Phenotypic and Genetic Analysis of ``chameleon,'''' a Paramecium Mutant with an Enhanced Sensitivity to Magnesium

Three mutant strains of Paramecium tetraurelia with an enhanced sensitivity to magnesium have been isolated. These new ``Chameleon' mutants result from partial- or codominant mutations at a single locus, Cha. Whereas the wild type responded to 5 mM Mg(2+) by swimming backward for 10-15 sec, Cha mutants responded with ~30 sec backward swimming. Electrophysiological analysis suggested that this behavior may be caused by slowing in the rate at which a Mg(2+)-specific ion conductance deactivates following membrane excitation. This would be consistent with an observed increase in the sensitivity of Cha mutants to nickel poisoning, since Ni(2+) is also able to enter the cell via this pathway. More extensive behavioral analysis showed that Cha cells also overresponded to Na(+), but there was no evidence for a defect in intracellular Ca(2+) homeostasis that might account for a simultaneous enhancement of both the Mg(2+) and Na(+) conductances. The possibility that the Cha locus may encode a specific regulator of the Mg(2+)- and Na(+)-permeabilities is considered.     

Acid resistance of Helicobacter pylori depends on the UreI membrane protein and an inner membrane proton barrier

ureI encodes an inner membrane protein of Helicobacter pylori. The role of the bacterial inner membrane and UreI in acid protection and regulation of cytoplasmic urease activity in the gastric microorganism was studied. The irreversible inhibition of urease when the organism was exposed to a protonophore (3,3',4', 5-tetrachlorsalicylanide; TCS) at acidic pH showed that the inner membrane protected urease from acid. Isogenic ureI knockout mutants of several H. pylori strains were constructed by replacing the ureI gene of the urease gene cluster with a promoterless kanamycin resistance marker gene (kanR). Mutants carrying the modified ureAB-kanR-EFGH operon all showed wild-type levels of urease activity at neutral pH in vitro. The mutants resisted media of pH > 4.0 but not of pH < 4.0. Whereas wild-type bacteria showed high levels of urease activity below pH 4.0, this ability was not retained in the ureI mutants, resulting in inhibition of metabolism and cell death. Gene complementation experiments with plasmid-derived H. pylori ureI restored wild-type properties. The activation of urease activity found in structurally intact but permeabilized bacteria treated with 0.01% detergent (polyoxy-ethylene-8-laurylether; C12E8), suggested a membrane-limited access of urea to internal urease at neutral pH. Measurement of 14C-urea uptake into Xenopus oocytes injected with ureI cRNA showed acid activation of uptake only in injected oocytes. Acceleration of urea uptake by UreI therefore mediates the increase of intracellular urease activity seen under acidic conditions. This increase of urea permeability is essential for H. pylori survival in environments below pH 4.0. ureI-independent urease activity may be sufficient for maintenance of bacterial viability above pH 4.0.     

Characterization of cadmium uptake in Lactobacillus plantarum and isolation of cadmium and manganese uptake mutants.

Two different Cd(2+) uptake systems were identified in Lactobacillus plantarum. One is a high-affinity, high-velocity Mn(2+) uptake system which also takes up Cd(2+) and is induced by Mn(2+) starvation. The calculated K(m) and V(max) are 0.26 microM and 3.6 micromol g of dry cell(-1) min(-1), respectively. Unlike Mn(2+) uptake, which is facilitated by citrate and related tricarboxylic acids, Cd(2+) uptake is weakly inhibited by citrate. Cd(2+) and Mn(2+) are competitive inhibitors of each other, and the affinity of the system for Cd(2+) is higher than that for Mn(2+). The other Cd(2+) uptake system is expressed in Mn(2+)-sufficient cells, and no K(m) can be calculated for it because uptake is nonsaturable. Mn(2+) does not compete for transport through this system, nor does any other tested cation, i.e., Zn(2+), Cu(2+), Co(2+), Mg(2+), Ca(2+), Fe(2+), or Ni(2+). Both systems require energy, since uncouplers completely inhibit their activities. Two Mn(2+)-dependent L. plantarum mutants were isolated by chemical mutagenesis and ampicillin enrichment. They required more than 5,000 times as much Mn(2+) for growth as the parental strain. Mn(2+) starvation-induced Cd(2+) uptake in both mutants was less than 5% the wild-type rate. The low level of long-term Mn(2+) or Cd(2+) accumulation by the mutant strains also shows that the mutations eliminate the high-affinity Mn(2+) and Cd(2+) uptake system.     

