Chromate-reducing properties of soluble flavoproteins from Pseudomonas putida and Escherichia coli

Cr(VI) (chromate) is a toxic, soluble environmental contaminant. Bacteria can reduce chromate to the insoluble and less toxic Cr(III), and thus chromate bioremediation is of interest. Genetic and protein engineering of suitable enzymes can improve bacterial bioremediation. Many bacterial enzymes catalyze one-electron reduction of chromate, generating Cr(V), which redox cycles, generating excessive reactive oxygen species (ROS). Such enzymes are not appropriate for bioremediation, as they harm the bacteria and their primary end product is not Cr(III). In this work, the chromate reductase activities of two electrophoretically pure soluble bacterial flavoproteins--ChrR (from Pseudomonas putida) and YieF (from Escherichia coli)-were examined. Both are dimers and reduce chromate efficiently to Cr(III) (kcat/Km = approximately 2 x 10(4) M(-1) x s(-1)). The ChrR dimer generated a flavin semiquinone during chromate reduction and transferred >25% of the NADH electrons to ROS. However, the semiquinone was formed transiently and ROS diminished with time. Thus, ChrR probably generates Cr(V), but only transiently. Studies with mutants showed that ChrR protects against chromate toxicity; this is possibly because it preempts chromate reduction by the cellular one-electron reducers, thereby minimizing ROS generation. ChrR is thus a suitable enzyme for further studies. During chromate reduction by YieF, no flavin semiquinone was generated and only 25% of the NADH electrons were transferred to ROS. The YieF dimer may therefore be an obligatory four-electron chromate reducer which in one step transfers three electrons to chromate and one to molecular oxygen. As a mutant lacking this enzyme could not be obtained, the role of YieF in chromate protection could not be directly explored. The results nevertheless suggest that YieF may be an even more suitable candidate for further studies than ChrR.     

Interactions of chromium with microorganisms and plants

Chromium is a highly toxic non-essential metal for microorganisms and plants. Due to its widespread industrial use, chromium (Cr) has become a serious pollutant in diverse environmental settings. The hexavalent form of the metal, Cr(VI), is considered a more toxic species than the relatively innocuous and less mobile Cr(III) form. The presence of Cr in the environment has selected microbial and plant variants able to tolerate high levels of Cr compounds. The diverse Cr-resistance mechanisms displayed by microorganisms, and probably by plants, include biosorption, diminished accumulation, precipitation, reduction of Cr(VI) to Cr(III), and chromate efflux. Some of these systems have been proposed as potential biotechnological tools for the bioremediation of Cr pollution. In this review we summarize the interactions of bacteria, algae, fungi and plants with Cr and its compounds.     

Mechanism of chromate reduction by the Escherichia coli protein, NfsA, and the role of different chromate reductases in minimizing oxidative stress during chromate reduction

Chromate [Cr(VI)] is a serious environmental pollutant, which is amenable to bacterial bioremediation. NfsA, the major oxygen-insensitive nitroreductase of Escherichia coli, is a flavoprotein that is able to reduce chromate to less soluble and less toxic Cr(III). We show that this process involves single-electron transfer, giving rise to a flavin semiquinone form of NfsA and Cr(V) as intermediates, which redox cycle, generating more reactive oxygen species (ROS) than a divalent chromate reducer, YieF. However, NfsA generates less ROS than a known one-electron chromate reducer, lipoyl dehydrogenase (LpDH), suggesting that NfsA employs a mixture of uni- and di-valent electron transfer steps. The presence of YieF, ChrR (another chromate reductase we previously characterized), or NfsA in an LpDH-catalysed chromate reduction reaction decreased ROS generation by c. 65, 40, or 20%, respectively, suggesting that these enzymes can pre-empt ROS generation by LpDH. We previously showed that ChrR protects Pseudomonas putida against chromate toxicity; here we show that NfsA or YieF overproduction can also increase the tolerance of E. coli to this compound.     

