Evaluation of transgenic tobacco plants expressing a bacterial Co-Ni transporter for acquisition of cobalt

Phytoremediation is a viable strategy for management of toxic wastes in a large area/volume with low concentrations of toxic elemental pollutants. With increased industrial use of cobalt and its alloys, it has become a major metal contaminant in soils and water bodies surrounding these industries and mining sites with adverse effects on the biota. A bacterial Co-Ni permease was cloned from Rhodopseudomonas palustris and introduced into Nicotiana tabacum to explore its potential for phytoremediation and was found to be specific for cobalt and nickel. The transgenic plants accumulated more cobalt and nickel as compared to control, whereas no significant difference in accumulation of other divalent ions was observed. The transgenic plants were evaluated for cobalt content and showed increased acquisition of cobalt (up to 5 times) as compared to control. The plants were also assessed for accumulation of nickel and found to accumulate up to 2 times more nickel than control. At the same initial concentration of cobalt and nickel, transgenic plant preferentially accumulated cobalt as compared to nickel. The present study is perhaps the first attempt to develop transgenic plants expressing heterologous Co transporter with an improved capacity to uptake cobalt.     

Identification of rcnA (yohM), a nickel and cobalt resistance gene in Escherichia coli

We report here on the isolation and primary characterization of the yohM gene of Escherichia coli. We show that yohM encodes a membrane-bound polypeptide conferring increased nickel and cobalt resistance in E. coli. yohM was specifically induced by nickel or cobalt but not by cadmium, zinc, or copper. Mutation of yohM increased the accumulation of nickel inside the cell, whereas cells harboring yohM in multicopy displayed reduced intracellular nickel content. Our data support the hypothesis that YohM is the first described efflux system for nickel and cobalt in E. coli. We propose rcnA (resistance to cobalt and nickel) as the new denomination of yohM.     

Dictyostelium discoideum cobB mutants show reduced heavy metal accumulation associated with gene amplification

Wild-type Dictyostelium discoideum cells grow- ing on non-toxic levels of nickel chloride or cobaltous chloride accumulate 2–3.5 times as much nickel and at least 1.5 times as much cobalt as cobB mutants. The cobB trait is dominant, confers unstable cobalt and nickel resistance and is correlated with the presence of up to 50 copies of a linear extrachromosomal DNA, approximately 100?kb in length, derived from linkage group III. Independent cobB mutants can be obtained by selection on medium containing either cobalt or nickel. The amplified DNA can be transferred to wild-type strains by electroporation. Strains with mutations at a second cobalt resistance locus, cobA, accumulate the same amount of cobalt, but more nickel than wild-type strains. Our results are consistent with the cobA mutant phenotype being due to internal sequestration of cobalt, and the cobB mutant phenotype being due to reduced net uptake of cobalt and nickel. Energy-dependent nickel export was detectable in wild-type and cobB mutant strains but its role in heavy metal resistance has not yet been proved.     

Cobalt and Nickel Stabilize Stem Cell Transcription Factor OCT4 through Modulating Its Sumoylation and Ubiquitination

Stem cell research can lead to the development of treatments for a wide range of ailments including diabetes, heart disease, aging, neurodegenerative diseases, spinal cord injury, and cancer. OCT4 is a master regulator of self-renewal of undifferentiated embryonic stem cells. OCT4 also plays a crucial role in reprogramming of somatic cells into induced pluripotent stem (iPS) cells. Given known vivo reproductive toxicity of cobalt and nickel metals, we examined the effect of these metals on expression of several stem cell factors in embryonic Tera-1 cells, as well as stem cells. Cobalt and nickel induced a concentration-dependent increase of OCT4 and HIF-1α, but not NANOG or KLF4. OCT4 induced by cobalt and nickel was due primarily to protein stabilization because MG132 stabilized OCT4 in cells treated with either metals and because neither nickel nor cobalt significantly modulated its steady-state mRNA level. OCT4 stabilization by cobalt and nickel was mediated largely through reactive oxygen species (ROS) as co-treatment with ascorbic acid abolished OCT4 increase. Moreover, nickel and cobalt treatment increased sumoylation and mono-ubiquitination of OCT4 and K123 was crucial for mediating these modifications. Combined, our observations suggest that nickel and cobalt may exert their reproductive toxicity through perturbing OCT4 activity in the stem cell compartment.     

