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

Hmiel, S. Paul; Snavely, M D; Miller, Charles G.; Maguire, Michael E.

doi:10.1128/jb.168.3.1444-1450.1986

Cited by 162 publications

(151 citation statements)

References 25 publications

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“…Accumulation of 57 Co 2ϩ was assayed as described previously (6,8,24) . All transport assays were performed at 37ЊC.…”

Section: Methodsmentioning

confidence: 99%

“…Plasmids were maintained in either E. coli DH5␣ or S. typhimurium JR501 (3). Plasmid complementation assays employed S. typhimurium MM281 as the host (7,8,24).…”

Section: Methodsmentioning

confidence: 99%

“…The third system, CorA, is constitutively expressed and mediates both influx and efflux of Mg . Under normal growth conditions, CorA is the dominant transporter (7,8), mediating as much as 99% of the total Mg 2ϩ accumulated. The CorA system can also facilitate the uptake of Co 2ϩ and Ni…”

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confidence: 99%

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Cloning and characterization of MgtE, a putative new class of Mg2+ transporter from Bacillus firmus OF4

1995

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The MM281 strain of Salmonella typhimurium which possesses mutations in each its three known Mg 2؉ transport systems and requires 100 mM Mg 2؉ for growth was used to screen a genomic library from the gram-positive alkaliphilic bacterium Bacillus firmus OF4 for clones that could restore the ability to grow without Mg 2؉ supplementation. . Mutant strains lacking a functional CorA transporter demonstrate a significant reduction in the ability to take up Mg 2ϩ as well as increased resistance to the cytotoxic effects of Co 2ϩ in the culture medium. This phenotype is not unique to S. typhimurium and E. coli. Mg 2ϩ transport mutants of Bacillus subtilis and Rhodobacter capsulatus that also exhibit resistance to Co 2ϩ have been described (11,13,19,20). This phenotype implies the presence of a CorA-like system in these organisms and has prompted the hypothesis that CorA is widely distributed throughout prokaryotes (22). As part of studies to examine the structure and distribution of CorA-like systems, an Mg 2ϩ transport-deficient strain of S. typhimurium was used to screen a plasmid library from the gram-positive alkaliphile Bacillus firmus OF4 (9, 10) for clones that could complement the corA mutation. In this study, we describe the isolation and characterization of the MgtE transport system, which complements the Mg 2ϩ uptake mutations in S. typhimurium and restores sensitivity to Co 2ϩ . Sequence analysis, however, indicates that the MgtE locus encodes a 34-kDa protein quite unlike CorA and appears to represent a new class of Mg 2ϩ transport system. MATERIALS AND METHODSBacterial strains and plasmids. The bacterial strains used in this study are listed in Table 1 with their sources. Plasmids were maintained in either E. coli DH5␣ or S. typhimurium JR501 (3). Plasmid complementation assays employed S. typhimurium MM281 as the host (7,8,24).Culture media and reagents. Luria-Bertani (LB) broth was routinely used as the complex growth medium. N-minimal broth was used as the minimal growth medium (16). Antibiotics were added to complex and minimal culture media, respectively, at the following concentrations (in micrograms per liter): sodium ampicillin, 100 and 30; tetracycline hydrochloride, 20 and 10; kanamycin sulfate, 50 and 100; chloramphenicol, 25 and 10. Growth of MM281 requires MgSO 4 supplementation at a final concentration of 100 mM in both complex and minimal culture media. Restriction endonucleases, T4 DNA ligase, calf intestinal alkaline phosphatase, Klenow fragment of DNA polymerase I, S1 nuclease, and T4 polynucleotide kinase were obtained from Life Technologies, Inc. (Gibco-BRL, Gaithersburg, Md.). Sequencing enzymes and associated biochemicals were obtained from U. S. Biochemicals Inc. (Cleveland, Ohio).57 Co 2ϩ was obtained from Amersham (Arlington, Ill.). Synthetic oligodeoxynucleotide primers were obtained from Oligo's Etc. (Wilsonville, Oreg.). Additional chemicals were obtained from standard suppliers.DNA manipulation. Plasmid DNA was prepared from 500-ml cultures with the Wizard Maxiprep DNA isolation...