水环境中Cr(Ⅵ)对鱼类毒性机理研究进展

随着工业,特别是不锈钢产业的迅速发展,环境中的铬污染问题日益严重,Cr(Ⅵ)能够很容易地透过细胞膜进入细胞和生物体内,其毒性远高于Cr(Ⅲ).铬作为变价金属,其在鱼类体内能够产生活性氧自由基(ROS),对机体产生氧化胁迫效应;此外,Cr(Ⅵ)在细胞还原作用下生成的中间产物\[Cr(Ⅴ)、Cr(Ⅳ)等\]会进一步和DNA结合,导致基因的损伤和突变,从而危害鱼类的生长发育和种群结构.本文在不同水平上,系统总结了Cr(Ⅵ)对鱼类的毒性效应,从多个层次(分子、细胞、组织、器官、个体）阐述了Cr(Ⅵ)的毒性作用机制和鱼类相应的毒性解毒机制,并对于Cr(Ⅵ)的毒性研究中尚不完全清楚、需要深入考察的方面进行了探讨.     

Hexavalent chromium reduction by bacteria from tannery effluent

Chromium is generated from several industrial processes. It occurs in different oxidation states, but Cr(III) and Cr(VI) are the most common ones. Cr(VI) is a toxic, soluble environmental contaminant. Some bacteria are able to reduce hexavalent chromium to the insoluble and less toxic Cr(III), and thus chromate bioremediation is of considerable interest. An indigenous chromium-reducing bacterial strain, Rb-2, isolated from a tannery water sample, was identified as Ochrobactrum intermedium, on the basis of 16S rRNA gene sequencing. The influence of factors like temperature of incubation, initial concentration of Cr, mobility of bacteria, and different carbon sources were studied to test the ability of the bacterium to reduce Cr(VI) under variable environmental conditions. The ability of the bacterial strain to reduce hexavalent chromium in artificial and industrial sewage water was evaluated. It was observed that the mechanism of resistance to metal was not due to the change in the permeability barrier of the cell membrane, and the enzyme activity was found to be inductive. Intracellular reduction of Cr(VI) was proven by reductase assay using cell-free extract. Scanning electron microscopy revealed chromium precipitates on bacterial cell surfaces, and transmission electron microscopy showed the outer as well as inner distribution of Cr(VI). This bacterial strain can be useful for Cr(VI) detoxification under a wide range of environmental conditions.     

Chromium (VI) Can Activate and Impair Antioxidant Defense System

The changes in glutathione-dependent cycle enzymes and catalase activities under Cr(VI)-induced oxidative stress were investigated in two distinct cell lines: L-41−human epithelial-like cells and HLF−fetal human diploid lung fibroblasts, which differ in tissue origin, proliferation, and antioxidant enzymes activities. The chromium concentrations from 1 to 5 μM cause nontoxic effects and activate antioxidant enzymes to overcome oxidative stress. In spite of some differences in the endogenous antioxidant activities, both cell lines reveal the same range of toxic concentrations (20–30 μM). The irreversible inhibition of glutathione-dependent antioxidant enzymes develops under toxic concentrations and serves as a marker of toxicity. The endogenous antioxidant activity influences time-dependent expression of Cr(VI) toxicity and the dynamics of antioxidant enzymes activity under nontoxic conditions. The cell antioxidant defense system is an important marker of the cell adaptive capacity under nontoxic and toxic conditions.     

The role of glutathione reductase in the cytotoxicity of chromium (VI) in isolated rat hepatocytes

Chromium (VI) is an environmental and occupational carcinogen, and it is accepted that intracellular reduction is necessary for DNA damage and cytotoxicity. We have investigated the interaction of Cr(VI) with hepatocytes in vitro to determine the contribution of various hepatic enzymes to the reduction of Cr(VI). Cr(VI) caused a dose-dependent decrease in cell viability and intracellular reduced glutathione (GSH) levels between 100 and 500 microM within 3 h exposure of hepatocytes. Both DT-diaphorase and cytochrome P450 play only a minor role in detoxifying Cr(VI) and/or its metabolites. (GSH) appears to act as a non-enzymatic reductant, reducing Cr(VI) to a toxic form. The evidence for this is two-fold. Firstly, GSH was depleted during the metabolism of Cr(VI) and, secondly, pretreatment of the cells with diethylmaleate to deplete GSH levels, partially protected the cells from Cr(VI) toxicity. Glutathione reductase appears to play an important role in the enzymatic reduction of Cr(VI) as inhibition of this enzyme by carmustine (BCNU) markedly protected the cells from cytotoxicity.     

