Extracellular Cr Research Articles

Enhancement of extracellular Cr(VI) reduction for anammox recovery using hydrazine: performance, pathways, and mechanism

Enhancement of extracellular Cr(VI) reduction for anammox recovery using hydrazine: performance, pathways, and mechanism

Exploration on the Cr(VI) resistance mechanism of a novel thermophilic Cr(VI)-reducing bacteria Anoxybacillus flavithermus ABF1 isolated from Tengchong geothermal region, China.

Hexavalent chromium resistance and reduction mechanisms of microorganism provide a critical guidance for Cr(VI) bioremediation. However, related researches are limited in mesophiles and deficient for thermophiles. In this work, a novel alkaline Cr(VI)-reducing thermophile Anoxybacillus flavithermus ABF1 was isolated from geothermal region. The mechanisms of Cr(VI) resistance and reduction were investigated. The results demonstrated that A. flavithermus ABF1 could survive in a wide temperature range from 50°C to 70°C and in pH range of 7.0-9.0. Strain ABF1 showed excellent growth activity and Cr(VI) removal performance when initial Cr(VI) concentration was lower than 200 mg L-1 . 93.71% of Cr(VI) was removed at initial concentration of 20 mg L-1 after 72 h. The majority of Cr(VI) was found to be reduced extracellularly by enzymes secreted by cells. XPS and Raman analysis further manifested that Cr2 O3 was the product of Cr(VI) reduction. Moreover, the Cr(VI) transportation-related gene cysP and Cr(VI) reduction-related gene azoR of A. flavithermus ABF1 played key roles in inhibiting Cr(VI) entering cells and promoting extracellular Cr(VI) reduction respectively. This work provides novel insight into the mechanisms of Cr(VI) resistance and detoxication of thermophiles, which leads to a promising alternative strategy for heavy metal bioremediation in areas with elevated temperature.

Enhancement effects of dissolved organic matter leached from sewage sludge on microbial reduction and immobilization of Cr(VI) by Geobacter sulfurreducens

Sewage sludge has a high concentration of dissolved organic matter (DOM) which contains compounds that can serve as electron donors or shuttles for metal reduction by dissimilatory metal reducing bacteria (DMRB). In this study, Cr(VI) removal by G. sulfurreducens, a common DMRB present in anaerobic soils, was examined in the presence or absence of sludge DOM. Two different types of sludge DOM were tested; composted sludge DOM (C-DOM) and anaerobically digested sludge DOM (A-DOM). Both sludge DOMs enhanced Cr(VI) reduction by G. sulfurreducens, but C-DOM was more effective likely because it had higher concentrations of humic substances that served as electron shuttles. Transcriptomic studies indicated that G. sulfurreducens utilizes several different mechanisms to tolerate chromium including extracellular Cr(VI) reduction and immobilization by outer membrane c-type cytochromes and electrically conductive pili, intracellular Cr(VI) reduction by triheme cytochromes and NAD(P)H FMN reductase proteins, and chromium efflux by several P-type ATPase and RND transporter proteins. Microscopy experiments also showed that Cr(III) crystals formed on the surface of the cells, indicating that extracellular Cr(VI) reduction and adsorption was involved in the chromium removal process. These results help provide insight into the potential use of sewage sludge as an additive to enhance the bioremediation of chromium contaminated soils.

Purification and Characterization of Extracellular Cr(VI) Reductase from Bacillus amyloliquefaciens (CSB 9) isolated from Chromite Mine Soil for Detoxification of Hexavalent Chromium

Chromate is widely used industrial pollutant which can be detoxified effectively using chromate reductase from chromium resistant bacteria. A soluble Cr(VI) reductase from Bacillus amyloliquefaciens (CSB 9) was extracted and purified to electrophoretic homogeneity through (NH4)2SO4 precipitation followed by dialysis and gel filtration chromatography with specific activity of 0.167 U/mg and 6% yield with 30.36-fold increase in purity. Based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the molecular weight of the purified enzyme was estimated to be ~ 116 KDa. Further, functional characterization with respect to pH, temperature and storage stability showed optimum Cr(VI) activity at 35 °C and pH 7.0 on standard analysis conditions. Using potassium dichromate as substrate, the enzyme kinetics showed maximum activity (Vmax) of 3.5 U/mL with its corresponding KM value of 27.78 μM and the second-order constant (Vmax/Km) 0.125 U/μM mL. The purified enzyme exhibited higher stability (μM/mg/min) when treated with additives such as metal ions K+ (1.3 ± 0.056), surfactant tween 80 (2.33 ± 0.52), inhibitor β-mercaptoethanol (0.67 ± 0.15) and organic solvent glycerol (1.33 ± 0.011). These remarkable qualities found with the chromate reductase produced by B. amyloliquefaciens (CSB 9) could make it an ideal candidate for bioremediation of hexavalent chromium under a wide range of environmental conditions.

