Intracellular chromium reduction

Two steps are involved in the uptake of Cr(VI): (1) the diffusion of the anion CrO4(2-) through a facilitated transport system, presumably the non-specific anion carrier and (2) the intracellular reduction of Cr(VI) to Cr(III). The intracellular reduction of Cr(VI), keeping the cytoplasmic concentration of Cr(VI) low, facilitates accumulation of chromate from extracellular medium into the cell. In the present paper, a direct demonstration of intracellular chromium reduction is provided by means of electron paramagnetic (spin) resonance (EPR) spectroscopy. Incubation of metabolically active rat thymocytes with chromate originates a signal which can be attributed to a paramagnetic species of chromium, Cr(V) or Cr(III). The EPR signal is originated by intracellular reduction of chromium since: (1) it is observed only when cells are incubated with chromate, (2) it is present even after extensive washings of the cells in a chromium-free medium; (3) it is abolished when cells are incubated with drugs able to reduce the glutathione pool, i.e., diethylmaleate or phorone; and (4) it is abolished when cells are incubated in the presence of a specific inhibitor of the anion carrier, 4-acetamido-4'-isothiocyanatostilbene-2-2'-disulfonic acid.     

Exogenously applied sulphate as a tool to investigate transport and reduction of chromate in the duckweed Spirodela polyrhiza

The accumulation of chromium in Spirodela polyrhiza was investigated in the presence and absence of exogenously applied sulphate. Precultivation (10 d) at minimum sulphate concentration (0.013 m m versus 1 m m in controls) enhanced the rate of chromium accumulation. This effect was caused by the increased number of sulphate transporters which transport chromate into cells. Chromate and sulphate compete for the available sulphate transporters. The kinetics of reduction Cr(VI)→Cr(V) was investigated by l -band electron paramagnetic resonance (EPR) spectroscopy. The kinetic model developed previously (Appenroth et al., Journal of Inorganic Biochemistry 78, 235–242, 2000) was refined and extended to include chromate transport and reduction in the presence of competing ions. The following conclusions were drawn from the fitting procedure: without simultaneously applied sulphate, the rate constant of Cr(VI) transport from apoplast into plant cells and the rate constant of Cr(VI) to Cr(V) reduction within the apoplast are comparable (7.0 versus 5.7 h⁻¹) demonstrating that these two processes are competing. Moreover, the rate constant of reduction Cr(V)→Cr(III) is much lower within cells than in apoplast (0.39 versus 7.0 h⁻¹) showing that Cr(V) is stabilized in the symplast. The rate of transport of Cr(VI) into plant cells is at least one order of magnitude higher than that of Cr(V) or Cr(III). The treatment with sulphate (10 m m ) decreases the rate constant of the transport of Cr(VI) into cells (2.0 h⁻¹) confirming the competition of chromate and sulphate for the same transporters. Simultaneously, the rate constant of Cr(V)→Cr(III) reduction is increased in the apoplast (by the factor of 3) and decreased in the symplast (by the factor of 5). Treatment with higher sulphate concentrations (100 m m ) increases the accumulation of chromium by enhancing the rate constant of Cr(VI) transport into cells leaving other processes essentially unchanged. We suggest that 100 m m sulphate opens a new pathway for chromate transport into cells.     

Chromate/nitrite interactions in Shewanella oneidensis MR-1: evidence for multiple hexavalent chromium [Cr(VI)] reduction mechanisms dependent on physiological growth conditions

