NRAMPs (natural resistance‐associated macrophage proteins) have been characterized in mammals as divalent transition metal transporters involved in iron metabolism and host resistance to certain pathogens. The mechanism of pathogen resistance is proposed to involve sequestration of Fe2+ and Mn2+, cofactors of both prokaryotic and eukaryotic catalases and superoxide dismutases, not only to protect the macrophage against its own generation of reactive oxygen species, but to deny the cations to the pathogen for synthesis of its protective enzymes. NRAMP homologues are also present in bacteria. We report the cloning and characterization of the single NRAMP genes in Escherichia coli and Salmonella enterica ssp. typhimurium, and the cloning of two distinct NRAMP genes from Pseudomonas aeruginosa and an internal fragment of an NRAMP gene in Burkholderia cepacia. The genes are designated mntH because the two enterobacterial NRAMPs encode H+‐stimulated, highly selective manganese(II) transport systems, accounting for all Mn2+ uptake in each species under the conditions tested. For S. typhimurium MntH, the Km for 54Mn2+ (≈ 0.1 µM) was pH independent, but maximal uptake increased as pH decreased. Monovalent cations, osmotic strength, Mg2+ and Ca2+ did not inhibit 54Mn2+ uptake. Ni2+, Cu2+ and Zn2+ inhibited uptake with Kis greater than 100 µM, Co2+ with a Ki of 20 µM and Fe2+ with a Ki that decreased from 100 µM at pH 7.6 to 10 µM at pH 5.5. Fe3+ and Pb2+ inhibited weakly, exhibiting Kis of 50 µM, while Cd2+ was a potent inhibitor with a Ki of about 1 µM. E. coli MntH had a similar inhibition profile, except that Kis were three‐ to 10‐fold higher. Both S. typhimurium and E. coli MntH also transport 55Fe2+ however, the Kms are equivalent to the Kis for Fe2+ inhibition of Mn2+ uptake, and are thus too high to be physiologically relevant. In both S. typhimurium and E. coli, mntH::lacZ constructs were strongly induced by hydrogen peroxide, weakly induced by EDTA and unresponsive to paraquat, consistent with the presence of Fur and OxyR binding sites in the promoters. Strains overexpressing mntH were more susceptible to growth inhibition by Mn2+ and Cd2+ than wild type, and strains lacking a functional mntH gene were more susceptible to killing by hydrogen peroxide. In S. typhimurium strain SL1344, mntH mutants showed no defect in invasion of or survival in cultured HeLa or RAW264.7 macrophage cells; however, expression of mntH::lacZ was induced severalfold by 3 h after invasion of the macrophages. S. typhimurium mntH mutants showed only a slight attenuation of virulence in BALB/c mice. Thus, the NRAMP Mn2+ transporter MntH and Mn2+ play a role in bacterial response to reactive oxygen species and possibly have a role in pathogenesis.

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.

No abstract

No abstract

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 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 Zn 2؉ , whereas DNA binding by TroR in vitro is Mn 2؉ -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 Zn 2؉ Treponema pallidum, the causative agent of syphilis, is a noncultivable sexually transmitted human pathogen that disseminates from a site of inoculation, usually within the genital area, to diverse organs where it can establish persistent, even lifelong, infection (1). Like all pathogens, T. pallidum presumably requires sequestered transition metals for vital structural and catalytic functions in proteins. Nevertheless, in contrast to other pathogenic bacteria in which specialized uptake systems for iron (2-4), and more recently for Zn 2ϩ and Mn 2ϩ (5-8), have been identified, there is little information about metal acquisition by the syphilis spirochete. The lack of techniques for in vitro cultivation and genetic manipulation of T. pallidum has greatly hindered investigation of treponemal physiology, including metal metabolism.Two recent developments have spawned interest in the processes by which T. pallidum acquires trace metals. One is the availability of the genomic sequence of the spirochete (9), which reveals that many genes encoding well characterized iron-containing proteins (e.g. cytochromes, superoxide dismutase, and tricarboxylic acid metalloenzymes) are lacking. The finding that T. pallidum expresses at least two experimentally confirmed iron-binding proteins, superoxide reductase (neelaredoxin) and rubredoxin, (10 -12), argues that the bacterium does require iron. The second impetus to study metal utilization by T. pallidum is provided by the discovery of the six gene tro (transport-related operon) ( Fig. 1) (13). The first gene of the operon, troA, encodes the solute binding protein (SBP) 1 component of an ATP-binding cassette transporter (13-15) belonging to a newly described cluster (cluster nine or C9) of transition metal permeases (5). In addition to encoding an ATPase (TroB), two cytoplasmic membrane permeases (TroC and TroD), and a DtxR-like metalloregulator (TroR), the operon also encodes the glycolytic enzyme phosphoglyceromutase (Gpm) (13). Because the T. pallidum Gpm has no requirement for metals (16), the physiological benefit of transcriptionally linking the spirochete's sole copy of this essential enzyme to the Tro transporter genes is unclear.The identity of the metal ligand(s) fo...

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.

The CorA transport system is the primary Mg2+ influx system of Salmonella typhimurium and Escherichia coli. The CorA protein has no homology to any other known family of proteins. It has an unusual membrane topology, with a large, soluble, highly charged periplasmic N-terminal domain with three transmembrane segments in a shorter, hydrophobic C-terminal domain. Previous phenotypic and molecular data had suggested that this transport system was widespread in the Bacteria. In this report we show that CorA is virtually ubiquitous in the Bacteria and Archaea, forming a distinct family of transport proteins. Genomic sequences to date have revealed at least 22 members of the CorA family in the Bacteria and the Archaea, with 6 more distant members in the yeasts. Only three of the smallest bacterial genomes lack a CorA homologue. Strikingly, phylogenetic analysis does not show clustering by related species or even within kingdom. Several species of Bacteria contain two or even three CorA paralogues. Within species, these paralogues are not closely related, however, and we suggest that they might have distinct transport functions. A multiple alignment suggests three extended consensus regions within the N-terminal soluble domain of CorA, which is predicted to be virtually all alpha-helical. A fourth consensus region includes the last 20 residues of the soluble domain and continues through the entire membrane domain. The first half of this last consensus domain may form an amphipathic alpha-helix that extends from the soluble domain into the first transmembrane segment. The degree of charge in the first transmembrane segment is quite variable, and we suggest that this transport family may include members with only two rather than three transmembrane segments. If so, this would place the N-terminal soluble domain on different sides of the membrane in different members of the family. We suggest that the CorA Mg2+ transport system forms the major Mg2+ uptake system in the Bacteria and Archaea but that some family members may have a function other than Mg2+ transport.