Reduction of hexavalent chromium by Exiguobacterium mexicanum isolated from chromite mines soil

Hexavalent chromium is a highly toxic element generated due to indiscriminate chromite mining in Sukinda, Odisha. In the present research investigation a relatively higher Cr(VI) resistant (900 mg L−1) bacterium CWB-54 was isolated from the chromite mine water. Based on the biochemical and molecular analysis the strain (CWB-54) was identified as Exiguobacterium mexicanum. When this bacterium was grown at 35 °C, 100 rpm, pH~8.0, and fructose as an electron donor, it could reduce the total hexavalent chromium (100 mg L−1) supplemented in the medium within 33 h of incubation period. Though experiment was carried out to study the effect of Mn, Ni, Cd, Hg and Zn on Cr(VI) reduction by the strain E. mexicanum it has been observed that in the presence of Cd and Hg, Cr(VI) reduction drastically decreased. Characterization of Cr(VI) reduced product by SEM-EDX and TEM analysis revealed intracellular and extracellular Cr(III) deposition in the bacterium, which is assumed to be Cr(OH)3 precipitate in nanometric size. But the extracellular chromate reductase enzyme production is found to be negligible as compared to the intracellular enzyme production. The increased concentration of Cr(VI) above (1000 mg L−1) also showed the genotoxic effect on the DNA. Several reports have been published on Exiguobacterium sp. on different scientific aspect but the current report on the reduction of toxic Cr(VI) by a new species E. mexicanum is a novel one which established the potentiality of this microorganism for a broad area of application.

Chemical-Assisted Microbially Mediated Chromium (Cr) (VI) Reduction Under the Influence of Various Electron Donors, Redox Mediators, and Other Additives: An Outlook on Enhanced Cr(VI) Removal.

Chromium (Cr) (VI) is a well-known toxin to all types of biological organisms. Over the past few decades, many investigators have employed numerous bioprocesses to neutralize the toxic effects of Cr(VI). One of the main process for its treatment is bioreduction into Cr(III). Key to this process is the ability of microbial enzymes, which facilitate the transfer of electrons into the high valence state of the metal that acts as an electron acceptor. Many underlying previous efforts have stressed on the use of different external organic and inorganic substances as electron donors to promote Cr(VI) reduction process by different microorganisms. The use of various redox mediators enabled electron transport facility for extracellular Cr(VI) reduction and accelerated the reaction. Also, many chemicals have employed diverse roles to improve the Cr(VI) reduction process in different microorganisms. The application of aforementioned materials at the contaminated systems has offered a variety of influence on Cr(VI) bioremediation by altering microbial community structures and functions and redox environment. The collective insights suggest that the knowledge of appropriate implementation of suitable nutrients can strongly inspire the Cr(VI) reduction rate and efficiency. However, a comprehensive information on such substances and their roles and biochemical pathways in different microorganisms remains elusive. In this regard, our review sheds light on the contributions of various chemicals as electron donors, redox mediators, cofactors, etc., on microbial Cr(VI) reduction for enhanced treatment practices.

