The Escherichia coli zupT (formerly ygiE) gene encodes a cytoplasmic membrane protein (ZupT) related to members of the eukaryotic ZIP family of divalent metal ion transporters. Previously, ZupT was shown to be responsible for uptake of zinc. In this study, we show that ZupT is a divalent metal cation transporter of broad substrate specificity. An E. coli strain with a disruption in all known iron uptake systems could grow in the presence of chelators only if zupT was expressed. Heterologous expression of Arabidopsis thaliana ZIP1 could also alleviate iron deficiency in this E. coli strain, as could expression of indigenous mntH or feoABC. Transport studies with intact cells showed that ZupT facilitates uptake of 55 Fe 2؉ similarly to uptake of MntH or Feo. Other divalent cations were also taken up by ZupT, as shown using 57 Co 2؉ . Expression of zupT rendered E. coli cells hypersensitive to Co 2؉ and sensitive to Mn 2؉ . ZupT did not appear to be metal regulated: expression of a ⌽(zupT-lacZ) operon fusion indicated that zupT is expressed constitutively at a low level.

The chemistry of Mg 2ϩ is unique among the biologically relevant cations. Compared with calcium, potassium, and sodium, Mg 2ϩ possesses the largest hydrated radius, the smallest ionic radius, and the greatest charge density (1). Its volume change from hydrated to atomic cation is almost 400-fold, whereas Ca 2ϩ , Na ϩ , and K ϩ are only 25-, 25-, and 4-fold, respectively. Waters of hydration are bound 3-4 orders of magnitude more tightly to Mg 2ϩ than to other common biological cations. The geometry of the hydration shell is octahedral with all six coordinate bond angles at angles of 90 Ϯ 6°. This contrasts with the far more flexible geometry of the Ca 2ϩ cation, which can tolerate bond angles of 90 Ϯ 35°(1-3).Study of Mg 2ϩ -dependent enzymes has revealed two types of interactions between Mg 2ϩ , enzymes, and ligands. Interaction can be through direct, inner sphere, coordination of a ligand to the metal ion or through an indirect, outer sphere, association with the bound water shell of the cation. In an outer sphere interaction, the relatively rigid geometry imposed by Mg 2ϩ

OH 44106-4965, USA Salmonella typhimurium has three distinct Mg2+ transport systems, the constitutive high-capacity CorA transporter and two P-type ATPases, MgtA and MgtB, whose transcription is repressed by normal concentrations of Mg2+ in the growth medium. The latter Mg2+-transporting ATPase is part of a two-gene operon, mgtCB, with mgtC encoding a 23 kDa protein of unknown function. Transcriptional regulation using fusions of the promoter regions of mgtA and mgtCB to luxAB showed a biphasic time and Mg2+ concentration dependence. Between 1 and 6 h after transfer to nitrogen minimal medium containing defined concentrations of Mg2+, transcription increased about 200-fold for mgtCB and up to 400-fold for mgtA, each with a half-maximal dependence on Mg2+ of 0 5 mM. Continued incubation revealed a second phase of increased transcription, up to 2000-fold for mgtCB and up to 10000-fold for mgtA. This secondary increase occurred between 6 and 9 h after transfer to defined medium for mgtCB but between 12 and 24 h for mgtA and had a distinct halfmaximal dependence for Mg2+ of 0.01 mM. A concomitant increase of at least 1000-fold in uptake of cation was seen between 8 and 24 h incubation with either system, showing that the transcriptional increase was followed by functional incorporation of large amounts of the newly synthesized transporter into the membrane. Regulation of transcription by Mg2+ was not dependent on a functional stationary-phase sigma factor encoded by rpoS, but it was dependent on the presence of a functional phoPQ two-component regulatory system. Whereas mgtCB was completely dependent on regulation via phoPQ, the secondary late Mg2+-dependent phase of mgtA transcription was still evident in strains carrying a mutation in either phoP or phoQ, albeit substantially diminished. Several divalent cations blocked the early phase of the increase in transcription elicited by the decrease in Mg2+ concentration, including cations that inhibit Mg2+ uptake (Co2+, NiZ+ and Mn2+) and those which do not (Ca2+ and Zn2+). In contrast, the second later phase of the transcriptional increase was not well blocked by any cation except those which inhibit uptake. Overall, the data suggest that at least two distinct mechanisms for transcriptional regulation of the mgtA and mgtCB loci exist.

Salmonella typhimurium contains two inducible Mgz+ transport systems, MgtA and MgtB, the latter encoded by a two-gene operon, mgtC6. Mg2+ deprivation of S. typhimurium increases transcription of both mgtA and mgtCB over a thousandfold and a similar increase occurs upon S. ephimurium invasion of epithelial cells. These increases are mediated by the phoPQ two-component signal transduction system, an essential system for 5. typhimurium virulence. It was therefore hypothesized that expression of MgtA and MgtCB is increased upon invasion of eukaryotic cells because of a lack of intravacuolar Mg? However, when S. typhimurium was grown a t pH 5.2. the capacity of the constitutive CorA transporter in mediating Mgz+ was greater than that a t pH 74. Furthermore, induction of mgtA and mgtCB transcription was greater in the presence of a wild-type corA allele than in its absence. This implies that intravacuolar 5. typhimurium could obtain sufficient Mg2* via the CorA system. The effect of acid pH on mgtA and mgtCf? transcription was also measured. Compared to induction at pH 74, exposure to pH 5.2 almost completely abolished induction of mgtA at low Mg2+ concentrations but diminished induction of m g K B only twofold. Adaptation of cells to acid pH by overnight growth resulted in normal levels of induction of mgtA and mgtCB at low Mg2+ concentrations. These results imply an additional level of regulation for mgtA that is not present for mgtCB. Conversely, repression of mgtA and mgtCB expression by increased extracellular Mgz* was relatively insensitive to acid.Transcription of both loci was strongly induced upon invasion of the Hep-2 or CMT-93 epithelial-like or J774 macrophage-like cell lines. However, the presence or absence of functional alleles of either or both mgtA or -5 had no effect on invasion efficiency or short-term survival of S. typhimurium within the eukaryotic cells. It was concluded that the strong Mgz*-dependent induction of mgtA and mgtCB upon invasion of eukaryotic cells is not required because 5. typhimurium lacks sufficient Mgz+ during eukaryotic cell invasion and initial intravacuolar growth.

No abstract