Purification and characterization of NADPH-dependent Cr(VI) reductase from Escherichia coli ATCC 33456

A soluble Cr(VI) reductase was purified from the cytoplasm of Escherichia coli ATCC 33456. The molecular mass was estimated to be 84 and 42 kDa by gel filtration and SDS-polyacrylamide gel electrophoresis, respectively, indicating a dimeric structure. The pI was 4.66, and optimal enzyme activity was obtained at pH 6.5 and 37 degrees C. The most stable condition existed at pH 7.0. The purified enzyme used both NADPH and NADH as electron donors for Cr(VI) reduction, while NADPH was the better, conferring 61%; higher activity than NADH. The Km values for NADPH and NADH were determined to be 47.5 and 17.2 micromol, and the Vmax values 322.2 and 130.7 micromol Cr(VI) min(-1)mg(-1) protein, respectively. The activity was strongly inhibited by N-ethylmalemide, Ag2+, Cd2+, Hg2+, and Zn2+. The antibody against the enzyme showed no immunological cross reaction with those of other Cr(VI) reducing strains.     

Simultaneous chromium reduction and phenol degradation in a coculture of Escherichia coli ATCC 33456 and Pseudomonas putida DMP-1.

In a defined coculture of a Cr(VI) reducer, Escherichia coli ATCC 33456, and a phenol degrader, Pseudomonas putida DMP-1, simultaneous reduction of Cr(VI) and degradation of phenol was observed. When Cr(VI) was present in the coculture, quantitative transformation of Cr(VI) into Cr(III) proceeded with simultaneous degradation of phenol. Cr(VI) reduction was correlated to phenol degradation in the coculture as demonstrated by a regression analysis of the cumulative Cr(VI) reduction and the cumulative phenol degradation. Both the rate and extent of Cr(VI) reduction and phenol degradation were significantly influenced by the population composition of the coculture. Although Cr(VI) reduction occurred as a result of E. coli metabolism, the rate of phenol degradation by P. putida may become a rate-limiting factor for Cr(VI) reduction at a low population ratio of P. putida to E. coli. Phenol degradation by P. putida was very susceptible to the presence of Cr(VI), whereas Cr(VI) reduction by E. coli was significantly influenced by phenol only when phenol was present at high concentrations (> 9 mM).     

Chromium(VI) reductase activity is associated with the cytoplasmic membrane of anaerobically grown Shewanella putrefaciens MR-1

C.R. MYERS, B.P. CARSTENS, W.E. ANTHOLINE and J.M. MYERS.2000. Shewanella putrefaciens MR-1 can reduce a diverse array of compounds under anaerobic conditions, including manganese and iron oxides, fumarate, nitrate, and many other compounds. These reductive processes are apparently linked to a complex electron transport system. Chromium (Cr) is a toxic and mutagenic metal and bacteria could potentially be utilized to immobilize Cr by reducing the soluble and bioavailable state, Cr(VI), to the insoluble and less bioavailable state, Cr(III). Formate-dependent Cr(VI) reductase activity was detected in anaerobically grown cells of S. putrefaciens MR-1, with highest specific activity in the cytoplasmic membrane. Both formate and NADH served as electron donors for Cr(VI) reductase, whereas l -lactate or NADPH did not support any activity. The addition of 10 μmol l⁻¹ FMN markedly stimulated formate-dependent Cr(VI) reductase, and the activity was almost completely inhibited by diphenyliodonium chloride, an inhibitor of flavoproteins. Cr(VI) reductase activity was also inhibited by p -chloromercuriphenylsulphonate, azide, 2-heptyl-4-hydroxyquinolone- N -oxide, and antimycin A, suggesting involvement of a multi-component electron transport chain which could include cytochromes and quinones. Cr(V) was detected by electron paramagnetic resonance (EPR) spectroscopy, suggesting a one-electron reduction as the first step.     

