Construction of a bacterial biosensor for zinc and copper and its application to the development of multifunctional heavy metal adsorption bacteria

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 μM and 26 μM, 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.     

Phytochelatins govern zinc/copper homeostasis and cadmium detoxification in Cuscuta campestris parasitizing Daucus carota

Cuscuta sp., known with the common name of “dodder”, is an obligate parasite capable of invading stems and leaves of a wide range of host plants. Dodder stem usually coils counterclockwise around the host and, within a few days, develops haustorial structures at each point of contact. As soon as dodder haustoria reach host vascular bundles, they start tapping water, photosynthates and minerals. Metal ions such as zinc (Zn) and copper (Cu) are essential for dodder growth and metabolism, although an exceedingly high (over-homeostatic) supply of these micronutrients can result in growth inhibition and cellular toxicity. Even more so, non-essential metals such as cadmium (Cd), if transferred from the host to the parasite, need to be neutralized by timely detoxification mechanisms. In this work, we showed that Cuscuta campestris Yuncker establishes effective haustorial connections with leaf petioles and blades of Daucus carota L. (carrot), with the consequent transfer of Cd and essential metals (such as Zn and Cu) from the host vascular bundles to the parasite. Following up to this point, we detected the presence in the parasite of significant amounts of glutathione and phytochelatins, even in the absence of Cd exposure. This suggests that thiol peptides in dodder might be particularly important for Zn and Cu homeostasis as well as for Cd detoxification. Finally, we demonstrated that dodder is capable of synthesizing phytochelatins on its own, rather than massively importing them from the host, and also provided evidence for the existence of an endogenous, constitutively expressed, dodder's phytochelatin synthase.     

The Schizosaccharomyces pombe Pccs protein functions in both copper trafficking and metal detoxification pathways

Because copper is both an essential cofactor and a toxic metal, different strategies have evolved to appropriately regulate its homeostasis as a function of changing environmental copper levels. In this report, we describe a metallochaperone-like protein from Schizosaccharomyces pombe that maintains the delicate balance between essentiality and toxicity. This protein, designated Pccs, has four distinct domains. SOD activity assays reveal that the first three domains of Pccs are necessary and sufficient to deliver copper to its target, copper-zinc superoxide dismutase (SOD1). Pccs domain IV, which is absent in Saccharomyces cerevisiae CCS1, contains seventeen cysteine residues, eight pairs of which are in a potential metal coordination arrangement, Cys-Cys. We show that S. cerevisiae ace1Delta mutant cells expressing the full-length Pccs molecule are resistant to copper toxicity. Furthermore, we demonstrate that the Pccs domain IV enhances copper resistance of the ace1Delta cells by an order of magnitude compared with that observed in the same strain expressing a pccs+ I-II-III allele encoding Pccs domains I-III. We consistently found that S. pombe cells disrupted in the pccs+ gene exhibit an increased sensitivity to copper and cadmium. Furthermore, we demonstrate that overexpression of pccs+ is associated with increased copper resistance in fission yeast cells. Taken together, our findings suggest that Pccs activates apo-SOD1 under copper-limiting conditions through the use of its first three domains and protects cells against metal ion toxicity via its fourth domain.     

Direct metal transfer between periplasmic proteins identifies a bacterial copper chaperone

Transition metals require exquisite handling within cells to ensure that cells are not harmed by an excess of free metal species. In gram-negative bacteria, copper is required in only small amounts in the periplasm, not in the cytoplasm, so a key aspect of protection under excess metal conditions is to export copper from the periplasm. Additional protection could be conferred by a periplasmic chaperone to limit the free metal species prior to export. Using isothermal titration calorimetry, we have demonstrated that two periplasmic proteins, CusF and CusB, of the Escherichia coli Cu(I)/Ag(I) efflux system undergo a metal-dependent interaction. Through the development of a novel X-ray absorption spectroscopy approach using selenomethionine labeling to distinguish the metal sites of the two proteins, we have demonstrated transfer of Cu(I) occurs between CusF and CusB. The interaction between these proteins is highly specific, as a homologue of CusF with a 51% identical sequence and a similar affinity for metal, did not function in metal transfer. These experiments establish a metallochaperone activity for CusF in the periplasm of gram-negative bacteria, serving to protect the periplasm from metal-mediated damage.     