Mechanisms of bacterial resistance to chromium compounds

Chromium is a non-essential and well-known toxic metal for microorganisms and plants. The widespread industrial use of this heavy metal has caused it to be considered as a serious environmental pollutant. Chromium exists in nature as two main species, the trivalent form, Cr(III), which is relatively innocuous, and the hexavalent form, Cr(VI), considered a more toxic species. At the intracellular level, however, Cr(III) seems to be responsible for most toxic effects of chromium. Cr(VI) is usually present as the oxyanion chromate. Inhibition of sulfate membrane transport and oxidative damage to biomolecules are associated with the toxic effects of chromate in bacteria. Several bacterial mechanisms of resistance to chromate have been reported. The best characterized mechanisms comprise efflux of chromate ions from the cell cytoplasm and reduction of Cr(VI) to Cr(III). Chromate efflux by the ChrA transporter has been established in Pseudomonas aeruginosa and Cupriavidus metallidurans (formerly Alcaligenes eutrophus) and consists of an energy-dependent process driven by the membrane potential. The CHR protein family, which includes putative ChrA orthologs, currently contains about 135 sequences from all three domains of life. Chromate reduction is carried out by chromate reductases from diverse bacterial species generating Cr(III) that may be detoxified by other mechanisms. Most characterized enzymes belong to the widespread NAD(P)H-dependent flavoprotein family of reductases. Several examples of bacterial systems protecting from the oxidative stress caused by chromate have been described. Other mechanisms of bacterial resistance to chromate involve the expression of components of the machinery for repair of DNA damage, and systems related to the homeostasis of iron and sulfur.     

Cr(III) Oxidation and Cr Toxicity in Cultures of the Manganese(II)-Oxidizing Pseudomonas putida Strain GB-1

Abstract

Mn oxides have long been considered the primary environmental oxidant of Cr(III), however, since most of the reactive Mn oxides in the environment are believed to be of biological origin, microorganisms may indirectly mediate Cr(III) oxidation and accelerate the rate over that seen in purely abiotic systems. In this study, we examined the ability of the Mn(II)-oxidizing bacterium, Pseudomonas putida strain GB-1, to oxidize Cr(III). Our results show that GB-1 cannot oxidize Cr(III) directly, but that in the presence of Mn(II), Cr(III) can be rapidly and completely oxidized. Growth studies suggest that in growth medium with few organics the resulting Cr(VI) may be less toxic to P. putida GB-1 than Cr(III), which is generally considered less hazardous. In addition, Cr(III) present during the growth of P. putida GB-1 appeared to cause iron stress as determined by the production of the fluorescent siderophore pyoverdine. When stressed by Fe limitation or Cr(III) toxicity, Mn(II) oxidation by GB-1 is inhibited.     

Chromate-Reducing Properties of Soluble Flavoproteins from Pseudomonas putida and Escherichia coli

