Escherichia coli xth mutants are hypersensitive to hydrogen peroxide.

Escherichia coli mutants lacking exonuclease III (xthA) are exceptionally sensitive to hydrogen peroxide. They are killed by H2O2 at 20 times the rate of wild-type bacteria and at 3 to 4 times the rate of recA cells. This is the first clear phenotypic sensitivity reported for xth- E. coli and should aid in clarifying peroxide-induced lethality and the in vivo role of exonuclease III.     

Two sources of endogenous hydrogen peroxide in Escherichia coli

Mechanisms of hydrogen peroxide generation in Escherichia coli were investigated using a strain lacking scavenging enzymes. Surprisingly, the deletion of many abundant flavoenzymes that are known to autoxidize in vitro did not substantially lessen overall H₂O₂ formation. However, H₂O₂ production diminished by 25–30% when NadB turnover was eliminated. The flavin‐dependent desaturating dehydrogenase, NadB uses fumarate as an electron acceptor in anaerobic cells. Experiments showed that aerobic NadB turnover depends upon its oxidation by molecular oxygen, with H₂O₂ as a product. This reaction appears to be mechanistically adventitious. In contrast, most desaturating dehydrogenases are associated with the respiratory chain and deliver electrons to fumarate anaerobically or oxygen aerobically without the formation of toxic by‐products. Presumably, NadB can persist as an H₂O₂‐generating enzyme because its flux is limited. The anaerobic respiratory enzyme fumarate reductase uses a flavoprotein subunit that is homologous to NadB and accordingly forms substantial H₂O₂ upon aeration. This tendency is substantially suppressed by cytochrome oxidase. Thus cytochrome d oxidase, which is prevalent among anaerobes, may diminish intracellular H₂O₂ formation by the anaerobic respiratory chain, whenever these organisms encounter oxygen. These two examples reveal biochemical and physiological arrangements through which evolution has minimized the rate of intracellular oxidant formation.     

Microsatellite instability induced by hydrogen peroxide in Escherichia coli

Damage to DNA by reactive oxygen species may be a significant source of endogenous mutagenesis in aerobic organisms. Using a selective assay for microsatellite instability in E. coli, we have asked whether endogenous oxidative mutagenesis can contribute to genetic instability. Instability of repetitive sequences, both in intronic sequences and within coding regions, is a hallmark of genetic instability in human cancers. We demonstrate that exposure of E. coli to low levels of hydrogen peroxide increases the frequency of expansions and deletions within dinucleotide repetitive sequences. Sequencing of the repetitive sequences and flanking non-repetitive regions in mutant clones demonstrated the high specificity for alterations with the repeats. All of the 183 mutants sequenced displayed frameshift alterations within the microsatellite repeats, and no base substitutions or frameshift mutations occurred within the flanking non-repetitive sequences. We hypothesize that endogenous oxidative damage to DNA can increase the frequency of strand slippage intermediates occurring during DNA replication or repair synthesis, and contribute to genomic instability.     

Repair response of Escherichia coli to hydrogen peroxide DNA damage.

The repair response of Escherichia coli to hydrogen peroxide-induced DNA damage was investigated in intact and toluene-treated cells. Cellular DNA was cleaved after treatment by hydrogen peroxide as analyzed by alkaline sucrose sedimentation. The incision step did not require ATP or magnesium and was not inhibited by N-ethylmaleimide (NEM). An ATP-independent, magnesium-dependent incorporation of nucleotides was seen after the exposure of cells to hydrogen peroxide. This DNA repair synthesis was not inhibited by the addition of NEM or dithiothreitol. In dnaB(Ts) strain CRT266, which is thermolabile for DNA replication, normal levels of DNA synthesis were found at the restrictive temperature (43 degrees C), showing that DNA replication was not necessary for this DNA synthesis. Density gradient analysis also indicated that hydrogen peroxide inhibited DNA replication and stimulated repair synthesis. The subsequent reformation step required magnesium, did not require ATP, and was not inhibited by NEM, in agreement with the synthesis requirements. This suggests that DNA polymerase I was involved in the repair step. Furthermore, a strain defective in DNA polymerase I was unable to reform its DNA after peroxide treatment. Chemical cleavage of the DNA was shown by incision of supercoiled DNA with hydrogen peroxide in the presence of a low concentration of ferric chloride. These findings suggest that hydrogen peroxide directly incises DNA, causing damage which is repaired by an incision repair pathway that requires DNA polymerase I.     

