The role of the natural polyamine putrescine in defense against oxidative stress in Escherichia coli

Putrescine up-regulated, in a concentration-dependent manner, the expression levels of the oxyR and katG genes of Escherichia coli cells exposed to hydrogen peroxide. Its stimulatory effect was more pronounced under conditions of strong oxidative stress. 1,4-Diamino-2-butanone, a specific inhibitor of putrescine synthesis, also inhibited oxyR expression under oxidative stress. When added to inhibited cells, putrescine relieved this inhibitory effect. Addition of putrescine to E. coli cultures exposed to oxidative stress led to increased cell survival.     

Role of Putrescine in the Regulation of the Expression of the Oxidative Stress Defense Genes of Escherichia coli

The role of putrescine in the adaptive response of Escherichia coligrown aerobically in synthetic M9 medium with glucose to the H₂O₂-induced oxidative stress was studied. Under oxidative stress, the expression of the single-copy reporter gene fusions oxyR"::lacZand katG"::lacZwas found to undergo biphasic changes, which were most pronounced in glucose-starved E. colicells. The concentration-dependent activating effect of putrescine on the expression of the OxyR regulon genes was maximum when theoxyRgene was inhibited by high concentrations of hydrogen peroxide.     

OxyR regulon controls lipid peroxidation-mediated oxidative stress in Escherichia coli

Membrane lipid peroxidation processes yield products that may react with DNA and proteins to cause oxidative modifications. The oxyR gene product regulates the expression of enzymes and proteins that are needed for cellular protection against oxidative stress. Upon exposure to tert-butylhydroperoxide (t-BOOH) and 2,2'-azobis (2-amidinopropane) hydrochloride (AAPH), which induce lipid peroxidation in membranes, the Escherichia coli oxyR overexpression mutant was much more resistant to lipid peroxidation-mediated cellular damage, when compared to the OxyR deletion mutant in regard to growth kinetics, viability, and DNA damage. The deletion of the OxyR gene in E. coli also resulted in increased susceptibility of superoxide dismutase to lipid peroxidation-mediated inactivation. The results indicate that the peroxidation of lipid is probably one of the important intermediary events in free radical-induced cellular damage. Also, the oxyR regulon plays an important protective role in lipid peroxidation-mediated cellular damage.     

Control of singlet oxygen-induced oxidative damage in Escherichia coli

Singlet oxygen ((1)O(2)) is a highly reactive form of molecular oxygen that may harm living systems by oxidizing critical cellular macromolecules. The oxyR gene product regulates the expression of the enzymes and proteins that are needed for cellular protection against oxidative stress. In this study, the role of oxyR in cellular defense against a singlet oxygen was investigated using Escherichia coli oxyR mutant strains. Upon exposure to methylene blue and visible light, which generates singlet oxygen, the oxyR overexpression mutant was much more resistant to singlet oxygen-mediated cellular damage when compared to the oxyR deletion mutant in regard to growth kinetics, viability and protein oxidation. Induction and inactivation of major antioxidant enzymes, such as superoxide dismutase and catalase, were observed after their exposure to a singlet oxygen generating system in both oxyR strains. However, the oxyR overexpression mutant maintained significantly higher activities of antioxidant enzymes than did the oxyR deletion mutant. These results suggest that the oxyR regulon plays an important protective role in singlet oxygen-mediated cellular damage, presumably through the protection of antioxidant enzymes.     

Oxidative and SOS-response in Escherichia coli and their interference]

Interference between the oxidative and SOS responses in Escherichia coli was studied. The oxidative response involves both reactive oxygen scavenging system and DNA repair systems which are distinct from either the SOS or adaptive response to alkylating agents. The oxyR gene is a positive regulatory gene for the oxidative response and controls at least 9 proteins which are induced by treatment with H2O2. This gene is not a portion of the SOS regulon that involves at least 17 different genes in E. coli and controls the SOS response--another inducible and nonspecific repair activity. The SOS response was measured in E. coli PQ37 by means of a sfiA: :lacZ operon fusion according to "SOS Chromotest" in a completely automated system "Bioscreen C" (Labsystems, Finland). Our data have shown that: 1) H2O2 was a potent inducer of sfiA gene--one of the SOS genes; 2) there was strong negative effect of the oxidative response on the subsequent induction of the SOS response. In common with our previous findings it should be concluded that there is an interference between the SOS response--on the one hand, and the adaptive and oxidative responses--on the other. The nonspecific heat shock response is proposed to be a main key in these interferences.     

Isolation and characterization of Escherichia coli strains containing new gene fusions (soi::lacZ) inducible by superoxide radicals.

