Thiobacillus ferrooxidans response to copper and other heavy metals: growth, protein synthesis and protein phosphorylation

Respirometric experiments demonstrated that the oxygen uptake by Thiobacillus ferrooxidans strain LR was not inhibited in the presence of 200 mM copper. Copper-treated and untreated cells from this T. ferrooxidans strain were used in growth experiments in the presence of cadmium, copper, nickel and zinc. Growth in the presence of copper was improved by the copper-treated cells. However, no growth was observed for these cells, within 190 h of culture, when cadmium, nickel and zinc were added to the media. Changes in the total protein synthesis pattern were detected by two-dimensional polyacrylamide gel electrophoresis for T. ferrooxidans LR cells grown in the presence of different heavy metals. Specific proteins were induced by copper (16, 28 and 42 kDa) and cadmium (66 kDa), whereas proteins that had their synthesis repressed were observed for all the heavy metals tested. Protein induction was also observed in the cytosolic and membrane fractions from T. ferrooxidans LR cells grown in the presence of copper. The level of protein phosphorylation was increased in the presence of this metal.     

Cadmium and Nickel Toxicity for <Emphasis Type="Italic">Sinapis alba</Emphasis> Plants Inoculated with Endophytic Strains of <Emphasis Type="Italic">Bacillus subtilis</Emphasis>

We studied the effect of cadmium and nickel on Sinapis alba L. plants inoculated with endophytic strains of Bacillus subtilis. It was shown that treatment of S. alba seeds with endophytic strains of bacteria B. subtilis improves plant resistance to the toxic effect of cadmium and nickel and reduces manifestation of oxidative stress in the presence of higher levels of metal ions in the above-ground part of plants. Anti-stress effect and the ability of endophytic strains of B. subtilis to intensify uptake of cadmium and nickel ions by S. alba plants may be used for phytoextraction of heavy metals and stimulation of plant growth in contaminated areas.     

Effects of heavy metals on Tetraselmis suecica: Ultrastructural and energy-dispersive X-ray spectroscopic studies

The influence of metal contamination on the marine alga Tetraselmis suecica was investigated at physiological and ultrastructural levels. For this analysis, the growth response of this microalga was studied after the addition of various concentrations of heavy metals (Cd, Cu). The concentration corresponding to 50% growth inhibition (IC₅₀) and the number of days per cell cycle (Td) studied, revealed that the toxic effects of copper are heavier than those of cadmium. In the case of copper contamination, the Td grows with increasing metal concentration in the culture medium, while it remains unchanged during the cadmium contamination. The toxicity of cadmium, only observed in the latency phase of growth, suggests an adaptation phenomenon of T suecica to this metal. Ultrastructural changes in response to pollutants were investigated; copper induced cytoplasmic vacuolisation, organelle changes, appearance of cells with multilayered cell walls and excretion of organic matter. In the case of cadmium contamination, ultrastructural changes mainly affected the osmiophilic vesicles, of which both number and volume increased with increasing metal concentration in the culture medium. The results of X-ray microanalysis revealed that Cd and Cu were strongly present in excreted organic matter and osmiophilic vesicles. The latter can be excreted during cell division, thus participating in detoxification processes. Intracellular cadmium incorporation proved that some toxic effects of this metal are a result of interaction with endogenous cellular constituents. In the case of copper contamination, the presence of copper in walls of a multilayered cell suggests that these structures constitute an additionnal adsorbing area for this element, reducing metal free concentration in the medium. Mechanisms of metal detoxification of Tetraselmis suecica are discussed.     

Mass balance of heavy metal uptake by encapsulated cultures ofKlebsiella aerogenes

Dialysis was employed as a method of speciating heavy metals in cultures of an extracellular polymer forming strain ofKlebsiella aerogenes. A noncapsulated strain of the same bacterium was used as a control, and a mass balance of copper, cadmium, cobalt, nickel, and manganese in batch culture at pH 4.5 and pH 6.8 and in continuous culture at pH 6.8 was constructed. Copper and cadmium were accumulated by the cell during rapid proliferation whereas all 5 metals were bound nonspecifically by extracellular polymer produced during stationary phase and at low dilution rates. The presence of extracellular polymer appeared to inhibit cellular uptake of nickel. At the lower pH, metal uptake was considerably reduced. The results are discussed in the context of metal removal in the activated sludge process of waste water treatment.     

