Functional expression of a bacterial heavy metal transporter in Arabidopsis enhances resistance to and decreases uptake of heavy metals

Large parts of agricultural soil are contaminated with lead (Pb) and cadmium (Cd). Although most environments are not heavily contaminated, the low levels observed nonetheless pose a high risk of heavy metal accumulation in the food chain. Therefore, approaches to develop plants with reduced heavy metal uptake are important. Recently, many transgenic plants with increased heavy metal resistance and uptake of heavy metals were developed for the purpose of phytoremediation. However, to reduce heavy metal in the food chain, plants that transfer less heavy metals to the shoot are required. We tested whether an Escherichia coli gene, ZntA, which encodes a Pb(II)/Cd(II)/Zn(II) pump, could be useful for developing plants with reduced heavy metal content. Yeast cells transformed with this gene had improved resistance to Pb(II) and Cd(II). In Arabidopsis plants transformed with ZntA, ZntA was localized at the plasma membrane and improved the resistance of the plants to Pb(II) and Cd(II). The shoots of the transgenic plants had decreased Pb and Cd content. Moreover, the transgenic protoplasts showed lower accumulation of Cd and faster release of preloaded Cd than wild-type protoplasts. These results show that a bacterial transporter gene, ZntA, can be functionally expressed in plant cells, and that that it may be useful for the development of crop plants that are safe from heavy metal contamination.     

High-Level Resistance to Cobalt and Nickel but Probably No Transenvelope Efflux: Metal Resistance in the Cuban Serratia marcescens Strain C-1

Molecular mechanisms underlying inducible cobalt and nickel resistance of a bacterial strain isolated from a Cuban serpentine deposit were investigated. This strain C-1 was assigned to Serratia marcescens by 16S rDNA analysis and DNA/DNA hybridization. Genes involved in metal resistance were identified by transposon mutagenesis followed by selection for cobalt- and nickel-sensitive derivatives. The transposon insertion causing the highest decrease in metal resistance was located in the ncrABC determinant. The predicted NcrA product was a NreB ortholog of the major facilitator protein superfamily and central for cobalt/nickel resistance in S. marcescens strain C-1. NcrA also mediated metal resistance in Escherichia coli and caused decreased accumulation of Co(II) and Ni(II) in this heterologous host. NcrB may be a regulatory protein. NcrC was a protein of the nickel–cobalt transport (NiCoT) protein family and necessary for full metal resistance in E. coli, but only when NcrA was also present. Without NcrA, NcrC caused a slight decrease in metal resistance and mediated increased accumulation of Ni(II) and Co(II). Because the cytoplasmic metal concentration can be assumed to be the result of a flow equilibrium of uptake and efflux processes, this interplay between metal uptake system NcrC and metal efflux system NcrA may contribute to nickel and cobalt resistance in this bacterium.     

The ATP hydrolytic activity of purified ZntA, a Pb(II)/Cd(II)/Zn(II)-translocating ATPase from Escherichia coli

ZntA, a soft metal-translocating P1-type ATPase from Escherichia coli, confers resistance to Pb(II), Cd(II), and Zn(II). ZntA was expressed as a histidyl-tagged protein, solubilized from membranes with Triton X-100, and purified to homogeneity. The soft metal-dependent ATP hydrolysis activity of purified ZntA was characterized. The activity was specific for Pb(II), Cd(II), Zn(II), and Hg(II), with the highest activity obtained when the metals were present as thiolate complexes of cysteine or glutathione. The maximal ATPase activity of ZntA was approximately 3 micromol/(mg x min) obtained with the Pb(II)-thiolate complex. In the absence of thiolates, Cd(II) inhibits ZntA above pH 6, whereas the Cd(II)-thiolate complexes stimulate activity, suggesting that a metal-thiolate complex is the true substrate in vivo. These results are consistent with the physiological role of ZntA as mediator of resistance to toxic concentrations of the divalent soft metals, Pb(II), Cd(II), and Zn(II), by ATP-dependent efflux. Our results confirm that ZntA is the first Pb(II)-dependent ATPase discovered to date.     

The Salmonella enterica sv. Typhimurium smvA,yddG and ompD (porin) genes are required for the efficient efflux of methyl viologen

In Gram-negative bacteria, a subset of inner membrane proteins in the major facilitator superfamily (MFS) acts as efflux pumps to decrease the intracellular concentrations of multiple toxic substrates and confers multidrug resistance. The Salmonella enterica sv. Typhimurium smvA gene encodes a product predicted to be an MFS protein most similar to QacA of Staphylococcus aureus. Like mutations in qacA, mutations in smvA confer increased sensitivity to methyl viologen (MV). Mutations in the adjacent ompD (porin) and yddG (drug/metabolite transporter) genes also confer increased sensitivity to MV, and mutations in smvA are epistatic to mutations in ompD or yddG for this phenotype. YddG and OmpD probably comprise a second efflux pump in which the OmpD porin acts as an outer membrane channel (OMC) protein for the efflux of MV and functions independently of the SmvA pump. In support of this idea, the pump dependent on YddG and OmpD has a different substrate specificity from the pump dependent on SmvA. Mutations in tolC, which encodes an OMC protein, confer increased resistance to MV. TolC apparently facilitates the import of MV, and a subset of OMC proteins including the OmpD porin and TolC may facilitate both import and export of distinct subsets of toxic substrates.     