Inhibition of hexavalent chromium [Cr(VI)] reduction due to nitrate and nitrite was observed during tests with Shewanella oneidensis MR-1 (previously named Shewanella putrefaciens MR-1 and henceforth referred to as MR-1). Initial Cr(VI) reduction rates were measured at various nitrite concentrations, and a mixed inhibition kinetic model was used to determine the kinetic parameters-maximum Cr(VI) reduction rate and inhibition constant [V(max,Cr(VI)) and K(i,Cr(VI))]. Values of V(max,Cr(VI)) and K(i,Cr(VI)) obtained with MR-1 cultures grown under denitrifying conditions were observed to be significantly different from the values obtained when the cultures were grown with fumarate as the terminal electron acceptor. It was also observed that a single V(max,Cr(VI)) and K(i,Cr(VI)) did not adequately describe the inhibition kinetics of either nitrate-grown or fumarate-grown cultures. The inhibition patterns indicate that Cr(VI) reduction in MR-1 is likely not limited to a single pathway, but occurs via different mechanisms some of which are dependent on growth conditions. Inhibition of nitrite reduction due to the presence of Cr(VI) was also studied, and the kinetic parameters V(max,NO2) and K(i,NO2) were determined. It was observed that these coefficients also differed significantly between MR-1 grown under denitrifying conditions and fumarate reducing conditions. The inhibition studies suggest the involvement of nitrite reductase in Cr(VI) reduction. Because nitrite reduction is part of the anaerobic respiration process, inhibition due to Cr(VI) might be a result of interaction with the components of the anaerobic respiration pathway such as nitrite reductase. Also, differences in the degree of inhibition of nitrite reduction activity by chromate at different growth conditions suggest that the toxicity mechanism of Cr(VI) might also be dependent on the conditions of growth. Cr(VI) reduction has been shown to occur via different pathways, but to our knowledge, multiple pathways within a single organism leading to Cr(VI) reduction has not been reported previously.     

Kinetics of Cr(VI) Removal by Iron Filings in Some Soils

In this research, kinetics of Cr(VI) reduction by iron filings was investigated through a batch study in seven different soils. Chromate reduction experiments were carried out for initial Cr(VI) concentrations ranging from 20 to 100 mgkg^?1 and iron filings dosage of 0 to 5% w/w. The experimental data were analyzed using various kinetic models including zero-order, pseudo first-order, power function, Elovich, and diffusion parabolic. Results showed that the Cr(VI) reduction efficiency in the presence of all studied soils increased with increasing iron filings dosage and decreased with increasing the initial Cr(VI) concentration. The reaction rates considerably depended on pH and were higher in acidic soils. The diffusion parabolic model was the best kinetic model as evidenced by the highest determination coefficient (r²) and the lowest standard error of the estimate (SE). The rate-limiting step(s) may be transport of chromate anions across a liquid film at the interface of soil-liquid, transport in liquid-filled macropores of iron filings aggregates, or diffusion in micropores and along the particle's surface.     

Chromate reduction by a pseudomonad isolated from a site contaminated with chromated copper arsenate

A pseudomonad (CRB5) isolated from a decommissioned wood preservation site reduced toxic chromate [Cr(VI)] to an insoluble Cr(III) precipitate under aerobic and anaerobic conditions. CRB5 tolerated up to 520 mg of Cr(VI) liter(-1) and reduced chromate in the presence of copper and arsenate. Under anaerobic conditions it also reduced Co(III) and U(VI), partially internalizing each metal. Metal precipitates were also found on the surface of the outer membrane and (sometimes) on a capsule. The results showed that chromate reduction by CRB5 was mediated by a soluble enzyme that was largely contained in the cytoplasm but also found outside of the cells. The crude reductase activity in the soluble fraction showed a K(m) of 23 mg liter(-1) (437 microM) and a V(max) of 0.98 mg of Cr h(-1) mg of protein(-1) (317 nmol min(-1) mg of protein(-1)). Minor membrane-associated Cr(VI) reduction under anaerobiosis may account for anaerobic reduction of chromate under nongrowth conditions with an organic electron donor present. Chromate reduction under both aerobic and anaerobic conditions may be a detoxification strategy for the bacterium which could be exploited to bioremediate chromate-contaminated or other toxic heavy metal-contaminated environments.     

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.     

