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+.     

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+.     

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.     

Magnesium transport in Salmonella typhimurium: characterization of magnesium influx and cloning of a transport gene

The influx of Mg2+ in Salmonella typhimurium LT-2 was studied by both kinetic and genetic techniques. Wild-type cells grown in a high MgSO4 concentration (10 mM) exhibited a Km of 15 microM for Mg2+ influx, with a Vmax of 0.25 nmol of Mg2+ per min per 10(8) cells. The apparent Km decreased to 3 microM, and the Vmax increased 60% after growth in a low MgSO4 concentration (10 microM). Co2+ was a simple competitive inhibitor (Ki = 30 microM) of Mg2+ influx in cells grown in high Mg2+ concentrations but blocked only a portion of the Mg2+ influx in cells grown in low Mg2+ concentrations. Co2+ influx exhibited kinetics similar to those of Mg2+ influx (Km = 30 microM; Vmax = 0.5 nmol of Co2+ per min per 10(8) cells) but was not affected by growth conditions. Co2+ influx was competitively inhibited by both Mg2+ and Mn2+. Mutations affecting Mg2+ uptake were isolated by selection for spontaneous resistance to toxic levels of Co2+. One class of mutants designated corA mapped at 84 min near metE with the following gene order: corA, metE, zie-3161::Tn10, pepQ. A second class designated corB mapped at 98 min near pyrB. Mg2+ influx was decreased in a corA mutant strain (relative to that of the wild type) when grown in high Mg2+ concentrations but was restored when grown in low Mg2+ concentrations. Co2+ transport was completely abolished by the corA mutation under all growth conditions. Recombinant plasmids carrying the corA region from either Escherichia coli K-12 or S. typhimurium complemented the corA mutation in S. typhimurium, restoring uptake of both Co2+ and Mg2+ and conferring sensitivity to Co2+. The S. typhimurium corA gene was localized to a restriction fragment of approximately 1.5 kilobases.     

Cloning of the mgtE Mg2+ transporter from Providencia stuartii and the distribution of mgtE in gram-negative and gram-positive bacteria.

The MM281 strain of Salmonella typhimurium possesses mutations in each of its three Mg2+ transport systems, requires 100 mM Mg2+ for growth, and was used to screen a genomic library from the gram-negative bacterium Providencia stuartii for clones that could restore the ability to grow without Mg2+ supplementation. The clones obtained also conferred sensitivity to Co2+, a phenotype similar to that seen with the S. typhimurium corA Mg2+ transport gene. The sequence of the cloned P. stuartii DNA revealed the presence of a single open reading frame, which was shown to express a protein with a gel molecular mass of 37 kDa in agreement with the deduced size of 34 kDa. Despite a phenotype similar to that of corA and the close phylogenetic relationship between P. stuartii and S. typhimurium, this new putative Mg2+ transporter lacks similarity to the CorA Mg2+ transporter and is instead homologous to MgtE, a newly discovered Mg2+ transport protein from the gram-positive bacterium Bacillus firmus OF4. The distribution of mgtE in bacteria was studied by Southern blot hybridization to PCR amplification products. In contrast to the ubiquity of the corA gene, which encodes the dominant constitutive Mg2+ influx system of bacteria, mgtE has a much more limited phylogenetic distribution.     

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: 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.     

Regulation of magnesium but not calcium transport by phorbol ester

Phorbol esters in the presence of Ca2+ apparently mimic diacylglycerol in activating protein kinase C. Resulting phosphorylations alter multiple cellular processes including inhibition of the action of Ca2+-mobilizing agonists. In contrast to this inhibition of Ca2+ mobilization, addition of 4 beta-phorbol 12-myristate 13-acetate (PMA) to murine S49 lymphoma cells stimulated Mg2+ influx severalfold without any detectable alteration of Mg2+ efflux or of Ca2+ influx or efflux. Stimulation of Mg2+ influx did not require extracellular Ca2+, was half-maximal at 10 nM PMA, and was characterized by a marked increase in the Vmax of Mg2+ influx without change in the Ka for Mg2+. Stimulation of Mg2+ influx was not mimicked by 4 alpha-phorbol didecanoate, which does not activate protein kinase C and was not the result of Na+/H+ exchange activity. The effect of PMA on Mg2+ influx was inhibited by the beta-adrenergic agonist (-)-isoproterenol, which we have previously shown to inhibit Mg2+ influx by a non-cyclic AMP-dependent mechanism (Maguire, M. E., and Erdos, J. J. (1980) J. Biol. Chem. 255, 1030-1035). Forskolin, a direct activator of adenylate cyclase, also inhibited PMA stimulation of Mg2+ influx, suggesting the presence of both cyclic AMP-dependent and -independent influences on Mg2+ influx. We have also previously demonstrated that Mg2+ influx occurs solely into a small subcytoplasmic pool (Grubbs, R. D., Collins, S. D., and Maguire, M. E. (1984) J. Biol. Chem. 259, 12184-12192). PMA did not alter this compartmentation; rather, it almost doubled the content of this cytosolic Mg2+ pool. These data indicate that, in addition to phorbol ester modulation of intracellular Ca2+ mobilization, substantial changes in Mg2+ flux and content occur. They further demonstrate that Mg2+ influx is regulated by a variety of stimuli. It seems likely that such alterations in Mg2+ flux and content would have physiological consequences.     

Cloning and nucleotide sequence of opdA, the gene encoding oligopeptidase A in Salmonella typhimurium.

