Abstract
The MM281 strain of Salmonella typhimurium which possesses mutations in each its three known Mg2+ transport systems and requires 100 mM Mg2+ 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 Mg2+ supplementation. Of the clones obtained, five also conferred sensitivity to Co2+, similar to the phenotype of mutants with mutations in the S. typhimurium corA Mg2+ transport locus. All five contained identical inserts by restriction analysis. Using 63Ni2+ as a surrogate for the unavailable 28Mg2+, the plasmid insert was shown to restore cation uptake with properties similar but not identical to those of the S. typhimurium CorA Mg2+ transporter. Sequence analysis of one clone identified a single open reading frame with multiple possible initiation sites. Deletion and mutation analysis identified a minimum open reading frame of 939 bp encoding a polypeptide with a predicted molecular mass of 34 kDa. Disruption of the open reading frame eliminated cation influx activity and restored resistance to Co2+. This putative transporter, designated MgtE, has no sequence similarity to any known protein including CorA and appears to represent a new class of Mg2+ transport system.
Full Text
The Full Text of this article is available as a PDF (269.3 KB).
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Altschul S. F., Gish W., Miller W., Myers E. W., Lipman D. J. Basic local alignment search tool. J Mol Biol. 1990 Oct 5;215(3):403–410. doi: 10.1016/S0022-2836(05)80360-2. [DOI] [PubMed] [Google Scholar]
- Bullas L. R., Ryu J. I. Salmonella typhimurium LT2 strains which are r- m+ for all three chromosomally located systems of DNA restriction and modification. J Bacteriol. 1983 Oct;156(1):471–474. doi: 10.1128/jb.156.1.471-474.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Grubbs R. D., Snavely M. D., Hmiel S. P., Maguire M. E. Magnesium transport in eukaryotic and prokaryotic cells using magnesium-28 ion. Methods Enzymol. 1989;173:546–563. doi: 10.1016/s0076-6879(89)73038-x. [DOI] [PubMed] [Google Scholar]
- Hmiel S. P., Snavely M. D., Florer J. B., Maguire M. E., Miller C. G. Magnesium transport in Salmonella typhimurium: genetic characterization and cloning of three magnesium transport loci. J Bacteriol. 1989 Sep;171(9):4742–4751. doi: 10.1128/jb.171.9.4742-4751.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Hmiel S. P., Snavely M. D., Miller C. G., Maguire M. E. Magnesium transport in Salmonella typhimurium: characterization of magnesium influx and cloning of a transport gene. J Bacteriol. 1986 Dec;168(3):1444–1450. doi: 10.1128/jb.168.3.1444-1450.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Ivey D. M., Guffanti A. A., Bossewitch J. S., Padan E., Krulwich T. A. Molecular cloning and sequencing of a gene from alkaliphilic Bacillus firmus OF4 that functionally complements an Escherichia coli strain carrying a deletion in the nhaA Na+/H+ antiporter gene. J Biol Chem. 1991 Dec 5;266(34):23483–23489. [PubMed] [Google Scholar]
- Ivey D. M., Krulwich T. A. Organization and nucleotide sequence of the atp genes encoding the ATP synthase from alkaliphilic Bacillus firmus OF4. Mol Gen Genet. 1991 Oct;229(2):292–300. doi: 10.1007/BF00272169. [DOI] [PubMed] [Google Scholar]
- Kyte J., Doolittle R. F. A simple method for displaying the hydropathic character of a protein. J Mol Biol. 1982 May 5;157(1):105–132. doi: 10.1016/0022-2836(82)90515-0. [DOI] [PubMed] [Google Scholar]
- Lusk J. E., Kennedy E. P. Magneisum transport in Escherichia coli. J Biol Chem. 1969 Mar 25;244(6):1653–1655. [PubMed] [Google Scholar]
- Maguire M. E., Snavely M. D., Leizman J. B., Gura S., Bagga D., Tao T., Smith D. L. Mg2+ transporting P-type ATPases of Salmonella typhimurium. Wrong way, wrong place enzymes. Ann N Y Acad Sci. 1992 Nov 30;671:244–256. doi: 10.1111/j.1749-6632.1992.tb43800.x. [DOI] [PubMed] [Google Scholar]
- Saier M. H., Jr Computer-aided analyses of transport protein sequences: gleaning evidence concerning function, structure, biogenesis, and evolution. Microbiol Rev. 1994 Mar;58(1):71–93. doi: 10.1128/mr.58.1.71-93.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Scribner H., Eisenstadt E., Silver S. Magnesium transport in Bacillus subtilis W23 during growth and sporulation. J Bacteriol. 1974 Mar;117(3):1224–1230. doi: 10.1128/jb.117.3.1224-1230.1974. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Smith D. L., Tao T., Maguire M. E. Membrane topology of a P-type ATPase. The MgtB magnesium transport protein of Salmonella typhimurium. J Biol Chem. 1993 Oct 25;268(30):22469–22479. [PubMed] [Google Scholar]
- Smith R. L., Banks J. L., Snavely M. D., Maguire M. E. Sequence and topology of the CorA magnesium transport systems of Salmonella typhimurium and Escherichia coli. Identification of a new class of transport protein. J Biol Chem. 1993 Jul 5;268(19):14071–14080. [PubMed] [Google Scholar]
- Snavely M. D., Florer J. B., Miller C. G., Maguire M. E. Magnesium transport in Salmonella typhimurium: 28Mg2+ transport by the CorA, MgtA, and MgtB systems. J Bacteriol. 1989 Sep;171(9):4761–4766. doi: 10.1128/jb.171.9.4761-4766.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Tabor S., Richardson C. C. Effect of manganese ions on the incorporation of dideoxynucleotides by bacteriophage T7 DNA polymerase and Escherichia coli DNA polymerase I. Proc Natl Acad Sci U S A. 1989 Jun;86(11):4076–4080. doi: 10.1073/pnas.86.11.4076. [DOI] [PMC free article] [PubMed] [Google Scholar]
- von Heijne G. Control of topology and mode of assembly of a polytopic membrane protein by positively charged residues. Nature. 1989 Oct 5;341(6241):456–458. doi: 10.1038/341456a0. [DOI] [PubMed] [Google Scholar]
- von Heijne G., Manoil C. Membrane proteins: from sequence to structure. Protein Eng. 1990 Dec;4(2):109–112. doi: 10.1093/protein/4.2.109. [DOI] [PubMed] [Google Scholar]
- von Heijne G. Membrane protein structure prediction. Hydrophobicity analysis and the positive-inside rule. J Mol Biol. 1992 May 20;225(2):487–494. doi: 10.1016/0022-2836(92)90934-c. [DOI] [PubMed] [Google Scholar]