The uptake of lead,zinc, cadmium,and copper by the pulmonate mollusc,Helix aspersa muller,and its relevance to the monitoring of heavy metal contamination of the environment

Summary The occurrence of lead, zinc, cadmium, and copper in individuals of Helix aspersa from two sites of varying degrees of contamination was studied. Zinc, cadmium, and copper were shown to increase in a linear fashion with animal weight. The rate of uptake for zinc and cadmium in particular was significantly greater at the more contaminated site. Statistical analysis of the data, using correlation and regression techniques provided information on apparent intermetallic effects.It is concluded that because metal uptake and body weight show a positive linear relationship only the use of animals of similar weight and/or size can be used for monitoring purposes. Even then, different patterns of uptake into different organs and interactions between metal uptakes are such as to seriously question the use of Helix, and other molluscs, for monitoring purposes unless specific organs from comparably sized and/or aged animals are used.     

Cadmium Accumulation and DNA Homology with Metal Resistance Genes in Sulfate-Reducing Bacteria

Cadmium resistance (0.1 to 1.0 mM) was studied in four pure and one mixed culture of sulfate-reducing bacteria (SRB). The growth of the bacteria was monitored with respect to carbon source (lactate) oxidation and sulfate reduction in the presence of various concentrations of cadmium chloride. Two strains Desulfovibrio desulfuricans DSM 1926 and Desulfococcus multivorans DSM 2059 showed the highest resistance to cadmium (0.5 mM). Transmission electron microscopy of the two strains showed intracellular and periplasmic accumulation of cadmium. Dot blot DNA hybridization using the probes for the smtAB, cadAC, and cadD genes indicated the presence of similar genetic determinants of heavy metal resistance in the SRB tested. DNA sequencing of the amplified DNA showed strong nucleotide homology in all the SRB strains with the known smtAB genes encoding synechococcal metallothioneins. Protein homology with the known heavy metal-translocating ATPases was also detected in the cloned amplified DNA of Desulfomicrobium norvegicum I1 and Desulfovibrio desulfuricans DSM 1926, suggesting the presence of multiple genetic mechanisms of metal resistance in the two strains.     

ZINC AND COPPER TOLERANCE OF AGROSTIS STOLONIFERA L. IN TISSUE CULTURE

Callus tissue was induced from shoot meristematic tissue and root tips of a clone of the grass Agrostis stolonifera tolerant to both zinc and copper, and from a control clone tolerant to neither metal. Growth of the callus tissue on media containing zinc and copper showed that tolerance to both metals was maintained in tissue culture. The pattern of metal uptake in tissue culture resembled uptake by whole plants in that tolerant tissue took up more metal than nontolerant tissue. Plants regenerated from callus had the same copper and zinc tolerance as the original parental clones regardless of time of growth in tissue culture and shoot or root origin of the tissue. The results support previous evidence that metal tolerance is genetically determined and acts at the cellular level.     

Susceptibility of Halobacteria to Heavy Metals

Sixty-eight halobacteria, including both culture collection strains and fresh isolates from widely differing geographical areas, were tested for susceptibility to arsenate, cadmium, chromium, cobalt, copper, lead, mercury, nickel, silver, and zinc ions by an agar dilution technique. The culture collection strains showed different susceptibilities, clustering into five groups. Halobacterium mediterranei and Halobacterium volcanii were the most metal tolerant, whereas Haloarcula californiae and Haloarcula sinaiiensis had the highest susceptibilities of the culture collection strains. Different patterns of metal susceptibility were found for all the halobacteria tested, and there was a uniform susceptibility to mercury and silver. All strains tested were multiply metal tolerant.     

