The glutathione-glutaredoxin system in Rhodobacter capsulatus: part of a complex regulatory network controlling defense against oxidative stress

Mutants with defects in components of the glutathione-glutaredoxin (GSH/Grx) system of Rhodobacter capsulatus were constructed to study its role in defense against oxidative stress and the redox-dependent formation of photosynthetic complexes. The lack of the glutaredoxin 3 gene (grxC) or the glutathione synthetase B gene (gshB) resulted in lower growth rates under aerobic conditions and higher sensitivity to oxidative stress, confirming the role of the GSH/Grx system in oxidative stress defense. Both mutants are highly sensitive to disulfide stress, indicating a major contribution of the GSH/Grx system to the thiol-disulfide redox buffer in the cytoplasm. Like mutations in the thioredoxin system, mutations in the GSH/Grx system affected the formation of photosynthetic complexes, which is redox dependent in R. capsulatus. Expression of the genes grxC, gshB, grxA for glutaredoxin 1, and gorA for glutathione reductase, all encoding components of the GSH/Grx system, was not induced by oxidative stress. Other genes, for which a role in oxidative stress was established in Escherichia coli, acnA, fpr, fur, and katG, were strongly induced by oxidative stress in R. capsulatus. Mutations in the grxC, and/or gshB, and/or trxC (thioredoxin 2) genes affected expression of these genes, indicating an interplay of the different defense systems against oxidative stress. The OxyR and the SoxRS regulons control the expression of many genes involved in oxidative stress defense in E. coli in response to H2O2 and superoxide, respectively. Our data and the available genome sequence of R. capsulatus suggest that a SoxRS system is lacking but an alternative superoxide specific regulator exists in R. capsulatus. While the expression of gorA and grxA is regulated by H2O2 in E. coli this is not the case in R. capsulatus, indicating that the OxyR regulons of these two species are significantly different.     

Stress-induced proteins of Agrobacterium tumefaciens

The pattern of proteins produced by bacteria represents the physiological state of the organism as well as the environmental conditions encountered. Environmental stress induces the expression of several regulons encoding stress proteins. Extensive information about the proteins which constitute these regulons (or stimulons) and their control is available for very few bacteria, such as the Gram-positive Bacillus subtilis and the Gram-negative Escherichia coli (gamma-proteobacteria) and is minimal for all other bacteria. Agrobacterium tumefaciens is a Gram-negative plant pathogen of the alpha-proteobacteria, which constitutes the main tool for plant recombinant genetics. Our previous studies on the control of chaperone-coding operons indicated that A. tumefaciens has unique features and combines regulatory elements from both B. subtilis and E. coli. Therefore, we examined the patterns of proteins induced in A. tumefaciens by environmental changes using two-dimensional gel electrophoresis and dual-channel image analysis. Shifts to high temperature, oxidative and mild acid stresses stimulated the expression of 97 proteins. The results indicate that most of these stress-induced proteins (80/97) were specific to one stress stimulon. Only 10 proteins appear to belong to a general stress regulon.     

Collaborative effects of Photobacterium CuZn superoxide dismutase (SODs) and human AP endonuclease in DNA repair and SOD-deficient Escherichia coli under oxidative stress

The defenses against free radical damage include specialized repair enzymes that correct oxidative damage in DNA and detoxification systems such as superoxide dismutases (SODs). These defenses may be coordinated genetically as global responses. We hypothesized that the expression of SOD and DNA repair genes would inhibit DNA damage under oxidative stress. Therefore, protection of Escherichia coli mutants deficient in SOD and DNA repair genes (sod-, xth-, and nfo-) was demonstrated by transforming the mutant strain with a plasmid pYK9 that encoded Photobacterium leiognathi CuZnSOD and human AP endonuclease. The results show that survival rates were increased in sod+ xth- nfo+ cells compared with sod- xth- ape-, sod- xth- ape-, and sod+ xth- ape- cells under oxidative stress generated with 0.1 mM paraquat or 3 mM H2O2. The data suggest that, at the least, SOD and DNA repair enzymes may collaborate on protection and repair of damaged DNA. Additionally, both enzymes are required for protection against free radicals.     

