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.     

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.     

Regulators of oxidative stress response genes in Escherichia coli and their functional conservation in bacteria

Oxidative stress, through the production of reactive oxygen species, is a natural consequence of aerobic metabolism. Escherichia coli has several major regulators activated during oxidative stress, including OxyR, SoxRS, and RpoS. OxyR and SoxR undergo conformation changes when oxidized in the presence of hydrogen peroxide and superoxide radicals, respectively, and subsequently control the expression of cognate genes. In contrast, the RpoS regulon is induced by an increase in RpoS levels. Current knowledge regarding the activation and function of these regulators and their dependent genes in E. coli during oxidative stress forms the scope of this review. Despite the enormous genomic diversity of bacteria, oxidative stress response regulators in E. coli are functionally conserved in a wide range of bacterial groups, possibly reflecting positive selection of these regulators. SoxRS and RpoS homologs are present and respond to oxidative stress in Proteobacteria, and OxyR homologs are present and function in H(2)O(2) resistance in a range of bacteria, from gammaproteobacteria to Actinobacteria. Bacteria have developed complex, adapted gene regulatory responses to oxidative stress, perhaps due to the prevalence of reactive oxygen species produced endogenously through metabolism or due to the necessity of aerotolerance mechanisms in anaerobic bacteria exposed to oxygen.     

Expression of the <Emphasis Type="Italic">yggE</Emphasis> gene protects <Emphasis Type="Italic">Escherichia coli</Emphasis> from potassium tellurite-generated oxidative stress

Potassium tellurite is highly toxic to most forms of life and specific bacterial tellurite defense mechanisms are not fully understood to date. Recent evidence suggests that tellurite would exert its toxic effects, at least in part, through the generation of superoxide anion that occurs concomitantly with intracellular tellurite (Te⁴⁺) reduction to elemental tellurium (Te^o). In this work the putative antioxidant role of YggE from Escherichia coli, a highly conserved protein in several bacterial species and whose function is still a matter of speculation, was studied. When exposed to tellurite, E. coli lacking yggE exhibited increased activity of superoxide dismutase and fumarase C, augmented levels of reactive oxygen species and high concentration of carbonyl groups in proteins. Upon genetic complementation with the homologous yggE gene these values were restored to those observed in the parental, isogenic, wild type strain, suggesting a direct participation of YggE in E. coli tolerance to tellurite.     

Oxidative stress involved in the antibacterial action of different antibiotics

Staphylococcus aureus and Escherichia coli sensitive to chloramphenicol incubated with this antibiotic suffered oxidative stress with increase of anion superoxide (O2-). This reactive species of oxygen was detected by chemiluminescence with lucigenin. S. aureus, E. coli, and Enterococcus faecalis sensitive to ciprofloxacin exhibited oxidative stress when they were incubated with this antibiotic while resistant strains did not show stimuli of O2-. Other bacteria investigated was Pseudomonas aeruginosa, strains sensitive to ceftazidime and piperacillin presented oxidative stress in presence of these antibiotics while resistant strains were not stressed. Higher antibiotic concentration was necessary to augment O2- in P. aeruginosa biofilm than in suspension, moreover old biofilms were resistant to oxidative stress caused by antibiotics. A ceftazidime-sensitive mutant of P. aeruginosa, coming from a resistant strain, exhibited higher production of O2- than wild type in presence of this antibiotic. There was relation between antibiotic susceptibility and production of oxidative stress.     

In vivo andin vitro cloning and phenotype characterization of tellurite resistance determinant conferred by plasmid pTE53 of a clinical isolate ofEscherichia coli

A determinant encoding resistance against potassium tellurite (Te(r)) was discovered in a clinical isolate of Escherichia coli strain KL53. The strain formed typical black colonies on solid LB medium with tellurite. The determinant was located on a large conjugative plasmid designated pTE53. Electron-dense particles were observed in cells harboring pTE53 by electron microscopy. X-Ray identification analysis identified these deposits as elemental tellurium and X-ray diffraction analysis showed patterns typical of crystalline structures. Comparison with JCPDS 4-0554 (Joint Committee on Powder Diffraction Standards) reference data confirmed that these crystals were pure tellurium crystals. In common with other characterized Te(r) determinants, accumulation studies with radioactively labeled tellurite showed that reduced uptake of tellurite did not contribute to the resistance mechanism. Tellurite accumulation rates for E. coli strain AB1157 harboring pTE53 were twice higher than for the plasmid-free host strain. In addition, no efflux mechanism was detected. The potassium tellurite resistance determinant of plasmid pTE53 was cloned using both in vitro and in vivo techniques in low-copy-number vectors pACYC184 and mini-Mu derivative pPR46. Cloning of the functional Te(r) determinant into high-copy cloning vectors pTZ19R and mini-Mu derivatives pBEf and pJT2 was not successful. During in vivo cloning experiments, clones with unusual "white colony" phenotypes were found on solid LB with tellurite. All these clones were Mucts62 lysogens. Their tellurite resistance levels were in the same order as the wild type strains. Clones with the "white" phenotype had a 3.6 times lower content of tellurium than the tellurite-reducing strain. Transformation of a "white" mutant with a recombinant pACYC184 based Te(r) plasmid did not change the phenotype. However, when one clone was cured from Mucts62 the "white" phenotype reverted to the wild-type "black" phenotype. It was suggested that the "white" phenotype was the result of an insertional inactivation of an unknown chromosomal gene by Mucts62, which reduced the tellurite uptake.     

