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.     

Inorganic cation transport and the effects on C4 dicarboxylate transport in Bacillus subtilis

The complex interrelationships between the transport of inorganic cations and C4 dicarboxylate were examined using mutants defective in potassium transport and retention, divalent cation transport, or phosphate transport. The potassium transport system, studied using 86Rb+ as a K+ analogue, kinetically appeared as a single system (Km 200 microM for Rb+, Ki 50 microM for K+), the activity of which was only slightly reduced in K+ retention mutants. Divalent cation transport, studied using 54Mn2+, 60Co2+, and 45Ca2+, was more complex being represented by at least two systems, one with a high affinity for Mn2+ (Km 2.5 microM) and a more general one of low affinity (Km 1.3-10 mM) for Mg2+, Mn2+, Ca/2+, and Co2+. Divalent cation transport was repressed by Mg2+, derepressed in K+ retention mutants, and defective in Co2+-resistant mutants. Phosphate was required for both divalent cation and succinate transport, and phosphate transport mutants (arsenate resistant) were found to be defective in both divalent cation and succinate transport. Divalent cations, especially Mg2+ and Co2+, decreased Km for succinate transport approximately 20-fold over that achieved with K+; neither cation was required stoichiometrically for succinate transport. The loss of divalent cation transport in cobalt-resistant mutants has been correlated with the loss of a 55,000 molecular weight membrane protein. Similarly, the loss of phosphate transport in arsenate-resistant mutants has been correlated with the loss of a 35,000 molecular weight membrane component.     

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.     

Activation by divalent cations of a Ca2+-activated K+ channel from skeletal muscle membrane

Several divalent cations were studied as agonists of a Ca2+-activated K+ channel obtained from rat muscle membranes and incorporated into planar lipid bilayers. The effect of these agonists on single-channel currents was tested in the absence and in the presence of Ca2+. Among the divalent cations that activate the channel, Ca2+ is the most effective, followed by Cd2+, Sr2+, Mn2+, Fe2+, and Co2+. Mg2+, Ni2+, Ba2+, Cu2+, Zn2+, Hg2+, and Sn2+ are ineffective. The voltage dependence of channel activation is the same for all the divalent cations. The time-averaged probability of the open state is a sigmoidal function of the divalent cation concentration. The sigmoidal curves are described by a dissociation constant K and a Hill coefficient N. The values of these parameters, measured at 80 mV are: N = 2.1, K = 4 X 10(-7) mMN for Ca2+; N = 3.0, K = 0.02 mMN for Cd2+; N = 1.45, K = 0.63 mMN for Sr2+; N = 1.7, K = 0.94 mMN for Mn2+; N = 1.1, K = 3.0 mMN for Fe2+; and N = 1.1 K = 4.35 mMN for Co2+. In the presence of Ca2+, the divalent cations Cd2+, Co2+, Mn2+, Ni2+, and Mg2+ are able to increase the apparent affinity of the channel for Ca2+ and they increase the Hill coefficient in a concentration-dependent fashion. These divalent cations are only effective when added to the cytoplasmic side of the channel. We suggest that these divalent cations can bind to the channel, unmasking new Ca2+ sites.     

Magnesium transport in Salmonella typhimurium: genetic characterization and cloning of three magnesium transport loci