Mechanism of action of and resistance to quinolones

Fluoroquinolones are an important class of wide‐spectrum antibacterial agents. The first quinolone described was nalidixic acid, which showed a narrow spectrum of activity. The evolution of quinolones to more potent molecules was based on changes at positions 1, 6, 7 and 8 of the chemical structure of nalidixic acid. Quinolones inhibit DNA gyrase and topoisomerase IV activities, two enzymes essential for bacteria viability. The acquisition of quinolone resistance is frequently related to (i) chromosomal mutations such as those in the genes encoding the A and B subunits of the protein targets (gyrA, gyrB, parC and parE), or mutations causing reduced drug accumulation, either by a decreased uptake or by an increased efflux, and (ii) quinolone resistance genes associated with plasmids have been also described, i.e. the qnr gene that encodes a pentapeptide, which blocks the action of quinolones on the DNA gyrase and topoisomerase IV; the aac(6′)‐Ib‐cr gene that encodes an acetylase that modifies the amino group of the piperazin ring of the fluoroquinolones and efflux pump encoded by the qepA gene that decreases intracellular drug levels. These plasmid‐mediated mechanisms of resistance confer low levels of resistance but provide a favourable background in which selection of additional chromosomally encoded quinolone resistance mechanisms can occur.     

SyrD is required for syringomycin production by Pseudomonas syringae pathovar syringae and is related to a family of ATP-binding secretion proteins

The syrD gene of Pseudomonas syringae pathovar syringae strain B301D-R was characterized and sequenced. The syrD open reading frame is 1695bp long and encodes a predicted protein, SyrD, of =63kDa. Database searches revealed that SyrD shares a high degree of similarity with the ATP-binding cassette (ABC) superfamily of transporter proteins which are responsible for specific nutrient uptake and for secretion of certain cellular products in prokaryotes, and for multiple drug resistance in mammals. The amino acid sequence homology between SyrD and the ABC proteins was greatest at the conserved residues which constitute the ATP-binding cassette of these proteins; these residues lie in the hydrophilic C-terminal half of SyrD. The N-terminus of SyrD is predicted to be hydrophobic and to contain six membrane-spanning α-helices. syrD mutants of strain B301D-R were significantly less virulent than other syr mutants, were deficient in four large polypeptides thought to be components of a syringomycin synthetase complex, and showed reduced expression of a syrB-lacZ reporter gene fusion in trans. It is proposed that SyrD is a cyto-plasmic membrane protein that functions as an ATP-driven efflux pump for the secretion of syringomycin.     

Identification of novel leads applying in silico studies for Mycobacterium multidrug resistant (MMR) protein

Multidrug efflux mechanism is the main cause of intrinsic drug resistance in bacteria. Mycobacterium multidrug resistant (MMR) protein belongs to small multidrug resistant family proteins (SMR), causing multidrug resistance to proton (H⁺)-linked lipophilic cationic drug efflux across the cell membrane. In the present work, MMR is treated as a novel target to identify new molecular entities as inhibitors for drug resistance in Mycobacterium tuberculosis. In silico techniques are applied to evaluate the 3D structure of MMR protein. The putative amino acid residues present in the active site of MMR protein are predicted. Protein–ligand interactions are studied by docking cationic ligands transported by MMR protein. Virtual screening is carried out with an in-house library of small molecules against the grid created at the predicted active site residues in the MMR protein. Absorption distribution metabolism and elimination (ADME) properties of the molecules with best docking scores are predicted. The studies with cationic ligands and those of virtual screening are analysed for identification of new lead molecules as inhibitors for drug resistance caused by the MMR protein.     

