Loss of cytosolic Mg2+ binding sites in the Thermotoga maritima CorA Mg2+ channel is not sufficient for channel opening.

The CorA Mg2+ channel is a homopentamer with five-fold symmetry. Each monomer consists of a large cytoplasmic domain and two transmembrane helices connected via a short periplasmic loop. In the Thermotoga maritima CorA crystal structure, a Mg2+ is bound between D89 of one monomer and D253 of the adjacent monomer (M1 binding site). Release of Mg2+ from these sites has been hypothesized to cause opening of the channel. We generated mutants to disrupt Mg2+ interaction with the M1 site. Crystal structures of the D89K/D253K and D89R/D253R mutants, determined to 3.05 and 3.3 Å, respectively, showed no significant structural differences with the wild type structure despite absence of Mg2+ at the M1 sites. Both mutants still appear to be in the closed state. All three mutant CorA proteins exhibited transport of 63Ni2+, indicating functionality. Thus, absence of Mg2+ from the M1 sites neither causes channel opening nor prevents function. We also provide evidence that the T. maritima CorA is a Mg2+ channel and not a Co2+ channel.

X-ray crystallography and isothermal titration calorimetry studies of the Salmonella zinc transporter ZntB.

The ZntB Zn(2+) efflux system is important for maintenance of Zn(2+) homeostasis in Enterobacteria. We report crystal structures of ZntB cytoplasmic domains from Salmonella enterica serovar Typhimurium (StZntB) in dimeric and physiologically relevant homopentameric forms at 2.3 Å and 3.1 Å resolutions, respectively. The funnel-like structure is similar to that of the homologous Thermotoga maritima CorA Mg(2+) channel and a Vibrio parahaemolyticus ZntB (VpZntB) soluble domain structure. However, the central α7 helix forming the inner wall of the StZntB funnel is oriented perpendicular to the membrane instead of the marked angle seen in CorA or VpZntB. Consequently, the StZntB funnel pore is cylindrical, not tapered, which may represent an "open" form of the ZntB soluble domain. Our crystal structures and isothermal titration calorimetry data indicate that there are three Zn(2+) binding sites in the full-length ZntB, two of which could be involved in Zn(2+) transport.

The phage shock protein PspA facilitates divalent metal transport and is required for virulence of Salmonella enterica sv. Typhimurium.

The phage shock protein (Psp) system is induced by extracytoplasmic stress and thought to be important for the maintenance of proton motive force. We investigated the contribution of PspA to Salmonella virulence. A pspA deletion mutation significantly attenuates the virulence of Salmonella enterica serovar Typhimurium following intraperitoneal inoculation of C3H/HeN (Ity(r) ) mice. PspA was found to be specifically required for virulence in mice expressing the natural resistance-associated macrophage protein 1 (Nramp1) (Slc11a1) divalent metal transporter, which restricts microbial growth by limiting the availability of essential divalent metals within the phagosome. Salmonella competes with Nramp1 by expressing multiple metal uptake systems including the Nramp-homologue MntH, the ABC transporter SitABCD and the ZIP family transporter ZupT. PspA was found to facilitate Mn(2+) transport by MntH and SitABCD, as well as Zn(2+) and Mn(2+) transport by ZupT. In vitro uptake of (54) Mn(2+) by MntH and ZupT was reduced in the absence of PspA. Transport-deficient mutants exhibit reduced viability in the absence of PspA when grown under metal-limited conditions. Moreover, the ZupT transporter is required for Salmonella enterica serovar Typhimurium virulence in Nramp1-expressing mice. We propose that PspA promotes Salmonella virulence by maintaining proton motive force, which is required for the function of multiple transporters mediating bacterial divalent metal acquisition during infection.

Cation selectivity by the CorA Mg2+ channel requires a fully hydrated cation.