Characterization of copper-inducible promoters regulated by CpxA/CpxR in Escherichia coli

The copper stimulon in Escherichia coli consists of four regulons, the CueR-, CusS/CusR-, CpxA/CpxR-, and YedV/YedW regulons. E. coli mutants defective in cpxRA showed higher sensitivity to copper than the wild type. A total of 15 promoters were found to be induced in E. coli culture upon exposure to copper in a CpxA/CpxR-dependent manner. After gel-shift and DNase I foot-printing analyses, a conserved tandem repeat of pentanucleotide sequence, GTAAA(N)(4-8)GTAAA, with a conserved A of 4-bp upstream of each pentamer, was identified to be the CpxR-binding site. The difference in the orientation and location of the CpxR box is discussed with respect to the regulation mechanism among CpxR-regulon genes.     

Inhibition of antioxidants and hyperthermia enhance bleomycin-induced cytotoxicity and lipid peroxidation in Chinese hamster ovary cells.

Regional hyperthermia has potential for human cancer treatment, particularly in combination with systemic chemotherapy or radiotherapy. Heat enhances the cytotoxic effect of certain anticancer agents such as bleomycin, but the mechanisms involved in cell killing are currently unknown. Bleomycin generates reactive oxygen species. It is likely that hyperthermia itself also increases oxidative stress in cells. We evaluate whether oxidative stress has a role in the mechanism of cell death caused by bleomycin and heat in Chinese hamster ovary cells. Heat (41 to 44 degrees C) increased cytotoxicity of bleomycin, evaluated by clonogenic cell survival. Decreased levels of cellular antioxidants should create an imbalance between prooxidant and antioxidant systems, thus enhancing cytotoxic responses to heat and to oxidant-generating drugs. We determine the involvement of four major cellular antioxidant defenses, superoxide dismutase (SOD), the glutathione redox cycle (GSH cycle), catalase, and glutathione S-transferase (GST), in cellular sensitivity to bleomycin, alone or combined with hyperthermia. These cellular defenses were inhibited by diethyldithiocarbamate, l-buthionine sulfoximine, aminotriazole, and ethacrynic acid, respectively. We show that levels of antioxidants (SOD, GSH cycle, and GST) affect cellular cytotoxic responses to bleomycin, at normal and elevated temperatures (41 to 44 degrees C), suggesting the involvement of oxidative stress. Bleomycin and iron caused oxidative damage to membrane lipids in intact cells, at 37 and 43 degrees C. Lipid peroxidation was evaluated by fluorescence detection of thiobarbituric acid-reactive products. There was an increase in damage to membrane lipids when the antioxidant defenses, SOD and catalase, were inhibited. The differing effects of antioxidant inhibitors on bleomycin-induced cytotoxicity and membrane lipid damage suggest that different mechanisms are involved in these two processes. However, free radicals appear to be involved in both cases. The marked sensitization of cells by diethyldithiocarbamate, to both bleomycin-induced cytotoxicity and lipid peroxidation, suggests that superoxide could be involved in both of these processes.     

Role of oxidative stress in Drosophila aging.

We review the role that oxidative damage plays in regulating the lifespan of the fruit fly, Drosophila melanogaster. Results from our laboratory show that the lifespan of Drosophila is inversely correlated to its metabolic rate. The consumption of oxygen by adult insects is related to the rate of damage induced by oxygen radicals, which are purported to be generated as by-products of respiration. Moreover, products of activated oxygen species such as hydrogen peroxide and lipofuscin are higher in animals kept under conditions of increased metabolic rate. In order to understand the in vivo relationship between oxidative damage and the production of the superoxide radical, we generated transgenic strains of Drosophila melanogaster that synthesize excess levels of enzymatically active superoxide dismutase. This was accomplished by P-element transformation of Drosophila melanogaster with the bovine cDNA for CuZn superoxide dismutase, an enzyme that catalyzes the dismutation of the superoxide radical to hydrogen peroxide and water. Adult flies that express the bovine SOD in addition to native Drosophila SOD are more resistant to oxidative stresses and have a slight but significant increase in their mean lifespan. Thus, resistance to oxidative stress and lifespan of Drosophila can be manipulated by molecular genetic intervention. In addition, we have examined the ability of adult flies to respond to various environmental stresses during senescence. Resistance to oxidative stress, such as that induced by heat shock, is drastically reduced in senescent flies. This loss of resistance is correlated with the increase in protein damage generated in old flies by thermal stress and by the insufficient protection from cellular defense systems which includes the heat shock proteins as well as the oxygen radical scavenging enzymes. Collectively, results from our laboratory demonstrate that oxidative damage plays a role in governing the lifespan of Drosophila during normal metabolism and under conditions of environmental stress.     