Survival of subsurface microorganisms exposed to UV radiation and hydrogen peroxide.

Aerobic and microaerophilic subsurface bacteria were screened for resistance to UV light. Contrary to the hypothesis that subsurface bacteria should be sensitive to UV light, the organisms studied exhibited resistance levels as efficient as those of surface bacteria. A total of 31% of the aerobic subsurface isolates were UV resistant, compared with 26% of the surface soil bacteria that were tested. Several aerobic, gram-positive, pigmented, subsurface isolates exhibited greater resistance to UV light than all of the reference bacterial strains tested except Deinococcus radiodurans. None of the microaerophilic, gram-negative, nonpigmented, subsurface isolates were UV resistant; however, these isolates exhibited levels of sensitivity similar to those of the gram-negative reference bacteria Escherichia coli B and Pseudomonas fluorescens. Photoreactivation activity was detected in three subsurface isolates, and strain UV3 exhibited a more efficient mechanism than E. coli B. The peroxide resistance of four subsurface isolates was also examined. The aerobic subsurface bacteria resistant to UV light tolerated higher levels of H2O2 than the microaerophilic organisms. The conservation of DNA repair pathways in subsurface microorganisms may be important in maintaining DNA integrity and in protecting the organisms against chemical insults, such as oxygen radicals, during periods of slow growth.     

Novel antioxidant role of alcohol dehydrogenase E from Escherichia coli

Alcohol dehydrogenase E (AdhE) is an Fe-enzyme that, under anaerobic conditions, is involved in dissimilation of glucose. The enzyme is also present under aerobic conditions, its amount is about one-third and its activity is only one-tenth of the values observed under anaerobic conditions. Nevertheless, its function in the presence of oxygen remained ignored. The data presented in this paper led us to propose that the enzyme has a protective role against oxidative stress. Our results indicated that cells deleted in adhE gene could not grow aerobically in minimal media, were extremely sensitive to oxidative stress and showed division defects. In addition, compared with wild type, mutant cells displayed increased levels of internal peroxides (even higher than those found in a Delta katG strain) and increased protein carbonyl content. This pleiotropic phenotype disappeared when the adhE gene was reintroduced into the defective strain. The purified enzyme was highly reactive with hydrogen peroxide (with a Ki of 5 microM), causing inactivation due to a metal-catalyzed oxidation reaction. It is possible to prevent this reactivity to hydrogen peroxide by zinc, which can replace the iron atom at the catalytic site of AdhE. This can also be achieved by addition of ZnSO4 to cell cultures. In such conditions, addition of hydrogen peroxide resulted in reduced cell viability compared with that obtained without the Zn treatment. We therefore propose that AdhE acts as a H2O2 scavenger in Escherichia coli cells grown under aerobic conditions.     

PMAP-23 triggers cell death by nitric oxide-induced redox imbalance in Escherichia coli

BackgroundAntibiotic resistance is a global problem and there is an urgent need to augment the arsenal against pathogenic bacteria. The emergence of different drug resistant bacteria is threatening human lives to be pushed toward the pre-antibiotic era. Antimicrobial peptides (AMPs) are a host defense component against infectious pathogens in response to innate immunity. PMAP-23, an AMP derived from porcine myeloid, possesses antibacterial activity. It is currently not clear how the antibacterial activity of PMAP-23 is manifested.MethodsThe disruptive effect of nitric oxide (NO) on the catalase activity, reactive oxygen species (ROS) production, DNA oxidation and apoptosis-like death were evaluated using the NO generation inhibitor.ResultsIn this investigation, PMAP-23 generates NO in a dose dependent manner. NO deactivated catalase and this antioxidant could not protect Escherichia coli against ROS, especially hydroxyl radical. This redox imbalance was shown to induce oxidative stress, thus leading to DNA strand break. Consequently, PMAP-23 treated E. coli cells resulted in apoptosis-like death. These physiological changes were inhibited when NO generation was inhibited. In the ΔdinF mutant, the levels of DNA strand break sharply increased and the cells were more sensitive to PMAP-23 than wild type.ConclusionOur data strongly indicates that PMAP-23 mediates apoptosis-like cell death through affecting intracellular NO homeostasis. Furthermore, our results demonstrate that DinF functioned in protection from oxidative DNA damage.General significanceThe identification of PMAP-23 antibacterial activity and mechanism provides a promising antibacterial agent, supporting the role of NO in cell death regulation.     

