Metal transport ATPase genes from Cupriavidus metallidurans CH34: a transcriptomic approach

Cupriavidus metallidurans strain CH34 is a multiple metal resistant β-proteobacterium isolated from the sediments of a zinc metallurgical plant. This strain possesses a large diversity of heavy-metal-resistance genes that are located mostly on two large plasmids, pMOL28 and pMOL30. The metal efflux ATPases constitute the major mechanism by which metal ions are removed and pumped out from the cytoplasmic pool of thiol groups. The genome of C. metallidurans CH34 contains eight such P1-ATPases. This high number of ATPases, compared with the P1-ATPase content in other micro-organisms, is a typical feature of the adaptation of C. metallidurans CH34 to metal-rich biotopes. We performed a phylogenetic analysis, and used quantitative PCR to study the metal-specific induction of the ATPases genes and, for some of them, their neighbouring genes. One of the ATPases, a homologue of the E. coli zntA gene, was characterized in detail by analysing the phenotypes of zntA mutants in different genetic backgrounds. In addition to the already-reported sensitivity to Zn(II), Cd(II), and Pb(II), zntA mutants also displayed a higher sensitivity to Tl(I) and Bi(III), indicating that the ZntA protein might play a role in the tolerance of very large cations.     

Synthesis,characterization, fluorescence and redox features of new <Emphasis Type="Italic">vic</Emphasis>-dioxime ligand bearing pyrene and its metal complexes

A new vic-dioxime ligand, N,N′-bis(aminopyreneglyoxime) (LH₂), and its copper(II), nickel(II) and cobalt(II) metal complexes were synthesized and characterized by elemental analyses, IR, UVVIS and ¹H and ¹³C NMR spectra (for the ligand). Mononuclear complexes were synthesized by a reaction of ligand (LH₂) and salts of Co(II), Ni(II), and Cu(II) in ethanol. The complexes have the metal-ligand ratio of 1: 2 and metals are coordinated by N,N′ atoms of vicinal dioximes. The ligand acts in a polydentate fashion bending through nitrogen atoms in the presence of a base, as do most vic-dioximes. Detection of a H-bonding in the Co(II), Ni(II), and Cu(II) complexes by IR revealed the square-planar MN₄ coordination of mononuclear complexes. Fluorescent properties of the ligand and its complexes arise from pyrene units conjugated with a vic-dioxime moiety. Fluorescence emission spectra of the ligand showed a drastic decrease in its fluorescence intensity upon metal binding. The electrochemical properties of the complexes were studied by the cyclic voltammetry technique. The nickel complex displayed an irreversible oxidation process while the copper complex exhibited a quasi-reversible oxidation and reduction processes based on the copper Cu(II)/Cu(III) and Cu(II)/Cu(I) couples, respectively.     

Mechanisms controlling the cellular accumulation of copper bis(thiosemicarbazonato) complexes

Copper (Cu) bis(thiosemicarbazonato) metal complexes [Cu(II)(btsc)s] have unique tumor-imaging and treatment properties and more recently have revealed potent neuroprotective actions in animal and cell models of neurodegeneration. However, despite the continued development of Cu(II)(btsc)s as potential therapeutics or diagnostic agents, little is known of the mechanisms involved in cell uptake, subcellular trafficking, and efflux of this family of compounds. Because of their high lipophilicity, it has been assumed that cellular accumulation is through passive diffusion, although this has not been analyzed in detail. The role of efflux pathways in cell homeostasis of the complexes is also largely unknown. In the present study, we investigated the cellular accumulation of the Cu(II)(btsc) complexes Cu(II)(gtsm) and Cu(II)(atsm) in human neuronal (M17) and glial (U87MG) cell lines under a range of conditions. Collectively, the data strongly suggested that Cu(II)(gtsm) and Cu(II)(atsm) may be taken into these cells by combined passive and facilitated (protein-carrier-mediated) mechanisms. This was supported by strong temperature-dependent changes to the uptake of the complexes and the influence of the cell surface protein on Cu accumulation. We found no evidence to support a role for copper-transporter 1 in accumulation of the compounds. Importantly, our findings also demonstrated that Cu from both Cu(II)(gtsm) and Cu(II)(atsm) was rapidly effluxed from the cells through active mechanisms. Whether this was in the form of released ionic Cu or as an intact metal complex is not known. However, this finding highlighted the difficulty of trying to determine the uptake mechanism of metal complexes when efflux is occurring concomitantly. These findings are the first detailed exploration of the cellular accumulation mechanisms of Cu(II)(btsc)s. The study delineates strategies to investigate the uptake and efflux mechanisms of metal complexes in cells, while highlighting specific difficulties and challenges that need to be considered before drawing definitive conclusions.     

