Influence of DNA adenine methylase on the sensitivity of Escherichia coli to near-ultraviolet radiation and hydrogen peroxide

Near-ultraviolet (NUV) radiation and hydrogen peroxide (H2O2) inactivation studies were performed on Escherichia coli K-12 DNA adenine methylation (dam) mutants and on cells that carry plasmids which overexpress Dam methylase. Lack of methylation resulted in increased sensitivity to NUV and H2O2 (a photoproduct of NUV). In a dam mutant carrying a dam plasmid, the levels of Dam enzyme and resistance to NUV and H2O2 were restored. However, using a multicopy dam+ plasmid strain, increasing the methylase above wildtype levels resulted in an increase in sensitivity of the cells rather than resistance.     

Alteration of bacteriophage attachment capacity by near-UV irradiation.

Near-UV (NUV) (300 to 400 nm) and far-UV (FUV) (254 nm) radiations damage bacteriophage by different mechanisms. Host cell reactivation, Weigle reactivation, and multiplicity reactivation were observed upon FUV, but not upon NUV irradiation. Also, the number of his+ recombinants increased with P22 bacteriophage transduction in Salmonella typhimurium after FUV, but not after NUV irradiation. This loss of reactivation and recombination after NUV irradiation was not necessarily due to host incapability to repair phage damage. Instead, the phage genome failed to enter the host cell after NUV irradiation. In the case of NUV-irradiated T7 phage, this was determined by genetic crosses with amber mutants, which demonstrated that either "all" or "none" of a T7 genome entered the Escherichia coli cell after NUV treatment. Further studies with radioactively labeled phage indicated that irradiated phage failed to adsorb to host cells. This damage by NUV was compared with the protein-DNA cross-link observed previously, when phage particles were irradiated with NUV in the presence of H2O2. H2O2 (in nonlethal concentration) acts synergistically with NUV so that equivalent phage inactivation is achieved by much lower irradiation doses.     

Effects of peroxide and catalase on near ultraviolet radiation sensitivity in Escherichia coli strains

The role of peroxide and catalase on NUV radiation sensitivity was examined in two repair competent E. coli strains, AB1157 and B/r. Exponential phase B/r is considerably more sensitive to NUV radiation than exponential phase AB1157. However, resistance to 5 mmol dm-3 H2O2 was induced in both AB1157 and B/r by pretreating growing cells with 30 mumol dm-3 H2O2. Pretreatment also induced resistance to broad-band NUV radiation in these strains. The addition of catalase to the post-irradiation plating medium increased survival to the same extent as that provided by pretreatment with 30 mumol dm-3 H2O2, in both strains. The NUV radiation sensitivity seen in B/r does not appear to be due to a deficiency in enzymes that scavenge H2O2, as a catalase deficient mutant, E. coli UM1, is more resistant to NUV radiation than B/r. Also, assays for H2O2 scavenging ability show little difference between AB1157 and B/r in this respect. Two hypotheses are put forward to account for the sensitivity of exponential phase B/r. Whilst it is apparent that peroxides and catalase do have a role in NUV radiation damage, it is clear that other factors also influence survival under certain conditions.     

Oxidative stress effects on conjugational recombination and mutation in catalase-deficient Escherichia coli

The objective of the present investigation was to determine the effects on genetic recombination and mutation in Escherichia coli of either endogenous increases in oxygen radicals resulting from catalase deficiencies, or exogenous increases resulting from H2O2 treatment. Using the classical paradigm of Escherichia coli bacterial conjugation, strains deficient in the production of hydroperoxidase I (HPI) and/or hydroperoxidase II (HPII) were used as recipients in Hfr x F- matings. 'Background' recombination rates, measured by the rate of appearance of threonine prototrophs, was similar to wild-type levels in the HPI-deficient (katG) strain, but were significantly decreased in HPII- (katE) mutants. The addition of relatively nontoxic H2O2 concentrations (0.25 mmoles dm-3) to the mating mixtures stimulated recombination rates in wild-type and katE strains, but decreased rates in katG and katEkatG strains. A 0.5 mmoles dm-3 concentration of H2O2 inhibited recombination rates in all strains. In order to gauge the level of recA-dependent 'SOS' processes occurring under the experimental conditions, 'background' mutation rates were determined in both fluctuation and forward mutation (thyA) assays. Mutation rates in aerobically-grown cultures were increased up to 2.2-fold in katG and katEkatG strains. Treatment with relatively nontoxic H2O2 concentrations elevated the thyA mutagenesis up to 8-fold in catalase-deficient cultures. Furthermore, these studies along with data presented elsewhere show that the SOS phenotype of katEkatG is more resistant than that of katG strains. These studies clearly show that cellular oxidative stress occurring from catalase deficiency interferes with normal DNA metabolism.     