Salmonella typhimurium strains lacking the CorA Mg2+ transport system retain Mg2+ transport and the ability to grow in medium containing a low concentration of Mg2+. Mutagenesis of a corA strain followed by ampicillin selection allowed isolation of a strain that required Mg2+-supplemented media for growth. This strain contained mutations in at least two loci in addition to corA, designated mgtA and mgtB (for magnesium transport). Strains with mutations at all three loci (corA, mgtA, and mgtB) exhibited no detectable Mg2+ uptake and required 10 mM Mg2+ in the medium for growth at the wild-type rate. A wild-type allele at any one of the three loci was sufficient to restore both Mg2+ transport and growth on 50 microM Mg2+. P22 transduction was used to map the mgt loci. The mgtA mutation was located to approximately 98 map units (cotransducible with pyrB), and mgtB mapped at about 80.5 map units (near gltC). A chromosomal library from S. typhimurium was screened for clones that complemented the Mg2+ requirement of a corA mgtA mgtB mutant. The three classes of plasmids obtained could each independently restore Mg2+ transport to this strain and corresponded to the corA, mgtA, and mgtB loci. Whereas the corA locus of S. typhimurium is analogous to the corA locus previously described for Escherichia coli, neither of the mgt loci described in this report appears analogous to the single mgt locus described in E. coli. Our data in this and the accompanying papers (M. D. Snavely, J. B. Florer, C. G. Miller, and M. E. Maguire, J. Bacteriol. 171:4752-4760, 4761-4766, 1989) indicate that the corA, mgtA, and mgtB loci of S. typhimurium represent three distinct systems that transport Mg2+.     

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.     

The mgtB Mg2+ transport locus of Salmonella typhimurium encodes a P-type ATPase.

The mgtB locus codes for one of three distinct Mg2+ transport systems of Salmonella typhimurium. The system encoded by the mgtB locus mediates Mg2+ influx only. The nucleotide sequence of a 4.6-kilobase fragment of DNA carrying mgtB has been determined. Two open reading frames were apparent. The most 5' (mgtC) could encode a hydrophobic protein of up to 25 kDa depending on which translation starts are used. A plasmid carrying this region downstream from a phage T7 promoter expresses a 22.5-kDa protein. The second open reading frame encoded a 101-kDa polypeptide (MgtB) consistent with our previous observation that a plasmid carrying the mgtB locus expresses a 102-kDa protein in maxicells. Insertions into either open reading frame abolished the ability of the plasmid to relieve the requirement for added Mg2+ and to restore Mg2+ uptake to a Mg2+ transport-deficient strain of S. typhimurium. The predicted amino acid sequence of MgtC showed no similarity to any other known protein. In contrast, the predicted sequence of MgtB indicated that it is a member of the family of cation transport P-type ATPases. Strikingly, however, MgtB was significantly more similar to eukaryotic Ca2(+)-ATPases than to prokaryotic P-type ATPases or other classes of eukaryotic P-type ATPases such as the Na+,K(+)-ATPase. MgtB is most closely related to Ca2(+)-ATPases of mammalian sarcoplasmic reticulum and yeast. A number of features of the Ca2(+)-ATPases thought to be important for cation transduction across the membrane are present in MgtB but not in other prokaryotic members of this enzyme family. Unlike the Ca2(+)-ATPases, however, which mediate efflux of cation from the cytosol, MgtB mediates influx of cation into the cytosol.     

Electrodiffusive magnesium transport across the intestinal brush border membrane of tilapia (Oreochromis mossambicus).

Mg2+ transport across the brush border of proximal intestinal epithelium of the teleost fish Oreochromis mossambicus was investigated, using 27Mg2+ to trace movement of Mg2+. Mg2+ uptake in brush border membrane vesicles was stimulated by a K+ diffusion potential (inside negative). Electrodiffusive Mg2+ transport obeyed simple Michaelis-Menten kinetics and was strongly temperature dependent, indicative of a carrier mechanism. The metal ion specificity of this electrodiffusive pathway (inhibition potency order: Co > Mn = Ni > La > Ca > Gd > Ba), predicts a specific role in Mg2+ transport. Competitive inhibition by Co(III) hexammine [Co(NH3)(6)(3+)] suggests that this transport system interacts with the solvated Mg ion. We propose that this novel transport system allows the uptake of Mg2+ across the apical brush border membrane, and is involved in transcellular Mg2+ transport. Consequently, the prevailing potential difference across the apical membrane represents a major driving force for intestinal Mg2+ absorption.     