Soil-modified carbon paste electrode: a useful tool in environmental assessment of heavy metal ion binding interactions

Carbon paste electrodes (CPEs) modified with different soils in their native form were prepared to create a soil-like solid phase suitable for application in studies of heavy metal ion uptake and binding interactions. The preparation of CPEs modified with five different soils was examined and their heavy metal ion uptake behavior investigated using a model Cu(II) aqueous solution. Metal ions were accumulated under open circuit conditions and were determined after a medium exchange using differential pulse anodic stripping voltammetry, applying pre-electrolysis at –0.7 V. The soil-modified CPE accumulation behavior, including the linearity of the current response versus Cu(II) concentration, the influence of the pH on the solution, and the uptake kinetics, was thoroughly investigated. The correlation between the soil-modified CPE uptake capability and the standard soil parameters, such as ion exchange capacity, soil pH, organic matter and clay content, were evaluated for all five examined soils. The influence of selected endogenous cations (K(I), Ca(II), Fe(III)) on the transfer of Cu(II) ions from a solution to the simulated soil solid phase was examined and is discussed. Preliminary examinations of the soil-modified CPE uptake behavior with some exogenous heavy metal ions of strong environmental interest (Pb(II), Hg(II), Cd(II) and Ag(I)) are also presented. This work demonstrates some attractive possibilities for the application of a soil-modified CPE in studying soil-heavy metal ion binding interactions, with a further potential use as a new environmental sensor appropriate for fast on-site testing of polluted soils.     

Soil-modified carbon paste electrode: a useful tool in environmental assessment of heavy metal ion binding interactions

Carbon paste electrodes (CPEs) modified with different soils in their native form were prepared to create a soil-like solid phase suitable for application in studies of heavy metal ion uptake and binding interactions. The preparation of CPEs modified with five different soils was examined and their heavy metal ion uptake behavior investigated using a model Cu(II) aqueous solution. Metal ions were accumulated under open circuit conditions and were determined after a medium exchange using differential pulse anodic stripping voltammetry, applying preelectrolysis at -0.7 V. The soil-modified CPE accumulation behavior, including the linearity of the current response versus Cu(II) concentration, the influence of the pH on the solution, and the uptake kinetics, was thoroughly investigated. The correlation between the soil-modified CPE uptake capability and the standard soil parameters, such as ion exchange capacity, soil pH, organic matter and clay content, were evaluated for all five examined soils. The influence of selected endogenous cations (K(I), Ca(II), Fe(III)) on the transfer of Cu(II) ions from a solution to the simulated soil solid phase was examined and is discussed. Preliminary examinations of the soil-modified CPE uptake behavior with some exogenous heavy metal ions of strong environmental interest (Pb(II), Hg(II), Cd(II) and Ag(I)) are also presented. This work demonstrates some attractive possibilities for the application of a soil-modified CPE in studying soil-heavy metal ion binding interactions, with a further potential use as a new environmental sensor appropriate for fist on-site testing of polluted soils.     

