Functional Characterization of the Co2+ Transporter AitP in Sinorhizobium meliloti: A New Player in Fe2+ Homeostasis.

Co2+ induces the increase of the labile-Fe pool (LIP) by Fe-S cluster damage, heme synthesis inhibition, and "free" iron import, which affects cell viability. The N2-fixing bacteria, Sinorhizobium meliloti, is a suitable model to determine the roles of Co2+-transporting cation diffusion facilitator exporters (Co-eCDF) in Fe2+ homeostasis because it has a putative member of this subfamily, AitP, and two specific Fe2+-export systems. An insertional mutant of AitP showed Co2+ sensitivity and accumulation, Fe accumulation and hydrogen peroxide sensitivity, but not Fe2+ sensitivity, despite AitP being a bona fide low affinity Fe2+ exporter as demonstrated by the kinetic analyses of Fe2+ uptake into everted membrane vesicles. Suggesting concomitant Fe2+-dependent induced stress, Co2+ sensitivity was increased in strains carrying mutations in AitP and Fe2+ exporters which did not correlate with the Co2+ accumulation. Growth in the presence of sublethal Fe2+ and Co2+ concentrations suggested that free Fe-import might contribute to Co2+ toxicity. Supporting this, Co2+ induced transcription of Fe-import system and genes associated with Fe homeostasis. Analyses of total protoporphyrin content indicates Fe-S cluster attack as the major source for LIP. AitP-mediated Fe2+-export is likely counterbalanced via a nonfutile Fe2+-import pathway. Two lines of evidence support this: (i) an increased hemin uptake in the presence of Co2+ was observed in wild-type (WT) versus AitP mutant, and (ii) hemin reversed the Co2+ sensitivity in the AitP mutant. Thus, the simultaneous detoxification mediated by AitP aids cells to orchestrate an Fe-S cluster salvage response, avoiding the increase in the LIP caused by the disassembly of Fe-S clusters or free iron uptake. IMPORTANCE Cross-talk between iron and cobalt has been long recognized in biological systems. This is due to the capacity of cobalt to interfere with proper iron utilization. Cells can detoxify cobalt by exporting mechanisms involving membrane proteins known as exporters. Highlighting the cross-talk, the capacity of several cobalt exporters to also export iron is emerging. Although biologically less important than Fe2+, Co2+ induces toxicity by promoting intracellular Fe release, which ultimately causes additional toxic effects. In this work, we describe how the rhizobia cells solve this perturbation by clearing Fe through a Co2+ exporter, in order to reestablish intracellular Fe levels by importing nonfree Fe, heme. This piggyback-ride type of transport may aid bacterial cells to survive in free-living conditions where high anthropogenic Co2+ content may be encountered.

Ursodeoxycholic Acid Binds PERK and Ameliorates Neurite Atrophy in a Cellular Model of GM2 Gangliosidosis.

The Unfolded protein response (UPR), triggered by stress in the endoplasmic reticulum (ER), is a key driver of neurodegenerative diseases. GM2 gangliosidosis, which includes Tay-Sachs and Sandhoff disease, is caused by an accumulation of GM2, mainly in the brain, that leads to progressive neurodegeneration. Previously, we demonstrated in a cellular model of GM2 gangliosidosis that PERK, a UPR sensor, contributes to neuronal death. There is currently no approved treatment for these disorders. Chemical chaperones, such as ursodeoxycholic acid (UDCA), have been found to alleviate ER stress in cell and animal models. UDCA's ability to move across the blood-brain barrier makes it interesting as a therapeutic tool. Here, we found that UDCA significantly diminished the neurite atrophy induced by GM2 accumulation in primary neuron cultures. It also decreased the up-regulation of pro-apoptotic CHOP, a downstream PERK-signaling component. To explore its potential mechanisms of action, in vitro kinase assays and crosslinking experiments were performed with different variants of recombinant protein PERK, either in solution or in reconstituted liposomes. The results suggest a direct interaction between UDCA and the cytosolic domain of PERK, which promotes kinase phosphorylation and dimerization.

Functional characterization of the CDF transporter SMc02724 (SmYiiP) in Sinorhizobium meliloti: Roles in manganese homeostasis and nodulation.