The Arabidopsis heavy metal P-type ATPase HMA5 interacts with metallochaperones and functions in copper detoxification of roots

Since copper (Cu) is essential in key physiological oxidation reactions, organisms have developed strategies for handling Cu while avoiding its potentially toxic effects. Among the tools that have evolved to cope with Cu is a network of Cu homeostasis factors such as Cu-transporting P-type ATPases that play a key role in transmembrane Cu transport. In this work we present the functional characterization of an Arabidopsis Cu-transporting P-type ATPase, denoted heavy metal ATPase 5 (HMA5), and its interaction with Arabidopsis metallochaperones. HMA5 is primarily expressed in roots, and is strongly and specifically induced by Cu in whole plants. We have identified and characterized plants carrying two independent T-DNA insertion alleles, hma5-1 and hma5-2. Both mutants are hypersensitive to Cu but not to other metals such as iron, zinc or cadmium. Interestingly, root tips from Cu-treated hma5 mutants exhibit a wave-like phenotype at early stages and later on main root growth completely arrests whereas lateral roots emerge near the crown. Accordingly, these lines accumulate Cu in roots to a greater extent than wild-type plants under Cu excess. Finally, yeast two-hybrid experiments demonstrate that the metal-binding domains of HMA5 interact with Arabidopsis ATX1-like Cu chaperones, and suggest a regulatory role for the plant-specific domain of the CCH Cu chaperone. Based on these findings, we propose a role for HMA5 in Cu compartmentalization and detoxification.     

Characterization of the denitrifying bacterial community in a full-scale rockwool biofilter for compost waste-gas treatment

The potential denitrification activity and the composition of the denitrifying bacterial community in a full-scale rockwool biofilter used for treating livestock manure composting emissions were analyzed. Packing material sampled from the rockwool biofilter was anoxically batch-incubated with 15N-labeled nitrate in the presence of different electron donors (compost extract, ammonium, hydrogen sulfide, propionate, and acetate), and responses were compared with those of activated sludge from a livestock wastewater treatment facility. Overnight batch-incubation showed that potential denitrification activity for the rockwool samples was higher with added compost extract than with other potential electron donors. The number of 16S rRNA and nosZ genes in the rockwool samples were in the range of 1.64–3.27 × 109 and 0.28–2.27 × 108 copies/g dry, respectively. Denaturing gradient gel electrophoresis analysis targeting nirK, nirS, and nosZ genes indicated that the distribution of nir genes was spread in a vertical direction and the distribution of nosZ genes was spread horizontally within the biofilter. The corresponding denitrifying enzymes were mainly related to those from Phyllobacteriaceae, Bradyrhizobiaceae, and Alcaligenaceae bacteria and to environmental clones retrieved from agricultural soil, activated sludge, freshwater environments, and guts of earthworms or other invertebrates. A nosZ gene fragment having 99% nucleotide sequence identity with that of Oligotropha carboxidovorans was also detected. Some nirK fragments were related to NirK from micro-aerobic environments. Thus, denitrification in this full-scale rockwool biofilter might be achieved by a consortium of denitrifying bacteria adapted to the intensely aerated ecosystem and utilizing mainly organic matter supplied by the livestock manure composting waste-gas stream.     

Ethylene removal efficiency and bacterial community diversity of a natural zeolite biofilter

To establish an economical and environmentally friendly technology for ethylene removal from horticultural facilities and industrial point sources, a bench-scale natural zeolite biofiltration system was developed in this study. The system was evaluated for its performance in removing ethylene from an artificially contaminated air stream and characterized for its bacterial diversity under varied ethylene concentrations, and in different spatial stages of the filter. The biofilter enabled to approximately 100% remove ethylene at loading rates of 0.26-3.76 g m⁻³ h⁻¹ when operated with inoculum containing enriched ethylene-degrading bacteria. The bacterial diversity and abundance varied with the height of the biofilter. Moreover, the occurrence and predominance of specific bacterial species varied with the concentrations of ethylene introduced into the biofilter, as observed by PCR-DGGE methods. Phylogenetic analysis indicated that the biofilter system supported a diverse community of ethylene-degrading bacteria, with high similarity to species in the classes Betaproteobacteria, Gammaproteobacteria, Bacilli, and Actinobacteria.     