Chromium metabolism characteristics of coexpression of ChrA and ChrT gene

ObjectiveSerratia sp. S2 is a wild strain with chromium resistance and reduction ability. Chromium(VI) metabolic-protein-coding gene ChrA and ChrT were cloned from Serratia sp. S2, and ligated with prokaryotic expression vectors pET-28a (+) and transformed into E. coli BL21 to construct ChrA, ChrT and ChrAT engineered bacteria. By studying the characteristics of Cr(VI) metabolism in engineered bacteria, the function and mechanism of the sole expression and coexpression of ChrA and ChrT genes were studied. MethodsUsing Serratia sp. S2 genome as template, ChrA and ChrT genes were amplified by PCR, and prokaryotic expression vectors was ligated to form the recombinant plasmid pET-28a (+)-ChrA, pET-28a (+)-ChrT and pET-28a (+)-ChrAT, and transformed into E. coli BL21 to construct ChrA, ChrT, ChrAT engineered bacteria. The growth curve, tolerance, and reduction of Cr(VI), the distribution of intracellular and extracellular Cr, activity of chromium reductase and intracellular oxidative stress in engineered bacteria were measured to explore the metabolic characteristics of Cr(VI) in ChrA, ChrT, ChrAT engineered bacteria. ResultsChrA, ChrT and ChrAT engineered bacteria were successfully constructed by gene recombination technology. The tolerance to Cr(VI) was Serratia sp. S2 > ChrAT ≈ ChrA > ChrT > Control (P < 0.05), and the reduction ability to Cr(VI) was Serratia sp. S2 > ChrAT ≈ ChrT > ChrA (P < 0.05). The chromium distribution experiments confirmed that Cr(VI) and Cr(III) were the main valence states. Effect of electron donors on chromium reductase activity was NADPH > NADH > non-NAD(P)H (P < 0.05). The activity of chromium reductase increased significantly with NAD(P)H (P < 0.05). The Glutathione and NPSH (Non-protein Sulfhydryl) levels of ChrA, ChrAT engineered bacteria increased significantly (P < 0.05) under the condition of Cr(VI), but there was no significant difference in the indexes of ChrT engineered bacteria (P > 0.05). ConclusionChrAT engineered bacteria possesses resistance and reduction abilities of Cr(VI). ChrA protein endows the strain with the ability to resist Cr(VI). ChrT protein reduces Cr(VI) to Cr(III) by using NAD(P)H as electronic donor. The reduction process promotes the production of GSH, GSSG and NPSH to maintain the intracellular reduction state, which further improves the Cr(VI) tolerance and reduction ability of ChrAT engineered bacteria.

Biotransformation of chromium (VI) by Bacillus sp. isolated from chromate contaminated landfill site

ABSTRACT A potent Cr (VI) resistant bacterial strain DHS-12(7) was isolated from tannery waste landfill site and identified as Bacillus sp. based on pheno- and genotypic methods. The maximum tolerable concentration (MTC) of Cr (VI) was found as 1000 and 250 ppm in nutrient-rich and minimal salt medium, respectively while significant variation of MTC and Cr (VI) reduction efficiency was observed in the solid and liquid medium. The complete reduction of 10 and 25 ppm Cr (VI) was recorded within 18 h and 24 h., respectively in the minimal salt medium and the reduction trend of the strain was bacterial growth-associated. The detection of almost invariable total Cr concentration and the presence of NADH-dependent chromate reductase activity in the cell-free extract, indicated the extracellular Cr (VI) reduction rather than intracellular uptake. Furthermore, chromate reductase (ChrR) gene was amplified, sequenced and the translated amino acid sequence of the ChrR gene showed a high degree of homology with chromate reductase of different organisms. Conclusively, the findings of the present study highlight that Bacillus sp. strain DHS-12(7) can be a potent bioremediation agent for detoxification of Cr (VI) from the contaminated environmental sites for ecological restoration.

Exploring novel Cr(VI) remediation genes for Cr(VI)-contaminated industrial wastewater treatment by comparative metatranscriptomics and metagenomics

