Mutants ofEscherichia coli sensitive to hydrogen peroxide

Barbado, Casimiro; Ramírez, Manuel; Blanco, M. Angel; López-Barea, Juán; Pueyo, Carmen

doi:10.1007/bf01577723

Cited by 14 publications

(9 citation statements)

References 10 publications

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“…In fact, glutathione appears dispensable in the defense of E. coli against H202 (14). Although other metabolic pathways for H202 detoxification may exist in E. coli (15,16), the quantitative importance of these alternative mechanisms is unclear. Therefore, that microbial catalase is reported to be an effective defense against relatively high concentrations of exogenous H202 is not surprising (17)(18)(19)(20)(21)(22)(23)(24). These earlier findings are, however, puzzling when one compares the ready diffusibility of H202 with the small cellular dimensions of most microbes.…”

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confidence: 48%

“…The accumulation of H202 by streptococci in aerobic culture was estimated by inoculating stationaryphase organisms into M9 medium/50 mM glucose at a concentration of 5 x 109 colony-forming units per ml (OD650 = 1.0). The culture was aerated (12 (17)(18)(19)(20)(21)(22)(23)(24), cat(+) and cat(-) E. coli should be equally susceptible to H202-mediated cytotoxicity. To test this hypothesis, we exposed dilute (500 organisms per ml) suspensions of stationary-phase cat(+) and cat(-) E. coli to 1.0 mM H202 and followed the kinetics of replication inactivation (hereafter referred to as killing).…”

Section: Methodsmentioning

confidence: 99%

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Multicellular oxidant defense in unicellular organisms.

Eaton

1992

Proc. Natl. Acad. Sci. U.S.A.

126

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Although catalase is thought to be a major defense against hydrogen peroxide (H202), the (17) were provided by P. C. Loewen (University ofManitoba). E. coli were grown statically in brain heart infusion broth or M9 minimal salts medium supplemented with 10 mM glucose (M9/glucose) (25) at 370C in room air overnight (18-20 hr) to early stationary-phase (OD600 = 2.2-2.4). Thiamin was not added to the growth medium because these strains of E. coli were phenotypically Thi+, probably from a spontaneous Thi--Thi+ reversion. Bacteria in midexponential growth were obtained by diluting these stationary-phase cultures 1:100 in prewarmed fresh brain heart infusion broth and incubated further until the OD6(o of the culture reached 0.5-1.0. Cells were harvested from the culture by centrifugation (10,000 x g for 10 min at 20C), washed thrice in cold phosphate-buffered saline (140 mM NaCI/10 mM Na2HPO4/10 mM KH2PO4, pH 7.2) or in M9/glucose before being resuspended in the indicated buffers, and the absorbance at 600 nm was measured. The correlation of OD6(, with bacterial concentration was affirmed by direct microscopic counts and by colonyforming units obtained by growth on LB agar. Bacterial stocks were maintained on LB agar plates, stored at 2-4C. The catalase status of colonies was constantly monitored by direct catalase assay and by H202 drop tests.Streptococcal strains were obtained from the American Type Culture Collection (Streptococcus sobrinus; 33478), G. Germaine (University of Minnesota) (Streptococcus mitis, 9811), and E. L. Thomas (University of Tennessee) (Streptococcus mutans OMZ176 and S. mutans GS5). Organisms were cultured in Todd-Hewitt broth statically at 370C either in room air (S. mutans) or in a candle extinction jar (S. sobrinus and S. mitis) and washed as described for E. coli.H202 King of E. cofi. Suspensions of E. coli were diluted to the concentration of 5 x 107 cells per ml (concentrated) or 500 cells per ml (dilute) in M9/glucose and prewarmed to 370C before being exposed to H202 at the indicated concentrations. Bacterial viability, or, more accurately, ability ofthe bacteria to replicate, was assayed by pour-plating diluted aliquots (in triplicate) of the H202/bacteria mixture in bovine catalase-supplemented (100 units per ml) LB agar at different times during incubation. Killing experiments were performed not only in pure cat(+) and pure cat(-) E. coli suspensions but also were carried out in mixtures of cat(+) and cat(-) E. coli as indicated. To differentiate cat(+) from cat(-) E. coli after H202 exposure of such mixtures, bacteria ofboth strains were made resistant to ampicillin by pUC118 (a derivation of Abbreviations: LB agar, Lennox L agar; M9, minimal salts medium; M9/glucose, M9/10 mM glucose; cat(-), catalase-deficient mutant; cat(+), wild-type strain CSH7. *To whom reprint requests should be addressed. 7924The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S...

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confidence: 48%

Section: Methodsmentioning

confidence: 99%

Multicellular oxidant defense in unicellular organisms.

Eaton

1992

Proc. Natl. Acad. Sci. U.S.A.