The CorA Mg(2+) channel is the primary uptake system in about half of all bacteria and archaea. However, the basis for its Mg(2+) selectivity is unknown. Previous data suggested that CorA binds a fully hydrated Mg(2+) ion, unlike other ion channels. The crystal structure of Thermotoga maritima CorA shows a homopentamer with two transmembrane segments per monomer connected by a short periplasmic loop. This highly conserved loop, (281)EFMPELKWS(289) in Salmonella enterica serovar Typhimurium CorA, is the only portion of the channel outside of the cell, suggesting a role in cation selectivity. Mutation of charged residues in the loop, E281 and K287, to any of several amino acids had little effect, demonstrating that despite conservation electrostatic interactions with these residues are not essential. While mutation of the universally conserved E285 gave a minimally functional channel, E285A and E285K mutants were the most functional, again indicating that the negative charge at this position is not a determining factor. Several mutations at K287 and W288 behaved anomalously in a transport assay. Analysis indicated that mutation of K287 and W288 disrupts cooperative interactions between distinct Mg(2+) binding sites. Overall, these results are not compatible with electrostatic interaction of the Mg(2+) ion with the periplasmic loop. Instead, the loop appears to form an initial binding site for hydrated Mg(2+), not for the dehydrated cation. The loop residues may function to accelerate dehydration of the before entry of Mg(2+) into the pore of the channel.

NinaB is essential for Drosophila vision but induces retinal degeneration in opsin-deficient photoreceptors.

In animals, visual pigments are essential for photoreceptor function and survival. These G-protein-coupled receptors consist of a protein moiety (opsin) and a covalently bound 11-cis-retinylidene chromophore. The chromophore is derived from dietary carotenoids by oxidative cleavage and trans-to-cis isomerization of double bonds. In vertebrates, the necessary chemical transformations are catalyzed by two distinct but structurally related enzymes, the carotenoid oxygenase beta-carotenoid-15,15'-monooxygenase and the retinoid isomerase RPE65 (retinal pigment epithelium protein of 65 kDa). Recently, we provided biochemical evidence that these reactions in insects are catalyzed by a single enzyme family member named NinaB. Here we show that in the fly pathway, carotenoids are mandatory precursors of the chromophore. After chromophore formation, the retinoid-binding protein Pinta acts downstream of NinaB and is required to supply photoreceptors with chromophore. Like ninaE encoding the opsin, ninaB expression is eye-dependent and is activated as a downstream target of the eyeless/pax6 and sine oculis master control genes for eye development. The requirement for coordinated synthesis of chromophore and opsin is evidenced by analysis of ninaE mutants. Retinal degeneration in opsin-deficient photoreceptors is caused by the chromophore and can be prevented by restricting its supply as seen in an opsin and chromophore-deficient double mutant. Thus, our study identifies NinaB as a key component for visual pigment production and provides evidence that chromophore in opsin-deficient photoreceptors can elicit retinal degeneration.

Comparative community genomics in the Dead Sea: an increasingly extreme environment.

Owing to the extreme salinity ( approximately 10 times saltier than the oceans), near toxic magnesium levels (approximately 2.0 M Mg(2+)), the dominance of divalent cations, acidic pH (6.0) and high-absorbed radiation flux rates, the Dead Sea represents a unique and harsh ecosystem. Measures of microbial presence (microscopy, pigments and lipids) indicate that during rare bloom events after exceptionally rainy seasons, the microbial communities can reach high densities. However, most of the time, when the Dead Sea level is declining and halite is precipitating from the water column, it is difficult to reliably measure the presence of microorganisms and their activities. Although a number of halophilic Archaea have been previously isolated from the Dead Sea, polar lipid analyses of biomass collected during Dead Sea blooms suggested that these isolates were not the major components of the microbial community of these blooms. In this study, in an effort to characterize the perennial microbial community of the Dead Sea and compare it with bloom assemblages, we performed metagenomic analyses of concentrated biomass from hundreds of liters of brine and of microbial material from the last massive Dead Sea bloom. The difference between the two conditions was reflected in community composition and diversity, in which the bloom was different and less diverse from the residual brine population. The distributional patterns of microbial genes suggested Dead Sea community trends in mono- and divalent cation metabolisms as well as in transposable elements. This may indicate possible mechanisms and pathways enabling these microbes to survive in such a harsh environment.

Mg(2+)-dependent gating of bacterial MgtE channel underlies Mg(2+) homeostasis.

