The CorA Mg2+ channel is required for the virulence of Salmonella enterica serovar typhimurium.

CorA is the primary Mg(2+) channel in Salmonella enterica serovar Typhimurium. A corA mutant is attenuated in mice and defective for invasion of and replication within epithelial cells. Microarray studies show that several virulence effectors are repressed in a corA mutant strain, which ultimately manifests itself as a decrease in virulence.

The MgtC virulence factor of Salmonella enterica serovar Typhimurium activates Na(+),K(+)-ATPase.

The mgtC gene of Salmonella enterica serovar Typhimurium encodes a membrane protein of unknown function that is important for full virulence in the mouse. Since mgtC is part of an operon with mgtB which encodes a Mg(2+)-transporting P-type ATPase, MgtC was hypothesized to function in ion transport, possibly in Mg(2+) transport. Consequently, MgtC was expressed in Xenopus laevis oocytes, and its effect on ion transport was evaluated using ion selective electrodes. Oocytes expressing MgtC did not exhibit altered currents or membrane potentials in response to changes in extracellular H(+), Mg(2+), or Ca(2+), thus ruling out a previously postulated function as a Mg(2+)/H(+) antiporter. However, addition of extracellular K(+) markedly hyperpolarized membrane potential instead of the expected depolarization. Addition of ouabain to block the oocyte Na(+),K(+)-ATPase completely prevented hyperpolarization and restored the normal K(+)-induced depolarization response. These results suggested that the Na(+),K(+)-ATPase was constitutively activated in the presence of MgtC resulting in a membrane potential largely dependent on Na(+),K(+)-ATPase. Consistent with the involvement of Na(+),K(+)-ATPase, oocytes expressing MgtC exhibited an increased rate of (86)Rb(+) uptake and had increased intracellular free [K(+)] and decreased free [Na(+)] and ATP. The free concentrations of Mg(2+) and Ca(2+) and cytosolic pH were unchanged, although the total intracellular Ca(2+) content was slightly elevated. These results suggest that the serovar Typhimurium MgtC protein may be involved in regulating membrane potential but does not directly transport Mg(2+) or another ion.

Transcriptional regulation of sitABCD of Salmonella enterica serovar Typhimurium by MntR and Fur.

Salmonella enterica serovar Typhimurium has two manganese transport systems, MntH and SitABCD. MntH is a bacterial homolog of the eukaryotic natural resistance-associated macrophage protein 1 (Nramp1), and SitABCD is an ABC-type transporter. Previously we showed that mntH is negatively controlled at the transcriptional level by the trans-acting regulatory factors, MntR and Fur. In this study, we examined the transcriptional regulation of sitABCD and compared it to the transcriptional regulation of mntH by constructing lacZ fusions to the promoter regions with and without mutations in putative MntR and/or Fur binding sites. The presence of Mn caused transcriptional repression of the sitABCD and mntH promoters primarily via MntR, but Fur was also capable of some repression in response to Mn. Likewise, Fe in the medium repressed transcription of both sit and mntH primarily via Fur, although MntR was also involved in this response. Transcriptional control by MntR and Fur was disrupted by site-specific mutations in the putative MntR and Fur binding sites, respectively. Transcription of the sit operon was also affected by the oxygen level and growth phase, but the increased expression observed under high oxygen conditions and higher cell densities is consistent with decreased availability of metals required for repression by the metalloregulatory proteins.

The Salmonella enterica serovar typhimurium divalent cation transport systems MntH and SitABCD are essential for virulence in an Nramp1G169 murine typhoid model.

Nramp1 is a transporter that pumps divalent cations from the vacuoles of phagocytic cells and is associated with the innate resistance of mice to diverse intracellular pathogens. We demonstrate that sitA and mntH, genes encoding high-affinity metal ion uptake systems in Salmonella enterica serovar Typhimurium, are upregulated when Salmonella is internalized by Nramp1-expressing macrophages and play an essential role in systemic infection of congenic Nramp1-expressing mice.

Emerging themes in manganese transport, biochemistry and pathogenesis in bacteria.

Though an essential trace element, manganese is generally accorded little importance in biology other than as a cofactor for some free radical detoxifying enzymes and in the photosynthetic photosystem II. Only a handful of other Mn2+-dependent enzymes are known. Recent data, primarily in bacteria, suggest that Mn2+-dependent processes may have significantly greater physiological importance. Two major classes of prokaryotic Mn2+ uptake systems have now been described, one homologous to eukaryotic Nramp transporters and one a member of the ABC-type ATPase superfamily. Each is highly selective for Mn2+ over Fe2+ or other transition metal divalent cations, and each can accumulate millimolar amounts of intracellular Mn2+ even when environmental Mn2+ is scarce. In Salmonella enterica serovar Typhimurium, simultaneous mutation of both types of transporter results in avirulence, implying that one or more Mn2+-dependent enzymes is essential for pathogenesis. This review summarizes current literature on Mn2+ transport, primarily in the Bacteria but with relevant comparisons to the Archaea and Eukaryota. Mn2+-dependent enzymes are then discussed along with some speculations as to their role(s) in cellular physiology, again primarily in Bacteria. It is of particular interest that most of the enzymes which interconvert phosphoglycerate, pyruvate, and oxaloacetate intermediates are either strictly Mn2+-dependent or highly stimulated by Mn2+. This suggests that Mn2+ may play an important role in central carbon metabolism. Further studies will be required, however, to determine whether these or other actions of Mn2+ within the cell are the relevant factors in pathogenesis.

