The unique glutathione reductase from Xanthomonas campestris: gene expression and enzyme characterization

The glutathione reductase gene, gor, was cloned from the plant pathogen Xanthomonas campestris pv. phaseoli. Its gene expression and enzyme characteristics were found to be different from those of previously studied homologues. Northern blot hybridization, promoter-lacZ fusion, and enzyme assay experiments revealed that its expression, unlike in Escherichia coli, is OxyR-independent and constitutive upon oxidative stress conditions. The deduced amino acid sequence shows a unique NADPH binding motif where the most highly conserved arginine residue, which is critical for NADPH binding, is replaced by glutamine. Interestingly, a search of the available Gor amino acid sequences from various sources, including other Xanthomonas species, revealed that this replacement is specific to the genus Xanthomonas. Recombinant Gor enzyme was purified and characterized, and was found to have a novel ability to use both, NADPH and NADH, as electron donor. A gor knockout mutant was constructed and shown to have increased expression of the organic peroxide-inducible regulator gene, ohrR.     

Enhanced sensitivity to oxidative stress in transgenic tobacco plants with decreased glutathione reductase activity leads to a decrease in ascorbate pool and ascorbate redox state

To investigate the possible mechanisms of glutathione reductase (GR) in protecting against oxidative stress, we obtained transgenic tobacco (Nicotiana tabacum) plants with 30–70% decreased GR activity by using a gene encoding tobacco chloroplastic GR for the RNAi construct. We investigated the responses of wild type and transgenic plants to oxidative stress induced by application of methyl viologen in vivo. Analyses of CO₂ assimilation, maximal efficiency of photosystem II photochemistry, leaf bleaching, and oxidative damage to lipids demonstrated that transgenic plants exhibited enhanced sensitivity to oxidative stress. Under oxidative stress, there was a greater decrease in reduced to oxidized glutathione ratio but a greater increase in reduced glutathione in transgenic plants than in wild type plants. In addition, transgenic plants showed a greater decrease in reduced ascorbate and reduced to oxidized ascorbate ratio than wild type plants. However, there were neither differences in the levels of NADP and NADPH and in the total foliar activities of monodehydroascorbate reductase and dehydroascorbate reductase between wild type and transgenic plant. MV treatment induced an increase in the activities of GR, ascorbate peroxidase, superoxide dismutase, and catalase. Furthermore, accumulation of H₂O₂ in chloroplasts was observed in transgenic plants but not in wild type plants. Our results suggest that capacity for regeneration of glutathione by GR plays an important role in protecting against oxidative stress by maintaining ascorbate pool and ascorbate redox state.     

The Escherichia coli btuE gene, encodes a glutathione peroxidase that is induced under oxidative stress conditions

Most aerobic organisms are exposed to oxidative stress. Looking for enzyme activities involved in the bacterial response to this kind of stress, we focused on the btuE-encoded Escherichia coli BtuE, an enzyme that shares homology with the glutathione peroxidase (GPX) family. This work deals with the purification and characterization of the btuE gene product.Purified BtuE decomposes in vitro hydrogen peroxide in a glutathione-dependent manner. BtuE also utilizes preferentially thioredoxin A to decompose hydrogen peroxide as well as cumene-, tert-butyl-, and linoleic acid hydroperoxides, confirming that its active site confers non-specific peroxidase activity. These data suggest that the enzyme may have one or more organic hydroperoxide as its physiological substrate.The btuE gene was induced when cells were exposed to oxidative stress elicitors that included potassium tellurite, menadione and hydrogen peroxide, among others, suggesting that BtuE could participate in the E. coli response to reactive oxygen species. To our knowledge, this is the first report describing a glutathione peroxidase in E. coli.     

Role of respiration and glutathione in cadmium-induced oxidative stress in Escherichia coli K-12

Cadmium is a widespread pollutant that has been associated with oxidative stress, but the mechanism behind this effect in prokaryotes is still unclear. In this work, we exposed two glutathione deficient mutants (ΔgshA and ΔgshB) and one respiration deficient mutant (ΔubiE) to a sublethal concentration of cadmium. The glutathione mutants show a similar increase in reactive oxygen species as the wild type. Experiments performed using the ΔubiE strain showed that this mutant is more resistant to cadmium ions and that Cd-induced reactive oxygen species levels were not altered. In the light of these facts, we conclude that the interference of cadmium with the respiratory chain is the cause of the oxidative stress induced by this metal and that, contrary to previously proposed models, the reactive oxygen species increase is not due to glutathione depletion, although this peptide is crucial for cadmium detoxification.     

