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

Abstract

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...

Functional characterization of human SLC41A1, a Mg2+transporter with similarity to prokaryotic MgtE Mg2+transporters

“…Clearly, SLC41A1 does not have the same cationic specificity as MgtE (16,17 Quantitative RT-PCR analysis was performed with SLC41A1 mRNA purified from the indicated tissues derived from mice maintained on a normal (0.5% by weight) or low-Mg 2ϩ (Ͻ0.01%) diet for 5 days. Mouse SLC41A1 and murine ␤-actin RNA were measured with real-time RT-PCR (AB7000, Applied Biosystems) using primers designed to mouse SLC41A1 and ␤-actin.…”

“…Computer analysis of the SLC41A1 protein structure reveals that it comprises 10 putative transmembrane domains, 2 of which are highly homologous to the integral membrane part of the prokaryote MgtE protein family. MgtE was initially identified in the bacteria Providencia stuartii and Bacillus firmus by Smith, Maguire, and their colleagues in their search for members of the CorA (cobalt resistance) family of Mg 2ϩ uptake transporters (15)(16)(17). On the basis of the similarity of SLC41A1 to MgtE, Wabakken et al (19) suggested that the human solute carrier SLC41A1 may be a eukaryotic Mg 2ϩ transporter.…”

Molecular identification of ancient and modern mammalian magnesium transporters

“…Clearly, cationic specificity was not the same for the SLC41 transporters as for MgtE (140). For example, SLC41A1 does not transport Co 2ϩ to any degree, and Ni 2ϩ is not a selective inhibitor of Mg 2ϩ transport.…”

“…The identification and characterization of plant, prokaryote, and yeast Mg 2ϩ transporters have been extensively reviewed elsewhere (35,70,73,138 transport (30,36,87,93,98,105,168). CorA is the primary Mg 2ϩ uptake transporter in most prokaryotes (140). It is constitutively expressed in bacteria and does not respond to external Mg 2ϩ levels (70).…”

Mg 2+ -sensing mechanism of Mg 2+ transporter MgtE probed by molecular dynamics study

“…Its Mg 2ϩ -transporting activity was demonstrated by a complementation assay and the uptake of a Co 2ϩ isotope in vivo (9). In bacteria, translation of its gene is regulated by a Mg 2ϩ -specific riboswitch (10).…”