Study of Bulk Membrane Transport of Some Heavy Metal Cations Using Three Macrocyclic Ligands Containing Oxygen and Nitrogen Heteroatoms

The transport experiments of Co²⁺, Ni²⁺, Cu²⁺, Zn²⁺, Cd²⁺, Ag⁺ and Pb²⁺ metal cations were carried out by dibenzo-18-crown-6 (DB18C6), dibenzyl-diaza-18-crown-6 (Dibenzyl-diaza-18C6) and di-tert-butyl-dibenzo-18-crown-6 (Di-tert-butyl-DB18C6) using chloroform (CHCl₃), 1,2-dichloroethane (1,2-DCE) and nitrobenzene (NB) organic solvents as liquid membranes. The source phase contained equimolar concentration of these metal cations and the source and receiving phases being buffered at pH=5 and pH=3, respectively. The obtained results show that the selectivity and the efficiency of transport for these heavy metal cations change with the nature of the ligand and also the organic solvents, which were used as liquid membranes in these experiments. A good selectivity was observed for silver (I) ion by dibenzyl-diaza-18C6 in all membrane systems. Dibenzo-18C6 and di-tert-butyl-DB18C6 showed the highest transport efficiency for cobalt (II) ion. The effect of stearic acid on transport efficiency was also investigated and the results show that the efficiency of transport of the heavy metal cations increases in the presence of this organic acid.     

Exopolysaccharide production by a marine cyanobacterium Cyanothece sp.

The adsorption and the hydrolytic action of purified cellulases of Trichoderma reesei, namely, cellobiohydrolase I (CBH I), endoglucanase II (EG II), and their core proteins, on steam-pretreated willow were compared. The two enzymes differed clearly in their adsorption and hydrolytic behavior. CBH I required the cellulose-binding domain (CBD) for efficient adsorption and hydrolysis, whereas EG II was able to adsorb to steam pretreated willow without its CBD. Absence of the CBD decreased the hydrolysis of cellulose by EG II, but the decrease was less pronounced than with CBH I. A linear relationship was observed between the amount of enzyme adsorbed and the degree of hydrolysis of cellulose only for CBHI. EG II and EG II core appeared to be able to hydrolyze only 1 to 2% of the substrate regardless of the amount of protein adsorbed.     

Purification and Biochemical Characterization of Two Xylanases from <Emphasis Type="Italic">Aspergillus sydowii</Emphasis> SBS 45

Two xylanases were isolated and purified from crude culture filtrate of Aspergillus sydowii SBS 45 after 9 days of growth on wheat bran containing 0.5% (w/v) birch wood xylan as the carbon source under solid-state fermentation. After a three-step purification scheme involving ammonium sulfate precipitation, gel filtration chromatography (Sephadex G-200), and anion exchange chromatography (DEAE-Sephadex A-50), xylanase I was purified 93.41 times, and xylanase II was purified 77.40 times with yields of 4.49 and 10.46, respectively. Molecular weights of xylanase I and II were 20.1 and 43 kDa, respectively, in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Optimum temperature was 50 degrees C, and optimum pH was 10.0 for both xylanase I and II. The Km value of xylanase I for birch wood xylan was 3.18 mg ml(-1) and for oat spelt xylan 6.45 mg ml(-1), while the Km value of xylanase II for birch wood xylan was 6.51 mg ml(-1) and for oat spelt xylan 7.69 mg ml(-1). Metal ions like Al3+, Ba2+, Ca2+, Na+, and Zn2+ enhanced the activity of xylanase I and II at 10 mM concentration. Among the additives, L-tryptophan enhanced the activity of xylanase I and II at 10-, 20-, and 30-mM concentrations. Both xylanases appeared to be glycoproteins.     

