Magnesium transport in Salmonella typhimurium: expression of cloned genes for three distinct Mg2+ transport systems.

In Salmonella typhimurium, the corA, mgtA, and mgtB loci are involved in active transport of Mg2+ (S. P. Hmiel, M. D. Snavely, C. G. Miller, and M. E. Maguire, J. Bacteriol. 168:1444-1450, 1988; S. P. Hmiel, M. D. Snavely, J. B. Florer, M. E. Maguire, and C. G. Miller, J. Bacteriol. 171:4742-4751, 1989). In this study, the gene products coded for by the corA, mgtA, and mgtB genes were identified by using plasmid expression in Escherichia coli maxicells. Complementation was assessed by introducing plasmids into a Mg2+-dependent corA mgtA mgtB strain and determining the ability of the plasmid to restore growth on medium without a Mg2+ supplement. Complementing plasmids containing corA expressed a 42-kilodalton (kDa) protein. This protein was not expressed by plasmids containing insertions or deletions that eliminated complementation. A plasmid containing mgtA expressed 37- and 91-kDa gene products. Data obtained with subclones and insertions in this plasmid indicated that plasmids expressing only the 91-kDa polypeptide complemented; plasmids that did not express this protein did not complement regardless of whether they expressed the 37-kDa protein. Plasmids carrying mgtB expressed a single protein of 102 kDa whose presence or absence correlated with the ability of the plasmid to complement the Mg2+-dependent triple mutant. Fractionation of labeled maxicells demonstrated that the 42-kDa corA, the 91-kDa mgtA, and the 102-kDa mgtB gene products are all tightly associated with the membrane, a location consistent with involvement in a transport process. These data provide further support the for existence of three distinct systems for Mg2+ transport in S. typhimurium.     

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.     

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.     

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.     

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.     

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.     

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.     

Properties of Escherichia coli mutants altered in calcium/proton antiport activity.

Mutants sensitive to growth inhibition by CaCl2 were found to have alterations in calcium uptake in everted membrane vesicles. These mutations map at different loci on the Escherichia coli chromosomes. A mutation at the calA locus results in vesicles which have two- to threefold higher levels of uptake activity than vesicles from wild-type cells. The calA mutation is phenotypically expressed as increased sensitivity to CaCl2 in a strain also harboring a mutation in the corA locus, which is involved in Mg2+ transport. The calA locus maps very close to purA and cycA at about min 97. The calB mutation results both in sensitivity to CaCl2 at pH 5.6 and in vesicles with diminished calcium transport capability. The CalB phenotype is also expressed only in a corA genetic background; the calB locus appears to map very near, yet separately from, the calA locus. When the cor+ allele is present, calA and calB mutations still result in a defect in calcium transport in vesicles. In addition, both calC and calD mutations result in vesicles with impaired calcium transport activity. calC is cotransducible with kdp and nagA, whereas calD is cotransducible with proC.     

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.     

Two outer membrane transport systems for vitamin B12 in Salmonella typhimurium.

The involvement of an outer membrane transport component for vitamin B12 uptake in Salmonella typhimurium, analogous to the btuB product in Escherichia coli, was investigated. Mutants of S. typhimurium selected for resistance to bacteriophage BF23 carried mutations at the btuB locus (butBS) (formerly called bfe, at the analogous map position as the E. coli homolog) and were defective in high-affinity vitamin B12 uptake. The cloned E. coli btuB gene (btuBE) hybridized to S. typhimurium genomic DNA and restored vitamin B12 transport activity to S. typhimurium btuBS mutants. An Mr-60,000 protein in the S. typhimurium outer membrane was repressed by growth with vitamin B12 and was eliminated in a btuBS mutant. The btuBS product thus appears to play the same role in vitamin B12 transport by S. typhimurium as does the E. coli btuBE product. A second vitamin B12 transport system that is not present in E. coli was found by cloning a fragment of S. typhimurium DNA that complemented btuB mutants for vitamin B12 utilization. In addition to this plasmid with a 6-kilobase insert of S. typhimurium DNA, vitamin B12 utilization by E. coli btuB strains required the btuC and btuD products, necessary for transport across the cytoplasmic membrane, but not the btuE or tonB product. The plasmid conferred low levels of vitamin B12-binding and energy-dependent transport activity but not susceptibility to phage BF23 or utilization of dicyanocobinamide. The cloned S. typhimurium DNA encoding this new transport system did not hybridize to the btuBE gene or to E. coli chromosomal DNA and therefore does not carry the S. typhimurium btuBS locus. Increased production of an Mr -84,000 polypeptide associated with the outer membrane was seen. The new locus appears to be carried on the large plasmid in most S. typhimurium strains. Thus S. typhimurium possesses both high- and low-affinity systems for uptake of cobalamins across the outer membrane.     

