Endogenous Superoxide Dismutase Levels Regulate Iron-Dependent Hydroxyl Radical Formation in Escherichia coli Exposed to Hydrogen Peroxide
01 Feb 1998-Journal of Bacteriology (American Society for Microbiology)-Vol. 180, Iss: 3, pp 622-625
TL;DR: The hypothesis that a resulting increase in .OH formation generated by Fenton chemistry is responsible for the observed enhancement of DNA damage and the increased susceptibility to H2O2-mediated killing seen in these mutants lacking SOD is supported.
Abstract: Aerobic organisms contain antioxidant enzymes, such as superoxide dismutase (SOD) and catalase, to protect them from both direct and indirect effects of reactive oxygen species, such as O2.- and H2O2. Previous work by others has shown that Escherichia coli mutants lacking SOD not only are more susceptible to DNA damage and killing by H2O2 but also contain larger pools of intracellular free iron. The present study investigated if SOD-deficient E. coli cells are exposed to increased levels of hydroxyl radical (.OH) as a consequence of the reaction of H2O2 with this increased iron pool. When the parental E. coli strain AB1157 was exposed to H2O2 in the presence of an alpha-(4-pyridyl-1-oxide)-N-tert-butyl-nitrone (4-POBN)-ethanol spin-trapping system, the 4-POBN-.CH(CH3)OH spin adduct was detectable by electron paramagnetic resonance (EPR) spectroscopy, indicating .OH production. When the isogenic E. coli mutant JI132, lacking both Fe- and Mn-containing SODs, was exposed to H2O2 in a similar manner, the magnitude of .OH spin trapped was significantly greater than with the control strain. Preincubation of the bacteria with the iron chelator deferoxamine markedly inhibited the magnitude of .OH spin trapped. Exogenous SOD failed to inhibit .OH formation, indicating the need for intracellular SOD. Redox-active iron, defined as EPR-detectable ascorbyl radical, was greater in the SOD-deficient strain than in the control strain. These studies (i) extend recent data from others demonstrating increased levels of iron in E. coli SOD mutants and (ii) support the hypothesis that a resulting increase in .OH formation generated by Fenton chemistry is responsible for the observed enhancement of DNA damage and the increased susceptibility to H2O2-mediated killing seen in these mutants lacking SOD.
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Dissertation•
23 May 2012
TL;DR: In this article, les donnees de sequencage des genomes ont revele a nouvelle famille de proteines UPF0079, comprenant des proteines de fonction inconnue qui sont exclusivement and largement presentes chez les bacteries and possedent un motif A de Walker dans leur sequence.
Abstract: Les donnees de sequencage des genomes ont revele une nouvelle famille de proteines UPF0079, comprenant des proteines de fonction inconnue qui sont exclusivement et largement presentes chez les bacteries et qui possedent un motif A de Walker dans leur sequence. La caracterisation biochimique et l'elucidation du role physiologique de cette famille contribueront a elargir nos connaissances en biologie fondamentale, et sont egalement un prealable vers le developpement de nouveaux composes antimicrobiens. Notre etude sur YdiB, un archetype de cette famille chez Bacillus subtilis a revele a la fois l‟autophosphorylation de YdiB et son activite de proteine kinase. L‟activite kinase de double specificite Ser/ Thr et Tyr de YdiB semble necessiter son oligomerisation et semble etre stimulee par des molecules basiques telles que des polyamines naturelles ou la poly-L-lysine. Les 10 residus les plus conserves chez cette famille ont ete etudies afin de mieux comprendre le mecanisme moleculaire de YdiB. Concernant la caracterisation fonctionnelle de la phosphorylation liee a YdiB, l‟etude de l‟operon ydiA-B-C-D-E de B. subtilis nous a permis de montrer que YdiB et YdiC fonctionnent comme un couple de proteine kinase/phosphatase de deux proteines substrats dont les fonctions seraient liees aux ribosomes, YdiD et YdiE. Une co-localisation partielle entre YdiB et les ribosomes a ete observee. En outre, YdiB est capable de phosphoryler des proteines ribosomiques appartennant aux deux sous-unites 50S et 30S, ainsi que deux GTPases impliquees dans la biogenese des ribosomes, EngA et EngB. Nous avons egalement demontre que EngA phosphorylee par YdiB est un substrat in vitro de la phosphatase YdiC. Enfin, base sur le phosphoproteome de Bacillus subtilis, des peptides mimant des sites de phosphorylation in vivo ont ete utilises. Certains entre eux sont phosphoryles in vitro par YdiB. Deux de ces peptides appartiennent a la superoxyde dismutase, SodA, dont l'activite in vitro et apres purification est regulee positivement via la phosphorylation par YdiB. Nous avons ensuite constate que les cellules de B. subtilis depourvues du gene ydiB sont plus sensibles aux agents oxidants tels que le paraquat ou la norfloxacine. Nous proposons que, in vivo, YdiB fonctionne comme une proteine kinase impliquee dans l‟activite et/ou la stabilite des ribosomes dans des conditions physiologiques normales, et YdiB contribuerait a proteger les cellules contre les dommages du stress oxydatif.