Gene fusions in Escherichia coli that showed increased beta-galactosidase expression in response to treatment with a superoxide radical (O2-) generator, methyl viologen (MV), were obtained. These fusions were constructed by using a Mud(Ap lac) phage to insert the lactose structural genes randomly into the E. coli chromosome. Ampicillin-resistant colonies were screened for increased expression of beta-galactosidase on X-Gal (5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside) plates containing MV at 1.25 micrograms/ml. Other O2- generators, menadione and plumbagin, also induced beta-galactosidase activity in these fusion strains. The induction by these drugs occurred only under aerobic conditions. Hyperoxygenation also elicited an induction of the fusions. On the other hand, no significant induction was observed with hydrogen peroxide and cumene hydroperoxide. The induction of these fusions by MV was not dependent on the peroxide stress control mediated by the oxyR gene or on the recA-dependent SOS system. These fusions were named soi (superoxide inducible)::lacZ. The induction of beta-galactosidase was significantly reduced by introducing a soxS::Tn10 locus into the fusion strains, indicating that the soi genes are members of the soxRS regulon. Five of the fusions were located in 6 to 26 min of the E. coli genetic map, while three fusions were located in 26 to 36 min, indicating that these fusions are not related to genes already known to be inducible by O2- under the control of soxRS. At least five mutants containing the soi::lacZ fusion were more sensitive to MV and menadione than the wild-type strain, suggesting that the products of these soi genes play an important role in protection against oxidative stress.     

Impact of nutritional factors on the proteome of intestinal Escherichia coli: induction of OxyR-dependent proteins AhpF and Dps by a lactose-rich diet

To study the impact of nutritional factors on protein expression of intestinal bacteria, gnotobiotic mice monoassociated with Escherichia coli K-12 were fed three different diets: a diet rich in starch, a diet rich in nondigestible lactose, and a diet rich in casein. Two-dimensional gel electrophoresis and electrospray-tandem mass spectrometry were used to identify differentially expressed proteins of bacteria recovered from small intestine and cecum. Oxidative stress response proteins such as AhpF, Dps, and Fur, all of which belong to the oxyR regulon, were upregulated in E. coli isolates from mice fed the lactose-rich diet. Luciferase reporter gene assays demonstrated that osmotic stress caused by carbohydrates led to the expression of ahpCF and dps, which was not observed in an E. coli ΔoxyR mutant. Growth of ahpCF and oxyR deletion mutants was strongly impaired when nondigestible sucrose was present in the medium. The wild-type phenotype could be restored by complementation of the deletions with plasmids containing the corresponding genes and promoters. The results indicate that some OxyR-dependent proteins play a major role in the adaptation of E. coli to osmotic stress. We conclude that there is an overlap of osmotic and oxidative stress responses. Mice fed the lactose-rich diet possibly had a higher intestinal osmolality, leading to the upregulation of OxyR-dependent proteins, which enable intestinal E. coli to better cope with diet-induced osmotic stress.     

Involvement of <Emphasis Type="Italic">soxRS</Emphasis> Regulon in Response of <Emphasis Type="Italic">Escherichia coli</Emphasis> to Oxidative Stress Induced by Hydrogen Peroxide

The effect of hydrogen peroxide on the activity of soxRS and oxyR regulon enzymes in different strains of Escherichia coli has been studied. Treatment of bacteria with 20 μM H₂O₂ caused an increase in catalase and peroxidase activities (oxyR regulon) in all strains investigated. It is shown for the first time that oxidative stress induced by hydrogen peroxide causes in some E. coli strains a small increase in activity of superoxide dismutase and glucose-6-phosphate dehydrogenase (soxRS regulon). This effect is cancelled by chloramphenicol, an inhibitor of protein synthesis in prokaryotes. The increase in soxRS regulon enzyme activities was not found in the strain lacking the soxR gene. These results provide evidence for the involvement of the soxRS regulon in the adaptive response of E. coli to oxidative stress induced by hydrogen peroxide. __________ Translated from Biokhimiya, Vol. 70, No. 11, 2005, pp. 1506–1513. Original Russian Text Copyright ? 2005 by Semchyshyn, Bagnyukova, Lushchak.     

Induction of the manganese-containing superoxide dismutase in Escherichia coli is independent of the oxidative stress (oxyR-controlled) regulon

The synthesis of manganese-superoxide dismutase in response to hydrogen peroxide and to paraquat was examined in strains of Escherichia coli with different mutations in the oxyR gene. Hydrogen peroxide treatment did not induce manganese-superoxide dismutase, but did induce the oxyR regulon. Paraquat induced this enzyme in a strain compromised in its ability to induce the defense response against oxidative stress (oxyR deletion) as well as in a strain that is constitutive and overexpresses the oxyR regulon. Catalase (HPI), but not manganese-superoxide dismutase, was over-expressed under anaerobic conditions in a strain harboring a constitutive oxyR mutation. The data clearly demonstrate that the induction of manganese-superoxide dismutase is independent of the oxyR-controlled regulon.     