The MgtE family of Mg(2+) transporters is ubiquitously distributed in all phylogenetic domains. Recent crystal structures of the full-length MgtE and of its cytosolic domain in the presence and absence of Mg(2+) suggested a Mg(2+)-homeostasis mechanism, in which the MgtE cytosolic domain acts as a 'Mg(2+) sensor' to regulate the gating of the ion-conducting pore in response to the intracellular Mg(2+) concentration. However, complementary functional analyses to confirm the proposed model have been lacking. Moreover, the limited resolution of the full-length structure precluded an unambiguous characterization of these regulatory divalent-cation-binding sites. Here, we showed that MgtE is a highly Mg(2+)-selective channel gated by Mg(2+) and elucidated the Mg(2+)-dependent gating mechanism of MgtE, using X-ray crystallographic, genetic, biochemical, and electrophysiological analyses. These structural and functional results have clarified the control of Mg(2+) homeostasis through cooperative Mg(2+) binding to the MgtE cytosolic domain.

The unique nature of mg2+ channels.

Considering the biological abundance and importance of Mg2+, there is a surprising lack of information regarding the proteins that transport Mg2+, the mechanisms by which they do so, and their physiological roles within the cell. The best characterized Mg2+ channel to date is the bacterial protein CorA, present in a wide range of bacterial species. The CorA homolog Mrs2 forms the mitochondrial Mg2+ channel in all eukaryotes. Physiologically, CorA is involved in bacterial pathogenesis, and the Mrs2 eukaryotic homolog is essential for cell survival. A second Mg2+ channel widespread in bacteria is MgtE. Its eukaryotic homologs are the SLC41 family of carriers. Physiological roles for MgtE and its homologs have not been established. Recently, the crystal structures for the bacterial CorA and MgtE Mg2+ channels were solved, the first structures of any divalent cation channel. As befits the unique biological chemistry of Mg2+, both structures are unique, unlike that of any other channel or transporter. Although structurally quite different, both CorA and MgtE appear to be gated in a similar manner through multiple Mg2+ binding sites in the cytosolic domain of the channels. These sites essentially serve as Mg2+ "sensors" of cytosolic Mg2+ concentration. Many questions about these channels remain, however, including the molecular basis of Mg2+ selectivity and the physiological role(s) of their eukaryotic homologs.

The CorA Mg2+ channel is required for the virulence of Salmonella enterica serovar typhimurium.

CorA is the primary Mg(2+) channel in Salmonella enterica serovar Typhimurium. A corA mutant is attenuated in mice and defective for invasion of and replication within epithelial cells. Microarray studies show that several virulence effectors are repressed in a corA mutant strain, which ultimately manifests itself as a decrease in virulence.

Regulation of CorA Mg2+ channel function affects the virulence of Salmonella enterica serovar typhimurium.

The CorA Mg(2+) channel is the primary source of intracellular Mg(2+) in Salmonella enterica serovar Typhimurium. In another study, we found that a strain lacking corA was attenuated in mice and also defective for invasion and replication within Caco-2 epithelial cells (K. M. Papp-Wallace, M. Nartea, D. G. Kehres, S. Porwollik, M. McClelland, S. J. Libby, F. C. Fang, and M. E. Maguire, J. Bacteriol. 190:6517-6523, 2008). Therefore, we further examined Salmonella interaction with Caco-2 epithelial cells. Inhibiting CorA acutely or chronically with a high concentration of a selective inhibitor, Co(III) hexaammine, had no effect on S. enterica serovar Typhimurium invasion of Caco-2 epithelial cells. Complementing the corA mutation with corA from various species rescued the invasion defect only if the complementing allele was functional and if it was evolutionarily similar to S. enterica serovar Typhimurium CorA. One explanation for these results could be that regulation of CorA function is needed for optimal virulence. Further experiments examining corA transcription, CorA protein content, CorA transport, and cell Mg(2+) content indicated that both CorA expression and CorA function are differentially regulated. Moreover, the rates of Mg(2+) influx via CorA are not closely correlated with either protein levels or Mg(2+) content. We conclude that loss of the CorA protein disrupts a regulatory network(s) with the ultimate phenotype of decreased virulence. This conclusion is compatible with the microarray results in our other study, which showed that loss of corA resulted in changes in transcription (and protein expression) in multiple metabolic pathways (Papp-Wallace et al., J. Bacteriol. 190:6517-6523, 2008). Further study of the regulation of CorA expression and function provides an opportunity to dissect the complexity of Mg(2+) homeostasis and its ties to virulence within the bacterium.

