Effect of complexing ligands on the surface adsorption, internalization, and bioresponse of copper and cadmium in a soil bacterium, Pseudomonas putida (original) (raw)
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Chemosphere, 2010
The distribution of cadmium (Cd) and copper (Cu) ions onto and within two soil pseudomonads, Pseudomonas putida strains KT2440 and Corvallis, was investigated using selective extraction procedures and modeled using Langmuir isotherms. Cadmium and Cu associated differently with the surface, periplasm and cytoplasm of the two strains. Both of these pseudomonad cells allowed more Cu to pass into the periplasmic space and to the cytoplasm than Cd. The distribution of Cu among the cellular spaces was solution concentration dependent, with limited amounts of Cu entering the cell at higher exposure concentrations. The Langmuir isotherm with a single binding site fit well to the observed data for Cu cell association. Cadmium was mainly found on the surface of the cells. The capacity of surface exchange sites for Cd increased with solution concentration, possibly indicating a modification of surface functional groups with ion concentration. This surface sorption behavior of Cd was best described using a two-site Langmuir model, whereas all other Cu and Cd associations were described using a one-site model. Although potentiometric titration identified differences in site densities for proton binding to the two strains, these differences were not consistently displayed with Cu and Cd surface interactions.
Environmental Toxicology and Chemistry, 2008
Increased levels of Cu in agricultural soils are of concern, because Cu toxicity may adversely affect important soil microorganisms, including pseudomonads. Because total metal concentrations correlate poorly with bioavailability and toxicity, a need exists for more information linking Cu speciation, bioavailability, and toxicity. The objective of the present study was to determine the bioavailability of different Cu complexes to Pseudomonas spp. A Cu-specific bioluminescent Pseudomonas fluorescens reporter strain was used to determine bioavailable Cu, which was operationally defined as those Cu species that induced expression of bioluminescence. Another strain of P. fluorescens, which continuously expressed bioluminescence, was used as a toxicity reporter. Experiments were performed in a defined aqueous medium containing 0.04 M Cu, which was amended with ethylenediaminetetraacetic acid (EDTA), citrate, or a well-characterized pool of dissolved organic matter (DOM). Bioluminescence emitted by the biosensors was related to data for Cu speciation obtained by geochemical modeling. Changes in Cu bioavailability in the presence of EDTA coincided with modeled changes in Cu 2ϩ activity, indicating that Cu-EDTA complexes were not bioavailable to the Cuspecific reporter. In contrast, changes of Cu bioavailability in the presence of citrate did not correspond to changes in Cu 2ϩ , indicating that Cu-citrate complexes were fully bioavailable to the reporter strain. Finally, the response of the Cu-reporter strain to Cu in the presence of DOM indicated that Cu formed bioavailable as well as unavailable complexes with DOM. We conclude that free Cu 2ϩ activity is a poor predictor of Cu bioavailability to Pseudomonas spp. in samples containing organic ligands.
Copper Speciation and Impacts on Bacterial Biosensors in the Pore Water of Copper-Contaminated Soils
Environmental Science & Technology, 2000
Knowledge of heavy metal speciation and its relationship with biological responses is important for the derivation of effects-based soil quality criteria. We determined soluble Cu concentrations and free Cu 2+ activities in the pore waters from 22 soils with total Cu varying from 19 to 8645 mg kg -1 . Pore water pCu 2+ () -log (Cu 2+ activity)) varied from 3.9 to 10.5 and was controlled by soil pH and total Cu concentration. The percentage of free Cu 2+ in total soluble Cu varied from 0.02 to 96% and was influenced strongly by pore water pH and, to a lesser extent, by dissolved organic C. In the pore waters with pH >6, the percentage of free Cu 2+ in total soluble Cu was lower than 1%. Using the default data base and with the fulvic acid content of DOC optimized at 69%, the equilibrium speciation program WHAM/Model VI gave estimates of pCu 2+ that agreed closely with measured values. Pore water samples were analyzed by two bioluminescence-based bacterial biosensors: Escherichia coli HB101 pUCD607 and Pseudomonas fluorescens 10586r pUCD607. The response of P. fluorescens correlated more closely with soil pore water pCu 2+ than with soluble Cu concentration, whereas pCu 2+ and soluble Cu fitted the response of E. coli equally well. The effect concentrations (EC 25 and EC 50 values) of pCu 2+ for the two biosensors were about 5.8 and 5.0, respectively. This is the first time that threshold values for Cu have been obtained for bacterial biosensors exposed to soil pore water from well-equilibrated contaminated soils.