Zinc(II) tolerance in Escherichia coli K-12: evidence that the zntA gene (o732) encodes a cation transport ATPase

A transposon (Tn 10 dCam) insertion mutant of Escherichia coli K-12 was isolated that exhibited hypersensitivity to zinc(II) and cadmium(II) and, to a lesser extent, cobalt(II) and nickel (II). The mutated gene, located between 75.5 and 76.2 min on the chromosome, is named zntA (for Zn(II) transport or tolerance). The metal-sensitive phenotype was complemented by a genomic DNA clone mapping at 3677.90–3684.60 kb on the physical map. Insertion of a kanamycin resistance (Kn^R) cassette at a Sal  I site in a subcloned fragment generated a plasmid that partially complemented the zinc(II)-sensitive phenotype. DNA sequence analysis revealed that the KnR cassette was located within the putative promoter region of an ORF ( o732 or yhhO ) predicted to encode a protein of 732 amino acids, similar to cation transport P-type ATPases in the Cpx-type family. Inverse PCR and sequence analysis revealed that the Tn 10 dCam element was located within o732 in the genome of the zinc(II)-sensitive mutant. The zntA mutant had elevated amounts of intracellular and cell surface-bound Zn(II), consistent with the view that zntA ⁺ encodes a zinc(II) efflux protein. Exposure of the z ntA mutant to cobalt(II) and cadmium(II) also resulted in elevated levels of intracellular and cell surface-bound metal ions.     

A peroxide-induced zinc uptake system plays an important role in protection against oxidative stress in Bacillus subtilis

In Bacillus subtilis, hydrogen peroxide (H2O2) induces expression of the PerR regulon including catalase (KatA), alkyl hydroperoxide reductase and the DNA-binding protein MrgA. We have identified the P-type metal-transporting ATPase ZosA (formerly YkvW) as an additional member of the perR regulon. Expression of zosA is induced by H2O2 and repressed by the PerR metalloregulatory protein, which binds to two Per boxes in the promoter region. Physiological studies implicate ZosA in Zn(II) uptake. ZosA functions together with two Zur-regulated uptake systems and one known efflux system to maintain Zn(II) homeostasis. ZosA is the major pathway for zinc uptake in cells growing with micromolar levels of Zn(II) that are known to repress the two Zur-regulated transporters. A perR mutant is sensitive to high levels of zinc, and this sensitivity is partially suppressed by a zosA mutation. ZosA is important for resistance to both H2O2 and the thiol-oxidizing agent diamide. This suggests that increased intracellular Zn(II) may protect thiols from oxidation. In contrast, catalase is critical for H2O2 resistance but does not contribute significantly to diamide resistance. Growth of cells with elevated zinc significantly increases resistance to high concentrations of H2O2, and this effect requires ZosA. Our results indicate that peroxide stress leads to the upregulation of a dedicated Zn(II) uptake system that plays an important role in H2O2 and disulphide stress resistance.     

The Arabidopsis Ethylene-Insensitive 2 gene is required for lead resistance

The Arabidopsis Ethylene-Insensitive 2 (EIN2) gene has been shown to be involved in the regulation of abiotic and biotic stresses, including ozone stress, high salt, oxidative stress and disease resistance. However, little is known about the role of EIN2 gene in lead (Pb) resistance in Arabidopsis. In this study, we showed that EIN2 gene is required for Pb(II) resistance in Arabidopsis. EIN2 gene was induced by Pb(II) treatment, and the ein2-1 mutant showed enhanced sensitivity to Pb(II). A higher Pb content was detected in ein2-1 plants than in wild-type plants when subjected to Pb(II) treatment, which was associated, at least in part, with reduction in expression of AtPDR12 gene, a pump excluding Pb(II) and/or Pb(II)-containing toxic compounds from the cytoplasm. Moreover, the ein2-1 mutation also impaired glutathione (GSH)-dependent Pb(II) resistance, which was related to constitutive reduction of express of GSH1 gene involved in GSH synthesis and consequently reduced GSH content. Taken together, all these results suggest that EIN2 gene mediates Pb(II) resistance, at least in part, through two distinct mechanisms, a GSH-dependent mechanism and a GSH-independent AtPDR12-mediated mechanism.     

Increased lead uptake and inhibition of ALAD-activity in isolated rat hepatocytes incubated with lead-diethyldithiocarbamate complex

Dithiocarbamates can form lipid soluble complexes with lead and are known to markedly increase tissue uptake of lead and potentiate toxic effects of lead in rats. Cellular effects of the interactions between lead and diethyldithiocarbamate were studied in primary cultures of rat hepatocytes. The cells were incubated with lead acetate (PbAc) or lead-diethyldithiocarbamate complex (Pb(DTC)2), labelled with 203Pb. The lipid soluble Pb(DTC)2 was rapidly taken up in the cells and after 30 min incubation the cellular levels of lead were approximately 40 times higher in cells incubated with Pb(DTC)2 than in cells incubated with a similar concentration of PbAc. The maximal cellular uptake of lead was reached after 4 h incubation with Pb(DTC)2, while incubation with PbAc caused a slow continuously increasing uptake of lead during the 20 h incubation. The enzyme delta-aminolevulinic acid dehydratase (ALAD) was inhibited to a much higher extent by Pb(DTC)2 compared to PbAc after incubations with similar concentrations of lead. Maximal inhibition of ALAD activity was reached at a cellular concentration of 0.5-1 nmol Pb/mg protein, irrespective of which form of lead was used in the incubation. Pb(DTC)2 was shown to inhibit ALAD activity also in vitro when incubated with purified ALAD enzyme. The rapid and high intracellular uptake and cellular response of Pb(DTC)2, shown in the present study, may explain the drastic effects of dithiocarbamates on lead distribution and toxicity previously shown in vivo.     