Magnesium Transport and Magnesium Homeostasis.

This review reviews the properties and regulation of the Salmonella enterica serovar Typhimurium and Escherichia coli transporters that mediate Mg2+ influx: CorA and the Mgt P-type ATPases. In addition, potential Mg2+ regulation of transcription and translation, largely via the PhoPQ two component system, is discussed. CorA proteins are a unique class of transporters and are widespread in the Bacteria and Archaea, with rather distant but functional homologs in eukaryotes. The Mgt transporters are highly homologous to other P-type ATPases but are more closely related to the eukaryotic H+ and Ca2+ ATPases than to most prokaryotic ATPases. Hundreds of homologs of CorA are currently known from genomic sequencing. In contrast, only when extracellular and possibly intracellular Mg2+ levels fall significantly is the expression of mgtA and mgtB induced. Topology studies using blaM and lacZ fusions initially indicated that the Salmonella serovar Typhimurium CorA contained three transmembrane (TM) segments; however, subsequent data obtained using a variety of approaches showed that the CorA superfamily of proteins have only two TMs at the extreme C terminus. PhoP-PhoQ is a two-component system consisting of PhoQ, the sensor/receptor histidine kinase, and PhoP, the response regulator/transcriptional activator. The expression of both mgtA and mgtCB in either E. coli or Salmonella serovar Typhimurium is markedly induced in a PhoPQ-dependent manner by low concentrations of Mg2+ in the medium. phoP and phoQ form an operon with two promoters in both E. coli and Salmonella serovar Typhimurium.

Bacterial homologs of eukaryotic membrane proteins: the 2-TM-GxN family of Mg(2+) transporters.

Magnesium is essential for all forms of life. It is the cofactor for many enzymes and plays a key role in many biological processes. Thus, the acquisition of Mg(2+) is crucial for cell survival. The best characterized Mg(2+) transporters to date belong to the 2-TM-GxN type family of transporters. The name indicates the two C-terminal transmembrane (TM) domains and a conserved GxN motif present in all members of this family towards the C-terminal end of TM1. In most members of the family, this conserved motif is generally YGMNF. The prototypical member of this family is CorA. Other characterized members of this family include Mrs2p, Alr, Mnr, AtMGT and ZntB. CorA is widely distributed throughout the prokaryotic world. It is the primary Mg(2+) uptake system in most bacteria and many Archaea. A homolog, Mrs2p, is a eukaryotic mitochondrial Mg(2+) channel. The Mrs2p related AtMGT transporters are found in plants and other eukaryotes. Alr1p and Mnr are Mg(2+) transporters found in the plasma membrane of many fungi. ZntB is a bacterial member of the 2-TM-GxN family but mediates efflux of Zn(2+) instead of influx of Mg(2+). The recent crystal structure of a bacterial CorA shows that the structure of this family is unlike that of any other class of transporter or channel currently known.

Magnesium, manganese, and divalent cation transport assays in intact cells.

Protocols are described for the measurement of radioisotopic cation influx and efflux in bacteria using Salmonella as a model system. Methods are discussed for both the use of primary radioisotopes for measurement, e.g., using 54Mn2+ to measure Mn2+ influx, and the use of surrogate radioisotopes when the primary radioisotope is not available, e.g., use of 63Ni2+ or 57Co2+ in place of 28Mg2+ to measure Mg2+ influx. Both vacuum filtration and centrifugation assays are described. In addition, the use and misuse of chelating agents is discussed.

The MgtC virulence factor of Salmonella enterica serovar Typhimurium activates Na(+),K(+)-ATPase.