羰基毒害在糖尿病晚期并发症中的作用

动物机体组织的氧化紧张损伤和老化交联是糖尿病晚期并发症中神经病变、动脉粥样硬化、风湿性关节炎、肾病变、白内障等老化慢性疾病的共同特征.对氧化紧张到底是糖尿病晚期并发症的初始原因还是糖尿病组织衰变的次级诱因提出了探讨.结果表明糖尿病患者机体和血浆中的糖氧化产物及脂类过氧化物的增多表明病患者体内氧化紧张的加强;然而,它们中的某些产物与氧化紧张并不相关;此外,其它一些氧化紧张的直接指标,如氨基酸的氧化,在糖尿病患者的皮肤胶原中也并没有增多.因此,对于糖尿病患者中蛋白质化学修饰的现象,用活性羰基的毒化超过其系统解毒能力的观点来解释更合适.这种在氧化或非氧化反应中形成的不饱和羰基化合物在体内总是以相对恒定的浓度存在,因而在糖尿病中,由羰基毒害引起组织中糖类和脂类衍生物的增长可以被认为是生物化学动力学的一种必然结果.     

CorA affects tolerance of Escherichia coli and Salmonella enterica serovar Typhimurium to the lactoperoxidase enzyme system but not to other forms of oxidative stress

The enzyme lactoperoxidase is part of the innate immune system in vertebrates and owes its antimicrobial activity to the formation of oxidative reaction products from various substrates. In a previous study, we have reported that, with thiocyanate as a substrate, the lactoperoxidase system elicits a distinct stress response in Escherichia coli MG1655. This response is different from but partly overlapping with the stress responses to hydrogen peroxide and to superoxide. In the current work, we constructed knockouts in 10 lactoperoxidase system-inducible genes to investigate their role in the tolerance of E. coli MG1655 to this antimicrobial system. Five mutations resulted in a slightly increased sensitivity, but one mutation (corA) caused hypersensitivity to the lactoperoxidase system. This hypersensitive phenotype was specific to the lactoperoxidase system, since neither the sensitivity to hydrogen peroxide nor to the superoxide generator plumbagin was affected in the corA mutant. Salmonella enterica serovar Typhimurium corA had a similar phenotype. Although corA encodes an Mg2+ transporter and at least three other inducible open reading frames belonged to the Mg2+ regulon, repression of the Mg stimulon by Mg2+ did not change the lactoperoxidase sensitivity of either the wild-type or corA mutant. Prior exposure to 0.3 mM Ni2+, which is also transported by CorA, strongly sensitized MG1655 but not the corA mutant to the lactoperoxidase system. Furthermore, this Ni2+-dependent sensitization was suppressed by the CorA-specific inhibitor Co(III) hexaammine. These results indicate that CorA affects the lactoperoxidase sensitivity of E. coli by modulating the cytoplasmic concentrations of transition metals that enhance the toxicity of the lactoperoxidase system.     

Proteome signatures for stress and starvation in Bacillus subtilis as revealed by a 2-D gel image color coding approach

In this paper we have defined proteome signatures of Bacillus subtilis in response to heat, salt, peroxide, and superoxide stress as well as after starvation for ammonium, tryptophan, glucose, and phosphate using the 2-D gel-based approach. In total, 79 stress-induced and 155 starvation-induced marker proteins were identified including 50% that are not expressed in the vegetative proteome. Fused proteome maps and a color coding approach have been used to define stress-specific regulons that are involved in specific adaptative functions (HrcA for heat, PerR and Fur for oxidative stress, RecA for peroxide, CymR and S-box for superoxide stress). In addition, starvation-specific regulons are defined that are involved in the uptake or utilization of alternative nutrient sources (TnrA, sigmaL/BkdR for ammonium; tryptophan-activated RNA-binding attenuation protein for tryptophan; CcpA, CcpN, sigmaL/AcoR for glucose; PhoPR for phosphate starvation). The general stress or starvation proteome signatures include the CtsR, Spx, sigmaL/RocR, sigmaB, sigmaH, CodY, sigmaF, and sigmaE regulons. Among these, the Spx-dependent oxidase NfrA was induced by all stress conditions indicating stress-induced protein damages. Finally, a subset of sigmaH-dependent proteins (sporulation response regulator, YvyD, YtxH, YisK, YuxI, YpiB) and the CodY-dependent aspartyl phosphatase RapA were defined as general starvation proteins that indicate the transition to stationary phase caused by starvation.     