Glutathione reductase activity and isoforms in leaves and roots of wheat plants subjected to cadmium stress

The behavior of glutathione reductase (GR, EC 1.6.4.2) activity and isoforms were analyzed in wheat (Triticum aestivum L.) leaves and roots exposed to a chronic treatment with a toxic cadmium (Cd) concentration. A significant growth inhibition (up to 55%) was found in leaves at 7, 14 and 21 days, whereas roots were affected (51%) only after three weeks. Wheat plants grown in the presence of 100microM Cd showed a time-dependent accumulation of this metal, with Cd concentration being 10-fold higher in roots than in leaves. Nevertheless, lipid peroxidation was augmented in leaves in all experiments, but not in roots until 21 days. Cadmium treatment altered neither the GR activity nor the isoform pattern in the leaves. However, GR activity increased 111% and 200% in roots at 7 and 14 days, respectively, returning to control levels after 21 days. Three GR isoforms were found in roots of control and treated plants, two of which were enhanced by Cd treatment at 7 and 14 days, as assessed by activity staining on native gels. The changes in the isoform pattern modified the global kinetic properties of GR, thereby decreasing significantly (2.5-fold) the Michaelis constant (K(m)) value for oxidized glutathione. Isozyme induction was not associated with an enhancement of GR mRNA and protein expression, indicating that post-translational modification could occur. Our data demonstrated that up-regulation of GR activity by the induction of distinctive isoforms occurs as a defense mechanism against Cd-generated oxidative stress in roots.     

Synechocystis Fe superoxide dismutase gene confers oxidative stress tolerance to Escherichia coli

The superoxide dismutase (SOD) gene (slr 1516) from the cyanobacterium Synechocystis sp. PCC 6803 was cloned and overexpressed in Escherichia coli BL 21 (DE3) using the pET-20b(+) expression vector. E. coli cells transformed with pET-SOD overexpressed the protein in cytosol, upon induction by isopropyl beta-D-thiogalactopyranoside (IPTG). The recombinant protein was purified to near homogeneity by gel filtration and ion-exchange chromatography. The SOD activity of the recombinant protein was sensitive to hydrogen peroxide and sodium azide, confirming it to be FeSOD. The pET-FeSOD transformed E. coli showed significantly higher SOD activity and tolerance to paraquat-mediated growth inhibition compared to the empty vector transformed cells. Based on these results it is suggested that overexpression of FeSOD gene from a heterologous source like Synechocystis sp. PCC 6803 may provide protection to E. coli against superoxide radical-mediated oxidative stress mediated by paraquat.     

大肠埃希菌中DPS对氧应激的抵抗

DPS是一种广泛存在于原核生物中的DNA结合蛋白,它能够在细菌乏营养等多种应激状态下为细菌提供保护。大肠埃希菌DPS已经被深入研究。本文从蛋白结构和铁隔离、DNA结合,铁氧化酶活性,调节基因表达四个方面介绍大肠埃希菌DPS的基本特性和作用机制。     

Characterization and transcriptional regulation of thioredoxin reductase 1 on exposure to oxidative stress inducing environmental pollutants in Chironomus riparius

We characterized thioredoxin reductase 1 (TrxR1) from Chironomus riparius (CrTrxR1) and studied its expression under oxidative stress. The full-length cDNA is 1820 bp long and contains an open reading frame (ORF) of 1488 bp. The deduced CrTrxR1 protein has 495 amino acids and a calculated molecular mass of 54.41 kDa and an isoelectric point of 6.15. There was a 71 bp 5’ and a 261 bp 3' untranslated region with a polyadenylation signal site (AATAAA). Homologous alignments showed the presence of conserved catalytic domain Cys-Val-Asn-Val-Gly-Cys (CVNVGC), the C-terminal amino acids ‘CCS’ and conserved amino acids required in catalysis. The expression of CrTrxR1 is measured using quantitative real-time PCR after exposure to 50 and 100 mg/L of paraquat (PQ) and 2, 10 and 20 mg/L of cadmium chloride (Cd). CrTrxR1 mRNA was upregulated after PQ exposure at all conditions tested. The highest level of CrTrxR1 expression was observed after exposure to 10 mg/L of Cd for 24 h followed by 20 mg/L for 48 h. Significant downregulation of CrTrxR1 was observed after exposure to 10 and 20 mg/L of Cd for 72 h. This study shows that the CrTrxR1 could be potentially used as a biomarker of oxidative stress inducing environmental contaminants.     