Chromate reduction by cell-free extract of Bacillus firmus KUCr1

Microbial enzymatic reduction of a toxic form of chromium [Cr(VI)] has been considered as an effective method for bioremediation of this metal. This study reports on the in vitro reduction of Cr(VI) using cell-free extracts from a Cr(VI) reducing Bacillus firmus KUCr1 strain. Chromium reductase was found to be constitutive and its activity was observed both in soluble cell fractions (S12 and S150 and membrane cell fraction (P150). The reductase activity of S12 fraction was found to be optimal at 40 microM Cr(VI) with enzyme concentration equivalent to 0.493 mg protein/ml. Enzyme activity was dependent on NADH or NADPH as electron donor; optimal temperature and pH for better enzyme activity were 70 degrees C and 5.6, respectively. The Km value of the reductase was 58.33 microM chromate having a V(max) of 11.42 microM/min/mg protein. The metabolic inhibitor like sodium azide inhibited reductase activity of membrane fraction of the cell-free extract. Metal ions like Cu2+, Co2+, Ni2+ and As3+ stimulated the enzyme but others, such as Ag+, Hg2+, Zn2+, Mn2+, Cd2+ and Pb2+, inhibited Cr(VI) reductase activity.     

Modeling simultaneous hexavalent chromium reduction and phenol degradation by a defined coculture of bacteria

Based on the kinetics of Cr(VI) reduction by Escherichia coli ATCC 33456 and phenol degradation by Pseudomonas putida DMP-1, a mathematical model is developed to describe simultaneous Cr(VI) reduction and phenol degradation in the coculture of the two species. The developed model incorporates the toxicity effects of Cr(VI) and phenol on phenol degradation and Cr(VI) reduction in the coculture. The model illustrates the inhibitory effects of phenol on Cr(VI) reduction and Cr(VI) toxicity toward phenol degradation. The model also reveals the recoveries of the activities of the repressed bacterial cells with continuous Cr(VI) reduction and phenol degradation in the coculture. The model is capable of predicting simultaneous Cr(VI) reduction and phenol degradation within a broad range of Cr(VI) and phenol concentrations and under an appropriate composition of populations. However, the model simulates lower concentrations of phenol than experimental observations once Cr(VI) is reduced to a low level (<7 mg/L). The model simulation for Cr(VI) also deviates from experimental data when P. putida is outnumbered by E. coli by a ratio of 1:5. (c) 1995 John Wiley & Sons, Inc.     

Hexavalent-chromium reduction by a chromate-resistantBacillus sp. strain

Bacillus strain QC1-2, isolated from a chromium-polluted zone, was selected by its high ability to both tolerate and reduce hexavalent chromium [Cr(VI)] to less-toxic trivalent chromium [Cr(III)]. Cell suspensions of strain QC1-2 rapidly reduced Cr(VI), in both aerobic and anaerobic conditions, to Cr(III) which remained in the supernatant. Cr(VI) reduction was dependent on the addition of glucose but sulfate, an inhibitor of chromate transport, had no effect. Studies with permeabilized cells and cell extracts showed that the Cr(VI) reductase of strain QC1-2 is a soluble NADH-dependent enzyme.     

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.     

Bacterial reduction of hexavalent chromium

Summary Cr(VI)-reducing bacteria are widespread and Cr(VI) reduction occurs under both aerobic and anaerobic conditions. Under aerobic conditions, both NADH and endogenous cell reserves may serve as the electron donor for Cr(VI) reduction. Under anaerobic conditions, electron transport systems containing cytochromes appear to be involved in Cr(VI) reduction. High cell densities are necessary to obtain a significant rate of Cr(VI) reduction. Cr(VI) reduction by bacteria may be inhibited by Cr(VI), oxygen, heavy metals, and phenolic compounds. The optimum pH and temperature observed for Cr(VI) reduction generally coincide with the optimal growth conditions of cells. The optimum redox potential for Cr(VI) reduction has not yet been established.     