A novel chromate reductase from Thermus scotoductus SA-01 related to old yellow enzyme

Bacteria can reduce toxic and carcinogenic Cr(VI) to insoluble and less toxic Cr(III). Thermus scotoductus SA-01, a South African gold mine isolate, has been shown to be able to reduce a variety of metals, including Cr(VI). Here we report the purification to homogeneity and characterization of a novel chromate reductase. The oxidoreductase is a homodimeric protein, with a monomer molecular mass of approximately 36 kDa, containing a noncovalently bound flavin mononucleotide cofactor. The chromate reductase is optimally active at a pH of 6.3 and at 65 degrees C and requires Ca(2+) or Mg(2+) for activity. Enzyme activity was also dependent on NADH or NADPH, with a preference for NADPH, coupling the oxidation of approximately 2 and 1.5 mol NAD(P)H to the reduction of 1 mol Cr(VI) under aerobic and anaerobic conditions, respectively. The K(m) values for Cr(VI) reduction were 3.5 and 8.4 microM for utilizing NADH and NADPH as electron donors, respectively, with corresponding V(max) values of 6.2 and 16.0 micromol min(-1) mg(-1). The catalytic efficiency (k(cat)/K(m)) of chromate reduction was 1.14 x 10(6) M(-1) s(-1), which was >50-fold more efficient than that of the quinone reductases and >180-fold more efficient than that of the nitroreductases able to reduce Cr(VI). The chromate reductase was identified to be encoded by an open reading frame of 1,050 bp, encoding a single protein of 38 kDa under the regulation of an Escherichia coli sigma(70)-like promoter. Sequence analysis shows the chromate reductase to be related to the old yellow enzyme family, in particular the xenobiotic reductases involved in the oxidative stress response.     

Reduction and precipitation of chromate by mixed culture sulphate-reducing bacterial biofilms

The ability of sulphate-reducing bacterial biofilms to reduce hexavalent chromium (Cr(VI)) to insoluble Cr(III), a process of environmental and biotechnological significance, was investigated. The reduction of chromate to insoluble form has been quantified and the effects of chromate on the carbon source utilization and sulphate-reducing activity of the bacterial biofilms evaluated. Using lactate as the carbon/energy source and in the presence of sulphate, reduction of 500 micromol l-1 Cr(VI) was monitored over a 48-h period where 88% of the total chromium was removed from solution. Mass balance calculations showed that ca 80% of the total chromium was precipitated out of solution with the bacterial biofilm retaining less than 10% of the chromium. Only ca 12% of the chromate added was not reduced to insoluble form. Although Cr(VI) did not have a significant effect on C source utilization, sulphate reduction was severely inhibited by 500 micromol-1 Cr(VI) and only ca 10% of the sulphate reducing activity detected in control biofilms occurred in the presence of Cr(VI). Low levels of sulphide were also produced in the presence of chromate, with control biofilms producing over 10-times more sulphide than Cr(VI)-exposed biofilms. Sulphide- or other chemically-mediated Cr(VI) reduction was not detected. The biological mechanism of Cr(VI) reduction is likely to be similar to that found in other sulphate-reducing bacteria.     

Proteogenomic and functional analysis of chromate reduction in Acidiphilium cryptum JF-5, an Fe(III)-respiring acidophile

Acidiphilium cryptum JF-5, an acidophilic iron-respiring Alphaproteobacterium, has the ability to reduce chromate under aerobic and anaerobic conditions, making it an intriguing and useful model organism for the study of extremophilic bacteria in bioremediation applications. Genome sequence annotation suggested two potential mechanisms of Cr(VI) reduction, namely, a number of c-type cytochromes, and a predicted NADPH-dependent Cr(VI) reductase. In laboratory studies using pure cultures of JF-5, an NADPH-dependent chromate reductase activity was detected primarily in soluble protein fractions, and a periplasmic c-type cytochrome (ApcA) was also present, representing two potential means of Cr(VI) reduction. Upon further examination, it was determined that the NADPH-dependent activity was not specific for Cr(VI), and the predicted proteins were not detected in Cr(VI)-grown cultures. Proteomic data did show measureable amounts of ApcA in cells grown with Cr(VI). Purified ApcA is reducible by menadiol, and in turn can reduce Cr(VI), suggesting a means to obtain electrons from the respiratory chain and divert them to Cr(VI). Electrochemical measurements confirm that Cr reduction by ApcA is pH dependent, with low pH being favored. Homology modeling of ApcA and comparison to a known Cr(VI)-reducing c-type cytochrome structure revealed basic amino acids which could interact with chromate ion. From these studies, it can be concluded that A. cryptum has the physiologic and genomic capability to reduce Cr(VI) to the less toxic Cr(III). However, the expected chromate reductase mechanism may not be the primary means of Cr(VI) reduction in this organism.     