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“…Accumulation of 57 Co 2ϩ was assayed as described previously (6,8,24) . All transport assays were performed at 37ЊC.…”

Section: Methodsmentioning

confidence: 99%

“…Plasmids were maintained in either E. coli DH5␣ or S. typhimurium JR501 (3). Plasmid complementation assays employed S. typhimurium MM281 as the host (7,8,24).…”

Section: Methodsmentioning

confidence: 99%

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Cloning and characterization of MgtE, a putative new class of Mg2+ transporter from Bacillus firmus OF4

1995

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“…MGT family proteins have been known as Mg transporters in both prokaryote and eukaryote (Hmiel et al, 1986;Li et al, 2001). There are 10 MGT homologs in the Arabidopsis genome and nine homologs in rice (Schock et al, 2000;Gebert et al, 2009).…”

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confidence: 99%

A Magnesium Transporter OsMGT1 Plays a Critical Role in Salt Tolerance in Rice

et al. 2017

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Salt stress is one of the major factors limiting rice (Oryza sativa) production globally. Although several transporters involved in salt tolerance have been identified in rice, the mechanisms regulating their transport activity are still poorly understood. Here, we show evidence that a rice Mg transporter OsMGT1 is required for salt tolerance probably by regulating transport activity of OsHKT1;5, a key transporter for the removal of Na + from the xylem sap at the root mature zone. Knockout of OsMGT1 did not affect total Na uptake, but increased Na concentration in the shoots and xylem sap, resulting in a significant increase in salt sensitivity at low external Mg 2+ concentration (20-200 mM). However, such differences were abolished at a higher Mg 2+ concentration (2 mM), although the total Na uptake was not altered. OsMGT1 was expressed in both the roots and shoots, but only that in the roots was moderately up-regulated by salt stress. Spatial expression analysis revealed that OsMGT1 was expressed in all root cells of the root tips but was highly expressed in the pericycle of root mature zone. OsMGT1 was also expressed in the phloem region of basal node, leaf blade, and sheath. When expressed in Xenopus laevis oocytes, the transport activity of OsHKT1;5 was enhanced by elevating external Mg 2+ concentration. Furthermore, knockout of OsHKT1;5 in osmgt1 mutant background did not further increase its salt sensitivity. Taken together, our results suggest that Mg 2+ transported by OsMGT1 in the root mature zone is required for enhancing OsHKT1;5 activity, thereby restricting Na accumulation to the shoots.

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“…The cytoplasmic domain of CorA is a seven-stranded parallel/antiparallel -sheet ( 2 1 3 7 6 5 4 ) sandwiched between two sets of -helices ( 1,2,3) and ( 4,5,6) (Fig. 1).…”

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confidence: 99%

Crystal structure of the CorA Mg2+ transporter

Лунин

Dobrovetsky²,

Khutoreskaya³

et al. 2006

Nature

236

301

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The magnesium ion, Mg 2+ , 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 Mg 2+ uptake system of most prokaryotes 1-3 and a functional homologue of the eukaryotic mitochondrial magnesium transporter 4 . 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 Å and 1.85Å 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 Mg 2+ ion was bound between monomers at a conserved site in the cytoplasmic domain, suggesting a mechanism to link gating of the pore to the intra-cellular concentration of Mg 2+ . The CorA magnesium transporter is a homopentamer with fivefold symmetry about a central pore and can be divided into three parts (Fig. 1). A carboxy-terminal transmembrane domain comprises two transmembrane helices from each monomer (Fig. 2). The middle portion resembles a funnel, narrow at the entrance ( 5 Å) and wide at the mouth ( 20Å), that is formed largely by a long -helix extension of the inner transmembrane helix. Finally, a large cytoplasmic domain lies exterior to the funnel.The cytoplasmic domain of CorA is a seven-stranded parallel/antiparallel -sheet ( 2 1 3 7 6 5 4 ) sandwiched between two sets of -helices ( 1, 2, 3) and ( 4, 5, 6) ( Fig. 1). The domain fold is unlike all other known structures of ion channels or transporters and constitutes a new protein fold (see Supplementary Information). This domain, solved in its soluble form at 1.85 Å resolution ( Supplementary Fig. S1), is linked to the transmembrane helices by the long 7 helix (residues 251-312), termed the stalk helix. The stalk helix kinks as it enters the membrane, extends through the membrane, forms the first transmembrane helix (TM1; residues 293-312) and harbours the 'YGMNF' signature sequence of CorA (residues 311-315) 5,6 ( Fig. 2 and Supplementary Fig. S2). The five TM1 helices (residues 293-312) form the pore. After a short extracellular seven-amino-acid loop, the TM2 helix (residues 326-345) returns back to the cytoplasm and ends in a highly conserved C-terminal KKKKWL motif (Fig. 3). In the current structure, neither the extracellular loop nor the final two amino acids could be resolved.The cytoplasmic domain shows the lowest sequence conservati...

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

Cited by 162 publications

References 25 publications

Cloning and characterization of MgtE, a putative new class of Mg2+ transporter from Bacillus firmus OF4

Cloning and characterization of MgtE, a putative new class of Mg2+ transporter from Bacillus firmus OF4

A Magnesium Transporter OsMGT1 Plays a Critical Role in Salt Tolerance in Rice

Crystal structure of the CorA Mg2+ transporter

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