Cytochromes of the trimethylamine N-oxide anaerobic respiratory pathway of Escherichia coli

Escherichia coli grown anaerobically with trimethylamine N-oxide (TMAO) as a terminal electron acceptor develops a new cytochrome pathway in addition to the aerobic respiratory pathways which are still formed. Formate, NADH, and possibly other substrates derived from glucose, supply electrons to this pathway. Cytochromes with alpha-absorption peaks at about 548, 552, 554 and 557 nm are rapidly reoxidized by TMAO in a reaction which is not inhibited by 2-n-heptyl -4-hydroxyquinone N-oxide. CuSO4 inhibits the reoxidation by TMAO of the first two of these cytochromes. This suggests that the pathway of electron transfer leading to the reduction of TMAO is: substrates leads to cytochromes 548,552 leads to cytochromes 554,557 leads to trimethylamine-N-oxide reductase leads to TMAO. These cytochromes, but not those of the aerobic respiratory pathways, are reoxidized by the membrane-impermeant oxidant ammonium persulfate in intact cells. This suggests that the cytochromes of the TMAO reduction pathway and/or trimethylamine-N-oxide reductase are situated at the periplasmic surface of the cytoplasmic membrane of E. coli.     

Reduction of diverse electron acceptors by Aeromonas hydrophila

Aeromonas hydrophila ATCC 7966 grew anaerobically on glycerol with nitrate, fumarate, Fe(III), Co(III), or Se(VI) as the sole terminal electron acceptor, but did not ferment glycerol. Final cell yields were directly proportional to the amount of terminal electron acceptor provided. Twenty-four estuarine mesophilic aeromonads were isolated; all reduced nitrate, Fe(III), or Co(III), and five strains reduced Se(VI). Dissimilatory Fe(III) reduction by A. hydrophila may involve cytochromes. Difference spectra obtained with whole cells showed absorption maxima at wavelengths characteristic of c-type cytochromes (419, 522, and 553 nm). Hydrogen-reduced cytochromes within intact cells were oxidized by the addition of Fe(III) or nitrate. Studies with respiratory inhibitors yielded results consistent with a respiratory chain involving succinate (flavin-containing) dehydrogenase, quinones and cytochromes, and a single Fe(III) reductase. Neither anaerobic respiration nor dissimilatory metal reduction by members of the genus Aeromonas have been reported previously. Received: 24 June 1997 / Accepted: 20 October 1997     

Modeling hexavalent chromium reduction in Escherichia coli 33456

A model based on te analysis of the mechanism of enzymatic reactions was developed to characterize the rate and extent of microbial reduction of hexavalent chromium in Escherichia coli 33456. A finite reduction capacity (R(c)) was proposed and incorporated into the enzymatic model to regulate the toxicity effect on cells due to the oxidizing power of Cr(VI). The parameter values were determined by nonlinear least-square analysis using experimental data of anaerobic cultures. The obtained parameters were then used to predict Cr(VI) reduction in aerobic cultures along with a modification term of uncompetitive inhibition from molecular oxygen. The applicability of the developed model was demonstrated through excellent prediction of the results of batch studies conducted over range of initial Cr(VI) concentrations, initial cell densities, and DO levels. A sensitivity analysis revealed that the parameters obtained using the experimental data were unique, and neither change in K(c), the half-velocity constant, at high initial Cr(VI) concentrations nor change in R(c), the reduction capacity, at low initial Cr(VI) concentrations was sensitive to model prediction. (c) 1994 John Wiley & Sons, Inc.     

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.     

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.     

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.     

Chromium reduction in Pseudomonas putida.

Reduction of hexavalent chromium (chromate) to less-toxic trivalent chromium was studied by using cell suspensions and cell-free supernatant fluids from Pseudomonas putida PRS2000. Chromate reductase activity was associated with soluble protein and not with the membrane fraction. The crude enzyme activity was heat labile and showed a Km of 40 microM CrO4(2-). Neither sulfate nor nitrate affected chromate reduction either in vitro or with intact cells.     

Chromium reduction in Pseudomonas putida

Reduction of hexavalent chromium (chromate) to less-toxic trivalent chromium was studied by using cell suspensions and cell-free supernatant fluids from Pseudomonas putida PRS2000. Chromate reductase activity was associated with soluble protein and not with the membrane fraction. The crude enzyme activity was heat labile and showed a Km of 40 microM CrO4(2-). Neither sulfate nor nitrate affected chromate reduction either in vitro or with intact cells.     

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.     