The mgtC gene of Salmonella enterica serovar Typhimurium encodes a membrane protein of unknown function that is important for full virulence in the mouse. Since mgtC is part of an operon with mgtB which encodes a Mg(2+)-transporting P-type ATPase, MgtC was hypothesized to function in ion transport, possibly in Mg(2+) transport. Consequently, MgtC was expressed in Xenopus laevis oocytes, and its effect on ion transport was evaluated using ion selective electrodes. Oocytes expressing MgtC did not exhibit altered currents or membrane potentials in response to changes in extracellular H(+), Mg(2+), or Ca(2+), thus ruling out a previously postulated function as a Mg(2+)/H(+) antiporter. However, addition of extracellular K(+) markedly hyperpolarized membrane potential instead of the expected depolarization. Addition of ouabain to block the oocyte Na(+),K(+)-ATPase completely prevented hyperpolarization and restored the normal K(+)-induced depolarization response. These results suggested that the Na(+),K(+)-ATPase was constitutively activated in the presence of MgtC resulting in a membrane potential largely dependent on Na(+),K(+)-ATPase. Consistent with the involvement of Na(+),K(+)-ATPase, oocytes expressing MgtC exhibited an increased rate of (86)Rb(+) uptake and had increased intracellular free [K(+)] and decreased free [Na(+)] and ATP. The free concentrations of Mg(2+) and Ca(2+) and cytosolic pH were unchanged, although the total intracellular Ca(2+) content was slightly elevated. These results suggest that the serovar Typhimurium MgtC protein may be involved in regulating membrane potential but does not directly transport Mg(2+) or another ion.

The structure of CorA: a Mg(2+)-selective channel.

The crystal structure of a closed form of the CorA Mg2+ transporter from Thermatoga maritima completes a set of representative structures of transport systems for all of the major biological elements, Mg2+, Ca2+, Na+, K+ and Cl-. The CorA monomer has a C-terminal membrane domain containing two transmembrane segments and a large N-terminal cytoplasmic soluble domain. In the membrane, CorA forms a homopentamer shaped like a funnel. Comparison of the structure of CorA with that of other ion channels and transporters suggests numerous common features, but, as might be predicted from the unique chemistry of the Mg2+ cation, the structure of CorA has several unusual features. Among these are initial binding in the periplasm of a fully hydrated Mg2+ ion; a ring of positive charge external to the ion-conduction pathway at the cytosolic membrane interface; and highly negatively charged helices in the cytosolic domain that appear capable of interacting with the ring of positive charge to facilitate Mg2+ entry. Finally, there are bound Mg2+ ions in the cytosolic domain that are well placed to control the interaction of the ring of positive charge and the negatively charged helices, and thus control Mg2+ entry.

Manganese transport and the role of manganese in virulence.

Two areas of research have recently converged to highlight important roles for Mn(2+) in pathogenesis: the recognition that both bacterial Nramp homologs and members of LraI family of proteins are Mn(2+) transporters. Their mutation is associated with decreased virulence of various bacterial species. Thus, Mn(2+) appears to be essential for bacterial virulence. This review describes what is currently known about Mn(2+) transport in prokaryotes and how prokaryotic Mn(2+) transport is regulated. Some of the phenotypes that arise when microorganisms lack Mn(2+) are then discussed, with an emphasis on those phenotypes involving pathogenesis. The concluding section describes possible enzymatic roles for Mn(2+) that might help explain why Mn(2+) is necessary for virulence.

Magnesium transporters: properties, regulation and structure.

The chemistry of Mg2+ is unique amongst biological cations, and the properties of Mg2+ transport systems reflect this chemistry. Prokaryotes carry three classes of Mg2+ transport systems: CorA, MgtA/B and MgtE. CorA and MgtE are widely distributed in both Eubacteria and Archaea, while the MgtA/B class is found primarily in the Eubacteria. Eukaryotic homologs of CorA, although clearly functional as Mg2+ transporters, have minimal sequence homology and include the Mrs2p mitochondrial Mg2+ channel and the ALR proteins of fungi. MgtE homologs are more recognizable in eukaryotes as the SLC41 class of transporters. The MgtA/B Mg2+ transporters belong to the P-type ATPase superfamily, but mediate Mg2+ influx down its electrochemical gradient rather than against the gradient as with other P-type ATPases. Their physiological role is not clear. CorA is the only Mg2+ transporter whose structure has been solved. It is a homopentamer with two transmembrane domains per monomer, the first of which forms the ion conduction pathway. Mg2+ transport involves first the binding of the fully hydrated cation to an extracellular binding loop connecting the transmembrane domains. Passage through the membrane involves no electrostatic interactions, but two cytosolic domains, one carrying extremely high concentrations of positive charge and the other negative charge appear to help control Mg2+ flux, in concert with an intracellular Mg2+ bound between domains of each monomer. Neither CorA nor MgtE appear to be transcriptionally regulated, implying they are primarily "housekeeping" genes. Nonetheless, mutation of the corA gene in Salmonella enterica serovar Typhimurium leads to attenuation of virulence and other defects, even though the strain carries two additional Mg2+ transporters and the mutant exhibits no Mg2+-dependent growth deficit.