126

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“…In this bacterium, GSH, GR and glutaredoxin are required for the reduction of ribonucleotides (Holmgren, 1985). Barbado et al (1983) demonstrated that a bacterial mutant deficient in GR activity had increased sensitivity to H2O2 relative to a catalase-deficient parental strain. Furthermore, there is evidence from work with E. coli mutants that GR is not essential for the reduction of glutathione in E. coli (Tuggle & Fuchs 1985) and that bacterial growth is unchanged in GR-deficient E. coli cells (Perham 1987).…”

Section: Results In Bacteriamentioning

confidence: 99%

Protection against oxygen radicals: an important defence mechanism studied in transgenic plants

Foyer¹,

Descourvieres²,

Kunert

1994

Plant Cell & Environment

1,107

566

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Free radicals and other active derivatives of oxygen are inevitable by‐products of biological redox reactions. Reduced oxygen species, such as hydrogen peroxide, the superoxide radical anion and hydroxyl radicals, inactivate enzymes and damage important cellular components. In addition, singlet oxygen, produced via formation of triplet state chlorophyll, is highly destructive. This oxygen species initiates lipid peroxidation, and produces lipid peroxy radicals and lipid hydroperoxides that are also very reactive. The increased production of toxic oxygen derivatives is considered to be a universal or common feature of stress conditions. Plants and other organisms have evolved a wide range of mechanisms to contend with this problem. The antioxidant defence system of the plant comprises a variety of antioxidant molecules and enzymes. Considerable interest has been focused on the ascorbate‐glutathione cycle because it has a central role in protecting the chloroplasts and other cellular compartments from oxidative damage. It is clear that the capacity and activity of the antioxidative defence systems are important in limiting photo‐oxidative damage and in destroying active oxygen species that are produced in excess of those normally required for signal transduction or metabolism. In our studies on this system, we became aware that the answers to many unresolved questions concerning the nature and regulation of the antioxidative defence system could not be obtained easily by either a purely physiological or purely biochemical approach. Transgenic plants offered us a means by which to achieve a more complete understanding of the roles of the enzymes involved in protection against stress of many types: environmental and man‐made. The ability to engineer plants which express introduced genes at high levels provides an opportunity to manipulate the levels of these enzymes, and hence metabolism in vivo. Studies on transformed plants expressing increased activities of single enzymes of the antioxidative defence system indicate that it is possible to confer a degree of tolerence to stress by this means. However, attempts to increase stress resistance by simply increasing the activity of one of the antioxidant enzymes have not always been successful presumably because of the need for a balanced interaction of protective enzymes. The study of these transformed plants has allowed a more complete understanding of the roles of individual enzymes in metabolism. Protection against oxidative stress has become a feasible objective through the application of molecular genetic techniques in conjunction with a biochemical and physiological approach.

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“…Glutathione reductase activity was assayed at 30°C as described by Pinto et al [1984]. Catalase activity was determined at 25°C following the procedures described by Beers and Sizer [1952] and Barbado et al [1983]. Superoxide dismutase activity was assayed at 25°C as described by Floh6 and Otting [ 19841.…”

Section: Cell-free Extracts and Biochemical Determinationsmentioning

confidence: 99%

Metal, mutagenicity, and biochemical studies on bivalve molluscs from Spanish coasts

Rodríguez‐Ariza

Abril

Navas

et al. 1992

Environ and Mol Mutagen

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Three species of marine bivalve molluscs (Chamelea gallina, Ruditapes decussatus, and Crassostrea gigas) have been studied in order to evaluate the levels of pollution on the South Atlantic Spanish littoral. Several transition metals (Cu, As, Cd, Sn, Hg, Pb) were determined as a general index of total contamination. Animals from putative contaminated areas exhibited higher metal contents than those from cleaner waters. C. gigas showed 5-20-fold higher total metal content than the other two species. The mutagenicity of ethanolic extracts was assayed by using both the His reversion and the Ara forward mutation tests. Mollusc tissues from the three species did not contain genotoxins active on TA98 (frameshift mutations) or TA100 (mainly G:C base-pair substitutions), but did contain direct-acting genotoxins of a polar nature and oxidative type. This was based on the following observations: 1) mammalian metabolic activation was not required for mutagenicity, 2) mutagens were eluted with the polar fraction from XAD-2 columns, and 3) mutagenic responses were observed with Salmonella typhimurium TA102 (A:T base-pair substitutions; sensitive to oxidative damages) and Escherichia coli catalase-deficient (AraR forward mutations) strains. No relevant differences were found in the mutagenicity of mollusc extracts from areas with different pollution levels. Otherwise, our data suggest that, in general, animals living in contaminated environments had fewer genotoxins of oxidative type than those from less polluted areas. Such a result might be explained by the observation of increased levels of a number of detoxifying and antioxidant enzymes, such as glutathione-S-transferase, glutathione-peroxidase, catalase, and superoxide dismutase. Thus, contaminated animals seem to be better protected against the oxidative damages induced by metals, in agreement with their lower malondialdehyde levels. To what extent the responsible mutagenic compounds are of endogenous origins, or "Nature's pesticides" (the major toxic chemicals ingested by phytoplankton filter-feeders), and/or the result of human activities remains to be determined.

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Mutants ofEscherichia coli sensitive to hydrogen peroxide

Cited by 14 publications

References 10 publications

Multicellular oxidant defense in unicellular organisms.

Multicellular oxidant defense in unicellular organisms.

Protection against oxygen radicals: an important defence mechanism studied in transgenic plants

Metal, mutagenicity, and biochemical studies on bivalve molluscs from Spanish coasts

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