Uptake of six heavy metals by oat as influenced by soil type and additions of cadmium,lead, zinc and copper

Summary The influence of heavy metal additions on availability and uptake of cadmium, lead, zinc, copper, manganese and iron by oat was studied. The experiments were carried out as pot experiments using sandy loam, sandy soil and organic soil. Selective extractants were used to remove metals held in different soil fractions.Lead and copper were preferently bound by organics and oxides, zinc by oxides and inorganics, and cadmium by inorganics and organics.Addition of cadmium to the soils resulted in higher cadmium concentrations in all plant parts but lower concentrations of lead, zinc, copper, manganese and iron, and the accumulation indexes of these metals were also lower when cadmium was added to the soil.Addition of cadmium plus lead, zinc and copper resulted in higher cadmium concentrations in leaves and straw of plants grown in sandy loam and sandy soil, but lower concentrations when plants were grown in organic soil as compared with the results when cadmium was added separately. The transfer of cadmium, lead, zinc and copper from soil to plant was greatest from sandy soil, and zinc and cadmium were more mobile in the plant than were lead and copper.Cadmium concentrations in leaves correlated significantly with CaCl₂ and CH₃COOH extractions in sandy loam and sandy soil and with CH₃COOH extractions in organic soil.Generally, the total metal uptake was lowest from organic soil.     

Activation of Methanogenesis by Cadmium in the Marine Archaeon Methanosarcina acetivorans

Methanosarcina acetivorans was cultured in the presence of CdCl₂ to determine the metal effect on cell growth and biogas production. With methanol as substrate, cell growth and methane synthesis were not altered by cadmium, whereas with acetate, cadmium slightly increased both, growth and methane rate synthesis. In cultures metabolically active, incubations for short-term (minutes) with 10 µM total cadmium increased the methanogenesis rate by 6 and 9 folds in methanol- and acetate-grown cells, respectively. Cobalt and zinc but not copper or iron also activated the methane production rate. Methanogenic carbonic anhydrase and acetate kinase were directly activated by cadmium. Indeed, cells cultured in 100 µM total cadmium removed 41–69% of the heavy metal from the culture and accumulated 231–539 nmol Cd/mg cell protein. This is the first report showing that (i) Cd²⁺ has an activating effect on methanogenesis, a biotechnological relevant process in the bio-fuels field; and (ii) a methanogenic archaea is able to remove a heavy metal from aquatic environments.     

Streptococcus pneumoniae Uses Glutathione To Defend against Oxidative Stress and Metal Ion Toxicity

The thiol-containing tripeptide glutathione is an important cellular constituent of many eukaryotic and prokaryotic cells. In addition to its disulfide reductase activity, glutathione is known to protect cells from many forms of physiological stress. This report represents the first investigation into the role of glutathione in the Gram-positive pathogen Streptococcus pneumoniae. We demonstrate that pneumococci import extracellular glutathione using the ABC transporter substrate binding protein GshT. Mutation of gshT and the gene encoding glutathione reductase (gor) increases pneumococcal sensitivity to the superoxide generating compound paraquat, illustrating the importance of glutathione utilization in pneumococcal oxidative stress resistance. In addition, the gshT and gor mutant strains are hypersensitive to challenge with the divalent metal ions copper, cadmium, and zinc. The importance of glutathione utilization in pneumococcal colonization and invasion of the host is demonstrated by the attenuated phenotype of the gshT mutant strain in a mouse model of infection.     

Enhanced Toxic Metal Accumulation in Engineered Bacterial Cells Expressing Arabidopsis thaliana Phytochelatin Synthase

Phytochelatins (PCs) are metal-binding cysteine-rich peptides, enzymatically synthesized in plants and yeasts from glutathione in response to heavy metal stress by PC synthase (EC 2.3.2.15). In an attempt to increase the ability of bacterial cells to accumulate heavy metals, the Arabidopsis thaliana gene encoding PC synthase (AtPCS) was expressed in Escherichia coli. A marked accumulation of PCs was observed in vivo together with a decrease in the glutathione cellular content. When bacterial cells expressing AtPCS were placed in the presence of heavy metals such as cadmium or the metalloid arsenic, cellular metal contents were increased 20- and 50-fold, respectively. We discuss the possibility of using genes of the PC biosynthetic pathway to design bacterial strains or higher plants with increased abilities to accumulate toxic metals, and also arsenic, for use in bioremediation and/or phytoremediation processes.     