Cr(VI) reduction in a chromate-resistant strain of Candida maltosa isolated from the leather industry

A Cr(VI)-resistant yeast was isolated from tanning liquors from a leather factory in Leon, Guanajuato, Mexico. Based on morphological and physiological analyses and the D1/D2 domain sequence of the 26S rDNA, the yeast was identified as Candida maltosa. Resistance of the strain to high Cr(VI) concentrations and its ability to chemically reduce chromium was studied. When compared to the three laboratory yeasts Candida albicans, Saccharomyces cerevisiae and Yarrowia lipolytica, the C. maltosa strain was found to tolerate chromate concentrations as high as 100 micro g/ml. In addition to this phenotypic trait, the C. maltosa strain showed ability to reduce Cr(VI). Chromate reduction occurred both in intact cells (grown in culture medium or in soil containing chromate) as well as in cell-free extracts. NADH-dependent chromate reductase activity was found associated with soluble protein and, to a lesser extent, with the membrane fraction.     

Cytochromes of the trimethylamine N-oxide anaerobic respiratory pathway of Escherichia coli

Escherichia coli grown anaerobically with trimethylamine N-oxide (TMAO) as a terminal electron acceptor develops a new cytochrome pathway in addition to the aerobic respiratory pathways which are still formed. Formate, NADH, and possibly other substrates derived from glucose, supply electrons to this pathway. Cytochromes with α-absorption peaks at about 548, 552, 554 and 557 nm are rapidly reoxidized by TMAO in a reaction which is not inhibited by 2-n-heptyl-4-hydroxyquinoneN-oxide. CuSO₄ inhibits the reoxidation by TMAO of the first two of these cytochromes. This suggests that the pathway of electron transfer leading to the reduction of TMAO is: substrates → cytochromes 548,552 → cytochromes 554,557 → trimethylamine-N-oxide reductase → TMAO. These cytochromes, but not those of the aerobic respiratory pathways, are reoxidized by the membrane-impermeant oxidant ammonium persulfate in intact cells. This suggests that the cytochromes of the TMAO reduction pathway and / or trimethylamine-N-oxide reductase are situated at the periplasmic surface of the cytoplasmic membrane of E. coli.     

Extracellular reduction of hexavalent chromium by cytochromes MtrC and OmcA of Shewanella oneidensis MR-1

To characterize the roles of cytochromes MtrC and OmcA of Shewanella oneidensis MR-1 in Cr(VI) reduction, the effects of deleting the mtrC and/or omcA gene on Cr(VI) reduction and the cellular locations of reduced Cr(III) precipitates were investigated. Compared to the rate of reduction of Cr(VI) by the wild type (wt), the deletion of mtrC decreased the initial rate of Cr(VI) reduction by 43.5%, while the deletion of omcA or both mtrC and omcA lowered the rate by 53.4% and 68.9%, respectively. In wt cells, Cr(III) precipitates were detected by transmission electron microscopy in the extracellular matrix between the cells, in association with the outer membrane, and inside the cytoplasm. No extracellular matrix-associated Cr(III) precipitates, however, were found in the cytochrome mutant cell suspension. In mutant cells without either MtrC or OmcA, most Cr(III) precipitates were found in association with the outer membrane, while in mutant cells lacking both MtrC and OmcA, most Cr(III) precipitates were found inside the cytoplasm. Cr(III) precipitates were also detected by scanning election microscopy on the surfaces of the wt and mutants without MtrC or OmcA but not on the mutant cells lacking both MtrC and OmcA, demonstrating that the deletion of mtrC and omcA diminishes the extracellular formation of Cr(III) precipitates. Furthermore, purified MtrC and OmcA reduced Cr(VI) with apparent k(cat) values of 1.2 ± 0.2 (mean ± standard deviation) and 10.2 ± 1 s(-1) and K(m) values of 34.1 ± 4.5 and 41.3 ± 7.9 μM, respectively. Together, these results consistently demonstrate that MtrC and OmcA are the terminal reductases used by S. oneidensis MR-1 for extracellular Cr(VI) reduction where OmcA is a predominant Cr(VI) reductase.