Validation of the Biotic Ligand Model in Metal Mixtures: Bioaccumulation of Lead and Copper
Environmental Science & Technology, 2010
The biotic ligand model (BLM) has the potential to predict biological effects and bioaccumulation in metal mixtures. Pb and Cu uptake by the green alga Chlamydomonas reinhardtii have been quantified in single-metal exposures and in metal mixtures in order to test some of the key assumptions of the BLM. Stability constants for the interaction of the metals with biological uptake sites were determined from measured shortterm internalization fluxes. In the absence of competition, a value of 10 5.8 M -1 was obtained for Cu, while 10 5.9 M -1 was obtained for Pb. Competition experiments did not show a straightforward antagonistic competition as would be predicted by the BLM. Only at high Cu 2+ concentrations (>1 µM) did Cu behave as a competitive inhibitor of Pb transport. Surprisingly, low concentrations of Cu 2+ had a synergistic effect on Pb uptake. Furthermore, Cu uptake was independent of Pb when Cu concentrations were below 10 -7 M. In order to explain the observed discrepancies with the BLM, membrane permeability and Cu transporter expression levels were probed. The expression of ctr2, a gene coding for a Cu transporter, increased significantly in the presence of Pb, indicating that bioaccumulation is much more dynamic than assumed in the equilibrium models.
Copper and cadmium: responses in Pseudomonas putida KT2440
Letters in Applied Microbiology, 2009
Aims: To compare responses of a soil bacterium to Cu and Cd. Methods and Results: In minimal medium, Cd caused a dose-dependent growth stasis of logarithmic phase cells of Pseudomonas putida, strain KT2440, whereas Cu did not compromise growth up to 10 mg l )1 . Proteomics showed changes in accumulation of both membrane and soluble proteins by 6 h of treatment; increased Krebs cycle enzymes were apparent. Transcript analysis showed Cd-and Cu-induced different genes. Cd-induced genes encoding the transcriptional regulator CzrR2; an outer membrane protein associated with lipopolysaccharide stability, H1; two oxidative stress protective proteins and the P-type ATPase, CadA2, associated with Cd 2+ efflux. The genes most responsive to Cu encoded the regulator CopR1 and the outer membrane resistance protein regulated by CopR1, CopB1; a putative porin, PorD and the Cu-binding protein, PacZ or CopZ, and CopA2. Conclusions: These findings support that a soil pseudomonad restricts internalization of the metals by using different sets of binding proteins and efflux pumps. Activation of mechanisms to protect against oxidative stress also was evident especially with Cd exposure. Significance and Impact of the Study: The differential cellular responses to Cd and Cu suggest that risk assessment for Cd and Cu should be different.
Process Biochemistry, 2012
In this study, we designed and applied molecular biosensors for heavy metals, zinc and copper, for use in bioremediation strategies. Bacteria utilize two component systems to sense changes in the environment by multiple signal components including heavy metals and control gene expression in response to changes in signal molecules. zraP and cusC promoters were selected from a genetic circuit of the ZraSR and CusSR two-component system and were fused to a dual-labeling reporter protein as an interactive biological component for zinc and copper to generate a signal from the constructed biosensor. The biosensor efficiently senses zinc and copper with a calculated detection limit of 16 M and 26 M, respectively, and was shown to be a sensitive and effective heavy metal monitoring bacterial system. To extend the application of the bacterial biosensor, we assembled a bioadsorption system that can trigger bacteria to sense and adsorb 13 ± 0.3 mg/L of zinc and 11.4 ± 0.42 mg/L of copper per gram of dry cell weight with induction at a concentration of 100 mg/L of the respective metal ion.