10 citations
TL;DR: Results clearly indicate that the oxyR mutation suppressed the pleiotropic effect of the B. multivorans fur mutant.
Abstract: Fur (ferric uptake regulator) is an iron-responsive transcriptional regulator in many bacterial species, and the fur mutant of Burkholderia multivorans ATCC 17616 exhibits pleiotropic phenotypes, such as an inability to efficiently use several carbon sources, as well as high sensitivity to hydrogen peroxide (H2O2), paraquat (a superoxide-producing compound) and nitric oxide (NO). To gain more insight into the pleiotropic role of the Fur protein of ATCC 17616, spontaneous suppressor mutants of the ATCC 17616 fur mutant that restored tolerance to NO were isolated and characterized in this study. The microarray-based comparative genomic analysis and subsequent sequencing analysis indicated that such suppressor mutants had a 2 bp deletion in the oxyR gene, whose orthologues encode H2O2-responsive transcriptional regulators in other bacterial species. The suppressor mutants and the reconstructed fur–oxyR double-deletion mutant showed indistinguishable phenotypes in that they were all (i) more resistant than the fur mutant to H2O2, superoxide, NO and streptonigrin (an iron-activated antibiotic) and (ii) able to use carbon sources that cannot efficiently support the growth of the fur mutant. These results clearly indicate that the oxyR mutation suppressed the pleiotropic effect of the B. multivorans fur mutant. The fur–oxyR double mutants were found to overexpress the KatG (catalase/peroxidase) and AhpC1 and AhpD (alkyl hydroperoxide reductase subunits C and D) proteins, and their enzymic activities to remove reactive oxygen and nitrogen species were suggested to be responsible for the suppression of phenotypes caused by the fur mutation.
9 citations
Cites background from "Endogenous Superoxide Dismutase Lev..."
...The hydroxyl radical then reacts with many biomolecules, and the bacterial cell death caused by H2O2 is due primarily to hydroxyl-radical-mediated DNA damage (Imlay & Linn, 1986, 1988; McCormick et al., 1998)....
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TL;DR: The crucial defense mechanisms, such as antimicrobial peptides or extracellular traps, typically activated within minutes of the initial infection phase are examined, which are termed the “immediate protective response” and raised the question of how the host limits microbial replication and invasion during this critical period.
Abstract: The innate immune system detects infection and tissue injury through different families of pattern-recognition receptors (PRRs), such as Toll-like receptors Most PRR-mediated responses initiate elaborate processes of signaling, transcription, translation, and secretion of effector mediators, which together require time to achieve Therefore, PRR-mediated processes are not active in the early phases of infection These considerations raise the question of how the host limits microbial replication and invasion during this critical period Here, we examine the crucial defense mechanisms, such as antimicrobial peptides or extracellular traps, typically activated within minutes of the initial infection phase, which we term the “immediate protective response” Deficiencies in different components of the immediate protective response are often associated with severe and recurrent infectious diseases in humans, highlighting their physiologic importance
7 citations
TL;DR: A review of the use of pharmacological ascorbate as a treatment for cancer has increased considerably since it was introduced by Cameron and Pauling in the 1970s as discussed by the authors, and the results of these studies are promising.
