Co(2+) and this protein possesses a relatively high turnover rate. A ctpJ deletion mutant accumulated Co(2+) , indicating that this ATPase controls cytoplasmic metal levels. In contrast, CtpD expression is induced by redox stressors and this protein displays a relatively low turnover rate. A ctpD mutant failed to accumulate metal, suggesting an alternative cellular function. ctpD is cotranscribed with two thioredoxin genes trxA (Rv1470), trxB (Rv1471), and an enoyl-coA hydratase (Rv1472), indicating a possible role for CtpD in the metallation of these redox-active proteins. Supporting this, in vitro metal binding assays showed that TrxA binds Co(2+) and Ni(2+) . Mutation of ctpD, but not ctpJ, reduced bacterial fitness in the mouse lung, suggesting that redox maintenance, but not Co(2+) accumulation, is important for growth in vivo.

A novel antimycobacterial compound acts as an intracellular iron chelator.

Efficient iron acquisition is crucial for the pathogenesis of Mycobacterium tuberculosis. Mycobacterial iron uptake and metabolism are therefore attractive targets for antitubercular drug development. Resistance mutations against a novel pyrazolopyrimidinone compound (PZP) that is active against M. tuberculosis have been identified within the gene cluster encoding the ESX-3 type VII secretion system. ESX-3 is required for mycobacterial iron acquisition through the mycobactin siderophore pathway, which could indicate that PZP restricts mycobacterial growth by targeting ESX-3 and thus iron uptake. Surprisingly, we show that ESX-3 is not the cellular target of the compound. We demonstrate that PZP indeed targets iron metabolism; however, we found that instead of inhibiting uptake of iron, PZP acts as an iron chelator, and we present evidence that the compound restricts mycobacterial growth by chelating intrabacterial iron. Thus, we have unraveled the unexpected mechanism of a novel antimycobacterial compound.

The mechanism of Cu+ transport ATPases: interaction with CU+ chaperones and the role of transient metal-binding sites.

Cu(+)-ATPases are membrane proteins that couple the hydrolysis of ATP to the efflux of cytoplasmic Cu(+). In cells, soluble chaperone proteins bind and distribute cytoplasmic Cu(+), delivering the ion to the transmembrane metal-binding sites in the ATPase. The structure of Legionella pneumophila Cu(+)-ATPase (Gourdon, P., Liu, X. Y., Skjørringe, T., Morth, J. P., Møller, L. B., Pedersen, B. P., and Nissen, P. (2011) Nature 475, 59-64) shows that a kinked transmembrane segment forms a "platform" exposed to the cytoplasm. In addition, neighboring invariant Met, Asp, and Glu are located at the "entrance" of the ion path. Mutations of amino acids in these regions of the Archaeoglobus fulgidus Cu(+)-ATPase CopA do not affect ATPase activity in the presence of Cu(+) free in solution. However, Cu(+) bound to the corresponding chaperone (CopZ) could not activate the mutated ATPases, and in parallel experiments, CopZ was unable to transfer Cu(+) to CopA. Furthermore, mutation of a specific electronegative patch on the CopZ surface abolishes the ATPase activation and Cu(+) transference, indicating that the region is required for the CopZ-CopA interaction. Moreover, the data suggest that the interaction is driven by the complementation of the electropositive platform in the ATPase and the electronegative Cu(+) chaperone. This docking likely places the Cu(+) proximal to the conserved carboxyl and thiol groups in the entrance site that induce metal release from the chaperone via ligand exchange. The initial interaction of Cu(+) with the pump is transient because Cu(+) is transferred from the entrance site to transmembrane metal-binding sites involved in transmembrane translocation.

A novel P(1B)-type Mn2+-transporting ATPase is required for secreted protein metallation in mycobacteria.