Transcriptional regulation of katE in Escherichia coli K-12

Escherichia coli produces two distinct species of catalase, hydroperoxidases I and II, which differ in kinetic properties and regulation. To further examine catalase regulation, a lacZ fusion was placed into one of the genes that is involved in catalase synthesis. Transductional mapping revealed the fusion to be either allelic with or very close to katE, a locus which together with katF controls the synthesis of the aerobically inducible hydroperoxidase (hydroperoxidase II). katE was expressed under anaerobic conditions at levels that were approximately one-fourth of those found in aerobically grown cells and was found to be induced to higher levels in early-stationary-phase cells relative to levels of exponentially growing cells under both anaerobic and aerobic conditions. katE was fully expressed in air and was not further induced when the growth medium was sparged with 100% oxygen. Expression of katE was unaffected by the addition of hydrogen peroxide or by the presence of additional lesions in oxyR or sodA, indicating that it is not part of the oxyR regulon. When katF::Tn10 was introduced into a katE::lacZ strain, beta-galactosidase synthesis was largely eliminated and was no longer inducible, suggesting that katF is a positive regulator of katE expression.     

Irreversible inactivation of purple acid phosphatase by hydrogen peroxide and ascorbate.

Treatment of the Cu(II)-Fe(III) derivative of pig allantoic fluid acid phosphatase with hydrogen peroxide caused irreversible inactivation of the enzyme and loss of half of the intensity of the visible absorption spectrum. Phosphate, a competitive inhibitor, protected against this inactivation, suggesting that it occurred as a result of a reaction at the active site. The native Fe(II)-Fe(III) enzyme was irreversibly inactivated by H2O2 to a much smaller extent than the Cu(II)-Fe(III) derivative, whereas the Zn(II)-Fe(III) derivative was stable to H2O2 treatment. The rates of inactivation of the Cu(II)-Fe(III) and Fe(II)-Fe(III) enzymes in the presence of H2O2 were increased by addition of ascorbate. These results suggest involvement of a Fenton-type reaction, generating hydroxyl radicals which react with essential active site groups. Experiments carried out on the Fe(II)-Fe(III) enzyme showed that irreversible inactivation by H2O2 in the presence of ascorbate obeyed pseudo first-order kinetics. A plot of kobs for this reaction against H2O2 concentration (at saturating ascorbate) was hyperbolic, giving kobs(max) = 0.41 +/- 0.025 min-1 and S0.5(H2O2) = 1.16 +/- 0.18 mM. A kinetic scheme is presented to describe the irreversible inactivation, involving hydroxyl radical generation by reaction of H2O2 with Fe(II)-Fe(III) enzyme, reduction of the product Fe(III)-Fe(III) enzyme by ascorbate and reaction of hydroxyl radical with an essential group in the enzyme.     

Escherichia coli strains carrying the cloned cytochrome d terminal oxidase complex are sensitive to near-UV inactivation.

To determine if membrane-bound cytochromes function as endogenous near-UV photosensitizers, strains containing the cloned cydA and cydB genes were tested for near-UV sensitivity. A strain containing both cloned genes overproduced cytochromes b558, b595, and d. Another strain containing only cloned cydB overproduced cytochrome b558. Both cytochrome-overproducing strains were hypersensitive to broad-spectrum near-UV inactivation. The presence of excess cytochromes did not affect sensitivity to far-UV radiation and provided protection against H2O2 inactivation.     