Simultaneous Simulations of Uptake in Plants and Leaching to Groundwater of Cadmium and Lead for Arable Land Amended with Compost or Farmyard Manure

The water budget of soil, the uptake in plants and the leaching to groundwater of cadmium (Cd) and lead (Pb) were simulated simultaneously using a physiological plant uptake model and a tipping buckets water and solute transport model for soil. Simulations were compared to results from a ten-year experimental field study, where four organic amendments were applied every second year. Predicted concentrations slightly decreased (Cd) or stagnated (Pb) in control soils, but increased in amended soils by about 10% (Cd) and 6% to 18% (Pb). Estimated plant uptake was lower in amended plots, due to an increase of K_d (dry soil to water partition coefficient). Predicted concentrations in plants were close to measured levels in plant residues (straw), but higher than measured concentrations in grains. Initially, Pb was mainly predicted to deposit from air into plants (82% in 1998); the next years, uptake from soil became dominating (30% from air in 2006), because of decreasing levels in air. For Cd, predicted uptake from air into plants was negligible (1–5%).     

Genetic and biochemical study of yeast acid phosphatases. XI. Gene ACP80 controls inorganic phosphate transport

Mutations in the gene ACP80 which simultaneously lead to constitutive synthesis of repressible acid phosphatases, resistance to 1 X 10(-3) M sodium arsenate and to the disturbance of phosphate uptake in the cell were obtained. Mutations suppressible by ochre suppressors in this gene were identified. The conclusion that the gene ACP80 encodes a protein which participates in the phosphate transport into the cell, was drawn. A selective method for isolation of mutations in this gene is proposed.     

TRK1 encodes a plasma membrane protein required for high-affinity potassium transport in Saccharomyces cerevisiae.

We identified a 180-kilodalton plasma membrane protein in Saccharomyces cerevisiae required for high-affinity transport (uptake) of potassium. The gene that encodes this putative potassium transporter (TRK1) was cloned by its ability to relieve the potassium transport defect in trk1 cells. TRK1 encodes a protein 1,235 amino acids long that contains 12 potential membrane-spanning domains. Our results demonstrate the physical and functional independence of the yeast potassium and proton transport systems. TRK1 is nonessential in S. cerevisiae and maps to a locus unlinked to PMA1, the gene that encodes the plasma membrane ATPase. Haploid cells that contain a null allele of TRK1 (trk1 delta) rely on a low-affinity transporter for potassium uptake and, under certain conditions, exhibit energy-dependent loss of potassium, directly exposing the activity of a transporter responsible for the efflux of this ion.     

Manganese(II) dynamics and distribution in glial cells cultured from chick cerebral cortex

The kinetics of manganese(II) ion uptake and efflux have been investigated using tracer⁵⁴Mn(II) with glial cells cultured from chick cerebral cortex in chemically defined medium. The initial velocity of Mn(II) uptake versus [Mn(II)] exhibit saturation, with an apparent S_0.518(±3) M. Both the rate and extent of Mn(II) uptake are inhibited by Ca(II), either added externally or preloaded into the glial cells. Preloading of glia with Mn(II) also inhibits the rate of external⁵⁴Mn(II) uptake. Zn(II) inhibits but Cu(II) activates Mn(II) uptake. Efflux of Mn(II) from preloaded cells occurs as a biphasic process, with rapid release of 30–40% of total cell Mn(II), then much slower release of the remainder. Permeabilization of cells with dextran sulfate also rapidly released ca. 30% of total cell Mn(II). High external Mn(II) enhanced both the rate and extent of Mn(II) efflux. CCCP, an uncoupler of oxidative phosphorylation, inhibited both Mn(II) uptake and efflux significantly, but addition of cyanide, ouabain, insulin, hydrocortisone, K⁺, or Nd(III) had no effect on either process. Taken together, these data suggest a model in which Mn(II) is brought across the plasma membrane by facilitated diffusion, binds to cytosolic protein sites, and is partitioned into the mitochondria by an active transport mechanism. The fact that the Mn(II) flux rates observed with cultured glia are much faster than those reported for overall uptake and efflux of brain Mn(II)in vivo suggests that the blood-brain barrier may play a significant role in determining these latter rates in whole animals.Supported in part by NIH grant GM-33358 and a Biomedical Research Support Grant from the NIH administered by Penn State.