Reduction of Hexavalent Chromium and Detection of Chromate Reductase (ChrR) in Stenotrophomonas maltophilia.
ABSTRACT: An Gram negative strain of S. maltophilia, indigenous to environments contaminated by Cr(VI) and identified by biochemical methods and 16S rRNA gene analysis, reduced chromate by 100%, 98-99% and 92% at concentrations in the 10-70, 80-300, and 500 mg/L range, respectively at pH 7 and temperature 37 °C. Increasing concentrations of Cr(VI) in the medium lowered the growth rate but could not be directly correlated with the amount of Cr(VI) reduced. The strain also exhibited multiple resistance to antibiotics and tolerance and resistance to various heavy metals (Ni, Zn and Cu), with the exception of Hg. Hexavalent chromium reduction was mainly associated with the soluble fraction of the cell evaluated with crude cell-free extracts. A protein of molecular weight around 25 kDa was detected on SDS-PAGE gel depending on the concentration of hexavalent chromium in the medium (0, 100 and 500 mg/L). In silico analysis in this contribution, revealed the presence of the chromate reductase gene ChrR in S. maltophilia, evidenced through a fragment of around 468 bp obtained experimentally. High Cr(VI) concentration resistance and high Cr(VI) reducing ability of the strain make it a suitable candidate for bioremediation.
Project description:<h4>Background</h4>Chromium is one of the most used toxic heavy metals. A large amount of chromium waste is discharged into the environment every year, causing serious environmental pollution, especially the pollution of soil and water by hexavalent chromium. Eliminating hexavalent chromium is the primary challenge to achieve a pollution-free environment.<h4>Objectives</h4>This study aims to understand the mechanism of <i>Pichia guilliermondii</i>'s reduction of hexavalent chromium through enzymatic characteristic, oxidative stress response, and reduction product.<h4>Material and methods</h4>The strain <i>Pichia guilliermondii</i> ZJH-1 was isolated and stored in our laboratory. The hexavalent chromium uses 1,5-diphenyl carbazide method (DPC) to measure. The UV spectrophotometer was used to measure the intracellular antioxidant enzyme activity, and the kit was used to measure the activity of catalase and glutathione reductase. The reduction products were analyzed by ultraviolet full-wavelength scanning and FTIR.<h4>Results</h4>The reduction of hexavalent chromium by ZJH-1 is accompanied by an increase in active oxygen and antioxidant levels. Chromate reductase mainly exists in the extracellular fluid, and the carboxyl, amide, hydroxide and other groups of the cell wall are involved in the bioremediation of Cr(VI) by complexing with Cr(VI) and Cr(III). After ZJH-1 was treated with different concentrations of Cr(VI), the expression of proteins with molecular weights of 15 kDa, 18 kDa, 35 kDa, 62 kDa, and 115 kDa increased significantly. This strain is the most suitable for chromate reductase (CChR). The optimum temperature is 40℃ and the optimum pH is 7.0. Cu2+ can enhance the activity of chromate reductase. At the optimum temperature and pH, the chromate reductase Km of this strain is 0.40 μmol and Vmax is 14.47 μmoL.L<sup>-1</sup>·min<sup>-1</sup>.<h4>Conclusions</h4>The bioremediation of Cr(VI) by <i>Pichia guilliermondii</i> ZJH-1 is attributable to the reduction product (Cr(III)) that can be removed in the precipitate and can be fixed on the cell surface and accumulated in the cell.
Project description:Hexavalent chromium reduction and accumulation by Acinetobacter AB1 isolated from Fez tanneries effluents were tested. The effects of some environmental factors such as pH, temperature, and exposure time on Cr(VI) reduction and resistance were investigated. We found that this strain was able to resist to concentrations as high as 400 mg/l of Cr(VI). Moreover, pH 10 and the temperature 30°C constitute favourable conditions to the growth and reduction of Acinetobacter AB1. Complete reduction of Cr(VI) was observed at low initial Cr(VI) concentrations of 50 mg/l after 72 h of incubation. Furthermore, Transmission electron microscope (TEM) analysis showed morphological changes in AB1 strain due 48H exposure to 100 mg/l chromate concentration and revealed circular electron dense (dark black point) inclusion within the cell cytoplasm suggesting chromium deposition within the cells.