Microbial remediation is a promising method to treat Cr(VI) in industrial wastewater. The remediation efficiency and stress-resistance ability of Cr(VI) remediation genes in microbes are the limiting factors for their application in industrial wastewater treatment. To screen novel highly efficient Cr(VI) remediation genes, comparative metatranscriptomic and metagenomic analyses were performed on long-term Cr(VI)-contaminated riparian soil with/without additional Cr(VI) treatment. The most suitable Cr(VI) treatment time was determined to be 30 min according to the high quality RNA yield and fold changes in gene expression. Six novel genes, which had complete open reading frames (ORFs) in metagenomic libraries, were identified from unculturable microbes. In the phenotypic functional assay, all novel genes enhanced the Cr(VI) resistance/reduction ability of E. coli. In the industrial wastewater treatment, E-mcr and E-gsr presented at least 50% Cr(VI) removal efficiencies in the presence of 200–600 μM of Cr(VI), without a decrease in efficiency over 17 days. The stress resistance assay showed that gsr increased the growth rate of E. coli by at least 30% under different extreme conditions, and thus, gsr was identified as a general stress-response gene. In the Cr valence distribution assay, E-mcr presented ~40 μM higher extracellular Cr (III) compared to E-yieF. Additionally, transmission electron microscopy (TEM) of E-mcr showed bulk black agglomerates on the cell surface. Thus, mcr was identified as a membrane chromate reductase gene. This research provides a new idea for studying novel highly efficient contaminant remediation genes from unculturable microbes.

Dynamic proteome responses to sequential reduction of Cr(VI) and adsorption of Pb(II) by Pannonibacter phragmitetus BB

Here, the microbial responses to Cr(VI) and Pb(II) with bio-removal of the metals in water by Pannonibacter phragmitetus BB were explored. The comparative bacterial proteomics showed that the intracellular and extracellular Cr(VI) reduction proteins, Pb(II) adsorption by the lipoprotein and sugar-related bacterial proteins, as well as Pb(II) precipitation by phosphate and OH− were vital to the bio-removal of Cr(VI) and Pb(II). Moreover, the influx and efflux channels of Cr(VI) and Cr(III), Pb(II) transporters, extracellular siderophores for Pb(II) complexation and antioxidant proteins enabled the strain BB to resist the toxicity of Cr(VI) and Pb(II). In addition, the dynamic expression levels of the proteins related to reduction and transportation of Cr(VI), and adsorption, transportation and complexation of Pb(II) were dependent on the corresponding metal, respectively. The anti-oxidative stress system, such as superoxide dismutase, and Na+/H+ antiporters played central roles in the protein–protein interaction network to resist and detoxify Cr(VI) and Pb(II). The results of our study provide a novel insight for the physiological responses of the strain BB to the combined stresses of Pb(II) and Cr(VI).

Chromium tolerance and accumulation in Aspergillus flavus isolated from tannery effluent

Cr(VI) tolerance in Aspergillus flavus, strain SFL, isolated from tannery effluent was measured and compared with a reference strain of A. flavus, A1120. On solid medium, SFL had a high level of Cr(VI) tolerance (1,600 mg/L), which was 16 times that of A1120 and greater than most previously analyzed fungal strains. When in 100 mg/L of Cr(VI), SFL completely depleted Cr(VI) within 72 h while A1120 depleted 85% of Cr(VI). SFL was more effective in reducing extracellular Cr(VI) than A1120. While A1120 showed greater biosorption of Cr(VI) than SFL, intracellular accumulation was approximately 50% greater in SFL and was more energy-dependent than A1120. Cr(VI) modified the external surface of the hyphae. Cr speciation detected the presence of only Cr(III), corresponding to Cr(OH)3 , which precipitated on the hyphal surface. Cr(VI) bound to the functional groups carboxyl, amine, and hydroxyl in both SFL and A1120. Transmission electron microscopy energy-dispersive X-ray detected Cr on the fungal wall and within membrane-bound organelles of the cytoplasm. In conclusion, the greater tolerance of SFL to Cr(VI) relative to A1120 is due to more effective energy-dependant uptake of Cr(VI) into the cell and increased capacity of SFL to store Cr in intracellular vacuoles compared with A1120.

Intracellular versus extracellular accumulation of Hexavalent chromium reduction products by Geobacter sulfurreducens PCA