Brazilian Journal of Aquatic Science and Technology, 2016
The increasing eutrophication and contamination of aquatic ecosystems motivates the study of interactions between natural dissolved organic matter (DOM), metals and the biota. Metals are mainly released into the environment by industrial processes, whereas organic materials through municipal sewage sludge. The association of these two processes and its effect on the environment poses unknown risks to aquatic communities. This investigation focused on copper speciation in the presence of aquatic heterotrophic bacteria and natural dissolved organic materials and demonstrated a relevant environmental consequence of heterotrophic bacteria related to copper speciation in aquatic systems. The experiments consisted of bacteria cultures to which natural dissolved organic materials and copper were added. Copper dynamics during bacteria growth was evaluated through the determination of free Cu2+ ions, total dissolved copper, copper internalized and adsorbed onto bacteria surface during incubation. The results showed a reduction of organic carbon and an increase of free Cu2+ ions in culture media. Significant ecological consequences are foreseen from the present results, since from less bioavailable forms of copper, bacteria released more toxic copper species into the environment.
Copper isotope fractionation during surface adsorption and intracellular incorporation by bacteria
Geochimica et Cosmochimica Acta, 2011
Copper isotopes may prove to be a useful tool for investigating bacteria-metal interactions recorded in natural waters, soils, and rocks. However, experimental data which attempt to constrain Cu isotope fractionation in biologic systems are limited and unclear. In this study, we utilized Cu isotopes (δ 65 Cu) to investigate Cu-bacteria interactions, including surface adsorption and intracellular incorporation. Experiments were conducted with individual representative species of Gram-positive (Bacillus subtilis) and Gram-negative (Escherichia coli) bacteria, as well as with wild-type consortia of microorganisms from several natural environments. Ph-dependent adsorption experiments were conducted with live and dead cells over the pH range 2.5-6. Surface adsorption experiments of Cu onto live bacterial cells resulted in apparent separation factors (Δ 65 Cu solution-solid = δ 65 Cu solution -δ 65 Cu solid ) ranging from +0.3‰ to +1.4‰ for B. subtilis and +0.2‰ to +2.6‰ for E. coli. However, because heat-killed bacterial cells did not exhibit this behavior, the preference of the lighter Cu isotope by the cells is probably not related to reversible surface adsorption, but instead is a metabolically-driven phenomenon. Adsorption experiments with heat-killed cells yielded apparent separation factors ranging from +0.3‰ to -0.69‰ which likely reflects fractionation from complexation with organic acid surface functional group sites. For intracellular incorporation experiments the lab strains and natural consortia preferentially incorporated the lighter Cu isotope with an apparent Δ 65 Cu solution-solid ranging from ~+1.0‰ to +4.4‰. Our results indicate that live bacterial cells preferentially sequester the lighter Cu isotope regardless of the experimental conditions. The fractionation mechanisms involved are likely related to active cellular transport and regulation, including the reduction of Cu(II) to Cu(I). Because similar intracellular Cu machinery is shared by fungi, plants, and higher organisms, the influence of biological processes on the δ 65 Cu of natural waters and soils is probably considerable.
Physicochemical Mechanisms of Trace Metal Bioaccumulation by Microorganisms
CHIMIA, 2002
Our research group is working towards understanding the fundamental physicochemical mechanisms of trace metal biouptake by aquatic microorganisms in natural systems. Research is currently focused on identifying under what conditions uptake fluxes are limited by physical (e.g. diffusion) as opposed to biological (transfer across biological membrane) processes. In addition, the complexation of the trace metals is being examined from both a thermodynamic (stability) and kinetic (lability) perspective in order to elucidate its effect on the overall uptake flux. Up-to-date information on the group, including a current publication list can be found at http://www.unige.ch/cabe/wilkinson.