Magnesium and the Role of mgtC in Growth of Salmonella typhimurium

In Salmonella typhimurium, there are three transport systems for Mg2+, CorA, MgtA, and MgtB. The corA gene is constitutively expressed, and the encoded 40-kDa protein product mediates both the influx and efflux of Mg2+ (10, 11, 23). The mgtA and mgtCB loci encode putative P-type ATPases (10, 13, 22). Although found in prokaryotes, MgtA and MgtB share greater similarity to the eukaryotic P-type ATPases, in particular to the sarcoplasmic reticulum Ca2+-ATPases, than to other prokaryotic P-type ATPases. Both loci are under control of the two-component PhoPQ regulatory system. PhoQ is a membrane sensor-kinase that at low Mg2+ concentrations phosphorylates PhoP to activate a variety of genes including mgtA and the mgtCB operon (8, 14, 15). Recent evidence has suggested, however, that there may be an additional mechanism for transcription of the mgtA locus (28). MgtA and MgtB, unlike CorA, mediate only the influx of magnesium and are produced under Mg2+-limiting conditions. The mgtCB locus encodes two proteins. MgtC is a hydrophobic protein with a predicted molecular mass of 22.5 kDa (25, 28). MgtB, the P-type ATPase, has a molecular mass of 102 kDa (22, 25). Although MgtB exhibits extensive sequence homology to members of the P-type ATPase superfamily, MgtC does not exhibit significant sequence homology to any proteins of known function in the current databases. Recently, the mgtCB locus has been identified as part of a new pathogenicity island, SPI-3 (3). Pathogenicity islands are segments of DNA containing virulence genes that are found in pathogenic organisms but absent from phylogenetically related nonpathogenic organisms. The mgtC gene is not necessary for S. typhimurium invasion or short-term survival in epithelial or macrophage cells (24, 28). It is, however, essential for long-term survival within the macrophage and for virulence in mice (3). A functional allele of mgtA is not required for survival within the macrophage, while inactivation of mgtB has only a small effect on survival. Since addition of 25 mM Mg2+ to the macrophage growth medium rescued the ability of an mgtC mutant strain to survive long-term within the macrophage, it was suggested that MgtC may be an additional Mg2+ transporter in S. typhimurium (3). In addition to a low Mg2+ concentration, S. typhimurium bacteria that invade epithelial and macrophage cells are also exposed to acid pH (1, 6, 7, 18). Exposure to acid also regulates gene expression of mgtA and mgtCB (2, 28). At pH 7.4, suspension of cells in N minimal medium without added Mg2+ induces transcription of mgtA and mgtCB more than 1,000-fold. At pH 5.2, low concentrations of Mg2+ fail to induce mgtA, but mgtCB induction is decreased only about twofold. If cells are adapted to pH 5.2 prior to Mg2+ starvation, there is no difference in mgtA or mgtCB induction properties compared to exposure at pH 7.4. Interestingly, a functional corA allele had a significant effect on transcription of these two loci. In strains lacking CorA and grown overnight at pH 5.2, induction of transcription at low Mg2+ levels was significantly diminished for both the mgtA and mgtCB loci. These data suggest that there is an acid component to induction of mgtA and mgtCB at low Mg2+ and that a functional corA allele is required for an optimal response to low Mg2+. In this context, it is of interest that Bearson et al. (2) have recently presented evidence that PhoPQ regulation of these loci is also responsive to acid. To elucidate MgtC’s role in a low-Mg2+ environment, we compared growth and Mg2+ transport of a strain dependent on the mgtCB operon for Mg2+ uptake with a strain with an intact mgtC gene but with mgtB inactivated. In Mg2+-starved cells, although Western blot analysis indicated that MgtC is produced and maintained, an mgtC+ strain exhibited no detectable uptake of 63Ni2+ or 57Co2+. This strain could grow at pH 7.4 in the absence of added Mg2+ only after a 24-h lag period and could not grow regardless of the level of extracellular Mg2+ at pH 5.2. We interpret these results to indicate that MgtC is not a functional Mg2+ transporter and that its expression does not facilitate survival at low pH.