The opdA gene (formerly called optA) of Salmonella typhimurium encodes a metallopeptidase, oligopeptidase A (OpdA), first recognized by its ability to cleave and allow utilization of N-acetyl-L-Ala4 (E. R. Vimr, L. Green, and C. G. Miller, J. Bacteriol. 153:1259-1265, 1983). Derivatives of pBR328 carrying the opdA gene were isolated and shown to express oligopeptidase activity at levels approximately 100-fold higher than that of the wild type. These plasmids complemented all of the phenotypes associated with opdA mutations (failure to use N-acetyl-L-Ala4, defective phage P22 development, and diminished endopeptidase activity). The opdA region of one of these plasmids (pCM127) was defined by insertions of Tn1000 (gamma delta), and these insertions were used as priming sites to determine the nucleotide sequence of a 2,843-bp segment of the insert DNA. This region contained an open reading frame coding for a 680-amino-acid protein, the N terminus of which agreed with that determined for purified OpdA. This open reading frame contained both a sequence motif typical of Zn2+ metalloproteases and a putative sigma 32 promoter. However, no induction was detected upon temperature shift by using a beta-galactosidase operon fusion. The predicted OpdA sequence showed similarity to dipeptidyl carboxypeptidase, the product of the S. typhimurium gene dcp, and to rat metallopeptidase EC 3.4.24.15., which is involved in peptide hormone processing.     

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.     

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.     

Plasmid recombination by the RecBCD pathway of Escherichia coli.

Previously, we demonstrated that exonuclease I-deficient strains of Escherichia coli accumulate high-molecular-weight linear plasmid concatemers when transformed with plasmids carrying the chi sequence (5'- GCTGGTGG-3') (M. M. Zaman and T. C. Boles, J. Bacteriol. 176:5093-5100, 1994). Since high-molecular weight linear DNA is believed to be the natural substrate for RecBCD-mediated recombination during conjugation (A. J. Clark and K. B. Low, p. 155-215, in K. B. Low, ed., The Recombination of Genetic Material, 1988), we analyzed the recombination frequencies of chi+ and chi0 plasmids in sbcB strains. Here, we report that chi sites stimulate plasmid recombination frequency by 16-fold in sbcB strains. Chi-stimulated plasmid recombination is dependent on RecBCD but is independent of RecF pathway genes. The distribution of recombination products suggests that high-molecular-weight linear plasmid DNA is a substrate for RecBCD-mediated recombination. Surprisingly, our data also suggest that chi+ plasmids also recombine by the RecBCD pathway in rec+ sbcB+ cells.     

Identification of polypeptides encoded by cloned pJM1 iron uptake DNA isolated from Vibrio anguillarum 775.

The XhoI fragment containing much of the iron uptake region of plasmid pJM1 was isolated from Vibrio anguillarum 775 and cloned into plasmid pBR322. Plasmid-encoded polypeptides were examined in maxicells of Escherichia coli, and transposon mutagenesis was used to map insertion mutations in the structural DNA encoding the OM2 polypeptide. Tn1000 insertions that mapped within OM2 and blocked maxicell expression of OM2 resulted in the loss of ferric iron-anguibactin receptor function when plasmids containing OM2:: Tn1000 insertions were introduced into V. anguillarum cells. Two iron-regulated polypeptides were identified in maxicell polypeptide profiles of E. coli SS201. A 20,000-dalton polypeptide was expressed in maxicells of SS201 grown under conditions of iron limitation but was barely detectable in profiles of SS201 cells that were grown under high-iron conditions. DNA encoding the 20,000-dalton polypeptide mapped downstream of and adjacent to the gene encoding OM2. DNA sequences required for production of a 46,000-dalton polypeptide mapped 4.5 kilobases downstream of the OM2 structural gene. The 46,000-dalton polypeptide was synthesized at high levels in E. coli SS201 maxicells grown under high-iron conditions, but synthesis of the protein was severely repressed under conditions of iron limitation. Iron-regulated expression of both proteins in maxicells of SS201 was relieved upon deletion of a 4.9-kilobase SalI-XhoI fragment of pJM1 DNA, which indicated that pJM1 DNA sequences present in the deleted fragment are required for regulated expression of both proteins in E. coli. Maxicells of SS201 harboring these deletion derivatives synthesized the 20,000-dalton polypeptide at very low constitutive levels and the 46,000-dalton polypeptide at high constitutive levels, regardless of the iron concentration of the growth medium. The observed regulation of the 20,000-dalton protein suggested that it might play a role either in siderophore biosynthesis or in the functional expression of OM2. The opposite regulatory pattern observed for the 46,000-dalton polypeptide suggested that it does not play a structural role in siderophore or OM2 biosynthesis, but the observed regulatory pattern might be expected if the 46,000-dalton protein played a negative regulatory role in siderophore biosynthesis.     

The Salmonella typhimurium LT2 uvrD gene is regulated by the lexA gene product.

The uvrD gene product apparently plays a role in the repair of UV damage, in mismatch repair, and in genetic recombination. A lower level of expression of the Salmonella typhimurium LT2 uvrD gene was observed in maxicells prepared from an Escherichia coli strain that contained a lexA+ plasmid than in maxicells prepared from an E. coli strain that lacked functional LexA protein. These results suggest that the uvrD+ gene is repressed by the LexA protein and is thus a member of the set of genes whose expression is increased by "SOS"-inducing treatments.