Drosophila Cu,Zn superoxide dismutase gene confers resistance to paraquat in Escherichia coli

Superoxide dismutase (SOD) is known to protect organisms from reactive oxygen metabolites. We tested the hypothesis that the Drosophila Cu,Zn SOD is capable of protecting Escherichia coli from oxidative damage caused by the herbicide paraquat. The Cu,Zn Sod gene of Drosophila sechellia was subcloned into pET-20b(+) expression vector. Transformation of E. coli with the constructed vector resulted in an overexpression of this eukaryotic superoxide dismutase, as evidenced by dramatically increased levels of the Cu,Zn SOD polypeptide in bacterial cytosolic extracts. As well, the E. coli transformants showed resistance to paraquat-mediated inhibition of growth and survival. Paraquat is known to promote formation of the superoxide radical anion inside cells and thus the data have been interpreted as indicating that the cloned superoxide dismutase provides protection in E. coli against damage attributable to free radicals.     

Molecular cloning of glutathione reductase cDNAs and analysis of GR gene expression in cowpea and common bean leaves during recovery from moderate drought stress

Two cDNAs of the enzyme glutathione reductase (GR; EC 1.6.4.2) encoding a dual-targeted isoform (dtGR) and a cytosolic isoform (cGR), were cloned from leaves of common bean (Phaseolus vulgaris L.). Moderate drought stress (Psi w=-1.5MPa) followed by re-watering was applied to common bean cultivars, one tolerant to drought (IPA), the other susceptible (Carioca) and to cowpea (Vigna unguiculata L. Walp) cultivars, one tolerant to drought (EPACE-1), and the other susceptible (1183). mRNA levels were much higher for PvcGR than for PvdtGR in all cases. Moderate drought stress induced an up-regulation of the expression of PvcGR in the susceptible cultivars. On the contrary, PvdtGR expression decreased. In the tolerant cowpea EPACE-1, GR gene expression remained stable under drought. During recovery from drought, an up-regulation of the two GR isoforms occurred, with a peak at 6-10h after re-hydration. This suggests that moderate drought stress may lead to a hardening process and acclimation tolerance. The role of GR isoforms in plant tolerance and capacity to recover from drought stress is discussed.     

Synthesis and properties of glutathione reductase in stressed peas

We have subjected peas (Pisum sativum L.) to four different oxidative stresses: cold conditions (4 °C) in conjunction with light, treatment with paraquat, fumigation with ozone, and illumination of etiolated seedlings (greening). In crude extracts of leaves from stressed plants, an increase (up to twofold) in activity of glutathione reductase (GR) was observed which was consistent with previous reports from several laboratories. In all cases, except for ozone fumigation, the increase in activity was not due to an elevation in the steady-state levels of GR protein. None of the applied stresses had any effect on steady-state levels of GR mRNA. In contrast to the small increase in GR activity, the K _m of GR for glutathione disulphide showed a marked decrease when determined for extracts of stressed leaves, compared with that from unstressed plants. This indicates that GR from stressed plants has an increased affinity for glutathione disulphide. The profile of GR activity bands fractionated on non-denaturing acrylamide gels varied for extracts from differently stressed leaves and when compared with GR from unstressed plants. The changes in GR-band profiles and the alteration in the kinetic properties are best explained as changes in the isoform population of pea GR in response to stress.Abbreviations GR glutathione reductase - GSSG glutathione disulphide - Rubisco Ribulose-1,5-bisphosphate carboxylase-oxygenase - RNase A/T1 ribonucleases A and T1 We are grateful to Prof. Alan Wellburn and Dr. Phil Beckett (Division of Biological Sciences, University of Lancaster, UK) for providing ozone-fumigated material and Dr. Jeremy Harbinson for providing material grown at 4° C. This work was supported by a grant-in-aid to the John Innes Institute from the Agricultural and Food Research Council. E.A.E. and C.E. gratefully acknowledge the support of a John Innes Foundation studentship and a European Molecular Biology Organisation Fellowship respectively.     