Hexavalent chromium (Cr(VI)) reduction by Geobacter sulfurreducens PCA was evaluated in batch experiments, and the form and amounts of intracellular and extra-cellular Cr(VI) reduction products were determined over time. The first-order Cr(VI) reduction rate per unit mass of cells was consistent for different initial cell concentrations, and approximately equal to (2.065 ± 0.389) x 10−9 mL CFU−1 h−1. A portion of the reduced Cr(VI) products precipitated on Geobacter cell walls as Cr(III) and was bound via carboxylate functional groups, a portion accumulated inside Geobacter cells, and another portion existed as soluble Cr(III) or organo-Cr(III) released to solution. A mass balance analysis of total chromium in aqueous media, on cell walls, and inside cells was determined as a function of time, and with different initial cell concentrations. Mass balances were between 92% and 98%, and indicated Cr(VI) reduction products accumulate more on cell walls and inside cells with time and with increasing initial cell concentration, as opposed to particulates in aqueous solution. Reduced Cr(VI) products both in solution and on cell surfaces appear to form organo-Cr(III) complexes, and our results suggest that such complexes are more stable to reoxidation than aqueous Cr(III) or Cr(OH)3. Chromium inside cells is also likely more stable to reoxidation, both because it can form organic complexes, and it is separated by the cell membrane from solution conditions. Hence, Cr(VI) reduction products in groundwater during bioremediation may become more stable against re-oxidation, and may pose a lower risk to human health, over time and with greater initial biomass densities.

Metal Respiratory Pathway-Independent Cr Isotope Fractionation during Cr(VI) Reduction by Shewanella oneidensis MR-1

Cr isotope fractionation during microbial reduction processes is commonly recognized as a promising tool in biogeochemistry and bioremediation. However, the mechanism of Cr isotope fractionation during microbial reduction is poorly understood. In this work, the relationship between the bacterial respiratory pathway and Cr isotope fractionation was investigated in Shewanella oneidensis MR-1. For comparison with the wild type, a mutant strain (ΔomcA/ΔmtrC) with a deficiency in extracellular Cr(VI) reduction was constructed by deleting the omcA and mtrC genes, which encode the terminal reductase during extracellular reduction. The magnitudes of Cr isotope fractionation (e) for Cr(VI) reduction by the wild type and ΔomcA/ΔmtrC were −2.42 ± 0.68‰ and −2.70 ± 0.22‰, respectively. Surprisingly, the e values were not significantly different between the two strains. This suggests that isotope fractionation is independent of the metal respiratory pathway during Cr(VI) reduction by S. oneidensis MR-1. Moreover, a th...

Evaluation of chromate reductase activity in the cell-free culture filtrate of Arthrobacter sp. SUK 1201 isolated from chromite mine overburden

Arthrobacter sp. SUK 1201, a chromate resistant and reducing bacterium isolated from chromite mine overburden of Sukinda valley, Odisha, India has been evaluated for its hexavalent chromium [Cr(VI)] reduction potential using cell-free culture filtrate as extracellular chromate reductase enzyme. Production of the enzyme was enhanced in presence of Cr(VI) and its reducing efficiency was increased with increasing concentration of Cr(VI). The Michaelis-Menten constant (Km) and the maximum specific velocity (Vmax) of the extracellular Cr(VI) reductase were calculated to be 54.03 μM Cr(VI) and 5.803 U mg−1 of protein respectively showing high affinity towards Cr(VI). The reducing activity of the enzyme was maximum at pH 6.5–7.5 and at a temperature of 35 °C and was dependent on NADH. The enzyme was tolerant to different metals such as Mn(II), Mg(II) and Fe(III) and was able to reduce Cr(VI) present in chromite mine seepage. These findings suggest that the extracellular chromate reductase of Arthrobacter sp. SUK 1201 has a great promise for use in Cr(VI) detoxification under different environmental conditions, particularly in the mining waste water treatment systems.

Chromium uptake and adsorption in marine phytoplankton – Implications for the marine chromium cycle

Using the radioisotope 51Cr, we investigated the controls of cellular Cr accumulation in an array of marine phytoplankton grown in environmentally relevant Cr concentrations (1–10nM). Given the affinity of Cr(III) for amorphous Fe-hydroxide mineral surfaces, and the formation of these mineral phases on the outside of phytoplankton cells, extracellular Cr was monitored in a model diatom species (Thalassiosira weissflogii) as extracellular Fe concentrations varied. Extracellular Cr in T. weissflogii increased with increasing extracellular Fe, demonstrating that Cr may be removed from seawater via extracellular adsorption to phytoplankton. Short-term Cr(VI) and Cr(III) uptake experiments performed with T. weissflogii demonstrated that Cr(III) was the primary oxidation state adsorbing to cells and being internalized by them. Cellular Cr:C ratios (<0.5μmolCrmolC−1) of the eight phytoplankton species surveyed were significantly lower than previously reported Cr:C ratios in marine particles with a high biogenic component (10–300μmolCrmolC−1). This indicates that Cr(III) likely accumulates in marine particles due to uptake and/or adsorption. Mass balance calculations demonstrate that surface water Cr deficits can be explained via loss of Cr(III) to exported particles, thereby providing a mechanism to account for the nutrient depth profile for Cr in modern seawater. Given the large fractionation of stable Cr isotopes during Cr(VI) reduction, Cr(III) associated with exported organic carbon is likely enriched in lighter isotopes. Most sedimentary Cr isotope studies have thus far neglected internal fractionating processes in the marine Cr cycle, but our data indicate that loss of Cr to exported particles may be traced in the sedimentary δ53Cr record.