In bacteria, membrane transporters of the cation diffusion facilitator (CDF) family participate in Zn(2+), Fe(2+), Mn(2+), Co(2+) and Ni(2+) homeostasis. The functional role during infection processes for several members has been shown to be linked to the specificity of transport. Sinorhizobium meliloti has two homologous CDF genes with unknown transport specificity. Here we evaluate the role played by the CDF SMc02724 (SmYiiP). The deletion mutant strain of SmYiiP (ΔsmyiiP) showed reduced in vitro growth fitness only in the presence of Mn(2+). Incubation of ΔsmyiiP and WT cells with sub-lethal Mn(2+) concentrations resulted in a 2-fold increase of the metal only in the mutant strain. Normal levels of resistance to Mn(2+) were attained by complementation with the gene SMc02724 under regulation of its endogenous promoter. In vitro, liposomes with incorporated heterologously expressed pure protein accumulated several transition metals. However, only the transport rate of Mn(2+) was increased by imposing a transmembrane H(+) gradient. Nodulation assays in alfalfa plants showed that the strain ΔsmyiiP induced a lower number of nodules than in plants infected with the WT strain. Our results indicate that Mn(2+) homeostasis in S. meliloti is required for full infection capacity, or nodule function, and that the specificity of transport in vivo of SmYiiP is narrowed down to Mn(2+) by a mechanism involving the proton motive force.

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 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.

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.

Bacterial transition metal P(1B)-ATPases: transport mechanism and roles in virulence.

P(1B)-type ATPases are polytopic membrane proteins that couple the hydrolysis of ATP to the efflux of cytoplasmic transition metals. This paper reviews recent progress in our understanding of the structure and function of these proteins in bacteria. These are members of the P-type superfamily of transport ATPases. Cu(+)-ATPases are the most frequently observed and best-characterized members of this group of transporters. However, bacterial genomes show diverse arrays of P(1B)-type ATPases with a range of substrates (Cu(+), Zn(2+), Co(2+)). Furthermore, because of the structural similarities among transitions metals, these proteins can also transport nonphysiological substrates (Cd(2+), Pb(2+), Au(+), Ag(+)). P(1B)-type ATPases have six or eight transmembrane segments (TM) with metal coordinating amino acids in three core TMs flanking the cytoplasmic domain responsible for ATP binding and hydrolysis. In addition, regulatory cytoplasmic metal binding domains are present in most P(1B)-type ATPases. Central to the transport mechanism is the binding of the uncomplexed metal to these proteins when cytoplasmic substrates are bound to chaperone and chelating molecules. Metal binding to regulatory sites is through a reversible metal exchange among chaperones and cytoplasmic metal binding domains. In contrast, the chaperone-mediated metal delivery to transport sites appears as a largely irreversible event. P(1B)-ATPases have two overarching physiological functions: to maintain cytoplasmic metal levels and to provide metals for the periplasmic assembly of metalloproteins. Recent studies have shown that both roles are critical for bacterial virulence, since P(1B)-ATPases appear key to overcome high phagosomal metal levels and are required for the assembly of periplasmic and secreted metalloproteins that are essential for survival in extreme oxidant environments.

A novel zinc binding system, ZevAB, is critical for survival of nontypeable Haemophilus influenzae in a murine lung infection model.

Nontypeable Haemophilus influenzae (NTHI) is a Gram-negative bacterial pathogen that causes upper and lower respiratory infections. Factors required for pulmonary infection by NTHI are not well understood. Previously, using high-throughput insertion tracking by deep sequencing (HITS), putative lung colonization factors were identified. Also, previous research indicates that secreted disulfide-dependent factors are important for virulence of H. influenzae. In the present study, HITS data were compared with an informatics-based list of putative substrates of the periplasmic oxidoreductase DsbA to find and characterize secreted virulence factors. This analysis resulted in identification of the "zinc binding essential for virulence" (zev) locus consisting of zevA (HI1249) and zevB (HI1248). NTHI mutants of zevA and zevB grew normally in rich medium but were defective for colonization in a mouse lung model. Mutants also exhibited severe growth defects in medium containing EDTA and were rescued by supplementation with zinc. Additionally, purified recombinant ZevA was found to bind to zinc with high affinity. Together, these data demonstrate that zevAB is a novel virulence factor important for zinc utilization of H. influenzae under conditions where zinc is limiting. Furthermore, evidence presented here suggests that zinc limitation is likely an important mechanism for host defense against pathogens during lung infection.

