The CorA Mg2+ transporter does not transport Fe2+

corA encodes the constitutively expressed primary Mg2+ uptake system of most eubacteria and many archaea. Recently, a mutation in corA was reported to make Salmonella enterica serovar Typhimurium markedly resistant to Fe2+-mediated toxicity. Mechanistically, this was hypothesized to be from an ability of CorA to mediate the influx of Fe2+. Consequently, we directly examined Fe2+ transport and toxicity in wild-type versus corA cells. As determined by direct transport assay, CorA cannot transport Fe2+ and Fe2+ does not potently inhibit CorA transport of 63Ni2+. Mg2+ can, relatively weakly, inhibit Fe2+ uptake, but inhibition is not dependent on the presence of a functional corA allele. Although excess Fe2+ was slightly toxic to S. enterica serovar Typhimurium, we were unable to elicit a significant differential sensitivity in a wild-type versus a corA strain. We conclude that CorA does not transport Fe2+ and that the relationship, if any, between iron toxicity and corA is indirect.     

Distribution of the CorA Mg2+ transport system in gram-negative bacteria.

The CorA Mg2+ transport system is the dominant constitutive uptake mechanism in Salmonella typhimurium and Escherichia coli. Southern blot hybridization and PCR techniques were used to screen a panel of 18 additional gram-negative bacterial species for corA homologs. Virtually all strains tested positive for the presence of corA. Thus, corA appears to be ubiquitous within gram-negative bacteria and is likely their major Mg2+ influx system.     

Magnesium transport in Salmonella typhimurium: 28Mg2+ transport by the CorA, MgtA, and MgtB systems.

Three loci in Salmonella typhimurium (corA, mgtA, and mgtB) code for components of distinct Mg2+ transport systems (S. P. Hmiel, M. D. Snavely, J. B. Florer, M. E. Maguire, and C. G. Miller, J. Bacteriol. 171:4742-4751, 1989). Strains carrying one wild-type and two mutant alleles of the three loci were constructed to study the kinetics and specificity of ion transport of each system in isolation. The transport systems had different Km and Vmax values for Mg2+ uptake, and each was inhibited by other divalent cations in a distinct rank order of potency: for CorA, Mg2+ greater than Mn2+ greater than Co2+ greater than Ni2+ greater than Ca2+; for MgtA, Zn2+ greater than or equal to Mg2+ greater than Ni2+ approximately Co2+ greater than Ca2+; and for MgtB, Mg2+ approximately Ni2+ approximately Ni2+ greater than Mn2+ much greater than Ca2+. Other differences among the three systems were apparent. The CorA transport system functioned as a Mg2+-Mg2+ exchange system, mediating both efflux and influx of Mg2+. Neither the MgtA nor the MgtB system could mediate Mg2+ efflux. Transport via the MgtB system was very temperature sensitive; Mg2+ was transported at 37 degrees C but not at 20 degrees C. The MgtA and the MgtB transport systems were found to be regulated by the extracellular concentration of Mg2+.     

Magnesium transport in Salmonella typhimurium: the influence of new mutations conferring Co2+ resistance on the CorA Mg2+ transport system

The CorA Mg2+ transport system of Salmonella typhimurium mediates both influx and efflux of Mg2+. Mutations at the corA locus (83.5 min) confer resistance to Co2+. Using transposon mutagenesis, three additional Co2+ resistance loci (corB, corC, and corD) were found and mapped to 55, 15, and 3min, respectively, on the S. typhimurium chromosome. No mutations corresponding to the reported corB locus at 95 min in Escherichia coli were obtained. The corB, corC, and corD mutations confer levels of Co2+ resistance intermediate between those of the wild-type and corA mutations. Isogenic strains were constructed containing combinations of transposon insertion mutations in each of the three Co(2+)-resistance loci to assess their influence on the CorA Mg2+ transport system. The Vmax and Km values for 28Mg2+ or for 57Co2+ and 63Ni2+ influx, analogues of Mg2+ transported by the CorA system, were changed less than twofold compared with the wild-type values, regardless of the mutation(s) present. However, while efflux of 28Mg2+ through the CorA system was decreased threefold in strains carrying one or two mutant alleles among corB, corC, or corD, efflux was completely abolished in either a corA or a corBCD strain. Thus, although the corA gene product is necessary and sufficient to mediate Mg2+ influx, Mg2+ efflux requires the presence of a wild-type allele of at least one of the corB, corC or corD loci.     