Neurotransmitter Transporter Family Including SLC6A6 and SLC6A13 Contributes to the 5‐Aminolevulinic Acid (ALA)‐Induced Accumulation of Protoporphyrin IX and Photodamage,through Uptake of ALA by Cancerous Cells

δ‐Aminolevulinic acid (ALA)‐induced protoporphyrin accumulation is widely used in the treatment of cancer, as photodynamic therapy (PDT). To clarify the mechanisms of ALA uptake by tumor cells, we have examined the ALA‐induced accumulation of protoporphyrin by the treatment of colon cancer DLD‐1 and epithelial cancer HeLa cells with γ‐aminobutyric acid (GABA)‐related compounds. When the cells were treated with GABA, taurine and β‐alanine, the level of protoporphyrin was decreased, suggesting that plasma membrane transporters involved in the transport of neurotransmitters contribute to the uptake of ALA. By transfection with neurotransmitter transporters SLC6A6, SLC6A8 and SLC6A13 cDNA, the ALA‐ and ALA methylester‐dependent accumulation of protoporphyrin markedly increased in HEK293T cells, dependent on an increase in the uptake of ALA. When ALA‐treated cells were exposed to white light, the extent of photodamage increased in SLC6A6‐ and SLC6A13‐expressing cells. Conversely, knockdown of SLC6A6 or SLC6A13 with siRNAs in DLD‐1 and HeLa cells decreased the ALA‐induced accumulation. The expression of SLC6A6 and SLC6A13 was found in some cancer cell lines. Immunohistochemical studies revealed that the presence of these transporters was elevated in colon cancerous cells. These results indicated that neurotransmitter transporters including SLC6A6 and SLC6A13 mediate the uptake of ALA and can play roles in the enhancement of ALA‐induced accumulation of protoporphyrin in cancerous cells.     

Accumulation of cadmium,lead, and nickel by fungal and wood biosorbents

Native fungal biomass of fungiAbsidia orchidis, Penicillium chrysogenum, Rhizopus arrhizus, Rhizopus nigricans, and modified spruce sawdust (Picea engelmanii) sequestered metals in the following decreasing preference pb>Cd>Ni. The highest metal uptake was q_max = 351 mg Pb/gA. orchidis biomass. P.chrysogenum biomass could accumulate cadmium best at 56 mg Cd/g. The sorption of nickel was the weakest always at < 5 mg Ni/g. The spruce sawdust was modified by crosslinking, oxidation to acidic oxoforms, and by substitution. The highest metal uptake was observed in phosphorylated sawdust reaching q_max = 224 mg Pb/g, 56 mg Cd/g, and 26 mg Ni/g. The latter value is comparable to the value of nickel sorption by wet commercial resin Duolite GT-73. Some improvement in metal uptake was also observed after reinforcement of fungal biomass.     

Biosorption of lead(II) from aqueous solutions by non-living algal biomass Oedogonium sp. and Nostoc sp.--a comparative study

Industrial wastewaters containing heavy metals pose a major environmental problem that needs to be remedied. The present study reports the ability of two non-living (dried) fresh water algae, Oedogonium sp. and Nostoc sp. to remove lead(II) from aqueous solutions in batch system under varying range of pH (2.99-7.04), contact time (5-300 min), biosorbent dose (0.1-0.8 g/L), and initial metal ion concentrations (100 and 200mg/L). The optimum conditions for lead biosorption are almost same for the two algal biomass Oedogonium sp. and Nostoc sp. (pH 5.0, contact time 90 and 70 min, biosorbent dose 0.5 g/L and initial Pb(II) concentration 200mg/L) however, the biomass of Oedogonium sp. was found to be more suitable than Nostoc sp. for the development of an efficient biosorbent for the removal of lead(II) from aqueous solutions, as it showed higher values of q(e) adsorption capacity (145.0mg/g for Oedogonium sp. and 93.5mg/g for Nostoc sp.). The equilibrium data fitted well in the Langmuir isotherms than the Freundlich isotherm, thus proving monolayer adsorption of lead on both the algal biomass. Analysis of data shows that the process involves second-order kinetics and thermodynamic treatment of equilibrium data shows endothermic nature of the adsorption process. The spectrum of FTIR confirms that the amino and carboxyl groups on the surface of algal biomass were the main adsorption sites for lead removal. Both the biosorbents could be regenerated using 0.1 mol/L HCl solution, with upto 90% recovery. The biosorbents were reused in five biosorption-desorption cycles without a significant loss in biosorption capacity. Thus, this study demonstrated that both the algal biomass could be used as an efficient biosorbents for the treatment of lead(II) bearing wastewater streams.     