Distinct functional roles of homologous Cu+ efflux ATPases in Pseudomonas aeruginosa.

In bacteria, most Cu(+) -ATPases confer tolerance to Cu by driving cytoplasmic metal efflux. However, many bacterial genomes contain several genes coding for these enzymes suggesting alternative roles. Pseudomonas aeruginosa has two structurally similar Cu(+) -ATPases, CopA1 and CopA2. Both proteins are essential for virulence. Expressed in response to high Cu, CopA1 maintains the cellular Cu quota and provides tolerance to this metal. CopA2 belongs to a subgroup of ATPases that are expressed in association with cytochrome oxidase subunits. Mutation of copA2 has no effect on Cu toxicity nor intracellular Cu levels; but it leads to higher H(2) O(2) sensitivity and reduced cytochrome oxidase activity. Mutation of both genes does not exacerbate the phenotypes produced by single-gene mutations. CopA1 does not complement the copA2 mutant strain and vice versa, even when promoter regions are exchanged. CopA1 but not CopA2 complements an Escherichia coli strain lacking the endogenous CopA. Nevertheless, transport assays show that both enzymes catalyse cytoplasmic Cu(+) efflux into the periplasm, albeit CopA2 at a significantly lower rate. We hypothesize that their distinct cellular functions could be based on the intrinsic differences in transport kinetic or the likely requirement of periplasmic partner Cu-chaperone proteins specific for each Cu(+) -ATPase.

The transport mechanism of bacterial Cu+-ATPases: distinct efflux rates adapted to different function.

Cu(+)-ATPases play a key role in bacterial Cu(+) homeostasis by participating in Cu(+) detoxification and cuproprotein assembly. Characterization of Archaeoglobus fulgidus CopA, a model protein within the subfamily of P(1B-1) type ATPases, has provided structural and mechanistic details on this group of transporters. Atomic resolution structures of cytoplasmic regulatory metal binding domains (MBDs) and catalytic actuator, phosphorylation, and nucleotide binding domains are available. These, in combination with whole protein structures resulting from cryo-electron microscopy analyses, have enabled the initial modeling of these transporters. Invariant residues in helixes 6, 7 and 8 form two transmembrane metal binding sites (TM-MBSs). These bind Cu(+) with high affinity in a trigonal planar geometry. The cytoplasmic Cu(+) chaperone CopZ transfers the metal directly to the TM-MBSs; however, loading both of the TM-MBSs requires binding of nucleotides to the enzyme. In agreement with the classical transport mechanism of P-type ATPases, occupancy of both transmembrane sites by cytoplasmic Cu(+) is a requirement for enzyme phosphorylation and subsequent transport into the periplasmic or extracellular milieus. Recent transport studies have shown that all Cu(+)-ATPases drive cytoplasmic Cu(+) efflux, albeit with quite different transport rates in tune with their various physiological roles. Archetypical Cu(+)-efflux pumps responsible for Cu(+) tolerance, like the Escherichia coli CopA, have turnover rates ten times higher than those involved in cuproprotein assembly (or alternative functions). This explains the incapability of the latter group to significantly contribute to the metal efflux required for survival in high copper environments.

Secreted amyloid precursor protein and holo-APP bind amyloid beta through distinct domains eliciting different toxic responses on hippocampal neurons.