CorA affects tolerance of Escherichia coli and Salmonella enterica serovar Typhimurium to the lactoperoxidase enzyme system but not to other forms of oxidative stress

The enzyme lactoperoxidase is part of the innate immune system in vertebrates and owes its antimicrobial activity to the formation of oxidative reaction products from various substrates. In a previous study, we have reported that, with thiocyanate as a substrate, the lactoperoxidase system elicits a distinct stress response in Escherichia coli MG1655. This response is different from but partly overlapping with the stress responses to hydrogen peroxide and to superoxide. In the current work, we constructed knockouts in 10 lactoperoxidase system-inducible genes to investigate their role in the tolerance of E. coli MG1655 to this antimicrobial system. Five mutations resulted in a slightly increased sensitivity, but one mutation (corA) caused hypersensitivity to the lactoperoxidase system. This hypersensitive phenotype was specific to the lactoperoxidase system, since neither the sensitivity to hydrogen peroxide nor to the superoxide generator plumbagin was affected in the corA mutant. Salmonella enterica serovar Typhimurium corA had a similar phenotype. Although corA encodes an Mg2+ transporter and at least three other inducible open reading frames belonged to the Mg2+ regulon, repression of the Mg stimulon by Mg2+ did not change the lactoperoxidase sensitivity of either the wild-type or corA mutant. Prior exposure to 0.3 mM Ni2+, which is also transported by CorA, strongly sensitized MG1655 but not the corA mutant to the lactoperoxidase system. Furthermore, this Ni2+-dependent sensitization was suppressed by the CorA-specific inhibitor Co(III) hexaammine. These results indicate that CorA affects the lactoperoxidase sensitivity of E. coli by modulating the cytoplasmic concentrations of transition metals that enhance the toxicity of the lactoperoxidase system.     

Cation hexaammines are selective and potent inhibitors of the CorA magnesium transport system

Cation hexaammines and related compounds are chemically stable analogs of the hydrated form of cations, particularly Mg(2+). We tested the ability of several of these compounds to inhibit transport by the CorA or MgtB Mg(2+) transport systems or the PhoQ receptor kinase for Mg(2+) in Salmonella typhimurium. Cobalt(III)-, ruthenium(II)-, and ruthenium(III)-hexaammines were potent inhibitors of CorA-mediated influx. Cobalt(III)- and ruthenium(III)chloropentaammines were slightly less potent inhibitors of CorA. The compounds inhibited uptake by the bacterial S. typhimurium CorA and by the archaeal Methanococcus jannaschii CorA, which bear only 12% identity in the extracellular periplasmic domain. Cation hexaammines also inhibited growth of S. typhimurium strains dependent on CorA for Mg(2+) uptake but not of isogenic strains carrying a second Mg(2+) uptake system. In contrast, hexacyano-cobaltate(III) and ruthenate(II)- and nickel(II)hexaammine had little effect on uptake. The inhibition by the cation hexaammines was selective for CorA because none of the compounds had any effect on transport by the MgtB P-type ATPase Mg(2+) transporter or the PhoQ Mg(2+) receptor kinase. These results demonstrate that cation hexaammines are potent and highly selective inhibitors of the CorA Mg(2+) transport system and further indicate that the initial interaction of the CorA transporter is with a fully hydrated Mg(2+) cation.     

Fluorescence measurements of free [Mg2+] by use of mag-fura 2 in Salmonella enterica

The Mg2+ fluorescent dye mag-fura 2, entrapped in cells or organelles, has frequently been used for dual excitation ratio-metric determinations of free ionic Mg2+ concentrations in eukaryotic, mostly mammalian cells. Here we report its successful application to measure free Mg2+ concentrations ([Mg2+]i) in Salmonella enterica cells. When kept in nominally Mg2+ free buffer (resting conditions), the [Mg2+]i of wild-type cells has been determined to be 0.9 mM. An increase in the external Mg2+ concentration ([Mg2+]e) resulted in a rapid increase of [Mg2+]i, saturating within a few seconds at about 1.5 mM with [Mg2+]e of 20 mM. In contrast, cells lacking the Mg2+ transport proteins CorA, MgtA, MgtB failed to show this rapid increase. Instead, their [Mg2+]i increased steadily over extended periods of time and saturated at concentrations below those of wild-type cells. Mg2+ uptake rates increased more than 15-fold when corA was overexpressed in these mutant cells. Uptake of Mg2+ into corA expressing cells was strongly stimulated by nigericin, which increased the membrane potential DeltaPsi at the expense of DeltapH, and drastically reduced by valinomycin, which decreased the membrane potential DeltaPsi. These results reveal mag-fura 2 as a useful indicator to measure steady-state [Mg2+]i values in resting bacterial cells and to determine Mg2+ uptake rates. They confirm the role of CorA as the major Mg2+ transport protein and reveal the membrane potential as driving force for Mg2+ uptake into S. enterica cells.     

The PhoP/PhoQ system and its role in Serratia marcescens pathogenesis

Serratia marcescens is able to invade, persist, and multiply inside nonphagocytic cells, residing in nonacidic, nondegradative, autophagosome-like vacuoles. In this work, we have examined the physiological role of the PhoP/PhoQ system and its function in the control of critical virulence phenotypes in S. marcescens. We have demonstrated the involvement of the PhoP/PhoQ system in the adaptation of this bacterium to growth on scarce environmental Mg(2+), at acidic pH, and in the presence of polymyxin B. We have also shown that these environmental conditions constitute signals that activate the PhoP/PhoQ system. We have found that the two S. marcescens mgtE orthologs present a conserved PhoP-binding motif and demonstrated that mgtE1 expression is PhoP dependent, reinforcing the importance of PhoP control in magnesium homeostasis. Finally, we have demonstrated that phoP expression is activated intracellularly and that a phoP mutant strain is defective in survival inside epithelial cells. We have shown that the Serratia PhoP/PhoQ system is involved in prevention of the delivery to degradative/acidic compartments.