Cr(VI) (chromate) is a toxic, soluble environmental contaminant. Bacteria can reduce chromate to the insoluble and less toxic Cr(III), and thus chromate bioremediation is of interest. Genetic and protein engineering of suitable enzymes can improve bacterial bioremediation. Many bacterial enzymes catalyze one-electron reduction of chromate, generating Cr(V), which redox cycles, generating excessive reactive oxygen species (ROS). Such enzymes are not appropriate for bioremediation, as they harm the bacteria and their primary end product is not Cr(III). In this work, the chromate reductase activities of two electrophoretically pure soluble bacterial flavoproteins—ChrR (from Pseudomonas putida) and YieF (from Escherichia coli)—were examined. Both are dimers and reduce chromate efficiently to Cr(III) (k_cat/K_m = ~2 × 10⁴ M⁻¹·s⁻¹). The ChrR dimer generated a flavin semiquinone during chromate reduction and transferred >25% of the NADH electrons to ROS. However, the semiquinone was formed transiently and ROS diminished with time. Thus, ChrR probably generates Cr(V), but only transiently. Studies with mutants showed that ChrR protects against chromate toxicity; this is possibly because it preempts chromate reduction by the cellular one-electron reducers, thereby minimizing ROS generation. ChrR is thus a suitable enzyme for further studies. During chromate reduction by YieF, no flavin semiquinone was generated and only 25% of the NADH electrons were transferred to ROS. The YieF dimer may therefore be an obligatory four-electron chromate reducer which in one step transfers three electrons to chromate and one to molecular oxygen. As a mutant lacking this enzyme could not be obtained, the role of YieF in chromate protection could not be directly explored. The results nevertheless suggest that YieF may be an even more suitable candidate for further studies than ChrR.     

Isolation of hexavalent chromium-reducing anaerobes from hexavalent-chromium-contaminated and noncontaminated environments

Hexavalent chromium [Cr(VI)], is a toxic, water-soluble contaminant present in many soils and industrial effluents. Bacteria from various soils were examined for Cr(VI) resistance and reducing potential. Microbes selected from both Cr(VI)-contaminated and-noncontaminated soils and sediments were capable of catalyzing the reduction of Cr(VI) to Cr(III) a less toxic, less water-soluble form of Cr, demonstrating the utility of using a selection strategy for indigenous Cr(VI)-reducing bacteria in a bioprocess. As a result, indigenous Cr(VI)- reducing microbes from contaminated sites should provide the means for developing a bioprocess to reduce Cr(VI) to Cr(III) in nonsterile effluents such as those from soil washes. This approach also avoids the contamination problems associated with pure cultures of allochthonous microorganisms. In addition the apparent ubiquity of Cr(VI)-reducing bacteria in soil and sediments indicates potential for in situ bioremediation of Cr(VI)-contaminated soils and ground water.     

Cr(VI) detoxification by Desulfovibrio vulgaris strain Hildenborough: microbe–metal interactions studies

Toxic heavy metals constitute a worldwide environmental pollution problem. Bioremediation technologies represent efficient alternatives to the classic cleaning-up of contaminated soil and ground water. Most toxic heavy metals such as chromium are less soluble and toxic when reduced than when oxidized. Sulfate-reducing bacteria (SRB) are able to reduce heavy metals by a chemical reduction via the production of H₂S and by a direct enzymatic process involving hydrogenases and c₃ cytochromes. We have previously reported the effects of chromate [Cr(VI)] on SRB bioenergetic metabolism and the molecular mechanism of the metal reduction by polyhemic cytochromes. In the current work, we pinpoint the bacteria–metal interactions using Desulfovibrio vulgaris strain Hildenborough as a model. The bacteria were grown in the presence of high Cr(VI) concentration, where they accumulated precipitates of a reduced form of chromium, trivalent chromium [Cr(III)], on their cell surfaces. Moreover, the inner and outer membranes exhibited precipitates that shared the spectroscopic signature of trivalent chromium. This subcellular localization is consistent with enzymatic metal reduction by cytochromes and hydrogenases. Regarding environmental significance, our findings point out the Cr(VI) immobilization mechanisms of SRB; suggesting that SRB are highly important in metal biogeochemistry.     