Selection and characterization of cadmium tolerant cells in tomato

Tomato (Lycopersicon esculentum cv. VFNT-Cherry)cell lines tolerant of to 5 mM cadmium (Cd) were selected by progressively elevating the level of CdCl₂ in the culture medium (the lethal concentration of Cd for unselected tomato cells is 400 μM). Cd tolerance was not lost during long-term culture (up to 12 months) in the absence of Cd stress. In all the cell lines examined, Cd uptake was rapid and Cd concentration within the cells exceeded that in the culture medium by several fold. While Cd included the synthesis and accumulation of phytochelatins (PCs) [poly[γ-glutamyl-cysteiny)glycine], little change has been observed in protein synthesis during short term Cd stress. PCs formed complexes with Cd. However, uptake and accumulation of Cd was not affected if PC synthesis was inhibited by treatment with buthionine sulfoximine. Selected and unselected cells were compared for their growth characteristics in the presence of various other metal ions. Cd tolerant cells showed a slightly higher tolerance of copper but not of mercury, zinc, lead or silver.     

Resistance to and uptake of heavy metals in mushrooms

Resistance levels to heavy metals in 21 mushrooms obtained mainly from Japan, but also from Thailand, were tested. The mushrooms were 7 Pleurotus species, 2 Pycnoporus and Pholiota species and one each of Agrocybe, Cryptoporus, Coriolus, Inonotus, Lampteromyces, Grifola, Flammulina, Lyophyllum, Agaricus, and Polyporus species. The Pleurotus species strains showed higher resistance to the heavy metals, copper, cadmium, zinc, nickel, cobalt, mercury than the other species. Pleurotus ostreatus exhibited the highest resistance to all these heavy metals. Pholiota species, Flammulina veltipes, Lyophyllum ulmarium, Agaricus bisporus and Polyporus arcularius were rather sensitive to all the metals tested. The accumulation of copper, zinc and cadmium in Pleurotus ostreatus was studied. The uptake of heavy metals into the mycelia of P. ostreatus increased proportionally to an increasing concentration of these metals in the medium.     

Respiratory inhibition by copper in Tetrahymena pyriformis GL

An excess of copper incorporated into Tetrahymena cells was mainly distributed in mitochondria, and inhibited oxygen uptake of Tetrahymena cells. The inhibition of oxygen uptake was clearly to copper uptake in mitochondria. Succinate was most favorable as a substrate stimulating oxygen uptake in mitochondria, and oxygen uptake was most strongly inhibited by copper (0.1 mM) in the presence of succinate among various substrates. The copper incorporated into mitochondria was in the fraction with the inner membranes. Succinate dehydrogenase (SDH) was inhibited at the lowest copper concentration (0.1 mM) among respiratory related enzymes. The redox potential of respiratory components was raised by copper. These results suggest that respiratory inhibition of Tetrahymena cells by copper may be mainly cause by inhibition of SDH as a FAD-protein and oxidation of electron carriers. At higher copper concentrations, MDH, cytochrome c reductase, and ATP synthesis were also inhibited. Growth inhibition may be due to these effects of copper in mitochondria. Mercury affected both oxygen uptake and SDH more strongly than copper. Zinc (0.1 mM) also affected oxygen uptake in mitochondria and a little in whole cells, however, it did not inhibit SDH. Cobalt, manganese, and nickel affected both oxygen uptake and SDH only a little at the same concentration (0.1 mM) as copper.     

Rescue of heavy metal effects on cell physiology of the algal model system Micrasterias by divalent ions

Recent studies have shown that metals such as copper, zinc, aluminum, cadmium, chromium, iron and lead cause severe dose-dependent disturbances in growth, morphogenesis, photosynthetic and respiratory activity as well as on ultrastructure and function of organelles in the algal model system Micrasterias denticulata ( Volland et al., 2011, Volland et al., 2012 and Andosch et al., 2012). In the present investigation we focus on amelioration of these adverse effects of cadmium, chromium and lead by supplying the cells with different antioxidants and essential micronutrients to obtain insight into metal uptake mechanisms and subcellular metal targets. This seems particularly interesting as Micrasterias is adapted to extremely low-concentrated, oligotrophic conditions in its natural bog environment.     