Superoxide protects Escherichia coli from bleomycin mediated lethality

Superoxide and its products, especially hydroxyl radical, were recently proposed to be instrumental in cell death following treatment with a wide range of antimicrobials. Surprisingly, bleomycin lethality to Escherichia coli was ameliorated by a genetic deficiency of superoxide dismutase or by furnishing the superoxide generator plumbagin. Rescue by plumbagin was similar in strains containing or lacking recA or with inactive, inducible, or constitutive soxRS regulons. Thus, superoxide interferes with bleomycin cytotoxicity in ways not readily explained by genetic pathways expected to protect from oxidative damage.     

Quantitative determination of ortho- and meta-tyrosine as biomarkers of protein oxidative damage in beta-thalassemia

Oxidative stress in thalassemia is caused by secondary iron overload and stems from blood transfusion and increased iron uptake. In this study, we hypothesized that levels of o- and m-tyrosine, products of hydroxyl radical attack on phenylalanine, would be elevated in beta-thalassemia (intermediate). This study represents the first report in which specific markers of protein oxidative damage have been quantified in thalassemia. We used GC/MS to assay o- and m-tyrosine at the femtomole level using only a few microliters of plasma. Levels of both markers were significantly higher in patients with beta-thalassemia than in controls and were positively correlated with serum ferritin, malondialdehyde, superoxide dismutase, glutathione peroxidase and glutathione. We conclude that o- and m-tyrosine are useful biomarkers of oxidative damage to proteins in thalassemia (intermediate) and may also be useful markers in other iron overload diseases. Positive correlations between o- and m-tyrosine levels and malondialdehyde as well as antioxidants such as superoxide dismutase, glutathione peroxidase and glutathione, are indicative of the broad impact of oxidative stress on blood plasma in thalassemia, with up-regulation of antioxidant proteins probably reflecting a homeostatic response to these increased stress levels.     

Lex marks the spot: the virulent side of SOS and a closer look at the LexA regulon

The SOS response that responds to DNA damage induces many genes that are under LexA repression. A detailed examination of LexA regulons using genome-wide techniques has recently been undertaken in both Escherichia coli and Bacillus subtilis. These extensive and elegant studies have now charted the extent of the LexA regulons, uncovered many new genes, and exposed a limited overlap in the LexA regulon between the two bacteria. As more bacterial genomes are analysed, more curiosities in LexA regulons arise. Several notable examples include the discovery of a LexA-like protein, HdiR, in Lactococcus lactis, organisms with two lexA genes, and small DNA damage-inducible cassettes under LexA control. In the cyanobacterium Synechocystis, genetic and microarray studies demonstrated that a LexA paralogue exerts control over an entirely different set of carbon-controlled genes and is crucial to cells facing carbon starvation. An examination of SOS induction evoked by common therapeutic drugs has shed new light on unsuspected consequences of drug exposure. Certain antibiotics, most notably fluoroquinolones such as ciprofloxacin, can induce an SOS response and can modulate the spread of virulence factors and drug resistance. SOS induction by beta-lactams in E. coli triggers a novel form of antibiotic defence that involves cell wall stress and signal transduction by the DpiAB two-component system. In this review, we provide an overview of these new directions in SOS and LexA research with emphasis on a few themes: identification of genes under LexA control, the identification of new endogenous triggers, and antibiotic-induced SOS response and its consequences.     

Investigation of the first events leading to loss of culturability during Escherichia coli starvation: future nonculturable bacteria form a subpopulation

In previous experiments we were able to separate, using a nondestructive separation technique, culturable and nonculturable bacteria, from a Luria-Bertani (LB) medium culture of Escherichia coli incubated for 48 h. We observed in the nonculturable bacterial population an increase in oxidative damage and up-induction of most defenses against reactive oxygen species (ROS), along with a decrease in cytoplasmic superoxide dismutases. In this study, using the same separation technique, we separated into two subpopulations a 10-h LB medium culture containing only culturable bacteria. For the first time, we succeeded in associating physical separation with physiological differences. Although the levels of defense against ROS (RpoS, RpoH, OxyR, and SoxRS regulons) and oxidative damage (carbonyl contents) were apparently the same, we found that bacteria in one subpopulation were more sensitive to LB medium starvation and to various stresses, such as phosphate buffer starvation, heat shock, and hydrogen peroxide exposure. Based on these results, we suggest that these physiological differences reflect uncharacterized bacterial modifications which do not directly involve defenses against ROS.