Response of antioxidant defense system to chromium (VI)-induced cytotoxicity in human diploid cells

The aim of this study is to establish antioxidant indicators of chromium toxicity in fetal human lung fibroblasts (HLF). The results obtained corroborate and develop our earlier observation of low-dose and long-term action of Cr(VI) on human cells in culture. In the case of a nontoxic chromium dose, temporary oxidative stress is overcome by increased activity of the antioxidant system with correlation to cell cycle re-entry. The toxic concentrations misbalance the cell antioxidant defense systems and cause irreversible growth arrest and massive cell death by apoptosis. Sub-toxicity is defined as toxicity stretched in time. The activity of GPx (glutathione peroxidase) is proposed as a biomarker of oxidative stress caused by Cr(VI), and the GR (glutathione reductase) inhibition is considered as a marker of the toxicity developed under the complex Cr(VI) action. In HLF cells the glutathione dependent defense system is the first system destroyed in response to toxic chromium action. Only the balance between SOD (superoxide dismutase) and H₂O₂ degrading enzymes (catalase and GPx), should play an important role in the fate of a cell, not individual enzymes.     

Cr(VI) Imposed Toxicity in Maize Seedlings Assessed in Terms of Disruption in Carbohydrate Metabolism

The present study examined the toxic effects of Cr(VI; 100, 250 and 500 μM) in maize seedlings by investigating the changes in carbohydrate metabolism after 48, 96, and 144 h of exposure. Cr-stress results in severe alterations in the contents of carbohydrates and reducing sugars and the activities of carbohydrate metabolizing enzymes, amylases, phosphatases and phosphorylases, and invertases in maize seedlings. Under Cr stress, the contents of carbohydrates and reducing sugars declined in roots, whereas an increase was noticed in leaves. The catalytic activity of carbohydrate metabolizing enzymes, except invertases, in roots declined in the presence of Cr(VI) in a concentration- and exposure time-dependent manner. In contrast, the activities of these enzymes were enhanced in leaves under Cr(VI) stress. The activity of invertases increased with increasing amount of Cr(VI) but declined with an increase in the time interval. In conclusion, our results show that carbohydrate metabolism is severely affected under Cr(VI) toxicity. The study suggests that Cr-induced perturbations in the carbohydrate metabolism are one of the factors resulting in growth inhibition under Cr(VI) stress.     

SPECTROSCOPIC DETERMINATION OF THE TOXICITY,ABSORPTION, REDUCTION,AND TRANSLOCATION OF Cr(VI) IN TWO MAGNOLIOPSIDA SPECIES

Hexavalent chromium is a contaminant highly mobile in the environment that is toxic for plants at low concentrations. In this work, the physiological response of Convolvulus arvensis and Medicago truncatula plants to Cr(VI) treatments was compared. C. arvensis is a potential Cr hyperaccumulator well adapted to semiarid conditions that biotransform Cr(VI) to the less toxic Cr(III). M. truncatula is a model plant well adapted to semiarid conditions with a well studied genetic response to heavy metal stress. The results demonstrated that C. arvensis is more tolerant to Cr toxicity and has a higher Cr translocation to the leaves. The inductively coupled plasma optical emission spectroscopy results showed that C. arvensis plants treated with 10 mg Cr(VI) L^–1 accumulated 1512, 210, and 131 mg Cr kg^–1 in roots, stems, and leaves, respectively. While M. truncatula plants treated with the same Cr(VI) concentration accumulated 1081, 331, and 44 (mg Cr kg^–1) in roots, stems, and leaves, respectively. Enzymatic assays demonstrated that Cr(VI) decreased ascorbate peroxidase activity and increased catalase activity in M. truncatula, while an opposite response was found in C. arvensis. The x-ray absorption spectroscopy studies showed that both plant species reduced Cr(VI) to the less toxic Cr(III).     

Physiological changes induced by chromium stress in plants: an overview

This article presents an overview of the mechanism of chromium (Cr) stress in plants. Toxic effects of Cr on plant growth and development depend primarily on its valence state. Cr(VI) is highly toxic and mobile whereas Cr(III) is less toxic. Cr-induced oxidative stress involves induction of lipid peroxidation in plants that cause severe damage to cell membranes which includes degradation of photosynthetic pigments causing deterioration in growth. The potential of plants with the adequacy to accumulate or to stabilize Cr compounds for bioremediation of Cr contamination has gained engrossment in recent years.     