Myriophyllum alterniflorum DC. Effect of low concentrations of copper and cadmium on somatic and photosynthetic endpoints: A chemometric approach

Aquatic plants can be a sensitive and cost effective route to understanding the effects of contaminants and assessing ecosystem health. Among the aquatic macrophytes, watermilfoil genus Myriophyllum could be used in ecological surveys as in situ biomonitors of water quality due to its ability to concentrate pollutants in their tissues and reflect the environmental pollution. The objectives of the present study were to evaluate the phytotoxicity of two heavy metals, Cu (essential micronutrient) and Cd (non-essential metal) alone or in mixture at realistic (low) concentration levels to a common European watermilfoil, Myriophyllum alterniflorum. ANOVA Kruskall–Wallis data indicate that effect of pH and mixture on copper or cadmium amounts in plants was statistically non significant. Correlation between variables was assessed using Spearman's rank order correlation. The main correlations were between: (i) time and node length, (ii) fresh mass and dry mass, (iii) chlorophyll A and carotenoids, and (iv) copper and cadmium in the mixture study. In addition to the Spearman correlations, data were analysed using the factor analysis. High positive loadings of mass (fresh weight and dry weight) along the factor 1 axis and high loadings (positive for copper and cadmium treatment, negative for the mixture) of plant growth (time, node length) along the factor 2 axis are evident. In conclusion, statistical estimate of the data revealed no strong significant relationships between the treatment (copper and cadmium in M. alterniflorum) and the somatic or photosynthetic endpoints studied. This lack of significant results could be explained by the low metal concentrations tested and this study failed to reproduce our first conclusions about the correlation between cadmium, chlorophyll A and carotenoids.     

AgsA response to cadmium and copper effects at different temperatures in Escherichia coli

Small heat shock proteins (sHsps), present from prokaryotes to eukaryotes, are a highly conserved molecular chaperone family. They play a crucial role in protecting organisms against cellular insults from single or multiple environmental stressors including heavy metal exposure, heat or cold shock, oxidative stress, desiccation, etc. Here, the toxicity of cadmium and copper, and their ability to modify the cellular growth rate at different temperatures in Escherichia coli cells were tested. Also, the response mechanism of the sHSP aggregation‐suppressing protein (AgsA) in such multiple stress conditions was investigated. The results showed that the half effect concentration (EC₅₀) of cadmium in AgsA‐transformed E. coli cells at 37°C, 42°C, and 50°C were 11.106, 29.50, and 4.35 mg/L, respectively, and that of the control cells lacking AgsA were 5.05, 0.93, and 0.18 mg/L, respectively, while the half effect concentration (EC₅₀) of copper in AgsA‐transformed E. coli cells at 37°C, 42°C, and 50°C were 27.3, 3.40, and 1.28 mg/L, respectively, and that of the control cells lacking AgsA were 27.7, 5.93, and 0.134 mg/L, respectively. The toxicities of cadmium and copper at different temperatures as observed by their modification of the cellular growth rate and inhibitory effects were in a dose‐dependent manner. Additionally, biochemical characterization of AgsA protein in cells subjected to cadmium and copper stresses at different temperatures implicated suppressed aggregation of cellular proteins in AgsA‐transformed E. coli cells. Altogether, our data implicate the AgsA protein as a sensitive protein‐based biomarker for metal‐induced toxicity monitoring.     

Comparative studies of the heavy metal uptake of whole cells and different types of cell walls from Chlorella fusca

Summary Short-term uptake of cadmium, copper and lead by whole cells, mother cell walls (mcw) and cell walls from cell homogenates (hcw) of Chlorella fusca were similar for all biosorbents: Adsorption equilibrium was reached five minutes after metal addition. Analogous, over 90% of the readily adsorbed metal ions could be desorbed by a 1 minute EDTA treatment. In all cases, short-term or long-term uptake and single ion solution or metal mixtures, whole cells showed the highest metal accumulation while hcw adsorbed more metal ions than mcw. The role of the cell wall for metal ion adsorption is discussed.