Activation of brain calcineurin phosphatase towards nonprotein phosphoesters by Ca2+, calmodulin, and Mg2+

Calcineurin purified from bovine brain was found to be active towards beta-naphthyl phosphate greater than p-nitrophenyl phosphate greater than alpha-naphthyl phosphate much greater than phosphotyrosine. In its native state, calcineurin shows little activity. It requires the synergistic action of Ca2+, calmodulin, and Mg2+ for maximum activation. Ca2+ and Ca2+ X calmodulin exert their activating effects by transforming the enzyme into a potentially active form which requires Mg2+ to express the full activity. Ni2+, Mn2+, and Co2+, but not Ca2+ or Zn2+, can substitute for Mg2+. The pH optimum, and the Vm and Km values of the phosphatase reaction are characteristics of the divalent cation cofactor. Ca2+ plus calmodulin increases the Vm in the presence of a given divalent cation, but has little effect on the Km for p-nitrophenyl phosphate. The activating effects of Mg2+ are different from those of the transition metal ions in terms of effects on Km, Vm, pH optimum of the phosphatase reaction and their affinity for calcineurin. Based on the Vm values determined in their respective optimum conditions, the order of effectiveness is: Mg2+ greater than or equal to Ni2+ greater than Mn2+ much greater than Co2+. The catalytic properties of calcineurin are markedly similar to those of p-nitrophenyl phosphatase activity associated with protein phosphatase 3C and with its catalytic subunit of Mr = 35,000, suggesting that there are common features in the catalytic sites of these two different classes of phosphatase.     

Effect of bivalent metal ions on the fluctuations of ion currents in rat liver mitochondria

Ions of bivalent metals are shown to arrange in the Sr2+ greater than Ca2+ greater than Ba2+ greater than Mn2+ series as to their ability to induce ion flow vibration in the rat liver mitochondria. Application of Sr2+ results in the most stable prolonged vibrations of ion flows in mitochondria. Ca2+, Ba2+ and Mn2+ induce slightly pronounced and intensively damped vibrations. The studied Mg2+, Co2+, Ni2+, Pb2+ Fe2+ cations have effect on valinomycin-induced K+ transport in mitochondria and do not induce vibrations. It is established that the ability of bivalent cations to induce vibrations is associated with the possibility of their transfer through the mitochondrion membrane and accumulation in the matrix. Inhibitors of the electrogenic Ca2+ transport in mitochondria produce the similar effect on vibrations induced by Sr2+, Ca2+, Ba2+ and Mn2+.     

Cadmium evokes inositol polyphosphate formation and calcium mobilization. Evidence for a cell surface receptor that cadmium stimulates and zinc antagonizes

Cd2+ and other divalent metals mobilized cell Ca2+ in human skin fibroblasts. The divalent metals produced a large spike in cytosolic free Ca2+ and strikingly increased net Ca2+ efflux similarly to bradykinin. One-tenth microM Cd2+ half-maximally increased 45Ca2+ efflux. The potency order of the Ca2+ mobilizing metals was: Cd2+ greater than Co2+ greater than Ni2+ greater than Fe2+ greater than Mn2+. Cd2+ probably acts at an extracellular site because loading the cells with a heavy metal chelator only slightly inhibited Cd2+-evoked 45Ca2+ efflux. Cd2+ increased [3H]inositol polyphosphates; [3H]inositol trisphosphate increased 4-fold in 15 s. Zn2+ reversibly blocked 45Ca2+ efflux evoked by Cd2+ but not that produced by bradykinin. Zn2+ competitively (Ki = approximately 0.4 microM) inhibited net Ca2+ efflux produced by Cd2+. Cd2+ also evoked Ca2+ mobilization in umbilical artery muscle, endothelial, and neuroblastoma cells, and the divalent cation agonist and antagonist specificities were similar to those in the fibroblasts. The divalent metals appear to trigger Ca2+ mobilization via a reversible interaction with an external site on the cell surface, which may be considered a "Cd2+ receptor."     