Transition metals are central for bacterial virulence and host defense. P(1B)-ATPases are responsible for cytoplasmic metal efflux and play roles either in limiting cytosolic metal concentrations or in the maturation of secreted metalloproteins. The P(1B)-ATPase, CtpC, is required for Mycobacterium tuberculosis survival in a mouse model (Sassetti, C. M., and Rubin, E. J. (2003) Genetic requirements for mycobacterial survival during infection. Proc. Natl. Acad. Sci. U.S.A. 100, 12989-12994). CtpC prevents Zn(2+) toxicity, suggesting a role in Zn(2+) export from the cytosol (Botella, H., Peyron, P., Levillain, F., Poincloux, R., Poquet, Y., Brandli, I., Wang, C., Tailleux, L., Tilleul, S., Charriere, G. M., Waddell, S. J., Foti, M., Lugo-Villarino, G., Gao, Q., Maridonneau-Parini, I., Butcher, P. D., Castagnoli, P. R., Gicquel, B., de Chastellièr, C., and Neyrolles, O. (2011) Mycobacterial P1-type ATPases mediate resistance to zinc poisoning in human macrophages. Cell Host Microbe 10, 248-259). However, key metal-coordinating residues and the overall structure of CtpC are distinct from Zn(2+)-ATPases. We found that isolated CtpC has metal-dependent ATPase activity with a strong preference for Mn(2+) over Zn(2+). In vivo, CtpC is unable to complement Escherichia coli lacking a functional Zn(2+)-ATPase. Deletion of M. tuberculosis or Mycobacterium smegmatis ctpC leads to cytosolic Mn(2+) accumulation but no alterations in other metals levels. Whereas ctpC-deficient M. tuberculosis is sensitive to extracellular Zn(2+), the M. smegmatis mutant is not. Both ctpC mutants are sensitive to oxidative stress, which might explain the Zn(2+)-sensitive phenotype of the M. tuberculosis ctpC mutant. CtpC is a high affinity/slow turnover ATPase, suggesting a role in protein metallation. Consistent with this hypothesis, mutation of CtpC leads to a decrease of Mn(2+) bound to secreted proteins and of the activity of secreted Fe/Mn-superoxide dismutase, particularly in M. smegmatis. Alterations in the assembly of metalloenzymes involved in redox stress response might explain the sensitivity of M. tuberculosis ctpC mutants to oxidative stress and growth and persistence defects in mice infection models.

Functional diversity of five homologous Cu+-ATPases present in Sinorhizobium meliloti.

Copper is an important element in host-microbe interactions, acting both as a catalyst in enzymes and as a potential toxin. Cu(+)-ATPases drive cytoplasmic Cu(+) efflux and protect bacteria against metal overload. Many pathogenic and symbiotic bacteria contain multiple Cu(+)-ATPase genes within particular genetic environments, suggesting alternative roles for each resulting protein. This hypothesis was tested by characterizing five homologous Cu(+)-ATPases present in the symbiotic organism Sinorhizobium meliloti. Mutation of each gene led to different phenotypes and abnormal nodule development in the alfalfa host. Distinct responses were detected in free-living S. meliloti mutant strains exposed to metal and redox stresses. Differential gene expression was detected under Cu(+), oxygen or nitrosative stress. These observations suggest that CopA1a maintains the cytoplasmic Cu(+) quota and its expression is controlled by Cu(+) levels. CopA1b is also regulated by Cu(+) concentrations and is required during symbiosis for bacteroid maturation. CopA2-like proteins, FixI1 and FixI2, are necessary for the assembly of two different cytochrome c oxidases at different stages of bacterial life. CopA3 is a phylogenetically distinct Cu(+)-ATPase that does not contribute to Cu(+) tolerance. It is regulated by redox stress and required during symbiosis. We postulated a model where non-redundant homologous Cu(+)-ATPases, operating under distinct regulation, transport Cu(+) to different target proteins.

Metal transport across biomembranes: emerging models for a distinct chemistry.

Transition metals are essential components of important biomolecules, and their homeostasis is central to many life processes. Transmembrane transporters are key elements controlling the distribution of metals in various compartments. However, due to their chemical properties, transition elements require transporters with different structural-functional characteristics from those of alkali and alkali earth ions. Emerging structural information and functional studies have revealed distinctive features of metal transport. Among these are the relevance of multifaceted events involving metal transfer among participating proteins, the importance of coordination geometry at transmembrane transport sites, and the presence of the largely irreversible steps associated with vectorial transport. Here, we discuss how these characteristics shape novel transition metal ion transport models.