Amyloid beta (Abeta) is a metabolic product of Abeta precursor protein (APP). Deposition of Abeta in the brain and neuronal degeneration are characteristic hallmarks of Alzheimer's disease (AD). Abeta induces neuronal degeneration, but the mechanism of neurotoxicity remains elusive. Increasing evidence implicates APP as a receptor-like protein for Abeta fibrils (fAbeta). In this study, we present further experimental support for the direct interaction of APP with fAbeta and for its involvement in Abeta neurotoxicity. Using recombinant purified holo-APP (h-APP), we have shown that it directly binds fAbeta. Employing deletion mutant forms of APP, we show that two different sequences are involved in the binding of APP to fAbeta. One sequence in the n-terminus of APP is required for binding of fAbeta to secreted APP (s-APP) but not to h-APP. In addition, the extracellular juxtamembrane Abeta-sequence mediates binding of fAbeta to h-APP but not to s-APP. Deletion of the extracellular juxtamembrane Abeta sequence abolishes abnormal h-APP accumulation and toxicity induced by fAbeta deposition, whereas deletions in the n-terminus of APP do not affect Abeta toxicity. These experiments show that interaction of toxic Abeta species with its membrane-anchored parental protein promotes toxicity in hippocampal neurons, adding further support to an Abeta-receptor-like function of APP directly implicated in neuronal degeneration in AD.

Electrofused giant protoplasts of Saccharomyces cerevisiae as a novel system for electrophysiological studies on membrane proteins.

Giant protoplasts of Saccharomyces cerevisiae of 10-35 microm in diameter were generated by multi-cell electrofusion. Thereby two different preparation strategies were evaluated with a focus on size distribution and "patchability" of electrofused protoplasts. In general, parental protoplasts were suitable for electrofusion 1-12 h after isolation. The electrophysiological properties of electrofused giant protoplasts could be analyzed by the whole-cell patch clamp technique. The area-specific membrane capacitance (0.66+/-0.07 microF/cm(2)) and conductance (23-44 microS/cm(2)) of giant protoplasts were consistent with the corresponding data for parental protoplasts. Measurements with fluorescein-filled patch pipettes allowed to exclude any internal compartmentalisation of giant protoplasts by plasma membranes, since uniform (diffusion-controlled) dye uptake was only observed in the whole-cell configuration, but not in the cell-attached formation. The homogeneous structure of giant protoplasts was further confirmed by the observation that no plasma membrane associated fluorescence was seen in the interior of giant cells after electrofusion of protoplasts expressing the light-activated cation channel Channelrhodopsin-2 (ChR2) linked to yellow fluorescent protein (YFP). Patch clamp analysis of the heterologously expressed ChR2-YFP showed typical blue light dependent, inwardly-directed currents for both electrofused giant and parental protoplasts. Most importantly, neither channel characteristics nor channel expression density was altered by electric field treatment. Summarising, multi-cell electrofusion increases considerably the absolute number of membrane proteins accessible in patch clamp experiments, thus presumably providing a convenient tool for the biophysical investigation of low-signal transporters and channels.

Transition metal transporters in rhizobia: tuning the inorganic micronutrient requirements to different living styles.

A group of bacteria known as rhizobia are key players in symbiotic nitrogen fixation (SNF) in partnership with legumes. After a molecular exchange, the bacteria end surrounded by a plant membrane forming symbiosomes, organelle-like structures, where they differentiate to bacteroids and fix nitrogen. This symbiotic process is highly dependent on dynamic nutrient exchanges between the partners. Among these are transition metals (TM) participating as inorganic and organic cofactors of fundamental enzymes. While the understanding of how plant transporters facilitate TMs to the very near environment of the bacteroid is expanding, our knowledge on how bacteroid transporters integrate to TM homeostasis mechanisms in the plant host is still limited. This is significantly relevant considering the low solubility and scarcity of TMs in soils, and the in crescendo gradient of TM bioavailability rhizobia faces during the infection and bacteroid differentiation processes. In the present work, we review the main metal transporter families found in rhizobia, their role in free-living conditions and, when known, in symbiosis. We focus on discussing those transporters which could play a significant role in TM-dependent biochemical and physiological processes in the bacteroid, thus paving the way towards an optimized SNF.

Defining the Roles of the Cation Diffusion Facilitators in Fe2+/Zn2+ Homeostasis and Establishment of Their Participation in Virulence in Pseudomonas aeruginosa.