Structural/functional analysis of the human OXR1 protein: identification of exon 8 as the anti-oxidant encoding function

ABSTRACT: BACKGROUND: The human OXR1 gene belongs to a class of genes with conserved functions that protect cells from reactive oxygen species (ROS). The gene was found using a screen of a human cDNA library by its ability to suppress the spontaneous mutator phenotype of an E. coli mutH nth strain. The function of OXR1 is unknown. The human and yeast genes are induced by oxidative stress and targeted to the mitochondria; the yeast gene is required for resistance to hydrogen peroxide. Multiple spliced isoforms are expressed in a variety of human tissues, including brain. RESULTS: In this report, we use a papillation assay that measures spontaneous mutagenesis of an E. coli mutM mutY strain, a host defective for oxidative DNA repair. Papillation frequencies with this strain are dependent upon a G->T transversion in the lacZ gene (a mutation known to occur as a result of oxidative damage) and are suppressed by in vivo expression of human OXR1. N-terminal, C-terminal and internal deletions of the OXR1 gene were constructed and tested for suppression of the mutagenic phenotype of the mutM mutY strain. We find that the TLDc domain, encoded by the final four exons of the OXR1 gene, is not required for papillation suppression in E. coli. Instead, we show that the protein segment encoded by exon 8 of OXR1 is responsible for the suppression of oxidative damage in E. coli. CONCLUSION: The protein segment encoded by OXR1 exon 8 plays an important role in the anti-oxidative function of the human OXR1 protein. This result suggests that the TLDc domain, found in OXR1 exons 12-16 and common in many proteins with nuclear function, has an alternate (undefined) role other than oxidative repair.     

Methionine sulfoxide reductases are essential for virulence of Salmonella typhimurium

Production of reactive oxygen species represents a fundamental innate defense against microbes in a diversity of host organisms. Oxidative stress, amongst others, converts peptidyl and free methionine to a mixture of methionine-S- (Met-S-SO) and methionine-R-sulfoxides (Met-R-SO). To cope with such oxidative damage, methionine sulfoxide reductases MsrA and MsrB are known to reduce MetSOs, the former being specific for the S-form and the latter being specific for the R-form. However, at present the role of methionine sulfoxide reductases in the pathogenesis of intracellular bacterial pathogens has not been fully detailed. Here we show that deletion of msrA in the facultative intracellular pathogen Salmonella (S.) enterica serovar Typhimurium increased susceptibility to exogenous H(2)O(2), and reduced bacterial replication inside activated macrophages, and in mice. In contrast, a ΔmsrB mutant showed the wild type phenotype. Recombinant MsrA was active against free and peptidyl Met-S-SO, whereas recombinant MsrB was only weakly active and specific for peptidyl Met-R-SO. This raised the question of whether an additional Met-R-SO reductase could play a role in the oxidative stress response of S. Typhimurium. MsrC is a methionine sulfoxide reductase previously shown to be specific for free Met-R-SO in Escherichia (E.) coli. We tested a ΔmsrC single mutant and a ΔmsrBΔmsrC double mutant under various stress conditions, and found that MsrC is essential for survival of S. Typhimurium following exposure to H(2)O(2,) as well as for growth in macrophages, and in mice. Hence, this study demonstrates that all three methionine sulfoxide reductases, MsrA, MsrB and MsrC, facilitate growth of a canonical intracellular pathogen during infection. Interestingly MsrC is specific for the repair of free methionine sulfoxide, pointing to an important role of this pathway in the oxidative stress response of Salmonella Typhimurium.     