Characterization of the DNA- and metal-binding properties of Vibrio anguillarum fur reveals conservation of a structural Zn(2+) ion

The ferric uptake regulator, Fur, represses iron uptake and siderophore biosynthetic genes under iron-replete conditions. Here we report in vitro solution studies on Vibrio anguillarum Fur binding to the consensus 19-bp Escherichia coli iron box in the presence of several divalent metals. We found that V. anguillarum Fur binds the iron box in the presence of Mn(2+), Co(2+), Cd(2+), and to a lesser extent Ni(2+) but, unlike E. coli Fur, not in the presence of Zn(2+). We also found that V. anguillarum Fur contains a structural zinc ion that is necessary yet alone is insufficient for DNA binding.     

Enhanced mercury biosorption by bacterial cells with surface-displayed MerR

The metalloregulatory protein MerR, which exhibits high affinity and selectivity toward mercury, was exploited for the construction of microbial biosorbents specific for mercury removal. Whole-cell sorbents were constructed with MerR genetically engineered onto the surface of Escherichia coli cells by using an ice nucleation protein anchor. The presence of surface-exposed MerR on the engineered strains enabled sixfold-higher Hg(2+) biosorption than that found in the wild-type JM109 cells. Hg(2+) binding via MerR was very specific, with no observable decline even in the presence of 100-fold excess Cd(2+) and Zn(2+). The Hg(2+) binding property of the whole-cell sorbents was also insensitive to different ionic strengths, pHs, and the presence of metal chelators. Since metalloregulatory proteins are currently available for a wide variety of toxic heavy metals, our results suggest that microbial biosorbents overexpressing metalloregulatory proteins may be used similarly for the cleanup of other important heavy metals.     

Roles of His-rich hpn and hpn-like proteins in Helicobacter pylori nickel physiology

Individual gene-targeted hpn and hpn-like mutants and a mutant with mutations in both hpn genes were more sensitive to nickel, cobalt, and cadmium toxicity than was the parent strain, with the hpn-like strain showing the most metal sensitivity of the two individual His-rich protein mutants. The mutant strains contained up to eightfold more urease activity than the parent under nickel-deficient conditions, and the parent strain was able to achieve mutant strain activity levels by nickel supplementation. The mutants contained 3- to 4-fold more and the double mutant about 10-fold more Ni associated with their total urease pools, even though all of the strains expressed similar levels of total urease protein. Hydrogenase activities in the mutants were like those in the parent strain; thus, hydrogenase is fully activated under nickel-deficient conditions. The histidine-rich proteins appear to compete with the Ni-dependent urease maturation machinery under low-nickel conditions. Upon lowering the pH of the growth medium from 7.3 to 5, the wild-type urease activity increased threefold, but the activity in the three mutant strains was relatively unaffected. This pH effect was attributed to a nickel storage role for the His-rich proteins. Under low-nickel conditions, the addition of a nickel chelator did not significantly affect the urease activity of the wild type but decreased the activity of all of the mutants, supporting a role for the His-rich proteins as Ni reservoirs. These nickel reservoirs significantly impact the active urease activities achieved. The His-rich proteins play dual roles, as Ni storage and as metal detoxification proteins, depending on the exogenous nickel levels.     

Functional reconstitution and characterization of the Arabidopsis Mg(2+) transporter AtMRS2-10 in proteoliposomes

Magnesium (Mg(2+)) plays critical role in many physiological processes. The mechanism of Mg(2+) transport has been well documented in bacteria; however, less is known about Mg(2+) transporters in eukaryotes. The AtMRS2 family, which consists of 10 Arabidopsis genes, belongs to a eukaryotic subset of the CorA superfamily proteins. Proteins in this superfamily have been identified by a universally conserved GlyMetAsn motif and have been characterized as Mg(2+) transporters. Some members of the AtMRS2 family, including AtMRS2-10, may complement bacterial mutants or yeast mutants that lack Mg(2+) transport capabilities. Here, we report the purification and functional reconstitution of AtMRS2-10 into liposomes. AtMRS2-10, which contains an N-terminal His-tag, was expressed in Escherichia coli and solubilized with sarcosyl. The purified AtMRS2-10 protein was reconstituted into liposomes. AtMRS2-10 was inserted into liposomes in a unidirectional orientation. Direct measurement of Mg(2+) uptake into proteoliposomes revealed that reconstituted AtMRS2-10 transported Mg(2+) without any accessory proteins. Mutation in the GMN motif, M400 to I, inactivated Mg(2+) uptake. The AtMRS2-10-mediated Mg(2+) influx was blocked by Co(III)hexamine, and was independent of the external pH from 5 to 9. The activity of AtMRS2-10 was inhibited by Co(2+) and Ni(2+); however, it was not inhibited by Ca(2+), Fe(2+), or Fe(3+). While these results indicate that AtMRS2-10 has similar properties to the bacterial CorA proteins, unlike bacterial CorA proteins, AtMRS2-10 was potently inhibited by Al(3+). These studies demonstrate the functional capability of the AtMRS2 proteins in proteoliposomes to study structure-function relationships.     