The effect of temperature or anoxia on Escherichia coli killing induced by hydrogen peroxide

The cytotoxicity of hydrogen peroxide in Escherichia coli was investigated after various conditions of drug exposure. Two modes of killing were detected following a 15-min challenge with H2O2 under either aerated or anoxic conditions. Mode one killing occurred at levels below 2.5 mM and mode two killing at concentrations higher than 10 mM. Whereas mode one killing was similar at the two conditions of drug exposure, mode two lethality differed in that aerated cells were more sensitive than anoxic cells. Independently of O2 tension the hydroxyl radical scavenger, thiourea, prevented mode two but not mode one killing by H2O2. Cells treated with the drug at ice temperature did not display mode one killing and mode two lethality occurred only at very high concentrations. We suggest that hydroxyl radicals mediate mode two but not mode one killing by H2O2.     

Activation of caspase 3 in HL-60 cells exposed to hydrogen peroxide

Recent studies have suggested that hydrogen peroxide (H2O2), a reactive compound formed endogenously in the breakdown of superoxide, may mediate the induction of apoptosis in various cell types in response to external stimuli. However, the role of H2O2 in the apoptotic pathway has not been clearly established. The purpose of this study was to determine if H2O2 treatment could induce apoptosis through the activation of caspases. Doses of H2O2 ranging from 10 microM to 100 microM, when added to HL-60 cells, resulted in the cleavage of poly(ADP-ribose) polymerase (PARP) from its native 113 Kd form to a processed 89 Kd fragment, indicative of cells undergoing apoptosis. PARP was predominantly in the fragmented form when doses of 20 microM and greater were used. A time course study of changes in PARP processing in H2O2-treated cells revealed that 10 and 50 microM H2O2 required 6 and 3 h, respectively, to specifically degrade PARP, suggesting that the H2O2-induced PARP cleavage is both time and concentration dependent. Since PARP is cleaved by CPP32 (caspase-3), we next determined if H2O2 was capable of effecting changes in CPP32 activity. The caspase activity was assayed using a colorimetric substrate, DEVD-pNa. Results of these experiments showed that H2O2 increased caspase activity at 3 h, corresponding to the time of appearance of fragmented PARP. Also, CPP32 activity and PARP processing were both significantly suppressed by caspase-3 inhibitors. Taken together, these results suggest that H2O2 mediates specific cleavage of PARP and possibly apoptosis by activating caspase 3.     

Copper ions mediate the lethality induced by hydrogen peroxide in low iron conditions in Escherichia coli

Iron ions mediate the formation of lethal DNA damage by hydrogen peroxide. However, when cells are depleted of iron ions by the treatment with iron chelators, DNA damage can still be detected. Here we show that the formation of such damage in low iron conditions is due to the participation of copper ions. Copper chelators can inhibit cell inactivation, DNA strand breakage and mutagenesis induced by hydrogen peroxide in cells pre-treated with iron chelators. The Fpg and UvrA proteins play an important role in the repair of DNA lesions formed in these conditions, as suggested by the great sensitivity of the uvrA and fpg mutant strains to the treatment when compared to the wild type strain.     

Bimodal pattern of killing of DNA-repair-defective or anoxically grown Escherichia coli by hydrogen peroxide.

Two modes of killing of Escherichia coli K-12 by hydrogen peroxide can be distinguished. Mode-one killing was maximal with hydrogen peroxide at a concentration of 1 to 2 mM. At higher concentrations the killing rate was approximately half maximal and was independent of H2O2 concentration but first order with respect to exposure time. Mode-one killing required active metabolism during the H2O2 challenge, and it resulted in sfiA-independent filamentation of both cells which survived and those which were killed by the challenge. This mode of killing was enhanced in xth, polA, recA, and recB strains and was accelerated in all strains by an unidentified, anoxia-induced cell function. A strain carrying both xth and recA mutations appeared to undergo spontaneous mode-one killing only under aerobic conditions. Mode-one killing appeared to result from DNA damage which normally occurs at a low, nonlethal level during aerobic growth. Mode-two killing occurred at higher doses of H2O2 and exhibited a multihit dependence on both H2O2 concentration and exposure time. Mode-two killing did not require active metabolism, and killed cells did not filament, although survivors demonstrated a dose-dependent growth lag. Strains with DNA-repair defects were not especially susceptible to mode-two killing.     