Dictyostelium discoideum cobB mutants show reduced heavy metal accumulation associated with gene amplification

 Wild-type Dictyostelium discoideum cells grow- ing on non-toxic levels of nickel chloride or cobaltous chloride accumulate 2–3.5 times as much nickel and at least 1.5 times as much cobalt as cobB mutants. The cobB trait is dominant, confers unstable cobalt and nickel resistance and is correlated with the presence of up to 50 copies of a linear extrachromosomal DNA, approximately 100 kb in length, derived from linkage group III. Independent cobB mutants can be obtained by selection on medium containing either cobalt or nickel. The amplified DNA can be transferred to wild-type strains by electroporation. Strains with mutations at a second cobalt resistance locus, cobA, accumulate the same amount of cobalt, but more nickel than wild-type strains. Our results are consistent with the cobA mutant phenotype being due to internal sequestration of cobalt, and the cobB mutant phenotype being due to reduced net uptake of cobalt and nickel. Energy-dependent nickel export was detectable in wild-type and cobB mutant strains but its role in heavy metal resistance has not yet been proved. Received: 21 December 1995/Accepted: 10 June 1996     

Comparative and functional genomic analysis of prokaryotic nickel and cobalt uptake transporters: evidence for a novel group of ATP-binding cassette transporters

The transition metals nickel and cobalt, essential components of many enzymes, are taken up by specific transport systems of several different types. We integrated in silico and in vivo methods for the analysis of various protein families containing both nickel and cobalt transport systems in prokaryotes. For functional annotation of genes, we used two comparative genomic approaches: identification of regulatory signals and analysis of the genomic positions of genes encoding candidate nickel/cobalt transporters. The nickel-responsive repressor NikR regulates many nickel uptake systems, though the NikR-binding signal is divergent in various taxonomic groups of bacteria and archaea. B(12) riboswitches regulate most of the candidate cobalt transporters in bacteria. The nickel/cobalt transporter genes are often colocalized with genes for nickel-dependent or coenzyme B(12) biosynthesis enzymes. Nickel/cobalt transporters of different families, including the previously known NiCoT, UreH, and HupE/UreJ families of secondary systems and the NikABCDE ABC-type transporters, showed a mosaic distribution in prokaryotic genomes. In silico analyses identified CbiMNQO and NikMNQO as the most widespread groups of microbial transporters for cobalt and nickel ions. These unusual uptake systems contain an ABC protein (CbiO or NikO) but lack an extracytoplasmic solute-binding protein. Experimental analysis confirmed metal transport activity for three members of this family and demonstrated significant activity for a basic module (CbiMN) of the Salmonella enterica serovar Typhimurium transporter.     

The production of phenazine antibiotics by the Pseudomonas aureofaciens strain with plasmid-controlled resistance to cobalt and nickel

Plasmid pBS501 responsible for the resistance of the wild-type Pseudomonas sp. BS501 (pBS501) to cobalt and nickel ions was conjugatively transferred to the rhizosphere Pseudomonas aureofaciens strain BS1393, which is able to synthesize phenazine antibiotics and to suppress a wide range of phytopathogenic microorganisms. The transconjugant P. aureofaciens BS1393 (pBS501) turned out to be resistant to cobalt and nickel with an MIC of 8 mM. When grown in a synthetic medium with 0.25 mM cobalt, the transconjugant accumulated 6 times more cobalt than the wild-type strain BS501 (pBS501) (1.2 and 0.2 microgram Co/mg protein). Electron microscopic studies showed that cobalt accumulates on the surface of transconjugant cells in the form of electron-opaque granules. In a culture medium with 2 mM cobalt or nickel, strain BS1393 produced phenazine-1-carboxylic acid in trace amounts. The transconjugant P. aureofaciens BS1393 (pBS501) produced this antibiotic in still smaller amounts. Unlike the parent strain BS1393, the transconjugant P. aureofaciens BS1393 (pBS501) was able to suppress in vitro the growth of the phytopathogenic fungus Gaeumannomyces graminis var. tritici 1818 in a medium containing 0.5 mM cobalt or nickel.     