A new pathway for heavy metal detoxification in animals. Phytochelatin synthase is required for cadmium tolerance in Caenorhabditis elegans

Increasing emissions of heavy metals such as cadmium, mercury, and arsenic into the environment pose an acute problem for all organisms. Considerations of the biochemical basis of heavy metal detoxification in animals have focused exclusively on two classes of peptides, the thiol tripeptide, glutathione (GSH, gamma-Glu-Cys-Gly), and a diverse family of cysteine-rich low molecular weight proteins, the metallothioneins. Plants and some fungi, however, not only deploy GSH and metallothioneins for metal detoxification but also synthesize another class of heavy metal binding peptides termed phytochelatins (PCs) from GSH. Here we show that PC-mediated heavy metal detoxification is not restricted to plants and some fungi but extends to animals by demonstrating that the ce-pcs-1 gene of the nematode worm Caenorhabditis elegans encodes a functional PC synthase whose activity is critical for heavy metal tolerance in the intact organism.     

Cellular mechanisms for heavy metal detoxification and tolerance.

Heavy metals such as Cu and Zn are essential for normal plant growth, although elevated concentrations of both essential and non-essential metals can result in growth inhibition and toxicity symptoms. Plants possess a range of potential cellular mechanisms that may be involved in the detoxification of heavy metals and thus tolerance to metal stress. These include roles for the following: for mycorrhiza and for binding to cell wall and extracellular exudates; for reduced uptake or efflux pumping of metals at the plasma membrane; for chelation of metals in the cytosol by peptides such as phytochelatins; for the repair of stress-damaged proteins; and for the compartmentation of metals in the vacuole by tonoplast-located transporters. This review provides a broad overview of the evidence for an involvement of each mechanism in heavy metal detoxification and tolerance.     

Reinvestigation of metal ion specificity for quinone cofactor biogenesis in bacterial copper amine oxidase

The topa quinone (TPQ) cofactor of copper amine oxidase is generated by copper-assisted self-processing of the precursor protein. Metal ion specificity for TPQ biogenesis has been reinvestigated with the recombinant phenylethylamine oxidase from Arthrobacter globiformis. Besides Cu2+ ion, some divalent metal ions such as Co2+, Ni2+, and Zn2+ were also bound to the metal site of the apoenzyme so tightly that they were not replaced by excess Cu2+ ions added subsequently. Although these noncupric metal ions could not initiate TPQ formation under the atmospheric conditions, we observed slow spectral changes in the enzyme bound with Co2+ or Ni2+ ion under the dioxygen-saturating conditions. Resonance Raman spectroscopy and titration with phenylhydrazine provided unambiguous evidence for TPQ formation by Co2+ and Ni2+ ions. Steady-state kinetic analysis showed that the enzymes activated by Co2+ and Ni2+ ions were indistinguishable from the corresponding metal-substituted enzymes prepared from the native copper enzyme (Kishishita, S., Okajima, T., Kim, M., Yamaguchi, H., Hirota, S., Suzuki, S., Kuroda, S., Tanizawa, K., and Mure, M. (2003) J. Am. Chem. Soc. 125, 1041-1055). X-ray crystallographic analysis has also revealed structural identity of the active sites of Co- and Ni-activated enzymes with Cu-enzyme. Thus Cu2+ ion is not the sole metal ion assisting TPQ formation. Co2+ and Ni2+ ions are also capable of forming TPQ, though much less efficiently than Cu2+.     

Decolorisation and detoxification of Direct Blue-15 by a bacterial consortium

Studies were carried out on decolorisation and biotransformation of the dye Direct Blue-15 into 3,3'-dimethoxybenzidine (O'-dianisidine) and a sulphonated derivative by a five-member bacterial consortium. Chromatographic studies revealed further complete biodegradation of 3,3'-dimethoxybenzidine coupled with release of ammonia, but the recalcitrant sulphonated derivative persisted. The microorganisms identified in the mixed consortium by 16S rDNA sequence analysis were Alcaligenes faecalis, Sphingomonas sp. EBD, Bacillus subtilis, Bacillus thuringiensis and Enterobacter cancerogenus. The cytotoxicity data showed a significant reduction in the toxicity (P<0.001) of the degraded dye as evidenced from the number of viable human polymorphonuclear leukocyte cells present.     