Chromate Interaction with the Chromate Reducing Actinobacterium Intrasporangium chromatireducens Q5-1

This study was conducted to determine the microbe-chromate [Cr(VI)] interaction and the effect of quinoid analogue anthraquinone-2-sulfonate (AQS) on aerobic Cr(VI) reduction by Intrasporangium chromatireducens Q5-1. The addition of redox mediator AQS, which might expedite the electron transfer, promoted Cr(VI) bioreduction. Addition of carbon sources, such as maltose, acetate, sucrose and lactose stimulated the AQS-promoted Cr(VI) reduction of strain Q5-1. Induction experiment clarified that the enzyme involves in the Cr(VI) reduction is constitutive. Energy-dispersive spectroscopy (EDS) spectra showed the existence of trace Cr distributed on the cell surface. X-ray photoelectron spectroscopy (XPS) analysis revealed that the Cr(III) complex was bound to the cell surface (0.87%, atomic percent). The spectra shifts detected by Fourier transform infrared (FTIR) spectroscopy indicated that Cr(III) was bound to the carbonyl and amide groups. In addition, Cr(VI) reduction by different cell fractions showed that Cr(VI) reduction was occurred extracellularly rather than intracellularly. The results disclosed that Cr(VI) detoxification of strain Q5-1 was mainly associated with extracellular Cr(VI) reduction process in combination with trace Cr(III) adsorption on the cell surface. A schematic figure depicting the interactions between strain Q5-1 and Cr(VI) was presented. This study enhanced the understanding of the microbe-Cr(VI) interaction mechanism and revealed the AQS-promoted aerobic Cr(VI) reduction of strain Q5-1. Such strain and quinoid analogue-mediated bacterial Cr(VI) reduction may facilitate the bioremediation for Cr(VI)-polluted environment.

Cr(VI) reduction by Enterobacter sp. DU17 isolated from the tannery waste dump site and characterization of the bacterium and the Cr(VI) reductase

Out of nineteen bacteria screened from the tannery waste dump site, the most effective isolate, strain DU17 was selected for Cr(VI) reduction process among the non-pathogenic once. Based on 16S rRNA gene sequence analysis, the bacterium was identified as Enterobacter sp. DU17. Its amplified Cr(VI) reductase gene showed maximum homology with flavoprotein of Enterobacter cloacae. Enterobacter sp. DU17 reduced Cr(VI) maximally at 37 °C and pH 7.0. Various co-metals, electron (e−) donors and inhibitors were tested to study their effect on Cr(VI) reduction. In presence (0.2% each) of glucose and fructose, Enterobacter sp. DU17 reduced Cr(VI) completely after 16 and 20 h, respectively. Since the concentration of total Cr was invariable after remediation as detected through AAS analysis, this experiment disclosed that responsible operation was associated with extracellular Cr(VI) reduction process rather than uptake mechanism. Multiple antibiotic resistance index of 0.08 for this bacterium was very low as compared to standard risk assessment value of 0.20. With high Cr(VI) reducing capability, non-pathogenicity and antibiotic sensitivity, Enterobacter sp. DU17 is found to be very efficient in removing Cr(VI) toxicity from the environment.