Influence of Surface Hydrophobic Groups on the Adsorption of Proteins onto Nonporous Polymeric Particles with Immobilized Metal Ions

Iminodiacetic acid (IDA) and octyl moieties were covalently bound on nonporous particles, which were prepared from dispersion polymerization of methyl methacrylate and glycidyl methacrylate. After being charged with copper ions, the IDA-bound particles could specifically adsorb deoxyribonuclease I (DNase I) through the affinity interaction between protein and immobilized metal ion. A mixed-ligand (metal–chelate and octyl–bound) support was obtained after hydrophobic (octyl) groups were also introduced to the particle surface. The affinity adsorption of DNase I on the copper–IDA chelate was influenced by interaction between the protein and the bound octyl group. Both the affinity and the hydrophobic interactions could be well described by the Langmuir isotherms. The equilibrium adsorption constants were estimated separately to be 0.96 and 0.50 liter g⁻¹ for affinity and hydrophobic bindings, respectively. For binding on mixed-ligand support, the adsorption constant was 0.45 liter g⁻¹. It was evident that both affinity and hydrophobic interactions are involved in the adsorption of proteins onto mixed-ligand particles. Desorption of the inactive proteins from the support was possible by increasing the hydrophobicity of the solution.     

Biosorption of copper(II) and cadmium(II) by a novel exopolysaccharide secreted from deep-sea mesophilic bacterium

The biosorption behaviors and mechanisms of a novel exopolysaccharide (EPS), which is secreted by a mesophilic bacterium (namely Wangia profunda (SM-A87)) isolated from deep-sea sediment, for heavy metals Cu(II) and Cd(II) have been studied in this paper. The effects of SM-A87 EPS concentration, solution pH and ionic strength on the metal uptake were investigated by employing batch adsorption techniques, respectively. The optimum biosorption capacities were observed at pH 5.0 for Cu(II) with 48.0 mg/g and pH 6.0 for Cd(II) with 39.75 mg/g, respectively. Addition of salts decreased Cu(II) or Cd(II) uptake in the order of K⁺ < Na⁺ < Ca²⁺. Langmuir and Freundlich models were employed to describe the biosorption equilibrium data, indicating the favorable biosorption occurs and larger biosorption capacity and intensity for Cu(II) than for Cd(II). The biosorption kinetics for both metals can be well described by pseudo-second-order kinetic model, compared with pseudo-first-order and intraparticle diffusion kinetic models. The competitive biosorption was also studied, indicating that in two-component solution with different metal ratios, the selective biosorption of SM-A87 EPS for Cu(II) was much higher than for Cd(II). The Fourier transform infrared spectroscopy (FT-IR) analysis indicated possible functional groups (e.g., OH, COO and COC) of SM-A87 EPS involved in metal biosorption process, which indicated the potential of using SM-A87 EPS as an effective sorbent for Cu(II) or Cd(II) removal from water.     

Reactivity of Thioglycolic Acid Physically and Chemically Bound to Silica Gel as New Selective Solid Phase Extractors for Removal of Heavy Metal Ions From Natural Water Samples

A method is presented for the immobilization of thioglycolic acid moiety on the surface of active silica gel via a simple and direct synthetic route and based on one step reaction procedure. Two-product solid phase extractors were successfully synthesized according to physical adsorption and chemical immobilization binding techniques, phases (I) and (II), respectively. The mode of interaction between the silanol group and the thioglycolic moiety was also discussed for both phases based on the infrared analysis studies. The thermal stability properties as well as the effect of buffer solutions on the percentage hydrolysis of the two silica gel phases were examined and revealed the high stability and superiority of silica phase (II) in these respects. The evaluation of the selectivity and metal uptake properties incorporated in these two silica gel phases were also studied and discussed for a series of divalent heavy metal ions under different controlling factors. The mmol/g values were found to be higher in case of phase (I). The selective removal and extraction of some heavy metal ions, viz . Cu(II), Zn(II), and Hg(II) from natural seawater samples was successfully accomplished with the percentage recovery values for the three tested metal ions in the range of 96.5-98.4 - 0.2-0.6%. The presence of higher concentrations of Na(I), K(I), Mg(II) and Ca(II) showed insignificant role or no matrix effect on such selective extraction process due to their 0% values of removal by these silica gel phases (I) and (II).     