Project description:Hexavalent chromium (Cr(VI)) is a serious environmental pollutant and human toxicant. Mammalian cells are very sensitive to chromate as they lack efficient chromate detoxifying strategy, e.g., chromate-reducing genes that are widely present in prokaryotes. To test whether introduction of prokaryotic chromate-reducing gene into mammalian cells could render higher chromate resistance, an Escherichia coli chromate-reducing gene yieF was transfected into human HepG2 cells. The expression of yieF was measured in stably transfected cells HepG2-YieF by quantitative RT-PCR and found up-regulated by 3.89-fold upon Cr(VI) induction. In chromate-reducing ability test, HepG2-YieF cells that harbored the reductase showed significantly higher reducing ability of Cr(VI) than HepG2 control cells. This result was further supported by the evidence of increased Cr(VI)-removing ability of crude cell extract of HepG2-YieF. Moreover, HepG2-YieF demonstrated 10% higher viability and decreased expression of GSH synthesizing enzymes under Cr(VI) stress. Subcellular localization of YieF was determined by tracing GFP-YieF fusion protein that was detected in both nucleus and cytoplasm by laser confocal microscopy. Altogether, this study successfully demonstrated that the expression of a prokaryotic Cr(VI)-reducing gene yieF endowed mammalian cell HepG2 with enhanced chromate resistance, which brought new insight of Cr(VI) detoxification in mammalian cells.
Project description:Numerous technologies and approaches have been used in the past few decades to remove hexavalent chromium (Cr[VI]) in wastewater and the environment. However, these conventional technologies are not economical and efficient in removing Cr(VI) at a very low concentration (1-100 ppm). As an alternative, the utilization of bioremediation techniques which uses the potential of microorganisms could represent an effective technique for the detoxification of Cr(VI). In this study, we reported a newly isolated bacterium identified as Acinetobacter radioresistens sp. NS-MIE from Malaysian agricultural soil. The chromate reduction potential of strain NS-MIE was optimized using RSM and ANN techniques. The optimum condition predicted by RSM for the bacterium to reduce hexavalent chromium occurred at pH 6, 10 g/L ppm of nutrient broth (NB) concentration and 100 ppm of chromate concentration while the optimum condition predicted by ANN is at pH 6 and 10 g/L of NB concentration and of 60 ppm of chromate concentration with chromate reduction (%) of 75.13 % and 96.27 %, respectively. The analysis by the ANN model shows better prediction data with a higher R2 value of 0.9991 and smaller average absolute deviation (AAD) and root mean square error (RMSE) of 0.33 % and 0.302 %, respectively. Validation analysis showed the predicted values by RSM and ANN were close to the validation values, whereas the ANN showed the lowest deviation, 2.57%, compared to the RSM. This finding suggests that the ANN showed a better prediction and fitting ability compared to the RSM for the nonlinear regression analysis. Based on this study, A. radioresistens sp. NS-MIE exhibits strong potential characteristics as a candidate for the bioremediation of hexavalent chromium in the environment.
Project description:The current study aimed to isolate and characterize a chromate-resistant bacterium from tannery effluent, able to reduce Cr(VI) aerobically at high pH and salinity. Environmental contamination by hexavalent chromium, Cr(VI), presents a serious public health problem. Enrichment led to the isolation of 12 bacteria displaying different degrees of chromate reduction. Phenotypic characterization and phylogenetic analysis based on 16S rDNA sequence comparison indicated that the most potent strain belonged to the genus Halomonas. The new strain designated as Halomonas sp. M-Cr was able to reduce 82% of 50 mg L-1 Cr(VI) in 48 h, concomitant with discolouring of yellow colour of the medium and formation of white insoluble precipitate of Cr(III). It exhibited growth up to 3500 mg L-1 Cr(VI), 20% NaCl and showed strong Cr(VI) reduction under alkaline condition, pH 10. Scanning electron microscopy revealed precipitation of chromium hydroxide on bacterial cell surfaces, which showed characteristic peak of chromium in energy-dispersive X-ray analysis. Plackett-Burman design was used to evaluate the influence of related parameters for enhancing Cr(VI) reduction. Glucose, yeast extract and KH2PO 4 were confirmed as significant variables in the medium. Data suggest Halomonas sp. M-Cr as a promising candidate for bioremediation of Cr(VI) contaminated effluents particularly in saline and alkaline environments. Up to our knowledge, this is the first report on isolation of haloalkaliphilic Halomonas sp. from tannery effluent.