Lysosomal and mitochondrial pathways in H2O2-induced apoptosis of alveolar type II cells

Increasing evidence suggests a role for apoptosis in the maintenance of the alveolar epithelium under normal and pathological conditions. However, the signaling pathways modulating alveolar type II (AT II) cell apoptosis remain poorly defined. Here we investigated the role of lysosomes as modulators of oxidant-mediated AT II cell apoptosis using an in vitro model of H(2)O(2)-stress. H(2)O(2) stress led to time-dependent increases in intracellular oxidants, mitochondrial membrane polarization, cytochrome c release, lysosomal rupture, and AT II cells apoptosis. Increased apoptosis was prevented by specific inhibition of the caspase cascade using the broad-spectrum caspase inhibitor z-VAD-fmk or a caspase 3 inhibitor, or by using functional inhibitors for cathepsin D (pepstatin A) or cathepsin B. Inhibition of cathepsin D also prevented mitochondrial permeabilization and cythocrome c release suggesting that lysosomal rupture precedes and is necessary for the activation of the mitochondrial pathway of cell death.     

The inactivation of mitochondrial F1 ATPase by H2O2 is mediated by iron ions not tightly bound in the protein.

Exposure to purified mitochondrial F1 ATPase to continuous flux of H2O2 resulted in significant loss (up to 60%) of the ATP hydrolytic activity. The presence of chelating agents including desferrioxamine or previous selective removal of the iron ions not tightly bound in the protein completely prevented the inactivation, whereas re-loading of the enzyme with F3+ restored the sensitivity to H2O2. A marked protective effect was provided as well by mannitol or by Cu,Zn superoxide dismutase. The results indicated the decomposition of H2O2 by redox-active iron-protein adducts as responsible for the enzyme inactivation, probably through site-directed generation of more highly reactive oxygen species. A possible role for iron associated to F1 component in the oxidation, aging and turnover of ATP synthase complex in vivo may be suggested on the basis on these results.     

Inactivation of glutamine synthetase by a purified rabbit liver microsomal cytochrome P-450 system

Several mixed-function oxidation systems catalyze inactivation of Escherichia coli glutamine synthetase and other key metabolic enzymes. In the presence of NADPH and molecular oxygen, highly purified preparations of cytochrome P-450 reductase and cytochrome P-450 (isozyme 2) from rabbit liver microsomes catalyze enzyme inactivation. The inactivation reaction is stimulated by Fe(III) or Cu(II) and is inhibited by catalase, Mn(II), Zn(II), histidine, and the metal chelators o-phenanthroline and EDTA. The inactivation of glutamine synthetase is highly specific and involves the oxidative modification of a histidine in each glutamine synthetase subunit and the generation of a carbonyl derivative of the protein which forms a stable hydrazone when treated with 2,4-dinitrophenylhydrazine. We have proposed that the mixed-function oxidation system (the cytochrome P-450 system) produces Fe(II) and H2O2 which react at the metal binding site on the glutamine synthetase to generate an activated oxygen species which oxidizes a nearby susceptible histidine. This thesis is supported by the fact that (a) Mn(II) and Zn(II) inhibit inactivation and also interfere with the reduction of Fe(III) to Fe(II) by the P-450 system; (b) Fe(II) and H2O2 (anaerobically), in the absence of a P-450 system, catalyze glutamine synthetase inactivation; (c) inactivation is inhibited by catalase; and (d) hexobarbital, which stimulates the rate of H2O2 production by the P-450 system, stimulates the rate of glutamine synthetase inactivation. Moreover, inactivation of glutamine synthetase by the P-450 system does not require complex formation because inactivation occurs when the P-450 components and the glutamine synthetase are separated by a semipermeable membrane. Also, if endogenous catalase is inhibited by azide, rabbit liver microsomes catalyze the inactivation of glutamine synthetase.     

Catalase HPI influences membrane permeability in Escherichia coli following near-UV stress

The katG gene in Escherichia coli encodes catalase HPI, which is involved in membrane transport and protects the cell during oxidative stress. Hydrogen peroxide (H2O2) induces synthesis of HPI. We examined the role of HPI in membrane permeability (proline uptake) following exposure to near-ultraviolet radiation (NUV). We found that NUV resulted in the same type of induction as H2O2. KatG::Tn10 cells experienced a large drop in uptake after NUV exposure, and levels remained low following incubation. A strain carrying a katG+ plasmid, however, showed considerably less decrease in uptake after NUV, and uptake quickly resumed upon incubation. Further, in an srd mutant which lacks 4-thiouracil, NUV resulted in only a small drop in proline uptake, which was immediately resumed.     