Abstract: Interest in the use of pharmacological ascorbate as a treatment for cancer has increased considerably since it was introduced by Cameron and Pauling in the 1970s. Recently, pharmacological ascorbate has been used in preclinical and early-phase clinical trials as a selective radiation sensitizer in cancer. The results of these studies are promising. This review summarizes data on pharmacological ascorbate (1) as a safe and efficacious adjuvant to cancer therapy; (2) as a selective radiosensitizer of cancer via a mechanism involving hydrogen peroxide; and (3) as a radioprotector in normal tissues. Additionally, we present new data demonstrating the ability of pharmacological ascorbate to enhance radiation-induced DNA damage in glioblastoma cells, facilitating cancer cell death. We propose that pharmacological ascorbate may be a general radiosensitizer in cancer therapy and simultaneously a radioprotector of normal tissue.
7 citations
References
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Book•
13 Jun 1985
TL;DR: 1. Oxygen is a toxic gas - an introduction to oxygen toxicity and reactive species, and the chemistry of free radicals and related 'reactive species'
Abstract: 1. Oxygen is a toxic gas - an introductionto oxygen toxicity and reactive species 2. The chemistry of free radicals and related 'reactive species' 3. Antioxidant defences Endogenous and Diet Derived 4. Cellular responses to oxidative stress: adaptation, damage, repair, senescence and death 5. Measurement of reactive species 6. Reactive species can pose special problems needing special solutions. Some examples. 7. Reactive species can be useful some more examples 8. Reactive species can be poisonous: their role in toxicology 9. Reactive species and disease: fact, fiction or filibuster? 10. Ageing, nutrition, disease, and therapy: A role for antioxidants?
21,528 citations
"Endogenous Superoxide Dismutase Lev..." refers background in this paper
...rapidly reacts with itself (dismutes) to form H2O2 (7)....
[...]
...Although the aerobic metabolism of bacteria optimally results in the near simultaneous four-electron reduction of O2 to H2O, a variable percentage of O2 reduction occurs initially via either one-electron reduction of O2 to superoxide (O2 ) or divalent reduction to H2O2 (7)....
[...]
TL;DR: It is proposed that the cell may also decrease such toxicity by diminishing available NAD(P)H and by utilizing oxygen itself to scavenge active free radicals into superoxide, which is then destroyed by superoxide dismutase.
Abstract: A major portion of the toxicity of hydrogen peroxide in Escherichia coli is attributed to DNA damage mediated by a Fenton reaction that generates active forms of hydroxyl radicals from hydrogen peroxide, DNA-bound iron, and a constant source of reducing equivalents. Kinetic peculiarities of DNA damage production by hydrogen peroxide in vivo can be reproduced by including DNA in an in vitro Fenton reaction system in which iron catalyzes the univalent reduction of hydrogen peroxide by the reduced form of nicotinamide adenine dinucleotide (NADH). To minimize the toxicity of oxygen radicals, the cell utilizes scavengers of these radicals and DNA repair enzymes. On the basis of observations with the model system, it is proposed that the cell may also decrease such toxicity by diminishing available NAD(P)H and by utilizing oxygen itself to scavenge active free radicals into superoxide, which is then destroyed by superoxide dismutase.
1,997 citations
"Endogenous Superoxide Dismutase Lev..." refers background in this paper
...Pretreatment of the JI132 (SOD-deficient) bacteria with DFO greatly reduced the magnitude of OH generation, confirming that it arose as a consequence of Fenton chemistry, as iron bound to DFO is no longer available for this redox chemistry (10)....
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TL;DR: Aerotolerant anaerobes, which survive exposure to air and metabolize oxygen to a limited extent but do not contain cytochrome systems, were found to be devoid of catalase activity but did exhibit superoxide dismutase activity.