Chromium in the environment: factors affecting biological remediation

Chromium, in the trivalent form (Cr(III)), is an important component of a balanced human and animal diet and its deficiency causes disturbance to the glucose and lipids metabolism in humans and animals. In contrast, hexavalent Cr (Cr(VI)) is highly toxic carcinogen and may cause death to animals and humans if ingested in large doses. Recently, concern about Cr as an environmental pollutant has been escalating due to its build up to toxic levels in the environment as a result of various industrial and agricultural activities. In this review, we present the state of knowledge about chromium mobility and distribution in the environment and the physiological responses of plants to Cr with the desire to understand how these processes influence our ability to use low cost, environmentally friendly biological remediation technologies to clean up Cr-contaminated soils, sediments, and waters. The use of biological remediation technologies such as bioremediation and phytoremediation for the cleanup of Cr-contaminated areas has received increasing interest from researchers worldwide. Several methods have been suggested and experimentally tested with varying degrees of success.     

The polycyclic aromatic hydrocarbon phenanthrene causes oxidative stress and alters polyamine metabolism in the aquatic liverwort Riccia fluitans L

The polycyclic aromatic hydrocarbon (PAH) phenanthrene (PHEN) is a highly toxic pollutant, commonly found in aquatic environments, the effects of which on aquatic plants have not been studied in depth. As PAHs are known to induce oxidative stress and recent studies have shown that polyamines (PAs) participate in the defence reactions protecting plants against environmental stresses, PA metabolism and oxidative damage were investigated in the aquatic form of the liverwort Riccia fluitans L. exposed to PHEN. Exposure of Riccia fluitans plants to PHEN at concentrations of 0.5 microm or less induced oxidative stress, but at a level from which plants could recover. Despite increased levels of enzymatic and non-enzymatic antioxidants, recovery appeared, at least in part, due to increased synthesis of PAs, achieved via increased activities of the enzymes arginine decarboxylase (ADC) and S-adenosylmethionine decarboxylase (SAMDC). Chemical inhibition of these enzymes inhibited plant recovery, while treatment with PAs aided recovery. Finally, as chloroplasts and the plasma membrane appeared to be key targets for PHEN-induced damage, the potential roles of PAs in protecting these cellular components were considered. How PAs could protect plant cells from serious environmental pollutants such as PHEN and could prevent oxidative stress is discussed.     

Development of a new Cr(VI)-biosorbent from agricultural biowaste

Among useless but abundant agricultural biowastes such as banana skin, green tea waste, oak leaf, walnut shell, peanut shell and rice husk, in this study, banana skin was screened as the most efficient biomaterial to remove toxic Cr(VI) from aqueous solution. X-ray photoelectron spectroscopy (XPS) study revealed that the mechanism of Cr(VI) biosorption by banana skin was its complete reduction into Cr(III) in both aqueous and solid phases and partial binding of the reduced-Cr(III), in the range of pH 1.5-4 tested. One gram of banana skin could reduce 249.6 (+/-4.2)mg of Cr(VI) at initial pH 1.5. Namely, Cr(VI)-reducing capacity of banana skin was four times higher than that of a common chemical Cr(VI)-reductant, FeSO(4).7H(2)O. To diminish undesirable/serious organic leaching from the biomaterial and to enhance removal efficiency of total Cr, its powder was immobilized within Ca-alginate bead. The developed Cr(VI)-biosorbent could completely reduce toxic Cr(VI) to less toxic Cr(III) and could remove almost of the reduced-Cr(III) from aqueous phase. On the basis of removal mechanisms of Cr(VI) and total Cr by the Cr(VI)-biosorbent, a kinetic model was derived and could be successfully used to predict their removal behaviors in aqueous phase. In conclusion, our Cr(VI)-biosorbent must be a potent candidate to substitute for chemical reductants as well as adsorbents for treating Cr(VI)-bearing wastewaters.