Heat shock inhibits the induced expression of the SOS genes and SoxRS regulons in Escherichia coli

Oxidative stress formed in Escherichia coli cells is known to bring about a complex induction of alternative DNA repair processes, including SOS, SoxRS, and heat-shock response (HSR). The modification by heat shock of the expression of sfiA and soxS genes induced by oxidative agents H2O2, menadione and 4-nitroquinoline-1-oxide (4NQO) was studied for the first time. Quantitative parameters of gene expression were examined in E. coli strains with fused genes (promoters) sfiA::lacZ and soxS::lacZ. The expression of these genes induced by cell treatment with H2O2, but not menadione or 4NQO, was shown to decrease selectively after exposure to heat shock. Since genetic activity of menadione and 4NQO depends mainly on the formation of superoxide anion O2-, it is assumed that the effect of selective inhibition by heat-shock of sfiA and soxS gene expression in experiments with H2O2 is connected with activity of DnaK heat shock protein, which, unlike other heat-shock proteins, cannot be induced by superoxide anion O2-.     

Induction of the SOS response by hydrogen peroxide in various Escherichia coli mutants with altered protection against oxidative DNA damage.

The induction of the SOS response by H2O2 was measured in Escherichia coli by means of a sfiA::lacZ operon fusion. The effects of mutations in genes involved in DNA repair or DNA metabolism on the SOS response were investigated. We found that in an uvrA mutant, H2O2 induced the SOS response at lower concentrations than in the uvr+ parent strain, indicating that some lesions induced by H2O2 may be repaired by the uvrABC-dependent excision repair system. A nth mutation, yielding deficiency in thymine glycol DNA glycosylase, had no detectable effect on SOS induction, indicating that thymine glycol, a DNA lesion expected to be induced by H2O2, does not participate detectably in the induction of the SOS response by this chemical under our conditions. H2O2 still induced the SOS response in a dnaC(Ts) uvrA double mutant under conditions in which no DNA replication proceeds, suggesting that this chemical induces DNA strand breaks. Induction of the SOS response by H2O2 was also assayed in various mutants affected in genes suspected to be important for protection against oxidative stress. Mutations in the catalase genes, katE and katG, had only minor effects. However, in an oxyR deletion mutant, in which the adaptative response to H2O2 does not occur, SOS induction occurred at much lower H2O2 concentrations than in the oxyR+ parent strain. These results indicate that some enzymes regulated by the oxyR gene are, under our conditions, more important than catalase for protection against the H2O2-induced DNA damages which trigger the SOS response.     

Evidence against a direct antimicrobial role of H2O2 in the infection of plants by Erwinia chrysanthemi

We have investigated the role of bacterial resistance to oxidative stress in pathogenesis. The oxyR gene from the pathogenic bacterium Erwinia chrysanthemi has been characterized. It is closely related to that found in Escherichia coli (88% overall amino acid identity). An E. chrysanthemi oxyR mutant strain was constructed by marker exchange. After induction with a sublethal dose of H2O2, this mutant was more sensitive to H2O2 and showed reduced levels of catalase and glutathione reductase activities, compared with the wild type. The oxyR mutant was unable to form individual colonies on agar plates unless catalase was added exogenously. However, it retained full virulence in potato tubers and tobacco leaves. These results suggest that the host-produced H2O2 has no direct antimicrobial effect on the interaction of E. chrysanthemi with the two plant species.     

Necessity of OxyR for the hydrogen peroxide stress response and full virulence in Ralstonia solanacearum

The plant pathogen Ralstonia solanacearum, which causes bacterial wilt disease, is exposed to reactive oxygen species (ROS) during tomato infection and expresses diverse oxidative stress response (OSR) genes during midstage disease on tomato. The R. solanacearum genome predicts that the bacterium produces multiple and redundant ROS-scavenging enzymes but only one known oxidative stress response regulator, OxyR. An R. solanacearum oxyR mutant had no detectable catalase activity, did not grow in the presence of 250 μM hydrogen peroxide, and grew poorly in the oxidative environment of solid rich media. This phenotype was rescued by the addition of exogenous catalase, suggesting that oxyR is essential for the hydrogen peroxide stress response. Unexpectedly, the oxyR mutant strain grew better than the wild type in the presence of the superoxide generator paraquat. Gene expression studies indicated that katE, kaG, ahpC1, grxC, and oxyR itself were each differentially expressed in the oxyR mutant background and in response to hydrogen peroxide, suggesting that oxyR is necessary for hydrogen peroxide-inducible gene expression. Additional OSR genes were differentially regulated in response to hydrogen peroxide alone. The virulence of the oxyR mutant strain was significantly reduced in both tomato and tobacco host plants, demonstrating that R. solanacearum is exposed to inhibitory concentrations of ROS in planta and that OxyR-mediated responses to ROS during plant pathogenesis are important for R. solanacearum host adaptation and virulence.