CopC protein from Pseudomonas syringae: intermolecular transfer of copper from both the copper(I) and copper(II) sites

The CopC protein from Pseudomonas syringae pathovar tomato is expressed as one of four proteins encoded by the operon CopABCD that is responsible for copper resistance. It is a small soluble molecule (10.5 kDa) with a beta-barrel structure and features two distinct copper binding sites, which are highly specific for Cu(I) (K(D) > or = 10(-)(13)) and Cu(II) (K(D) approximately 10(-)(15)). These dissociation constants were estimated via ligand competition experiments monitored by electronic spectral and fluorescence probes. The chemistries of the two copper sites are interdependent. When the Cu(II) site is empty, the Cu(I) ion is oxidized by air, but when both sites are occupied, the molecule is stable in air. The availability of an unoccupied site of higher affinity induces intermolecular transfer of either Cu(I) or Cu(II) while maintaining free copper ion concentrations in solution at sub-picomolar levels. This intriguing copper chemistry is consistent with the proposed role of CopC as a copper carrier in the oxidizing periplasmic space. These properties would allow it to exchange either Cu(I) or Cu(II) with its putative partners CopA, CopB, and CopD, contrasting with the role of the Cu(I) (only) chaperones found in the reducing cytoplasm.     

Ion/Molekülreaktionen negativ geladener KomplexeSchiffscher Basen mit potentiellen Ligandmolekülen in der Gasphase

Ion/molecule reactions of four coordinateSchiff base complexes under negative ion chemical ionization conditions have been studied. The complex metal ions consisted of cobalt(II), nickel(II) and copper(II).Schiff base ligands with different donor strengths were employed. The gas mixtures used contained 90% methane and 10% of the gases O₂, NO or CO. The spectra showed intense molecular negative ions, formed by secondary electron capture processes. Secondary ions were formed via ion/molecule reactions between the parent molecular negative ion and added gas molecules to giveMLX ^–,X=O₂, NO, CO;L=Schiff base ligand,M=Co(II) or Ni(II). Consistent with former investigations, secondary ion formation was not found for the copper compounds. Influence of the central metal ion as well as the ligand donor strength on the ion/molecule reactions are discussed. From the results obtained a mechanism of the secondary ion formation is suggested.

     

Electrochemical study on the keto-enol tautomerization of p-hydroxyphenylpyruvic acid in aqueous solution

The keto-enol tautomerization of p-hydroxyphenylpyruvic acid (pHPP) in aqueous solutions and the complexation reaction between enolic pHPP and boric acid have been studied by electrochemical techniques including linear sweep voltammetry (LSV), pulse voltammetry, and cyclic voltammetry (CV), combining with UV spectrometry. Electrochemical techniques reveal that in aqueous solution, there are two tautomers of pHPP: enolic form and ketonic form; the former exists mainly in freshly prepared pHPP solution, and the latter exists mainly in equilibrium solution. Both enolic and ketonic pHPP are electroactive. The electrochemical oxidation of enolic pHPP gives rise to two anodic waves, I(a) and II(a), while the electrochemical oxidation of ketonic pHPP only results in the observation of the second wave II(a). The oxidation process I(a) is revealed to be associated with the quasi-reversible, two-electron two-proton oxidation of "C=C"group at the side chain of enolic pHPP, and the oxidation process II(a) is proposed to result from the irreversible oxidation of phenolic hydroxyl group. It is observed that in aqueous solution, enolic pHPP can quickly complex with boric acid to yield enol-borate complex that can also oxidize at a glassy carbon electrode to yield an anodic wave.