Metal complexes with schiff bases obtained from salicylaldehyde derivatives and 2-aminoethylpyridine

Cobalt(II), cobalt(III), nickel(II), copper(II) and palladium(II) complexes with N-2-(2-pyridyl)ethylring-substituted salicylideneiminates (abbreviated as X-Sal-2-Epy) were synthesized. In addition to Co^III (H-Sal-2-Epy)₃, the complexes of the formula M^II(X-Sal-2-Epy)₂·nH₂O were obtained in crystals. The cobalt(III) complex is diamagnetic and has an electronic absorption spectrum typical of the six-coordinate, octahedral cobalt(III) complex. The cobalt(II) complexes in the solid state show electronic spectra typical of the six-coordinate cobalt(II) complexes. Electronic spectra also indicate that the nickel(II) complexes in the solid state and in non-donor solvents are six-coordinate, octahedral. In the cobalt(II) and nickel(II) complexes, the ligand X-Sal-2-Epy functions as terdentates, while in the cobalt(III) complex it acts as a bidentate ligand. The results are compared with those reported previously for related ligands.     

Chemical Forms of Nickel and Cobalt in Phloem of Ricinus communis

Investigations were performed to study the chemical form in which nickel and cobalt are transported in sieve tubes. Ricinus communis plants were used since they allow easy collection of relatively pure phloem sap. In the experiments labelled nickel or cobalt were given to the nutrient solution. The tapped phloem exudate was separated by gel filtration on Sephadex or by paper electrophoresis. Nickel and cobalt were bound to complexing organic compounds with a molecular weight in the range of 1000—5000. The overall charge of the nickel complex was negative, and the bulk of cobalt also appeared to complex to a compound with negative overall charge.     

Cloning of pMOL28-encoded nickel resistance genes and expression of the genes in Alcaligenes eutrophus and Pseudomonas spp.

The 163-kilobase-pair (kb) plasmid pMOL28, which determines inducible resistance to nickel, cobalt, chromate, and mercury salts in its native host Alcaligenes eutrophus CH34, was transferred to a derivative of A. eutrophus H16 and subjected to cloning procedures. After Tn5 transposon mutagenesis, restriction endonuclease analysis, and DNA-DNA hybridization, two DNA fragments, a 9.5-kb KpnI fragment and a 13.5-kb HindIII fragment (HKI), were isolated. HKI contained EK1, the KpnI fragment, as a subfragment flanked on both sides by short regions. Both fragments were ligated into the suicide vector pSUP202, the broad-host-range vector pVK101, and pUC19. Both fragments restored a nickel-sensitive Tn5 mutant to full nickel and cobalt resistance. The hybrid plasmid pVK101::HKI expressed full nickel resistance in all nickel-sensitive derivatives, either pMOL28-deficient or -defective, of the native host CH34. The hybrid plasmid pVK101::HKI also conferred nickel and cobalt resistance to A. eutrophus strains H16 and JMP222, Alcaligenes hydrogenophilus, Pseudomonas putida, and Pseudomonas oleovorans, but to a lower level of resistance. In all transconjugants the metal resistances coded by pVK101::HKI were expressed constitutively rather than inducibly. The hybrid plasmid metal resistance was not expressed in Escherichia coli. DNA sequences responsible for nickel resistance in newly isolated strains showed homology to the cloned pMOL28-encoded nickel and cobalt resistance determinant.     

Transformation of Fe,Mn, Co,and Ni compounds in humic podzols at different moisture

Incubation of humic podzol at soil moisture of 60 and 100% field capacity (FC) and after addition of peat and glucose increased the content of nickel and cobalt compounds in the water-soluble, exchangeable, organic matter-bound, and amorphous iron-bound fractions. At the same time, the content of elements bound to crystallized iron compounds decreased twofold and fourfold at 60 and 100% FC, respectively. The content of cobalt and nickel decreased in the residual fraction by 25 and 50%, respectively. The transformation of cobalt and nickel in soil is closely related to the transformation of iron and manganese compounds as well as to redox processes. The lowest pH and redox potential (RP) as well as the highest increase in the mobility of the elements was observed after soil incubation with glucose at 100% FC.     

