Ronald L. Smith

ZupT Is a Zn(II) Uptake System in Escherichia coli

Escherichia coli zupT (ygiE), encoding a ZIP family member, mediated zinc uptake. Growth of cells disrupted in both zupT and the znuABC operon was inhibited by EDTA at a much lower concentration than a single mutant or the wild type. Cells expressing ZupT from a plasmid exhibited increased uptake of (65)Zn(2+).

ZntB Is a Novel Zn2+ Transporter in Salmonella enterica Serovar Typhimurium

A Zn2+ transport system encoded by the zntB locus of Salmonella enterica serovar Typhimurium has been identified. The protein encoded by this locus is homologous to the CorA family of Mg2+ transport proteins and is widely distributed among the eubacteria. Mutations at zntB confer an increased sensitivity to the cytotoxic effects of Zn2+ and Cd2+, a phenotype that suggests that the encoded protein mediates the efflux of both cations. A direct analysis of transport activity identified a capacity for Zn2+ efflux. These data identify ZntB as a zinc efflux pathway in the enteric bacteria and assign a new function to the CorA family of cation transporters.

Magnesium transport in Salmonella typhimurium: regulation of mgtA and mgtCB during invasion of epithelial and macrophage cells.

Salmonella typhimurium contains two inducible Mg2+ transport systems, MgtA and MgtB, the latter encoded by a two-gene operon, mgtCB. Mg2+ deprivation of S. typhimurium increases transcription of both mgtA and mgtCB over a thousandfold and a similar increase occurs upon S. typhimurium invasion of epithelial cells. These increases are mediated by the phoPQ two-component signal transduction system, an essential system for S. 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 Mg2+. However, when S. typhimurium was grown at pH 5.2, the capacity of the constitutive CorA transporter in mediating Mg2+ was greater than that at pH 7.4. 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 S. typhimurium could obtain sufficient Mg2+ via the CorA system. The effect of acid pH on mgtA and mgtCB transcription was also measured. Compared to induction at pH 7.4, exposure to pH 5.2 almost completely abolished induction of mgtA at low Mg2+ concentrations but diminished induction of mgtCB 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 Mg2+ 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 mgtCB had no effect on invasion efficiency or short-term survival of S. typhimurium within the eukaryotic cells. It was concluded that the strong Mg(2+)-dependent induction of mgtA and mgtCB upon invasion of eukaryotic cells is not required because S. typhimurium lacks sufficient Mg2+ during eukaryotic cell invasion and initial intravacuolar growth.

Magnesium transport in Salmonella typhimurium: biphasic magnesium and time dependence of the transcription of the mgtA and mgtCB loci

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 Mg(2+)-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 10,000-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 half-maximal 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 Mg(2+)-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+, Ni2+ 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.

The CorA Mg2+ Transport Protein of Salmonella typhimurium MUTAGENESIS OF CONSERVED RESIDUES IN THE THIRD MEMBRANE DOMAIN IDENTIFIES A Mg2+ PORE

The CorA transport system is the major Mg2+ influx pathway for bacteria and the Archaea. CorA contains three C-terminal transmembrane segments. No conserved charged residues are apparent within the membrane, suggesting that Mg2+ influx does not involve electrostatic interactions. We have mutated conserved residues within the third transmembrane segment to identify sites involved in transport. Mutation of conserved aromatic residues at either end of the membrane segment to alternative aromatic amino acids did not affect total cation uptake or cation affinity. Mutation to alanine greatly diminished uptake with little change in cation affinity implying that the conserved aromatic residues play a structural role in stabilizing this membrane segment of CorA at the interface between the bilayer and the aqueous environment. In contrast, mutation of Tyr292, Met299, and Tyr307 greatly altered the transport properties of CorA. Y292F, Y292S, Y292C, or Y292I mutations essentially abolished transport, without effect on expression or membrane insertion. M299C and M299A mutants exhibited a decrease in cation affinity for Mg2+, Co2+, or Ni2+ of 10–50-fold without a significant change in uptake capacity. Mutations at Tyr307 had no significant effect on cation uptake capacity; however, the affinity of Y307F and Y307A mutations for Mg2+and Co2+ was decreased 3–10-fold, while affinity for Ni2+ was unchanged compared with the wild type CorA. In contrast, the affinity of the Y307S mutant for all three cations was decreased 2–5-fold. Projection of the third transmembrane segment as an α-helix suggests that Tyr292, Met299, and Tyr307 all reside on the same face of the α-helix. We interpret the transport data to suggest that a hydroxyl group is important at Tyr307, and that these three residues interact with Mg2+ during transport, forming part of the cation pore or channel within CorA.

Membrane Topology of the ZntB Efflux System of Salmonella enterica Serovar Typhimurium

The membrane topology of the ZntB Zn(2+) transport protein of Salmonella enterica serovar Typhimurium was determined by constructing deletion derivatives of the protein and genetically fusing them to blaM or lacZ cassettes. The enzymatic activities of the hybrid proteins indicate that ZntB is a bitopic integral membrane protein consisting largely of two independent domains. The first 266 amino acids form a large, highly charged domain within the cytoplasm, while the remaining 61 residues form a small membrane domain containing two membrane-spanning segments. The overall orientation towards the cytoplasm is consistent with the ability of ZntB to facilitate zinc efflux.