Construction and characterization of a photosynthetic bacterium genetically engineered for Hg2+ uptake

A recombinant photosynthetic bacterium, Rhodopseudomonas palustris, was constructed to simultaneously express mercury transport system and metallothionein for Hg(2+) removal from heavy metal wastewater. The effects of essential process parameters, including pH, ionic strength and presence of co-ions on Hg(2+) uptake were evaluated. The results showed that compared with wild type R. palustris, recombinant strain displayed stronger resistance to toxic Hg(2+), and its Hg(2+) binding capacity was enhanced threefolds. In the range of pH 4-10, recombinant R. palustris maintained effective accumulation of Hg(2+). The presence of 10 mg L(-1) Mg(2+), Ca(2+), Zn(2+) or Ni(2+) did not significantly influence Hg(2+) bioaccumulation by recombinant R. palustris from solutions containing 0.2 mg L(-1) Hg(2+), while Na(+) and Cd(2+) posed serious adverse effect on Hg(2+) uptake. Furthermore, EDTA treatment experiment confirmed that different from wild type R. palustris that mainly absorbed Hg(2+) on the cell surface, recombinant R. palustris transported most of the bound Hg(2+) into the cells.     

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+).     

Escherichia coli HypA is a zinc metalloprotein with a weak affinity for nickel

The hyp operon encodes accessory proteins that are required for the maturation of the [NiFe] hydrogenase enzymes and, in some organisms, for the production of urease enzymes as well. HypA or a homologous protein is required for nickel insertion into the hydrogenase precursor proteins. In this study, recombinant HypA from Escherichia coli was purified and characterized in vitro. Metal analysis was used to demonstrate that HypA simultaneously binds stoichiometric Zn(2+) and stoichiometric Ni(2+). Competition experiments with a metallochromic indicator reveal that HypA binds zinc with nanomolar affinity. Spectroscopic analysis of cobalt-containing HypA provides evidence for a tetrathiolate coordination sphere, suggesting that the zinc site has a structural role. In addition, HypA can exist as several oligomeric complexes and the zinc content modulates the quaternary structure of the protein. Fluorescence titration experiments demonstrate that HypA binds nickel with micromolar affinity and that the presence of zinc does not dramatically affect the nickel-binding activity. Finally, complex formation between HypA and HypB, another accessory protein required for nickel insertion, was observed. These experiments suggest that HypA is an architectural component of the hydrogenase metallocenter assembly pathway and that it may also have a direct role in the delivery of nickel to the hydrogenase large subunit.     

Nic1p, a relative of bacterial transition metal permeases in Schizosaccharomyces pombe, provides nickel ion for urease biosynthesis

The Schizosaccharomyces pombe genome sequencing project identified an open reading frame (O74869 and O74912, named Nic1p in the present study) with significant similarity to members of a family of bacterial transition metal permeases. These uptake systems transport Ni(2+) ion with extremely high affinity across the bacterial cytoplasmic membrane, but they differ in selectivity toward divalent transition metal cations. An S. pombe mutant harboring an interrupted nic1 allele (nic1-1) was strongly impaired in (63)Ni(2+) uptake in the presence of a high molar ratio of Mg(2+) relative to Ni(2+), conditions that reflect the natural situation. Under these conditions, the nic1-1 mutant contained only background activities of the nickel-dependent cytoplasmic enzyme urease and could not catabolize urea. Among a series of divalent transition metal cations tested (Cd(2+), Co(2+), Cu(2+), Mn(2+), and Zn(2+)), only Co(2+) caused considerable inhibition of Nic1p-mediated Ni(2+) uptake. On the other hand, experiments with (57)Co(2+) (at nm concentrations) did not show significant differences in Co(2+) uptake between the nic1-1 mutant and the parental strain. Our data suggest that Nic1p acts as a plasma-membrane nickel transporter in fission yeast, a finding that invites searches for isologous counterparts in higher eukaryotes.     

In vivo and in vitro kinetics of metal transfer by the Klebsiella aerogenes urease nickel metallochaperone, UreE