Mechanisms of cadmium detoxification in cattail (Typha angustifolia L.)

Cadmium (Cd) is a widespread heavy metal pollutant and environmental and human health hazard, which may be partially resolved using green and cost-effective phytoremediation techniques. However, the efficiency of phytoremediation is often limited by the small biomass of Cd-hyperaccumulator plants. Although cattail (Typha angustifolia L.) is tolerant of heavy metals and has a high biomass, there is little information available on its detoxification mechanisms for heavy metals, especially Cd. In the present study we investigated the tolerance of cattail to Cd and mechanisms involved in its Cd detoxification. Our results show that: (a) cattail is tolerant of Cd; (b) the root Casparian band, cell wall, vacuole, glutathione (GSH), and glutathione peroxidase (GPX) play important roles in Cd detoxification; and (c) mechanisms of Cd detoxification differ in leaf cell cytoplasm (mainly a GSH-related antioxidant defense system) and root cell cytoplasm (mainly a GSH-related chelation system). In summary, cattail possesses multiple detoxification mechanisms for Cd and is a promising species for phytoremediation of Cd-polluted environments.     

Utilization of various agricultural wastes for activated carbon preparation and application for the removal of dyes and metal ions from aqueous solutions

Activated carbons were prepared from the agricultural solid wastes, silk cotton hull, coconut tree sawdust, sago waste, maize cob and banana pith and used to eliminate heavy metals and dyes from aqueous solution. Adsorption of all dyes and metal ions required a very short time and gave quantitative removal. Experimental results show all carbons were effective for the removal of pollutants from water. Since all agricultural solid wastes used in this investigation are freely, abundantly and locally available, the resulting carbons are expected to be economically viable for wastewater treatment.     

Heavy metal stress. Activation of distinct mitogen-activated protein kinase pathways by copper and cadmium

Excessive amounts of heavy metals adversely affect plant growth and development. Whereas some regions naturally contain high levels of heavy metals, anthropogenic release of heavy metals into the environment continuously increases soil contamination. The presence of elevated levels of heavy metal ions triggers a wide range of cellular responses including changes in gene expression and synthesis of metal-detoxifying peptides. To elucidate signal transduction events leading to the cellular response to heavy metal stress we analyzed protein phosphorylation induced by elevated levels of copper and cadmium ions as examples for heavy metals with different physiochemical properties and functions. Exposure of alfalfa (Medicago sativa) seedlings to excess copper or cadmium ions activated four distinct mitogen-activated protein kinases (MAPKs): SIMK, MMK2, MMK3, and SAMK. Comparison of the kinetics of MAPK activation revealed that SIMK, MMK2, MMK3, and SAMK are very rapidly activated by copper ions, while cadmium ions induced delayed MAPK activation. In protoplasts, the MAPK kinase SIMKK specifically mediated activation of SIMK and SAMK but not of MMK2 and MMK3. Moreover, SIMKK only conveyed MAPK activation by CuCl(2) but not by CdCl(2). These results suggest that plants respond to heavy metal stress by induction of several distinct MAPK pathways and that excess amounts of copper and cadmium ions induce different cellular signaling mechanisms in roots.     

The composition of pyrophosphate heavy metal detoxification granules in barnacles

A major detoxification mechanism involving the binding of excess metals to inorganic, insoluble metalliferous granules occurs in many invertebrates. X-ray microanalysis of granules extracted from the barnacles Elminius modestus Darwin and Semibalanus balanoides (L.) (Crustacea: Cirripedia) indicated that one granule type was predominant. Examples of this granule type usually contained Fe and P, or Zn and S, or a combination of all four elements as major constituents. The predominant form of P in the granules was pyrophosphate, P₂O⁴⁻₇, with oprthophosphate and other polyphosphates present in lower amounts. The S was present as organic S. Biochemical analysis showed high levels of Ca, Fe, Mg, K and Zn in the granules with Cu and Ag present in smaller quantities. Considerable variation was shown between granules.