214 REGULATION OF CELLULAR CREATINE HOMEOSTASIS: A ROLE FOR THIOREDOXIN INTERACTING PROTEIN (TXNIP)

Background Creatine (Cr) uptake across the plasma membrane is particularly important in excitable cells that cannot synthesise Cr, such as cardiomyocytes, where creatine has a role in energy buffering and transport. Uptake is tightly controlled via a specific transmembrane Creatine Transporter (CrT), while cellular loss is slow, passive, and non-enzymatic. Earlier work has suggested that CrT activity is subject to substrate inhibition, but, that this requires de novo synthesis of unidentified protein(s). The purpose of this study was to identify this protein and to verify its role as a potential endogenous inhibitor of the CrT. Methods and Results To achieve this we took a global gene array approach under varying conditions of cellular [Cr], and verified our findings using an in vitro assay of Cr-uptake using a radiolabelled 14 Cr method. 3T3 fibroblasts stably over-expressing CrT were incubated for 2-5 hours with either low (250 µM) or saturating (5 mM) Cr, with and without cycloheximide (CHX) to inhibit protein synthesis. Cr uptake plateaued at 3 hours in response to saturating levels of extracellular Cr (5mM) and was 33% ( P P =0.036). Quantitative RT-PCR verified up-regulation of Txnip mRNA (61% increase; P=0.002 ) and immunoblotting detected a 45% increase in Txnip protein after 3hr of Cr incubation ( P=0.01 ). Treatment of cells with either 250 µM Cr or 5mM taurine, did not result in Txnip mRNA or protein up-regulation, confirming specificity for saturating Cr. Small interfering RNA against Txnip (siTxnip) reduced mRNA levels by 50% and attenuated the increase in Txnip gene expression in response to 5mM Cr (17% in siTxnip versus 45% in control cells). This was sufficient to maintain CrT activity at control levels during high [Cr], i.e. substrate inhibition was completely abolished. The in vitro observations were tested in vivo in relation to altered left ventricular [Cr]. Cr-deficient mice, due to deletion of the Cr biosynthetic enzyme guanidinoacetate methyl-transferase (GAMT −/− ), had LV Txnip mRNA 35% lower than wild-types. Conversely, mice with cardiac overexpression of CrT had elevated LV [Cr] (mean 124 versus 74nmol/mg protein in WT) associated with a 58% increase in Txnip mRNA and 29% more Txnip protein when compared to wild-type littermates. Conclusion Our work suggests a novel role for Txnip in the endogenous regulation of CrT activity in cardiomyocytes, thereby contributing to modulation of Cr homeostasis. The molecular mechanisms involved in the CrT-Txnip interaction, merit further elucidation.

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.

The pro-oxidant chromium(VI) inhibits mitochondrial complex I, complex II, and aconitase in the bronchial epithelium: EPR markers for Fe–S proteins

Hexavalent chromium (Cr(VI)) compounds (e.g., chromates) are strong oxidants that readily enter cells, where they are reduced to reactive Cr species that also facilitate reactive oxygen species generation. Recent studies demonstrated inhibition and oxidation of the thioredoxin system, with greater effects on mitochondrial thioredoxin (Trx2). This implies that Cr(VI)-induced oxidant stress may be especially directed at the mitochondria. Examination of other redox-sensitive mitochondrial functions showed that Cr(VI) treatments that cause Trx2 oxidation in human bronchial epithelial cells also result in pronounced and irreversible inhibition of aconitase, a TCA cycle enzyme that has an iron–sulfur (Fe–S) center that is labile with respect to certain oxidants. The activities of electron transport complexes I and II were also inhibited, whereas complex III was not. Electron paramagnetic resonance (EPR) studies of samples at liquid helium temperature (10 K) showed a strong signal at g = 1.94 that is consistent with the inhibition of electron flow through complex I and/or II. A signal at g = 2.02 was also observed, which is consistent with oxidation of the Fe–S center of aconitase. The g = 1.94 signal was particularly intense and remained after extracellular Cr(VI) was removed, whereas the g = 2.02 signal declined in intensity after Cr(VI) was removed. A similar inhibition of these activities and analogous EPR findings were noted in bovine airways treated ex vivo with Cr(VI). Overall, the data support the hypothesis that Cr(VI) exposure has deleterious effects on a number of redox-sensitive core mitochondrial proteins. The g = 1.94 signal could prove to be an important biomarker for oxidative damage resulting from Cr(VI) exposure. The EPR spectra simultaneously showed signals for Cr(V) and Cr(III), which verify Cr(VI) exposure and its intracellular reductive activation.