Abstract: The distribution of catalase and superoxide dismutase has been examined in various micro-organisms. Strict anaerobes exhibited no superoxide dismutase and, generally, no catalase activity. All aerobic organisms containing cytochrome systems were found to contain both superoxide dismutase and catalase. Aerotolerant anaerobes, which survive exposure to air and metabolize oxygen to a limited extent but do not contain cytochrome systems, were found to be devoid of catalase activity but did exhibit superoxide dismutase activity. This distribution is consistent with the proposal that the prime physiological function of superoxide dismutase is protection of oxygen-metabolizing organisms against the potentially detrimental effects of the superoxide free radical, a biologically produced intermediate resulting from the univalent reduction of molecular oxygen.
974 citations
"Endogenous Superoxide Dismutase Lev..." refers background in this paper
...Most bacteria, including Escherichia coli, contain superoxide dismutase (SOD) and catalase as means of eliminating O2 z2 and H2O2, respectively (16, 17)....
[...]
TL;DR: This presentation discusses the role of catalytic metals in free radical-mediated oxidations, ascorbate as both a pro-oxidant and an antioxidant, use of asCorbate to determine adventitious catalytic metal concentrations, and uses of ascorBate radical as a marker of oxidative stress.
Abstract: Trace levels of transition metals can participate in the metal-catalyzed Haber-Weiss reaction (superoxide-driven Fenton reaction) as well as catalyze the oxidation of ascorbate. Generally ascorbate is thought of as an excellent reducing agent; it is able to serve as a donor antioxidant in free radical-mediated oxidation processes. However, as a reducing agent it is also able to reduce redox-active metals such as copper and iron, thereby increasing the pro-oxidant chemistry of these metals. Thus ascorbate can serve as both a pro-oxidant and an antioxidant. In general, at low ascorbate concentrations, ascorbate is prone to be a pro-oxidant, and at high concentrations, it will tend to be an antioxidant. Hence there is a crossover effect. We propose that the "position" of this crossover effect is a function of the catalytic metal concentration. In this presentation, we discuss: (1) the role of catalytic metals in free radical-mediated oxidations; (2) ascorbate as both a pro-oxidant and an antioxidant; (3) catalytic metal catalysis of ascorbate oxidation; (4) use of ascorbate to determine adventitious catalytic metal concentrations; (5) use of ascorbate radical as a marker of oxidative stress; and (6) use of ascorbate and iron as free radical pro-oxidants in photodynamic therapy of cancer.
851 citations
TL;DR: In this article, the authors show that the level of loose iron in severely superoxide-stressed cells greatly exceeds that of unstressed cells, and that both growth defects and DNA damage caused by superoxide ensue from its ability to damage a subset of iron-sulfur clusters.
Abstract: Superoxide promotes hydroxyl-radical formation and consequent DNA
damage in cells of all types. The long-standing hypothesis that it
primarily does so by delivering electrons to adventitious iron on DNA
was refuted by recent studies in Escherichia coli.
Alternative proposals have suggested that superoxide may accelerate
oxidative DNA damage by leaching iron from storage proteins or enzymic
[4Fe-4S] clusters. The released iron might then deposit on the
surface of the DNA, where it could catalyze the formation of DNA
oxidants using other electron donors. The latter model is affirmed by
the experiments described here. Whole-cell electron paramagnetic
resonance demonstrated that the level of loose iron in
superoxide-stressed cells greatly exceeds that of unstressed cells.
Bacterial iron storage proteins were not the major source for free
iron, since superoxide also increased iron levels in mutants lacking
these iron storage proteins. However, overproduction of an enzyme
containing a labile [4Fe-4S] cluster dramatically increased the free
iron content of cells when they were growing in air. The rates of
spontaneous mutagenesis and DNA damage from exogenous
H2O2 increased commensurately. It is striking
that both growth defects and DNA damage caused by superoxide ensue from
its ability to damage a subset of iron–sulfur clusters.
803 citations