Transporters of the cation diffusion facilitator (CDF) family form dimers that export transition metals from the cytosol. The opportunistic pathogen Pseudomonas aeruginosa encodes three homologous CDF genes, czcD (PA0397), aitP (PA1297), and yiiP (PA3963). The three proteins are required for virulence in a plant host model. Disruption of the aitP gene leads to higher Fe2+ and Co2+ sensitivity together with an intracellular accumulation of these ions and to a decreased survival in presence of H2O2. Strains lacking czcD and yiiP showed low Zn2+ sensitivity. However, in iron-rich media and in the presence of Zn2+ these strains secreted higher levels of the iron chelator pyoverdine. Disruption of czcD and yiiP in a non-pyoverdine producer strain and lacking the Zn2+-transporting ATPase, increased the Zn2+ sensitivity and the accumulation of this ion. Most importantly, independent of the pyoverdine production strains lacking CzcD or YiiP, presented lower resistance to imipenem, ciprofloxacin, chloramphenicol, and gentamicin. These observations correlated with a lower survival rate upon EDTA-lysozyme treatment and overexpression of OprN and OprD porins. We hypothesize that while AitP is an Fe2+/Co2+ efflux transporter required for Fe2+ homeostasis, and ultimately redox stress handling, CzcD, and YiiP export Zn2+ to the periplasm for proper Zn2+-dependent signaling regulating outer membrane stability and therefore antibiotic tolerance.

Mechanisms of copper homeostasis in bacteria.

Copper is an important micronutrient required as a redox co-factor in the catalytic centers of enzymes. However, free copper is a potential hazard because of its high chemical reactivity. Consequently, organisms exert a tight control on Cu(+) transport (entry-exit) and traffic through different compartments, ensuring the homeostasis required for cuproprotein synthesis and prevention of toxic effects. Recent studies based on biochemical, bioinformatics, and metalloproteomics approaches, reveal a highly regulated system of transcriptional regulators, soluble chaperones, membrane transporters, and target cuproproteins distributed in the various bacterial compartments. As a result, new questions have emerged regarding the diversity and apparent redundancies of these components, their irregular presence in different organisms, functional interactions, and resulting system architectures.

Periplasmic response upon disruption of transmembrane Cu transport in Pseudomonas aeruginosa.

Pseudomonas aeruginosa, an opportunistic pathogen, has two transmembrane Cu(+) transport ATPases, CopA1 and CopA2. Both proteins export cytoplasmic Cu(+) into the periplasm and mutation of either gene leads to attenuation of virulence. CopA1 is required for maintaining cytoplasmic copper levels, while CopA2 provides copper for cytochrome c oxidase assembly. We hypothesized that transported Cu(+) ions would be directed to their destination via specific periplasmic partners and disruption of transport should affect the periplasmic copper homeostasis. Supporting this, mutation of either ATPase gene led to large increments in periplasmic cuproprotein levels. Toward identifying the proteins participating in this cellular response the periplasmic metalloproteome was resolved in non-denaturing bidimensional gel electrophoresis, followed by X-ray fluorescence visualization and identification by mass-spectrometry. A single spot containing the electron shuttle protein azurin was responsible for the observed increments in cuproprotein contents. In agreement, lack of either Cu(+)-ATPase induced an increase in azu transcription. This is associated with an increase in the expression of anr and rpoS oxidative stress response regulators, rather than cueR, a copper sensing regulator. We propose that azurin overexpression and accumulation in the periplasm is part of the cellular response to cytoplasmic oxidative stress in P. aeruginosa.

Identifying metalloproteins through X-ray fluorescence mapping and mass spectrometry.

Metals are critical and dynamic components of biochemistry. To understand their roles, we greatly need tools to identify the ligands that bind them within the complexity of natural systems. This work describes the development of methods that not only detect and distinguish metals, but also characterize the proteins that bind them. We describe an approach to expose, identify and quantify metalloproteins in complex mixtures by sequential non-denaturing 2D-gel electrophoresis (2D GE)/X-ray Fluorescence (XRF) and tandem mass spectrometry (MS/MS) in the same spot of a sample. We first apply the development of 2D GE/XRF to Shewanella oneidensis MR-1, a well-studied system, and verify our electrophoretic approach. Then, we identified a novel periplasmic zinc protein in Pseudomonas aeruginosa PAO1 through 2D GE/XRF followed by MS/MS. The identity and function of this protein was verified through a gene mutation experiment.