The ABC-transporter AtmA is involved in nickel and cobalt resistance of <Emphasis Type="Italic">Cupriavidus metallidurans</Emphasis> strain CH34

Cupriavidus metallidurans CH34 genome contains an ortholog of Atm1p named AtmA (Rmet_0391, YP_582546). In Saccharomyces cerevisiae, the ABC-type transport system Atm1p is involved in export of iron–sulfur clusters from mitochondria into the cytoplasm for assembly of cytoplasmic iron–sulfur containing proteins. An ∆atmA mutant of C. metallidurans was sensitive to nickel and cobalt but not iron cations. AtmA increased also resistance to these cations in Escherichia coli strains that carry deletions of the genes for other nickel and cobalt transport systems. In C. metallidurans, atmA expression was not significantly induced by nickel and cobalt, but repressed by zinc. AtmA was purified as a 70 kDa protein after expression in E. coli. ATPase activity of AtmA was stimulated by nickel and cobalt.     

Relationship between Serum Nickel and Homocysteine Concentration in Hemodialysis Patients

Severe hyperhomocysteinemia (HHC) is associated with atherosclerosis. In hemodialysis (HD) patients, one of the main causes of death is cardiovascular disease. In animals, trace elements such as cobalt, copper, iron, and nickel ameliorated vitamin B(12) deficiency-induced HHC. However, correlations between plasma total homocysteine (tHcy) and trace elements in HD patients have not been investigated. Therefore, tHcy, folate, vitamin B(12), trace elements (cobalt, copper, iron, and nickel), and some laboratory parameters such as serum total protein, albumin, transferrin, ferritin, C-reactive protein (CRP), and interleukin-6 concentrations were determined in 122 hemodialysis patients. When patients were divided into groups according to their tHcy, we found no significant differences in concentrations of cobalt, copper, and total protein, while nickel was higher, and folate, vitamin B(12), and iron were lower in patients with lower than higher tHcy. In univariate regression analysis, tHcy negatively correlated with concentrations of folate (r = -0.302, p < 0.006), vitamin B(12) (r = -0.347, p < 0.0001), nickel (r = -0.289, p < 0.006), and CRP (r = -0.230, p < 0.02) and positively with serum albumin (r = 0.316, p < 0.0004) and hemoglobin (r = 0.329, p < 0.0001) values. No relationship between tHcy and serum concentrations of cobalt, copper, iron, or other laboratory parameters was found in HD patients. The effect of cobalt and nickel on homocysteine production was assessed in human peripheral mononuclear cells (PBMCs). Nickel but not cobalt at concentrations found in HD patients significantly inhibited homocysteine, cysteine, and S-adenosylhomocysteine production in human PBMCs. These results suggest that nickel might also be involved in the regulation of the methionine-folate cycle in humans, as was demonstrated in animal experiments.     

MD-2 Determinants of Nickel and Cobalt-Mediated Activation of Human TLR4

Recent findings unexpectedly revealed that human TLR4 can be directly activated by nickel ions. This activation is due to the coordination of nickel by a cluster of histidine residues on the ectodomain of human TLR4, which is absent in most other species. We aimed to elucidate the role of MD-2 in the molecular mechanism of TLR4/MD-2 activation by nickel, as nickel binding site on TLR4 is remote from MD-2, which directly binds the endotoxin as the main pathological activator of TLR4. We identified MD-2 and TLR4 mutants which abolished TLR4/MD-2 receptor activation by endotoxin but could nevertheless be significantly activated by nickel, which acts in synergy with LPS. Human TLR4/MD-2 was also activated by cobalt ions, while copper and cadmium were toxic in the tested concentration range. Activation of TLR4 by cobalt required MD-2 and was abolished by human TLR4 mutations of histidine residues at positions 456 and 458. We demonstrated that activation of TLR4 by nickel and cobalt ions can trigger both the MyD88-dependent and the –independent pathway. Based on our results we propose that predominantly hydrophobic interactions between MD-2 and TLR4 contribute to the stabilization of the TLR4/MD-2/metal ion complex in a conformation that enables activation.     