Crystal structure of the CorA Mg2+ transporter.

The magnesium ion, Mg2+, is essential for myriad biochemical processes and remains the only major biological ion whose transport mechanisms remain unknown. The CorA family of magnesium transporters is the primary Mg2+ uptake system of most prokaryotes and a functional homologue of the eukaryotic mitochondrial magnesium transporter. Here we determine crystal structures of the full-length Thermotoga maritima CorA in an apparent closed state and its isolated cytoplasmic domain at 3.9 A and 1.85 A resolution, respectively. The transporter is a funnel-shaped homopentamer with two transmembrane helices per monomer. The channel is formed by an inner group of five helices and putatively gated by bulky hydrophobic residues. The large cytoplasmic domain forms a funnel whose wide mouth points into the cell and whose walls are formed by five long helices that are extensions of the transmembrane helices. The cytoplasmic neck of the pore is surrounded, on the outside of the funnel, by a ring of highly conserved positively charged residues. Two negatively charged helices in the cytoplasmic domain extend back towards the membrane on the outside of the funnel and abut the ring of positive charge. An apparent Mg2+ ion was bound between monomers at a conserved site in the cytoplasmic domain, suggesting a mechanism to link gating of the pore to the intracellular concentration of Mg2+.

The metal permease ZupT from Escherichia coli is a transporter with a broad substrate spectrum.

The Escherichia coli zupT (formerly ygiE) gene encodes a cytoplasmic membrane protein (ZupT) related to members of the eukaryotic ZIP family of divalent metal ion transporters. Previously, ZupT was shown to be responsible for uptake of zinc. In this study, we show that ZupT is a divalent metal cation transporter of broad substrate specificity. An E. coli strain with a disruption in all known iron uptake systems could grow in the presence of chelators only if zupT was expressed. Heterologous expression of Arabidopsis thaliana ZIP1 could also alleviate iron deficiency in this E. coli strain, as could expression of indigenous mntH or feoABC. Transport studies with intact cells showed that ZupT facilitates uptake of 55Fe2+ similarly to uptake of MntH or Feo. Other divalent cations were also taken up by ZupT, as shown using 57Co2+. Expression of zupT rendered E. coli cells hypersensitive to Co2+ and sensitive to Mn2+. ZupT did not appear to be metal regulated: expression of a Phi(zupT-lacZ) operon fusion indicated that zupT is expressed constitutively at a low level.

Transcriptional regulation of sitABCD of Salmonella enterica serovar Typhimurium by MntR and Fur.

Salmonella enterica serovar Typhimurium has two manganese transport systems, MntH and SitABCD. MntH is a bacterial homolog of the eukaryotic natural resistance-associated macrophage protein 1 (Nramp1), and SitABCD is an ABC-type transporter. Previously we showed that mntH is negatively controlled at the transcriptional level by the trans-acting regulatory factors, MntR and Fur. In this study, we examined the transcriptional regulation of sitABCD and compared it to the transcriptional regulation of mntH by constructing lacZ fusions to the promoter regions with and without mutations in putative MntR and/or Fur binding sites. The presence of Mn caused transcriptional repression of the sitABCD and mntH promoters primarily via MntR, but Fur was also capable of some repression in response to Mn. Likewise, Fe in the medium repressed transcription of both sit and mntH primarily via Fur, although MntR was also involved in this response. Transcriptional control by MntR and Fur was disrupted by site-specific mutations in the putative MntR and Fur binding sites, respectively. Transcription of the sit operon was also affected by the oxygen level and growth phase, but the increased expression observed under high oxygen conditions and higher cell densities is consistent with decreased availability of metals required for repression by the metalloregulatory proteins.