Project description:Hexavalent chromium-resistant Ochrobactrum intermedium BCR400 was isolated from chromium contaminated soil collected from Vadodara, Gujarat. It reduced 100 mg Cr(VI)/L completely in 52 h with initial Cr(VI) reduction rate of 1.98 mg/L/h. The Cr(VI) reduction rate decreased with increase in Cr(VI) concentration from 100 to 500 mg/L. The addition of anthraquinone-2-sulphonic acid (AQS) to culture O. intermedium BCR400 significantly enhanced its chromium reduction rate. The activation energy of AQS-mediated Cr(VI) reduction (120.69 KJ/mol) was 1.1-fold lower than non-mediated Cr(VI) reduction. An increase in the activities of quinone reductase and chromate reductase in cells grown in presence of AQS/AQS + Cr(VI) suggests their role in reduction of Cr(VI) by O. intermedium. Both chromate reductase and quinone reductase activities were FAD independent, required NADH as reductant, displayed maximum activity at pH (7.0) and temperature (30 °C). Thus Cr(VI) bioremediation potential of O. intermedium can be enhanced by augmentation of system with AQS as redox mediator.
Project description:Hexavalent chromium [Cr(VI)] is a soluble carcinogen that has caused widespread contamination of soil and water in many industrial nations. Bacteria have the potential to aid remediation as certain strains can catalyze the reduction of Cr(VI) to insoluble and less toxic Cr(III). Here, we examine Cr(VI) reducing Microbacterium spp. (Cr-K1W, Cr-K20, Cr-K29, and Cr-K32) isolated from contaminated sediment (Seymore, Indiana) and show varying chromate responses despite the isolates' phylogenetic similarity (i.e., identical 16S rRNA gene sequences). Detailed analysis identified differences based on genomic metabolic potential, growth and general metabolic capabilities, and capacity to resist and reduce Cr(VI). Taken together, the discrepancies between the isolates demonstrate the complexity inter-strain variation can have on microbial physiology and related biogeochemical processes.
Project description:Hexavalent chromium [Cr(VI)] damages DNA and causes cancer, but it is unclear which DNA damage responses (DDRs) most critically protect cells from chromate toxicity. Here, genome-wide quantitative functional profiling, DDR measurements and genetic interaction assays in Schizosaccharomyces pombe reveal a chromate toxicogenomic profile that closely resembles the cancer chemotherapeutic drug camptothecin (CPT), which traps Topoisomerase 1 (Top1)-DNA covalent complex (Top1cc) at the 3' end of single-stand breaks (SSBs), resulting in replication fork collapse. ATR/Rad3-dependent checkpoints that detect stalled and collapsed replication forks are crucial in Cr(VI)-treated cells, as is Mus81-dependent sister chromatid recombination (SCR) that repairs single-ended double-strand breaks (seDSBs) at broken replication forks. Surprisingly, chromate resistance does not require base excision repair (BER) or interstrand crosslink (ICL) repair, nor does co-elimination of XPA-dependent nucleotide excision repair (NER) and Rad18-mediated post-replication repair (PRR) confer chromate sensitivity in fission yeast. However, co-elimination of Tdp1 tyrosyl-DNA phosphodiesterase and Rad16-Swi10 (XPF-ERCC1) NER endonuclease synergistically enhances chromate toxicity in top1? cells. Pnk1 polynucleotide kinase phosphatase (PNKP), which restores 3'-hydroxyl ends to SSBs processed by Tdp1, is also critical for chromate resistance. Loss of Tdp1 ameliorates pnk1? chromate sensitivity while enhancing the requirement for Mus81. Thus, Tdp1 and PNKP, which prevent neurodegeneration in humans, repair an important class of Cr-induced SSBs that collapse replication forks.