Na+/Ca2+ antiport in cultured arterial smooth muscle cells. Inhibition by magnesium and other divalent cations

Cultured smooth muscle cells from rat aorta were loaded with Na+, and Na+/Ca2+ antiport was assayed by measuring the initial rates of 45Ca2+ influx and 22Na+ efflux, which were inhibitable by 2',4'-dimethylbenzamil. The replacement of extracellular Na+ with other monovalent ions (K+, Li+, choline, or N-methyl-D-glucamine) was essential for obtaining significant antiport activity. Mg2+ competitively inhibited 45Ca2+ influx via the antiporter (Ki = 93 +/- 7 microM). External Ca2+ or Sr2+ stimulated 22Na+ efflux as would be expected for antiport activity. Mg2+ did not stimulate 22Na+ efflux, which indicates that Mg2+ is probably not transported by the antiporter under the conditions of these experiments. Mg2+ inhibited Ca2+-stimulated 22Na+ efflux as expected from the 45Ca2+ influx data. The replacement of external N-methyl-D-glucamine with K+, but not other monovalent ions (choline, Li+), decreased the potency of Mg2+ as an inhibitor of Na+/Ca2+ antiport 6.7-fold. Other divalent cations (Co2+, Mn2+, Cd2+, Ba2+) also inhibited Na+/Ca2+ antiport activity, and high external potassium decreased the potency of each by 4.3-8.6-fold. The order of effectiveness of the divalent cations as inhibitors of Na+/Ca2+ antiport (Cd2+ greater than Mn2+ greater than Co2+ greater than Ba2+ greater than Mg2+) correlated with the closeness of the crystal ionic radius to that of Ca2+.     

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.     

Mutants in three genes affecting transport of magnesium in Escherichia coli: genetics and physiology.

Mutants in three genes affecting two Mg2+ transport systems are described. System I, for which Co2+, Mn2+, and Mg2+ are substrates, is inactive in corA mutants corB mutants express system I after growth on high (10 mM) Mg2+ but not low (0.1 mM) Mg2+. Both corA and corB mutants are resistant to Co2+ or Mn2+. corA mutants are sensitive to CA2+. Transport system II is specific for Mg2+ and is repressed by growth on 10 mM Mg2+. mgt mutations inactivate system II. Growth on mgt mutants in normal except on very low (1 muM) concentrations of Mg2+, corA mgt strains exhibit no high-affinity, energy-dependent transport of Mg2+ and require 10 mM Mg2+ for optimal growth. The three genes are not linked. The corA locus is contransducible with ilv at 75 min, corB is cotransducible with pyrB at 85 min, and mgt is cotransducible with malB and mel at 81 min on the genetic map.     

Photosensory transduction in the flagellated alga, Euglena gracilis I. Action of divalent cations, Ca2+ antagonists and Ca2+ ionophore on motility and photobehavior.

1. The flagellated alga, Euglena gracilis, swims forward essentially in a straight path under constant light intensity. Strong motility of the cells can be supported by Mg2+ alone but optimum motility is found in the presence of Mg2+, Ca2+ and K+. 2. Ca2+, Co2+, Mn2+ and Ba2+ induce a concentration-dependent increase in the rate at which the cells change the direction of their swimming path (a klinokinesis). Ni2+ immobilizes the flagellum. 3. On perception of a reduction ('step-down stimulus') in blue light intensity in their environment, Euglena rotate in place (tumble) for a finite period (the step-down photophobic response). 4. The duration of the tumbling is enhanced in the presence of divalent cations following the series Ca2+ greater than Ba2+ greater than Mn2+ greater than Co2+ greater than Mg2+ = Ni2+ = 0. 5. Neither the tumbling response in the presence of low concentrations of Ca2+ or the Ca2+-stimulated response is altered by verapamil (a Ca2+ conductance antagonist). The Ca2+ conductance/active transport antagonist, ruthenium red, is also inactive. 6. The Ca2+ ionophore, A23187, has little effect on flagellar activity in the absence of extracellular Ca2+. However, in the presence of A23187, Ca2+ induces a specific light-independent, concentration-dependent discontinuous tumbling response of the cells. 7. The data support a role for Ca2+ and Mg2+ in control of flagellar activity. However, blue light-induced tumbling behavior would not appear to be the direct result of a light-mediated alteration in the Ca2+ conductance of the flagellar membrane to affect flagellar reorientation. The results are discussed in connection with previous theories on control of flagella activity in green alga.