逆境胁迫对油菜谷胱甘肽还原酶基因表达及其酶活性的影响

利用cDNA末端快速分离(RACE)技术从陇油6号油菜中克隆得到一个新的谷胱甘肽还原酶基因GR2,全长2073 bp,开放阅读框1692 bp,编码563个氨基酸,预测蛋白质分子量为60.7 kDa,等电点7.9.实时荧光定量PCR分析表明:GR2基因在油菜根、茎、叶中均有表达,其中在叶中表达量最高.GR1和GR2基因的转录以及谷胱甘肽还原酶(GR)活性受到低温、高温、干旱、高盐胁迫的诱导,表明油菜谷胱甘肽还原酶在抵御低温、高温、干旱、高盐胁迫过程中发挥重要作用.脱落酸(ABA)预处理后再进行上述胁迫处理,与单独上述胁迫相比,GR1和GR2基因的转录以及GR活性水平明显上升,表明ABA可以诱导GR1和GR2基因表达和GR酶活性.MAPKK抑制剂U0126预处理后再进行上述胁迫处理,与单独上述胁迫相比,GR1和GR2基因的转录以及GR活性水平明显下降,表明U0126对GR1、GR2基因表达以及GR酶活性有抑制作用.     

Co-expression of monodehydroascorbate reductase and dehydroascorbate reductase from Brassica rapa effectively confers tolerance to freezing-induced oxidative stress

Plants are exposed to various environmental stresses and have therefore developed antioxidant enzymes and molecules to protect their cellular components against toxicity derived from reactive oxygen species (ROS). Ascorbate is a very important antioxidant molecule in plants, and monodehydroascorbate reductase (MDHAR; EC 1.6.5.4) and dehydroascorbate reductase (DHAR; EC 1.8.5.1) are essential to regeneration of ascorbate for maintenance of ROS scavenging ability. The MDHAR and DHAR genes from Brassica rapa were cloned, transgenic plants overexpressing either BrMDHAR and BrDHAR were established, and then, each transgenic plant was hybridized to examine the effects of co-expression of both genes conferring tolerance to freezing. Transgenic plants co-overexpressing BrMDHAR and BrDHAR showed activated expression of relative antioxidant enzymes, and enhanced levels of glutathione and phenolics under freezing condition. Then, these alteration caused by co-expression led to alleviated redox status and lipid peroxidation and consequently conferred improved tolerance against severe freezing stress compared to transgenic plants overexpressing single gene. The results of this study suggested that although each expression of BrMDHAR or BrDHAR was available to according tolerance to freezing, the simultaneous expression of two genes generated synergistic effects conferring improved tolerance more effectively even severe freezing.     

Bacteriocin of Enterococcus from lactoserum able to cause oxidative stress in Staphylococcus aureus

The effect of a bacteriocin of Enterococcus on the oxidative metabolism of sensitive bacteria was investigated through the detection of oxidative stress by chemiluminescence (CL). The bacteriocin named EntB was purified to study the action on Staphylococcus aureus isolated from cosmetic. Chromatographic separation of EntB indicated different states of oligomerization with molecular weights multiple of 12,000Da monomeric form. The monomer purified by ion exchange was studied in its capacity to affect the oxidative metabolism of S. aureus, which showed increase of anion superoxide (O(2)(-)) when incubated with EntB. This effect was compared to the action of EntB on leukocytes as an assay of toxicity. EntB did not generate significant oxidative stress in leukocytes. Pyoverdin, a leukotoxic pigment of Pseudomonas fluorescens, was taken as reference, and it was found that this pigment caused similar oxidative stress to EntB in S. aureus; however, pyoverdin generated high production of anion superoxide (O(2)(-)) in leukocytes, while EntB did not increase the level of O(2)(-).