Project description:BACKGROUND: Chromium is a toxic heavy metal, which primarily exists in two inorganic forms, Cr(VI) and Cr(III). Chromate [Cr(VI)] is carcinogenic, mutational, and teratogenic due to its strong oxidizing nature. Biotransformation of Cr(VI) to less-toxic Cr(III) by chromate-resistant and reducing bacteria has offered an ecological and economical option for chromate detoxification and bioremediation. However, knowledge of the genetic determinants for chromate resistance and reduction has been limited so far. Our main aim was to investigate chromate resistance and reduction by Bacillus cereus SJ1, and to further study the underlying mechanisms at the molecular level using the obtained genome sequence. RESULTS: Bacillus cereus SJ1 isolated from chromium-contaminated wastewater of a metal electroplating factory displayed high Cr(VI) resistance with a minimal inhibitory concentration (MIC) of 30 mM when induced with Cr(VI). A complete bacterial reduction of 1 mM Cr(VI) was achieved within 57 h. By genome sequence analysis, a putative chromate transport operon, chrIA1, and two additional chrA genes encoding putative chromate transporters that likely confer chromate resistance were identified. Furthermore, we also found an azoreductase gene azoR and four nitroreductase genes nitR possibly involved in chromate reduction. Using reverse transcription PCR (RT-PCR) technology, it was shown that expression of adjacent genes chrA1 and chrI was induced in response to Cr(VI) but expression of the other two chromate transporter genes chrA2 and chrA3 was constitutive. In contrast, chromate reduction was constitutive in both phenotypic and gene expression analyses. The presence of a resolvase gene upstream of chrIA1, an arsenic resistance operon and a gene encoding Tn7-like transposition proteins ABBCCCD downstream of chrIA1 in B. cereus SJ1 implied the possibility of recent horizontal gene transfer. CONCLUSION: Our results indicate that expression of the chromate transporter gene chrA1 was inducible by Cr(VI) and most likely regulated by the putative transcriptional regulator ChrI. The bacterial Cr(VI)-resistant level was also inducible. The presence of an adjacent arsenic resistance gene cluster nearby the chrIA1 suggested that strong selective pressure by chromium and arsenic could cause bacterial horizontal gene transfer. Such events may favor the survival and increase the resistance level of B. cereus SJ1.
Project description:An efficient keratinolytic strain of Stenorophomonas maltophilia KB13 was isolated from feather disposal site of Bilaspur, Chhattisgarh, India. The strain could metabolize 10 g/l chicken feathers as sole source of carbon and nitrogen. Soluble protein, amino acid, and cysteine content were found to be maximum (690.6 ± 8.7, 688.9 ± 9.12 and 21 ± 0.36 µg/ml, respectively) at late logarithmic phase of growth. Protease and keratinase activity reached its maximum level (103.26 ± 7.09 and 178.5 ± 9.10 U/ml) at the 4th day of incubation. The feather protein hydrolysate (FPH) obtained after degradation of chicken feathers was utilized to reduce hexavalent chromium. About 78.4 ± 2.4 and 63.6 ± 2.2 % reduction of 50 and 100 mg/l Cr(VI), respectively, was observed after 60 min of incubation with FPH. Further, there was no effect of autoclaved FPH on Cr(VI) reduction indicating that any bacterial enzyme was not involved in reduction process. Cr(VI) reduction was significantly inhibited by 10 mm Hg2+ ions indicating the role of sulfur-containing amino acids in reduction process. FTIR analysis confirmed that chromium reduction occurred due to oxidation of amino acids cysteine and cystine. This study shows that FPH arising after feather degradation can be employed as a potential candidate for the reduction of hexavalant chromium.