Iron storage proteins are essential for the survival and pathogenesis of Mycobacterium tuberculosis in THP-1 macrophages and the guinea pig model of infection

Iron is one of the crucial elements required for the growth of Mycobacterium tuberculosis. However, excess free iron becomes toxic for the cells because it catalyzes the production of reactive oxygen radicals, leading to oxidative damage. Hence, it is essential for the pathogen to have the ability to store intracellular iron in an iron-rich environment and utilize it under iron depletion. M. tuberculosis has two iron storage proteins, namely BfrA (Rv1876; a bacterioferritin) and BfrB (Rv3841; a ferritin-like protein). However, the demonstration of biological significance requires the disruption of relevant genes and the evaluation of the resulting mutant for its ability to survive in the host and cause disease. In this study, we have disrupted bfrA and bfrB of M. tuberculosis and demonstrated that these genes are crucial for the storage and supply of iron for the growth of bacteria and to withstand oxidative stress in vitro. In addition, the bfrA bfrB double mutant (H37Rv ΔbfrA ΔbfrB) exhibited a marked reduction in its ability to survive inside human macrophages. Guinea pigs infected with H37Rv ΔbfrA ΔbfrB exhibited a marked diminution in the dissemination of the bacilli to spleen compared to that of the parental strain. Moreover, guinea pigs infected with H37Rv ΔbfrA ΔbfrB exhibited significantly reduced pathological damage in spleen and lungs compared to that of animals infected with the parental strain. Our study clearly demonstrates the importance of these iron storage proteins in the survival and pathogenesis of M. tuberculosis in the host and establishes them as attractive targets for the development of new inhibitors against mycobacterial infections.     

The Geobacillus stearothermophilus V iscS gene, encoding cysteine desulfurase, confers resistance to potassium tellurite in Escherichia coli K-12

Many eubacteria are resistant to the toxic oxidizing agent potassium tellurite, and tellurite resistance involves diverse biochemical mechanisms. Expression of the iscS gene from Geobacillus stearothermophilus V, which is naturally resistant to tellurite, confers tellurite resistance in Escherichia coli K-12, which is naturally sensitive to tellurite. The G. stearothermophilus iscS gene encodes a cysteine desulfurase. A site-directed mutation in iscS that prevents binding of its pyridoxal phosphate cofactor abolishes both enzyme activity and its ability to confer tellurite resistance in E. coli. Expression of the G. stearothermophilus iscS gene confers tellurite resistance in tellurite-hypersensitive E. coli iscS and sodA sodB mutants (deficient in superoxide dismutase) and complements the auxotrophic requirement of an E. coli iscS mutant for thiamine but not for nicotinic acid. These and other results support the hypothesis that the reduction of tellurite generates superoxide anions and that the primary targets of superoxide damage in E. coli are enzymes with iron-sulfur clusters.     

Prooxidant activity of the superoxide dismutase (SOD)-mimetic EUK-8 in proliferating and growth-arrested Escherichia coli cells

Numerous studies have aimed to alleviate oxidative stress in a wide range of organisms by increasing superoxide dismutase (SOD) activity. However, experimental approaches have yielded contradictory evidence, and kinetics models have shown that increases in SOD activity may increase, decrease, or not change hydrogen peroxide (H2O2) production, depending on the balance of the various processes that produce and consume superoxide (O2-). In this study we tested whether administration of EUK-8, a synthetic mimetic of the SOD enzyme, can protect starving Escherichia coli cells against stasis-induced oxidative stress. Surprisingly, administration of EUK-8 to starving E. coli cells enhances the production of reactive oxygen species (ROS), resulting in a massive increase of oxidative damage and replicative death of the bacteria. Our results confirm that manipulation of ROS levels by increasing SOD activity does not necessarily result in a consequent decline of oxidative stress and can yield opposite results in a relatively simple model system such as starving E. coli cells.     

Acclimation to singlet oxygen stress in Chlamydomonas reinhardtii

In an aerobic environment, responding to oxidative cues is critical for physiological adaptation (acclimation) to changing environmental conditions. The unicellular alga Chlamydomonas reinhardtii was tested for the ability to acclimate to specific forms of oxidative stress. Acclimation was defined as the ability of a sublethal pretreatment with a reactive oxygen species to activate defense responses that subsequently enhance survival of that stress. C. reinhardtii exhibited a strong acclimation response to rose bengal, a photosensitizing dye that produces singlet oxygen. This acclimation was dependent upon photosensitization and occurred only when pretreatment was administered in the light. Shifting cells from low light to high light also enhanced resistance to singlet oxygen, suggesting an overlap in high-light and singlet oxygen response pathways. Microarray analysis of RNA levels indicated that a relatively small number of genes respond to sublethal levels of singlet oxygen. Constitutive overexpression of either of two such genes, a glutathione peroxidase gene and a glutathione S-transferase gene, was sufficient to enhance singlet oxygen resistance. Escherichia coli and Saccharomyces cerevisiae exhibit well-defined responses to reactive oxygen but did not acclimate to singlet oxygen, possibly reflecting the relative importance of singlet oxygen stress for photosynthetic organisms.