Phosphate relieves chromium toxicity in Arabidopsis thaliana plants by interfering with chromate uptake

Soil contamination by hexavalent chromium [Cr(VI) or chromate] due to anthropogenic activities has become an increasingly important environmental problem. Mineral nutrients such as phosphate (Pi), sulfate and nitrate have been reported to attenuate Cr(VI) toxicity, but the underlying mechanisms remain to be clarified. Here, we show that chromate activates the expression of low-Pi inducible reporter genes AtPT1 and AtPT2 in Arabidopsis thaliana transgenic seedlings. Primary-root growth was inhibited by 60 % in AtPT2::uidA-expressing seedlings upon exposure to 140-μM Cr(VI). However, increasing the Pi and sulfate supply to the seedlings that were experiencing Cr(VI) toxicity completely and partially restored the root growth, respectively. This effect correlated with the Cr(VI)-induced AtPT2::uidA expression being completely reversed by addition of Pi. To evaluate whether the nutrient supply may affect the endogenous level of Cr in plants grown under toxic Cr(VI) levels, the contents of Cr were measured (by ICP-MS analyses) in seedlings treated with Cr and with or without Pi, sulfate or nitrate. It was found that Cr accumulation increases tenfold in plants treated with 140-μM Cr(VI) without modifying the phosphorus concentration in the plant. In contrast, the supply of Pi specifically decreased the Cr content to levels similar to those found in seedlings grown in medium without chromate. Taken together, these results show that in A. thaliana seedlings the uptake of Cr(VI) is reduced by Pi. Moreover, our data indicate that Pi and sulfate supplements may be useful in strategies for handling Cr-contaminated soils.     

The chromate resistance phenotype of some yeast mutants correlates with a lower level of Cr(V)-species generated in the extra-cellular medium

The paper describes the selection of chromate-resistant mutants of the yeast Pichia guilliermondii with a higher chromate-reducing activity and reports the EPR-study of Cr(V)-generation in the extra-cellular medium during the reduction of chromate by the yeast culture. It is shown that the reduction of chromate to Cr(III) species runs through the extra-cellular generation of Cr(V)-intermediate(s), thus supporting the assumption about the existence of an extra-cellular pathway of Cr(VI)-reduction. Furthermore, it is demonstrated that the chromate-resistance phenotype of tested mutants correlates with a lower stationary level of Cr(V)-species in the medium. It is thus suggested that isolated mutants can be used as sources of Cr(III)-biocomplexes due to their ability to effectively reduce chromate to Cr(III)-chelates with potential pharmacological applications.     

Hexavalent chromate reduction by alkaliphilic Amphibacillus sp. KSUCr3 is mediated by copper-dependent membrane-associated Cr(VI) reductase

The present study was aimed to localize and characterize hexavalent chromate [Cr(VI)] reductase activity of the extreme alkaliphilic Amphibacillus sp. KSUCr3 (optimal growth pH 10.5). The resting cells were able to reduce about 62 % of the toxic heavy metal Cr(VI) at initial concentration of 200 μM within 30 min. Cell permeabilization resulted in decrease of Cr(VI) reduction in comparison to untreated cells. Enzymatic assays of different sub-cellular fractions of Amphibacillus sp. KSUCr3 demonstrated that the Cr(VI) reductase was mainly associated with the membranous fraction and expressed constitutively. In vitro studies of the crude enzyme indicated that copper ion was essential for Cr(VI) reductase activity. In addition, Ca2? and Mn2? slightly stimulated the chromate reductase activity. Glucose was the best external electron donor, showing enhancement of the enzyme activity by about 3.5-fold. The K (m) and V (max) determined for chromate reductase activity in the membranous fraction were 23.8 μM Cr(VI) and 72 μmol/min/mg of protein, respectively. Cr(VI) reductase activity was maximum at 40 °C and pH 7.0 and it was significantly inhibited in the presence of disulfide reducers (2-mercaptoethanol), ion chelating agent (EDTA), and respiratory inhibitors (CN and Azide). Complete reduction of 100 and 200 μM of Cr(VI) by membrane associated enzyme were observed within 40 and 180 min, respectively. However, it should be noted that biochemical characterization has been done with crude enzyme only, and that final conclusion can only be drawn with the purified enzyme.     