Ferrous iron uptake by a magnesium transport system is toxic for Escherichia coli and Salmonella typhimurium.

At low magnesium concentrations, Escherichia coli and Salmonella typhimurium LT2 accumulate ferrous iron independent of the ferrous iron transport system feo. Mutant strains with mutations in the magnesium transport gene corA accumulated less ferrous iron than the parent strains. corA+ and corA strains also differed in their sensitivity to ferrous iron under oxic conditions. corA mutants were more resistant to ferrous iron than their parent corA+ strains. Part of the ferrous iron accumulated can be chased by the addition of magnesium. Much less iron was chased when ferric iron was taken up by the siderophore ferrichrome. These results may indicate that the intracellular metabolism of the iron taken up by these systems differs and that it depends on the uptake route of the iron.     

Genetic characterization and molecular cloning of the tripeptide permease (tpp) genes of Salmonella typhimurium.

Of the three bacterial peptide transport systems only one, the oligopeptide permease, has been characterized in any detail. We have now isolated Salmonella typhimurium mutants deficient in a second transport system, the tripeptide permease (Tpp), using the toxic peptide alafosfalin. Alafosfalin resistance mutations map at three loci, the gene encoding peptidase A (pepA) and two transport-defective loci, tppA and tppB. Locus tppA has been mapped to 74 min on the S. typhimurium chromosome, cotransducible with aroB, and is a positive regulator of tppB. Locus tppB maps at 27 min in the cotransduction gap between purB and pyrF. We cloned tppB, the structural locus for the tripeptide permease. Two simple methods are described for mapping the location of cloned DNA fragments on the chromosome of S. typhimurium.     

Mapping and characterization of the nad genes in Salmonella typhimurium LT-2.

An ampicillin enrichment technique was used to isolate 39 nicotinic acid-requiring mutants of Salmonella typhimurium LT-2. Using interrupted-mating and transductional mapping procedures, three loci, designated nadA, nadB, and nadC, were identified. These loci mapped at 33, 82, and 6 min, respectively, on the S. typhimurium linkage map. The arrangement of the loci on the Salmonella linkage map corresponded closely to the nadA, nadB, and nadC loci on the Escherichia coli K-12 linkage map, indicating that the de novo pathway to nicotinamide adenine dinucleotide and the genes governing the enzymes involved in this pathway in S. typhimurium are very similar to those in E. coli. Evidence is also presented which indicates that the product of the nadC locus in S. typhimurium LT-2 is the enzyme quinolinic acid phosphoribosyltransferase. All nadC mutants of S. typhimurium secreted between 2 and 8 mumol of quinolinic acid per 100 ml of secretion medium. In addition, none of the nadC mutants isolated were able to grow in 10(-3) M quinolinic acid, whereas all nadA and nadB mutants of S. typhimurium grew well in the presence of quinolinic acid. Transductional crosses between nadB mutants provided evidence suggestive of more than one locus in the nadB region.     

Insertional inactivation of determinants for Mg2+ and Co2+ transport as a tool for screening recombinant Lactococcus species clones

Insertional inactivation of the plasmid-encoded determinants for Mg(2+) and Co(2+) transport, orf18/corA, provides a tool for screening recombinant clones in Lactococcus, based on the observation that overexpression of orf18/corA results in cell growth inhibition on certain concentrations of CoCl(2). The lacticin 3147 immunity gene, ltnI, was used to insertionally inactivate orf18/corA. The resulting clones were capable of growth on concentrations of CoCl(2) that were inhibitory to the parent strain. Since only 3 of 17 lactococcal starters naturally harbor corA, the system has potential as a screen for selecting recombinant lactococcal clones.