The urease accessory protein encoded by ureE from Klebsiella aerogenes is proposed to deliver Ni(II) to the urease apoprotein during enzyme activation. Native UreE possesses a histidine-rich region at its carboxyl terminus that binds several equivalents of Ni(2+); however, a truncated form of this protein (H144*UreE) binds only 2 Ni(2+) per dimer and is functionally active (Brayman, T. G., and Hausinger, R. P. (1996) J. Bacteriol. 178, 5410-5416). The urease activation kinetics were studied in vivo by monitoring the development of urease activity upon adding Ni(2+) to spectinomycin-treated Escherichia coli cells that expressed the complete K. aerogenes urease gene cluster with altered forms of ureE. Site-specific alterations of H144*UreE decrease the rate of in vivo urease activation, with the most dramatic changes observed for the H96A, H110A, D111A, and H112A substitutions. Notably, urease activity in cells producing H96A/H144*UreE was lower than cells containing a ureE deletion. Prior studies had shown that H110A and H112A variants each bound a single Ni(2+) per dimer with elevated K(d) values compared with control H144*UreE, whereas the H96A and D111A variants bound 2 Ni(2+) per dimer with unperturbed K(d) values (Colpas, G. J., Brayman, T. G., Ming, L.-J., and Hausinger, R. P. (1999) Biochemistry 38, 4078-4088). To understand why cells containing the latter two proteins showed reduced rates of urease activation, we characterized their metal binding/dissociation kinetics and compared the results to those obtained for H144*UreE. The truncated protein was shown to sequentially bind two Ni(2+) with k(1) approximately 18 and k(2) approximately 100 M(-1) s(-1), and with dissociation rates k(-1) approximately 3 x 10(-3) and k(-2) approximately 10(-4) s(-1). Similar apparent rates of binding and dissociation were noted for the two mutant proteins, suggesting that altered H144*UreE interactions with Ni(2+) do not account for the changes in cellular urease activation. These conclusions are further supported by in vitro experiments demonstrating that addition of H144*UreE to urease apoprotein activation mixtures inhibited the rate and extent of urease formation. Our results highlight the importance of other urease accessory proteins in assisting UreE-dependent urease maturation.     

A histidine-rich and cysteine-rich metal-binding domain at the C terminus of heat shock protein A from Helicobacter pylori: implication for nickel homeostasis and bismuth susceptibility

HspA, a member of the GroES chaperonin family, is a small protein found in Helicobacter pylori with a unique histidine- and cysteine-rich domain at the C terminus. In this work, we overexpressed, purified, and characterized this protein both in vitro and in vivo. The apo form of the protein binds 2.10 +/- 0.07 Ni(2+) or 1.98 +/- 0.08 Bi(3+) ions/monomer with a dissociation constant (K(d)) of 1.1 or 5.9 x 10(-19) microm, respectively. Importantly, Ni(2+) can reversibly bind to the protein, as the bound nickel can be released either in the presence of a chelating ligand, e.g. EDTA, or at an acidic pH (pH((1/2)) 3.8 +/- 0.2). In contrast, Bi(3+) binds almost irreversibly to the protein. Both gel filtration chromatography and native electrophoresis demonstrated that apo-HspA exists as a heptamer in solution. Unexpectedly, binding of Bi(3+) to the protein altered its quaternary structure from a heptamer to a dimer, indicating that bismuth may interfere with the biological functions of HspA. When cultured in Ni(2+)-supplemented M9 minimal medium, Escherichia coli BL21(DE3) cells expressing wild-type HspA or the C-terminal deletion mutant clearly indicated that the C terminus might protect cells from high concentrations of external Ni(2+). However, an opposite phenomenon was observed when the same E. coli hosts were grown in Bi(3+)-supplemented medium. HspA may therefore play a dual role: to facilitate nickel acquisition by donating Ni(2+) to appropriate proteins in a nickel-deficient environment and to carry out detoxification via sequestration of excess nickel. Meanwhile, HspA can be a potential target of the bismuth antiulcer drug against H. pylori.     

Functional characterization and Me ion specificity of a Ca-citrate transporter from Enterococcus faecalis

Secondary transporters of the bacterial CitMHS family transport citrate in complex with a metal ion. Different members of the family are specific for the metal ion in the complex and have been shown to transport Mg(2+)-citrate, Ca(2+)-citrate or Fe(3+)-citrate. The Fe(3+)-citrate transporter of Streptococcus mutans clusters on the phylogenetic tree on a separate branch with a group of transporters found in the phylum Firmicutes which are believed to be involved in anaerobic citrate degradation. We have cloned and characterized the transporter from Enterococcus faecalis EfCitH in this cluster. The gene was functionally expressed in Escherichia coli and studied using right-side-out membrane vesicles. The transporter catalyzes proton-motive-force-driven uptake of the Ca(2+)-citrate complex with an affinity constant of 3.5 microm. Homologous exchange is catalyzed with a higher efficiency than efflux down a concentration gradient. Analysis of the metal ion specificity of EfCitH activity in right-side-out membrane vesicles revealed a specificity that was highly similar to that of the Bacillus subtilis Ca(2+)-citrate transporter in the same family. In spite of the high sequence identity with the S. mutans Fe(3+)-citrate transporter, no transport activity with Fe(3+) (or Fe(2+)) could be detected. The transporter of E. faecalis catalyzes translocation of citrate in complex with Ca(2+), Sr(2+), Mn(2+), Cd(2+) and Pb(2+) and not with Mg(2+), Zn(2+), Ni(2+) and Co(2+). The specificity appears to correlate with the size of the metal ion in the complex.