Cometabolism of Cr(VI) by Shewanella oneidensis MR-1 produces cell-associated reduced chromium and inhibits growth

Microbial reduction is a promising strategy for chromium remediation, but the effects of competing electron acceptors are still poorly understood. We investigated chromate (Cr(VI)) reduction in batch cultures of Shewanella oneidensis MR-1 under aerobic and denitrifying conditions and in the absence of an additional electron acceptor. Growth and Cr(VI) removal patterns suggested a cometabolic reduction; in the absence of nitrate or oxygen, MR-1 reduced Cr(VI), but without any increase in viable cell counts and rates gradually decreased when cells were respiked. Only a small fraction (1.6%) of the electrons from lactate were transferred to Cr(VI). The 48-h transformation capacity (Tc) was 0.78 mg (15 micromoles) Cr(VI) reduced. [mg protein](-1) for high levels of Cr(VI) added as a single spike. For low levels of Cr(VI) added sequentially, Tc increased to 3.33 mg (64 micromoles) Cr(VI) reduced. [mg protein](-1), indicating that it is limited by toxicity at higher concentrations. During denitrification and aerobic growth, MR-1 reduced Cr(VI), with much faster rates under denitrifying conditions. Cr(VI) had no effect on nitrate reduction at 6 microM, was strongly inhibitory at 45 microM, and stopped nitrate reduction above 200 microM. Cr(VI) had no effect on aerobic growth at 60 microM, but severely inhibited growth above 150 microM. A factor that likely plays a role in Cr(VI) toxicity is intracellular reduced chromium. Transmission electron microscopy (TEM) and electron energy loss spectroscopy (EELS) of denitrifying cells exposed to Cr(VI) showed reduced chromium precipitates both extracellularly on the cell surface and, for the first time, as electron-dense round globules inside cells.     

Chromium resistance strategies and toxicity: what makes <Emphasis Type="Italic">Ochrobactrum tritici</Emphasis> 5bvl1 a strain highly resistant

Large-scale industrial use of chromium (Cr) resulted in widespread environmental contamination with hexavalent chromium (Cr(VI)). The ability of microorganisms to survive in these environments and detoxify chromate requires the presence of specific resistance systems. Several Cr(VI) resistant species, belonging to a variety of genera, have been isolated in recent years. Ochrobactrum tritici strain 5bvl1 is a model for a highly Cr(VI)-resistant and reducing microorganism, with different strategies to cope with chromium. The strain contains the transposon-located (TnOtChr) chromate resistance genes chrB, chrA, chrC, chrF. The chrB and chrA genes were found to be essential for the establishment of high resistance but not chrC or chrF genes. Other mechanisms involved in chromium resistance in this strain were related to strategies such as specific or unspecific Cr(VI) reduction, free-radical detoxifying activities, and repairing DNA damage. Expression of the chrB, chrC or chrF genes was related to increased resistance to superoxide-generating agents. Genetic analyses also showed that, the ruvB gene is related to chromium resistance in O. tritici 5bvl1. The RuvABC complex probably does not form when ruvB gene is interrupted, and the repair of DNA damage induced by chromium is prevented. Aerobic or anaerobic chromate reductase activity and other unspecific mechanisms for chromium reduction have been identified in different bacteria. In the strain O. tritici 5bvl1, several unspecific mechanisms were found. Dichromate and chromate have different effects on the physiology of the chromium resistant strains and dichromate seems to be more toxic. Toxicity of Cr(VI) was evaluated by following growth, reduction, respiration, glucose uptake assays and by comparing cell morphology.     