Synthesis, characterisation, X-ray structure and biological activity of three new 5-formyluracil thiosemicarbazone complexes

Three new complexes of transition metals as copper, nickel and cobalt with 5-formyluracil thiosemicarbazone (H3ut) have been synthesised and characterised by single-crystal X-ray diffraction. In all compounds the ligand behaves as SNO terdentate. In the copper complex the coordination geometry is square pyramidal with the ligand lying on the basal plane and two water molecules that complete the metal environment, the nickel compound is surrounded by six donor atoms (three of the ligand, two water oxygen atoms and a chlorine atom) in an octahedral fashion, and cobalt also shows an octahedral geometry but determined only by two terdentate ligand molecules. These three compounds have been tested on human leukemic cell lines K562 and CEM. The nickel and cobalt complexes have demonstrated low activity in cell growth, while the copper complex that is more active has been tested also on a third leukemic human cell line (U937), but it was not able to induce apoptosis on all cell lines.     

Study on reaction mechanism of bioleaching of nickel and cobalt from lateritic chromite overburdens

Depletion of high-grade ores and presence of significant quantities of metals in low-grade oxide ores has enforced to utilize the overburdens (COB) and wastes (low-grade ores) generated during mining operations. The impact of ore mineralogy and mineral–microbe interaction during bioleaching could not be ignored. Seeking to the need, a systematic study was performed to establish the reaction mechanism involved for recovery of nickel and cobalt from chromite overburden (COB), Sukinda, Orissa using pure culture of Aspergillus niger. Mineralogical analysis reveals a complete conversion of goethite into hematite phase leading to exposure of nickel particles into the micro-pores and cracks developed in the matrix which was initially found to be intertwined in the goethite lattice. As a result, it became more susceptible to attack by the fungal bio acids which in turn accelerate the dissolution rate. Organic acids like oxalic and citric acids were detected in the culture filtrate using HPLC. TEM analysis of the leached samples shows that nickel dissolute into the solution leaving a porous space in the matrix of the hematite by forming nickel oxalate or nickel citrate. Kinetics of the nickel bioleaching was studied to support the mechanism of the reaction. It was observed that the initial rate of reaction follows the chemical control dissolution reaction where as the later part fits to shrinking core model. 18% of nickel and 37.8% of cobalt was recovered from pre-treated COB at 2.5% pulp-density with 10% (v/v) fungal inoculum at 30 °C within 25 days in shake flask while 32.5% of nickel and 86% of cobalt was recovered in bioreactor.     

TRACE-ELEMENT TOXICITIES IN OAT PLANTS

Excessive amounts of nickel, cobalt, chromium, copper, zinc, manganese, molybdenum and aluminium in nutrient solutions supplied to oat plants in sand culture produce ( a ) chlorosis and ( b ) other symptoms specific to the element involved. The specific symptoms are distinct for each metal, although those of cobalt and nickel might be confused.
The toxic effects of nickel, cobalt, copper, zinc, manganese and molybdenum are associated with high concentrations of the element in the leaf tissue, but this is not always so with chromium and aluminium.
The toxic effects of nickel, chromium, copper and molybdenum are associated with a reduced nitrogen content of the plant. Nickel, cobalt, chromium, zinc and manganese increase the concentration of phosphorus in the tissue whilst aluminium decreases it, probably to a deficiency level.
Aluminium reduces the intensity of toxic symptoms produced by nickel—probably by reducing the uptake of nickel and phosphorus. Copper effectively reduces the leaf necrosis produced by nickel, but not the nickel content of the leaf tissue; it is suggested that one factor in nickel toxicity may be inhibition of one or more functions of copper. The other elements slightly increase chlorosis and some increase necrosis.
The order of activitjl of the elements in producing chlorosis is found to be Ni>Cu>Co>Cr>Zn>Mo>Mn. This order, which is related to that giving yield reduction and is similar to the order of stability of metal complexes, is discussed in relation to induced iron deficiency.