The Treponema pallidum tro operon encodes a multiple metal transporter, a zinc-dependent transcriptional repressor, and a semi-autonomously expressed phosphoglycerate mutase.

The Treponema pallidum tro operon encodes an ABC transporter (TroABCD), a transcriptional repressor (TroR), and the essential glycolytic enzyme phosphoglycerate mutase (Gpm). The apparently discordant observations that the solute binding protein (TroA) binds Zn2+, whereas DNA binding by TroR in vitro is Mn2+-dependent, have generated uncertainty regarding the identities of the ligand(s) and co-repressor(s) of the permease. Moreover, this operonic structure suggests that Gpm expression, and hence glycolysis, the sole source of ATP for the bacterium, would be suspended during TroR-mediated repression. To resolve these discrepancies, we devised an experimental strategy permitting a more direct assessment of Tro operon function and regulation. We report that (i) apo-TroA has identical affinities for Zn2+ and Mn2+; (ii) the Tro transporter expressed in Escherichia coli imports Zn2+, Mn2+, and possibly iron; (iii) TroR represses transporter expression in E. coli at significantly lower concentrations of Zn2+ than of Mn2+; and (iv) TroR-mediated repression causes a disproportionately greater down-regulation of the transporter genes than of gpm. The much higher concentrations of Zn2+ than of Mn2+ in human body fluids suggests that Zn2+ is both the primary substrate and co-repressor of the permease in vivo. Our data also indicate that Gpm expression and, therefore, glycolysis would not be abrogated when T. pallidum encounters high Zn2+ levels.

Regulation of Salmonella enterica serovar Typhimurium mntH transcription by H(2)O(2), Fe(2+), and Mn(2+).

MntH, a bacterial homolog of mammalian natural resistance associated macrophage protein 1 (Nramp1), is a primary transporter for Mn(2+) influx in Salmonella enterica serovar Typhimurium and Escherichia coli. S. enterica serovar Typhimurium MntH contributes to H(2)O(2) resistance and is important for full virulence. Consistent with its phenotype and function, mntH is regulated at the transcriptional level by both H(2)O(2) and substrate cation. We have now identified three trans-acting regulatory factors and the three corresponding cis-acting mntH promoter motifs that mediate this regulation. In the presence of hydrogen peroxide, mntH is activated by OxyR, acting through an OxyR-binding motif centered just upstream of the likely -35 RNA polymerase-binding site. In the presence of Fe(2+), mntH is repressed primarily by Fur, acting through a Fur-binding motif overlapping the -35 region. In the presence of Mn(2+), mntH is repressed primarily by the Salmonella equivalent of E. coli b0817, a distant homolog of the Bacillus subtilis manganese transport repressor, MntR, acting through an inverted-repeat motif located between the likely -10 polymerase binding site and the ribosome binding site. E. coli b0817 was recently shown to bind the identical inverted-repeat motif in the E. coli mntH promoter and hence has been renamed MntR (S. I. Patzer and K. Hantke, J. Bacteriol. 183:4806-4813, 2001). Using Deltafur, DeltamntR, and Deltafur DeltamntR mutant strains as well as mutations in the Fur- and MntR-binding motif elements, we found that Fe(2+) can also mediate repression through the Mn(2+) repressor MntR.

SitABCD is the alkaline Mn(2+) transporter of Salmonella enterica serovar Typhimurium.

MntH, a bacterial homolog of the mammalian natural resistance-associated macrophage protein 1 (Nramp1), is a primary Mn(2+) transporter of Salmonella enterica serovar Typhimurium and Escherichia coli. S. enterica serovar Typhimurium MntH expression is important for full virulence; however, strains carrying an mntH deletion are only partially attenuated and display no obvious signs of Mn(2+) deficiency. We noted that promoter sequences for mntH and for the putative Fe(2+) transporter sitABCD appeared to have the same regulatory element responsive to Mn(2+) and so hypothesized that sitABCD could transport Mn(2+) with high affinity. We have now characterized transport by SitABCD in S. enterica serovar Typhimurium using (54)Mn(2+) and (55)Fe(2+) and compared its properties to those of MntH. SitABCD mediates the influx of Mn(2+) with an apparent affinity (K(a)) identical to that of MntH, 0.1 microM. It also transports Fe(2+) but with a K(a) 30 to 100 times lower, 3 to 10 microM. Inhibition of (54)Mn(2+) transport by Fe(2+) and of (55)Fe(2+) transport by Mn(2+) gave inhibition constants comparable to each cation's K(a) for influx. Since micromolar concentrations of free Fe(2+) are improbable in a biological system, we conclude that SitABCD functions physiologically as a Mn(2+) transporter. The cation inhibition profiles of SitABCD and MntH are surprisingly similar for two structurally and energetically unrelated transporters, with a Cd(2+) K(i) of approximately 1 microM and a Co(2+) K(i) of approximately 20 microM and with Ni(2+), Cu(2+), and Fe(3+) inhibiting both transporters only at concentrations of >0.1 mM. The one difference is that Zn(2+) exhibits potent inhibition of SitABCD (K(i) = 1 to 3 microM) but inhibits MntH weakly (K(i) > 50 microM). We have previously shown that MntH transports Mn(2+) most effectively under acidic conditions. In sharp contrast, SitABCD has almost no transport capacity at acid pHs and optimally transports Mn(2+) at slightly alkaline pHs. Overall, coupled with evidence that each transporter is multiply but distinctly regulated at the transcriptional level, the distinct transport properties of MntH versus SitABCD suggest that each transporter may be specialized for Mn(2+) uptake in different physiological environments.