Factors affecting catalase level and sensitivity to hydrogen peroxide in Escherichia coli.

Composition of the culture medium, growth phase, and temperature play important roles in the sensitivity of Escherichia coli to H2O2. The medium and growth phase affected the sensitivity of the cells to H2O2 by modifying the amount of catalase synthesized by them, whereas the effect of temperature was due to the thermolability of the enzyme. Since catalase is unstable in the presence of its substrate, the correlation between the catalase level in the cells and their sensitivity to H2O2 could be observed only when the H2O2 concentration was not excessive in proportion to the amount of catalase.     

Chronic low dose exposure to hydrogen peroxide changes sensitivity of V79 cells to different damaging agents

Chinese hamster V79 cells were repeatedly exposed to a low dose of hydrogen peroxide (H2O2) over several weeks and then exposed to H2O2, cisplatin or ultraviolet (UV) light. Cell killing was examined by colony formation, following these treatments. It was seen that cells conditioned by multiple low doses of H2O2 showed resistance to killing in case of H2O2 and cisplatin but the sensitivity to UV light was same as the control cells. Apoptosis was also determined in these cells after the same treatments. UV light failed to induce apoptosis in both conditioned and in control cells, but in case of cells treated with H2O2 and with cisplatin, there was less apoptosis in the conditioned cells compared to the control cells. From our observation we can say that the enhanced survival of cells after treatment with H2O2 or cisplatin could be due to inhibition of apoptosis.     

Mutagenesis and stress responses induced in Escherichia coli by hydrogen peroxide

Killing of Escherichia coli by hydrogen peroxide proceeds by two modes. Mode one killing appears to be due to DNA damage, has a maximum near 1 to 3 mM H2O2, and requires active metabolism during exposure. Mode two killing is due to uncharacterized damage, occurs in the absence of metabolism, and exhibits a classical multiple-order dose-response curve up to at least 50 mM H2O2 (J. A. Imlay and S. Linn, J. Bacteriol. 166:519-527, 1986). H2O2 induces the SOS response in proportion to the degree of killing by the mode one pathway, i.e., induction is maximal after exposure to 1 to 3 mM H2O2. Mutant strains that cannot induce the SOS regulon are hypersensitive to peroxide. Analysis of the sensitivities of mutants that are deficient in individual SOS-regulated functions suggested that the SOS-mediated protection is due to the enhanced synthesis of recA protein, which is rate limiting for recombinational DNA repair. Specifically, strains wholly blocked in both SOS induction and DNA recombination were no more sensitive than mutants that are blocked in only one of these two functions, and strains carrying mutations in uvrA, -B, -C, or -D, sfiA, umuC or -D, ssb, or dinA, -B, -D, -F, -G, -H, -I, or -J were not abnormally sensitive to killing by H2O2. After exposure to H2O2, mutagenesis and filamentation also occurred with the dose response characteristic of SOS induction and mode one killing, but these responses were not dependent on the lexA-regulated umuC mutagenesis or sfiA filamentation functions, respectively. Exposure of E. coli to H2O2 also resulted in the induction of functions under control of the oxyR regulon that enhance the scavenging of active oxygen species, thereby reducing the sensitivity to H2O2. Catalase levels increased 10-fold during this induction, and katE katG mutants, which totally lack catalase, while not abnormally sensitive to killing by H2O2 in the naive state, did not exhibit the induced protective response. Protection equal to that observed during oxyR induction could be achieved by the addition of catalase to cultures of naive cells in an amount equivalent to that induced by the oxyR response. Thus, the induction of catalase is necessary and sufficient for the observed oxyR-directed resistance to killing by H2O2. Although superoxide dismutase appeared to be uninvolved in this enhanced protective response, sodA sodB mutants, which totally lack superoxide dismutase, were especially sensitive to mode one killing by H2O2 in the naive state. gshB mutants, which lack glutathione, were not abnormally sensitive to killing by H2O2.     