Evaluation of in vitro Cr(VI) reduction potential in cytosolic extracts of three indigenous Bacillus sp. isolated from Cr(VI) polluted industrial landfill

Three efficient Cr(VI) reducing bacterial strains were isolated from Cr(VI) polluted landfill and characterized for in vitro Cr(VI) reduction. Phylogenetic analysis using 16S rRNA gene sequencing revealed that the newly isolated strains G1DM20, G1DM22 and G1DM64 were closely related to Bacillus cereus, Bacillus fusiformis and Bacillus sphaericus, respectively. The suspended cultures of all Bacillus sp. exhibited more than 85% reduction of 1000 microM Cr(VI) within 30 h. The suspended culture of Bacillus sp. G1DM22 exhibited an ability for continuous reduction of 100 microM Cr(VI) up to seven consecutive inputs. Assays with the permeabilized cells and cell-free extracts from each of Bacillus sp. demonstrated that the hexavalent chromate reductase activity was mainly associated with the soluble fraction of cells and expressed constitutively. The Cr(VI) reduction by the cell-free extracts of Bacillus sp. G1DM20 and G1DM22 was maximum at 30 degrees C and pH 7 whereas, Bacillus sp. G1DM64 exhibited maximum Cr(VI) reduction at pH 6. Addition of 1mM NADH enhanced the Cr(VI) reductase activity in the cell-free extracts of all three isolates. Amongst all three isolates tested, crude cell-free extracts of Bacillus sp. G1DM22 exhibited the fastest Cr(VI) reduction rate with complete reduction of 100 microM Cr(VI) within 100 min. The apparent K(m) and V(max) of the chromate reductase activity in Bacillus sp. G1DM22 were determined to be 200 microM Cr(VI) and 5.5 micromol/min/mg protein, respectively. The Cr(VI) reductase activity in cell-free extracts of all the isolates was stable in presence of different metal ions tested except Hg(2+) and Ag(+).     

In vitro interaction of mutagenic chromium (VI) with red blood cells

The interaction of mutagenic Cr(VI) with red blood cells has been studied by ESR spectroscopy. Signals of two Cr(V) species are observed almost immediately after contacting red cells with chromate(VI) aqueous solution at pH 7.4. The signal at go = 1.985, which decays within one hour, is attributed to a Cr(V) complex formed by glutathione due its reducing and chelating ability. The other signal at go = 1.979, which is distinctly more persistent, may indicate that some immobilization of the formed Cr(V) ions takes place on the macromolecular cell components, e.g. glycoproteins.     

A membrane-associated protein with Cr(VI)-reducing activity from Thermus scotoductus SA-01

A membrane-associated chromate reductase from Thermus scotoductus SA-01 has been purified to apparent homogeneity and shown to couple the reduction of Cr(VI) to NAD(P)H oxidation, with a preference towards NADH. The chromate reductase is a homodimer with a monomeric molecular weight of 48 kDa and a noncovalently bound FAD coenzyme. The enzyme is optimally active at a pH of 6.5 and 65 degrees C with a K(m) of 55.5+/-4.2 microM and a V(max) of 2.3+/-0.1 micromol Cr(VI) min(-1) mg(-1) protein. The catalytic efficiency (k(cat)/K(m)) of the enzyme was found to be comparable to that found for quinone reductases but more efficient than the nitroreductases. N-terminal sequencing and subsequent screening of a genomic library of T. scotoductus revealed an ORF of 1386 bp, homologous (84%) to the dihydrolipoamide dehydrogenase gene of Thermus thermophilus HB8. These results extend the knowledge of chromate reductases mediating Cr(VI) reduction via noncovalently bound or free redox-active flavin groups and the activity of dihydrolipoamide dehydrogenases towards physiologically unrelated substrates.     

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.