Oxidation of glutathione to its thiyl free radical metabolite by prostaglandin H synthase. A potential endogenous substrate for the hydroperoxidase

The oxidation of glutathione to a thiyl radical by prostaglandin H synthase was investigated. Ram seminal vesicle microsomes, in the presence of arachidonic acid, oxidized glutathione to its thiyl-free radical metabolite, which was detected by ESR using the spin trap 5,5-dimethyl-1-pyrroline-N-oxide. Oxidation of glutathione was dependent on arachidonic acid and inhibited by indomethacin. Peroxides also supported oxidation, indicating that the oxidation was by prostaglandin hydroperoxidase. Glutathione served as a reducingcofactor for the reduction of 15-hydroperoxy-5,8,11,13-eicosatetraenoic acid to 15-hydroxy-5,8,11,13-eicosatetraenoic acid at 1.5-2 times the nonenzymatic rate. Although purified prostaglandin H synthase in the presence of either H2O2 or 15-hydroperoxy-5,8,11,13-eicosatetraenoic acid oxidized glutathione to a thiyl radical, arachidonic acid did not support glutathione oxidation. Glutathione also inhibited cyclooxygenase activity as determined by measuring oxygen incorporation into arachidonic acid. Reverse-phase high pressure liquid chromatography analysis of the arachidonic acid metabolites indicated that the presence of glutathione in an incubation altered the metabolite profile. In the absence of the cofactor, the metabolites were PGD2, PGE2, and 15-hydroperoxy-PGE2 (where PG indicates prostaglandin), while in the presence of glutathione, the only metabolite was PGE2. These results indicate that glutathione not only serves as a cofactor for prostaglandin E isomerase but is also a reducing cofactor for prostaglandin H hydroperoxidase. Assuming that glutathione thiyl-free radical observed in the trapping experiments is involved in the enzymatic reduction of 15-hydroperoxy-5,8,11,13-eicosatetraenoic acid to 15-hydroxy-5,8,11,13-eicosatetraenoic acid, then a 1-electron donation from glutathione to prostaglandin hydroperoxidase is indicated.     

Inactivation of Trypanosoma cruzi and Crithidia fasciculata topoisomerase I by Fenton systems

Fenton systems (H(2)O(2)/Fe(II) or H(2)O(2)/Cu(II)) inhibited Trypanosoma cruzi and Crithidia fasciculata topoisomerase I activity. About 61-71% inactivation was produced by 25 microM Fe(II) or Cu(II) with 3.0 mM H(2)O(2). Thiol compounds and free radical scavengers prevented Fenton system effects, depending on the topoisomerase assayed. With the T. cruzi enzyme, reduced glutathione (GSH), dithiothreitol (DTT), cysteine and N-acetyl-L-cysteine (NAC) entirely prevented the effect of the H(2)O(2)/Fe(II) system; mannitol protected 37%, whereas histidine and ethanol were ineffective. With C. fasciculata topoisomerase, GSH, DTT and NAC protected 100%, cysteine, histidine and mannitol protected 28%, 34% and 48%, respectively, whereas ethanol was ineffective. With the H(2)O(2)/Cu(II) system and T. cruzi topoisomerase, DTT and histidine protected 100% and 60%, respectively, but the other assayed protectors were less effective. Similar results were obtained with the C. fasciculata enzyme. Topoisomerase inactivation by the H(2)O(2)/Fe(II) or H(2)O(2)/Cu(II) systems proved to be irreversible since it was not reversed by the more effective enzyme protectors. It is suggested that topoisomerases could act either as targets of 'reactive oxygen species' (ROS) generated by Fenton systems or bind the corresponding metal ions, whose redox cycling would generate reactive oxygen species in situ.     