A tetrahedral coordination of Zinc during transmembrane transport by P-type Zn(2+)-ATPases.

Zn(2+) is an essential transition metal required in trace amounts by all living organisms. However, metal excess is cytotoxic and leads to cell damage. Cells rely on transmembrane transporters, with the assistance of other proteins, to establish and maintain Zn(2+) homeostasis. Metal coordination during transport is key to specific transport and unidirectional translocation without the backward release of free metal. The coordination details of Zn(2+) at the transmembrane metal binding site responsible for transport have now been established. Escherichia coli ZntA is a well-characterized Zn(2+)-ATPase responsible for intracellular Zn(2+) efflux. A truncated form of the protein lacking regulatory metal sites and retaining the transport site was constructed. Metrical parameters of the metal-ligand coordination geometry for the zinc bound isolated form were characterized using x-ray absorption spectroscopy (XAS). Our data support a nearest neighbor ligand environment of (O/N)(2)S(2) that is compatible with the proposed invariant metal coordinating residues present in the transmembrane region. This ligand identification and the calculated bond lengths support a tetrahedral coordination geometry for Zn(2+) bound to the TM-MBS of P-type ATPase transporters.

Role in metal homeostasis of CtpD, a Co²âº transporting P(1B4)-ATPase of Mycobacterium smegmatis.

Genetic studies in the tuberculosis mouse model have suggested that mycobacterial metal efflux systems, such as the P(1B4)-ATPase CtpD, are important for pathogenesis. The specificity for substrate metals largely determines the function of these ATPases; however, various substrates have been reported for bacterial and plant P(1B4)-ATPases leaving their function uncertain. Here we describe the functional role of the CtpD protein of Mycobacterium smegmatis. An M. smegmatis mutant strain lacking the ctpD gene was hypersensitive to Co²âº and Ni²âº and accumulated these metals in the cytoplasm. ctpD transcription was induced by both Co²âº and superoxide stress. Biochemical characterization of heterologously expressed, affinity-purified CtpD showed that this ATPase is activated by Co²âº, Ni²âº and to a lesser extend Zn²âº (20% of maximum activity). The protein was also able to bind one Co²âº, Ni²âº or Zn²âº to its transmembrane transport site. These observations indicate that CtpD is important for Co²âº and Ni²âº homeostasis in M. smegmatis, and that M. tuberculosis CtpD orthologue could be involved in metal detoxification and resisting cellular oxidative stress by modulating the intracellular concentration of these metals.

Evolution and diversity of periplasmic proteins involved in copper homeostasis in gamma proteobacteria.

BACKGROUND: Different systems contributing to copper homeostasis in bacteria have been described in recent years involving periplasmic and transport proteins that provide resistance via metal efflux to the extracellular media (CopA/Cue, Cus, Cut, and Pco). The participation of these proteins in the assembly of membrane, periplasmic and secreted cuproproteins has also been postulated. The integration and interrelation of these systems and their apparent redundancies are less clear since they have been studied in alternative systems. Based on the idea that cellular copper is not free but rather it is transferred via protein-protein interactions, we hypothesized that systems would coevolve and be constituted by set numbers of essential components. RESULTS: By the use of a phylogenomic approach we identified the distribution of 14 proteins previously characterized as members of homeostasis systems in the genomes of 268 gamma proteobacteria. Only 3% of the genomes presented the complete systems and 5% of them, all intracellular parasites, lacked the 14 genes. Surprisingly, copper homeostatic pathways did not behave as evolutionary units with particular species assembling different combinations of basic functions. The most frequent functions, and probably because of its distribution the most vital, were copper extrusion from the cytoplasm to the periplasm performed by CopA and copper export from the cytoplasm to the extracellular space performed by CusC, which along with the remaining 12 proteins, assemble in nine different functional repertoires. CONCLUSIONS: These observations suggest complex evolutionary dynamics and still unexplored interactions to achieve copper homeostasis, challenging some of the molecular transport mechanism proposed for these systems.