Superoxide anions are required for the inactivation ofEscherichia coli induced by high concentrations of hydrogen peroxide

The wild-type strain and mutants ofEscherichia coli lacking Mn-superoxide dismutase (Sod A) or Fe-superoxide dismutase (Sod B) are compared for their sensitivity to the H₂O₂ insult (exposure for 15 min at 37°C, in M9 salts). Whereas mode one killing is similar in superoxide dismutase mutants and wild-type cells, the latter strain appears to be more resistant than the former ones to mode two lethality. Furthermore, Sod B cells, as well as wild-type cells but unlike Sod A cells, are capable of reversing the toxicity of the oxidant (even in the presence of chloramphenicol), this effect being observed by gradually increasing the H₂O₂ concentration from 2.5 to 10 mM. It is concluded that (a) superoxide ions may not be involved in the production of mode one killing by H₂O₂, although further experiments are needed to validate or modify this hypothesis; (b) superoxide ions mediate mode two killing by H₂O₂, possibly by reducing trivalent iron to the divalent form; and (c) the intervening zone of partial resistance observed in wild-type and Sod B cells exposed to intermediate H₂O₂ concentrations is not a consequence of Mn-superoxide dismutase induction; it would appear, however, that cells lacking this superoxide dismutase isoenzyme are not proficient in this acquired response.     

Changes in the electro-orientation of Escherichia coli cells exposed to disinfectants

Various disinfectants were shown to influence the frequency dependence of Escherichia coli electro-orientation. Cell inactivation by different agents was found to decrease the effect at high frequencies (5 X 10(5)-5 X 10(6) Hz). The decrease should be attributed to the fact that the barrier properties of membranes are disorganized and the equivalent electric conductivity of cells drops down. The microbiological control of the bactericidal action produced by disinfectants fits in well with changes in the electro-orientation of bacterial cells at these frequencies.     

Mechanism of thermoresistance in Escherichia coli cells exposed to temperature elevation

The influence of media with different osmotic pressure (NaCl water solution) and chloramphenicol (10 micrograms/ml) on the survival, permeability, and survival curve shape of Escherichia coli B/r and E. coli Bs-1 cells, heated up to 50, 52, and 60 degrees C was investigated. As shown, the survival curve of cells heated up to 60 degrees C in isotonic conditions was characterized by exponential shape, while the survival curves of cells heated up to 50 and 52 degrees C consisted of two components characterizing thermosensitive and thermoresistant parts of cell population. Hypertonic conditions of heat at 52 degrees C decreased cell lethality and permeability. In this case, survival curves were characterized by exponential shape. Chloramphenicol was shown to protect against damaging action of heat at 50 degrees C and not to affect the viability of cells heated at 52 and 60 degrees C. It is proposed that the increase of cell thermoresistance with heat dose elevation at 50 and 52 degrees C in isotonic conditions, which is accompanied by the appearance of thermotolerant components on survival curves, may be associated with accommodational cell reactions. The essence of these reactions consists in stabilization of the osmotic cell homeostasis.     

Enhanced recovery of injured Escherichia coli by compounds that degrade hydrogen peroxide or block its formation

Escherichia coli LSUFS was injured either by freezing at -10 degrees C or by heating at 57 degrees C for 12 min. Surviving cells were recovered on nonselective tryptone-glucose extract agar and selective violet red bile agar supplemented with compounds that degrade hydrogen peroxide or block its formation. Various concentrations of the following compounds were tested: sodium pyruvate, 3,3'-thiodipropionic acid, catalase, ascorbic acid, potassium permanganate, sodium thioglycolate, dimethylsulfoxide, ethoxyquin, n-propyl gallate, alpha-tocopherol sodium metabisulfite, and ferrous sulfate. Sodium pyruvate and 3,3'-thiodipropionic acid, when added to either medium, significantly (P greater than 0.01) increased recovery of injured cells. More than 90% of the heat-injured cells and 40 to 90% of the freeze-injured cells failed to grow on unsupplemented tryptone-glucose extract agar. Supplementation of violet red bile agar increased recovery, but the counts remained considerably lower than the tryptone-glucose extract agar counts. The repair detection procedure of Speck et al. (M. Speck, B. Ray, R. Read, Jr., Appl. Microbiol. 29:549-550, 1975) was greatly improved by the addition of pyruvate or 3,3'-thiodipropionic acid. However, when this improved repair detection procedure was applied to foods, pyruvate-supplemented media showed some false-positives. We therefore recommend that 3,3'-thiodipropionic acid be used to supplement media in the repair detection procedure.     

Effect of hydrogen peroxide in low and superlow concentrations on DNA structure

Hydrogen peroxide in low concentrations have effect on DNA structural characteristics both in solution at 38 degrees C and in vivo, in mice organs.