Regulation of transcription of katE and katF in Escherichia coli

Fusion plasmids with lacZ under the control of the katE (encoding catalase or hydroperoxidase HPII) and katF (encoding a sigma factor-like protein required for katE expression) promoters were constructed. Expression from both katE and katF promoters was low in rich medium but elevated in poor medium during log-phase growth. Furthermore, the slowdown in growth as cells entered the stationary phase in rich medium, a result of carbon source depletion, was associated with an increase in katE and katF expression. A simple reduction in the carbon source level as the cells entered the stationary phase was not responsible for the increase in expression, because transferring the culture to a medium with no glucose did not induce expression from either promoter. Spent rich medium from stationary-phase cells was capable of inducing expression, as were simple aromatic acids such as benzoate, o-hydroxybenzoate, and p-aminobenzoate added to new medium. Anaerobiosis did not cause an increase in expression, nor did it significantly change the pattern of expression. Regardless of the medium, katF expression was always turned on before or coincidently with katE expression; in the presence of benzoate katF was fully induced, whereas katE was only partially induced, suggesting that a factor in addition to KatF protein was involved in katE expression. During prolonged aerobic incubation, cells lacking katF died off more rapidly than did cells lacking either katE or katG.     

Hyperoxia increases H2O2 production by brain in vivo

Hyperoxia and hyperbaric hyperoxia increased the rate of cerebral hydrogen peroxide (H2O2) production in unanesthetized rats in vivo, as measured by the H2O2-mediated inactivation of endogenous catalase activity following injection of 3-amino-1,2,4-triazole. Brain catalase activity in rats breathing air (0.2 ATA O2) decreased to 75, 61, and 40% of controls due to endogenous H2O2 production at 30, 60, and 120 min, respectively, after intraperitoneal injection of 3-amino-1,2,4-triazole. The rate of catalase inactivation increased linearly in rats exposed to 0.6 ATA O2 (3 ATA air), 1.0 ATA O2 (normobaric 100% O2) and 3.0 ATA O2 (3 ATA 100% O2) compared with 0.2 ATA O2 (room air). Catalase inactivation was prevented by pretreatment of rats with ethanol (4 g/kg), a competitive substrate for the reactive catalase-H2O2 intermediate, compound I. This confirmed that catalase inactivation by 3-amino-1,2,4-triazole was due to formation of the catalase-H2O2 intermediate, compound I. The linear rate of catalase inactivation allows estimates of the average steady-state H2O2 concentration within brain peroxisomes to be calculated from the formula: [H2O2] = 6.6 pM + 5.6 ATA-1 X pM X [O2], where [O2] is the concentration of oxygen in ATA that the rats are breathing. Thus the H2O2 concentration in brains of rats exposed to room air is calculated to be about 7.7 pM, rises 60% when O2 tension is increased to 100% O2, and increases 300% at 3 ATA 100% O2, where symptoms of central nervous system toxicity first become apparent. These studies support the concept that H2O2 is an important mediator of O2-induced injury to the central nervous system.     

Role of cloned carotenoid genes expressed in Escherichia coli in protecting against inactivation by near-UV light and specific phototoxic molecules.

Genes controlling carotenoid synthesis were cloned from Erwinia herbicola and expressed in an Escherichia coli strain. Carotenoids protect against high fluences of near-UV (NUV; 320 to 400 nm) but not against far-UV (200-300 nm). Protection of E. coli cells was not observed following treatment with either psoralen or 8-methoxypsoralen plus NUV. However, significant protection of cells producing carotenoids was observed with three photosensitizing molecules activated by NUV (alpha-terthienyl, harmine, and phenylheptatriyne) which are thought to have the membrane as an important lethal target. Protection of carotenoid-producing cells against inactivation was not observed with acridine orange plus visible light but was seen with toluidine blue O plus visible light.     

Acceleration of P/C-type inactivation in voltage-gated K(+) channels by methionine oxidation

Oxidation of amino acid residues causes noticeable changes in gating of many ion channels. We found that P/C-type inactivation of Shaker potassium channels expressed in Xenopus oocytes is irreversibly accelerated by patch excision and that this effect was mimicked by application of the oxidant H(2)O(2), which is normally produced in cells by the dismutase action on the superoxide anion. The inactivation time course was also accelerated by high concentration of O(2). Substitution of a methionine residue located in the P-segment of the channel with a leucine largely eliminated the channel's sensitivity to patch excision, H(2)O(2), and high O(2). The results demonstrate that oxidation of methionine is an important regulator of P/C-type inactivation and that it may play a role in mediating the cellular responses to hypoxia/hyperoxia.