©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Metabolic Sources of Hydrogen Peroxide in Aerobically Growing Escherichia coli(*)

Beatriz González-Flecha (§) , Bruce Demple (¶)

From the (1) Department of Molecular and Cellular Toxicology, Harvard School of Public Health, Boston, Massachusetts 02115

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Exposure of cells to hydrogen peroxide (HO) mediates adaptive responses or oxidative damage, depending on the magnitude of the challenge. Determining the threshold for peroxide-mediated oxidative stress thus requires quantitation of the changes in endogenous HO production. The intracellular steady-state concentrations of HO were measured in intact Escherichia coli under different conditions. Compounds that block electron transport at NADH dehydrogenase (rotenone) or between ubiquinone and cytochrome b (antimycin) showed that univalent reduction of O can occur at these sites in vivo to form superoxide anion (O), in agreement with reports for mammalian mitochondria. Mutational inactivation of different components of the respiratory chain showed that HO production also depended on the energy status of the cell and on the arrangement of respiratory chain components corresponding to particular growth conditions. Production rates for O and HO were linearly related to the number of active respiratory chains that reached maximal values during exponential growth. In the strains defective in respiratory chain components, catalase activity was regulated to compensate for changes in the HO production rates, which maintained intracellular HO at 0.1-0.2 µM during aerobic growth over a wide range of cell densities. The expression of a katG`::lacZ fusion (reporting transcriptional control of the catalase-hydroperoxidase I gene) was increased by HO given either as a pulse or as a steady production. This response not only depended on the type and severity of the stimulus but was also strongly influenced by the growth phase of the cells.


INTRODUCTION

Reactive by-products of oxygen, superoxide anion radicals (O), hydrogen peroxide (HO), and hydroxyl radicals (HO), are derived from sequential univalent reductions of molecular oxygen. These agents are produced continuously in aerobically growing cells (Chance et al., 1979). In eukaryotic cells, the respiratory chain and cytochrome P-450 seem to be the significant intracellular sources of O (Chance et al., 1979). HO is produced by the superoxide dismutase-catalyzed dismutation of O in mitochondria and in the cytosol and by flavin oxidases in peroxisomes (Chance et al., 1979). Intracellular O and HO are kept at acceptably low concentrations by the action of antioxidant enzymes such as superoxide dismutase, catalase, and other peroxidases (Chance et al., 1979; Sies, 1991).

``Oxidative stress'' refers to imbalances between the production and disposal of oxygen radicals (Gerschman et al., 1954; Sies, 1991). Oxidative stress has been associated with aging (Harman, 1991), carcinogenesis (Cerutti, 1985), and diverse clinical situations such as Alzheimer's disease (Luft, 1994; Yan, 1994) and cell damage due to ischemia-reperfusion (González-Flecha et al., 1993). Oxidative stress is also exploited as a cytotoxic weapon during phagocytosis (Babior, 1991). HO can be excreted by the acatalasic bacterium Streptococcus sanguis in amounts sufficient to prevent the growth of other organisms (Holmberg and Hallander, 1973). In plants, hydrogen peroxide from an oxidative burst in pathogen-infected cells may act as a signal for the induction of resistance in adjacent cells (Levine et al., 1994). Environmental agents such as ionizing or near-UV radiations or numerous compounds that generate intracellular O (e.g. paraquat, plumbagin, and menadione) can cause oxidative stress (Sies, 1991; Kappus and Sies, 1981).

Genetic responses to oxidative stress occur in bacteria (Demple, 1991), yeast (Jamieson, 1994), and mammalian cell lines (Amstad et al., 1994; Keyse and Tyrrel, 1989; Schulze-Osthoff and Baeuerle, 1994). Escherichia coli cells possess a specific defense against peroxides mediated by the transcriptional activator OxyR and another against superoxide, controlled by the two-stage soxRS system (Hidalgo and Demple, 1995). The OxyR regulon includes catalase-hydroperoxidase I, encoded by katG, a NADPH-dependent alkyl hydroperoxidase, encoded by ahpFC, glutathione reductase, encoded by gorA, a protective DNA binding protein, encoded by dps, and several other genes (Hidalgo and Demple, 1995). The expression of these genes is elevated in E. coli exposed to 5-100 µM HO (Demple and Halbrook, 1983; Storz et al., 1990; Demple, 1991; Hidalgo and Demple, 1995).

Despite intensive study of adaptive responses of bacteria to oxidative stress as cited above, no systematic analysis of the effects of growth state or the physiological threshold of oxidative stress required to trigger these responses has been reported. Imlay and Fridovich (1991a) estimated a steady-state O concentration of 10M in E. coli by measuring the rate of superoxide production in isolated membranes. We present here a study of the physiological production and disposal of HO in intact E. coli, measured by the rapid equilibration of intracellular HO (which passes freely through membranes) (Chance et al., 1979) with the surrounding medium. We have also analyzed intracellular sources of oxygen radicals, the effect of different types of oxidative stress on the steady-state HO concentration, and the growth-dependent variation in the extent of oxyR-regulated HO response.


MATERIALS AND METHODS

Reagents

Antimycin A, ampicillin, rotenone, D-glucose, bovine cytochrome c type III, tetracycline, NADH, succinate, carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP)() , scopoletin, bovine serum albumin, horseradish peroxidase type VI, glucose oxidase, bovine superoxide dismutase, and bovine catalase were purchased from Sigma.

Bacterial Strains and Growth Conditions

lists the strains used in these studies. The bacteriophage RS45[ bla`-`lacZ lacYA] (Simons et al., 1987) was used to insert the katG`::lacZ fusion into chromosomal DNA by recombination in MC4100 carrying plasmid pAQ24 (katG`::lacZ) (Tartaglia et al., 1989). The construction was carried out as described by Simons et al.(1987). Briefly, a culture of MC4100/pAQ24 was infected with a RS45 lysate; recombination between RS45 and the plasmids within the homologous lacZYA-bla region yielded a [(katG`::lacZ)] fusion and a still incomplete bla gene. Recombinant phages were screened and identified by their Lac phenotype (blue plaques on LB agar supplemented with 40 µg/ml 5-bromo-4-chloro-3-indol--D-galactopyranose). A plaque-purified isolate, AQ24, was integrated into the att site in the chromosome of the Lac strains RK4936 and TA4112. The resulting lysogens, BGF931 and BGF933, were identified by their Lac Amp phenotype.

For growth, strains were inoculated into LB broth (Miller, 1992) containing the appropriate antibiotic and incubated at 37 °C for 12-16 h with gentle shaking (200 rpm). The saturated cultures were diluted 100-fold into fresh LB and incubated for the indicated times at 37 °C with shaking at 200 rpm in flasks of volume 10-20-fold greater than the culture. Antibiotics were used at the following concentrations (in µg/ml): ampicillin, 100; tetracyclin, 12.5; kanamycin, 50; and chloramphenicol, 25.

Hydrogen Peroxide and Superoxide Anion Measurements

Intracellular concentrations of HO and HO production rates were measured as described previously (González-Flecha and Demple, 1984) by the horseradish peroxidase-scopoletin method (Boveris, 1994). Antimycin and rotenone, inhibitors of electron transport, and FCCP, an uncoupler, were used at final concentrations of 1, 0.5, and 0.2 µM, respectively. Bacterial suspensions (10 cells ml in phosphate-buffered saline (PBS)) were incubated with the indicated compound for 10 min at 0-4 °C, washed twice with fresh PBS, and assayed as described (González-Flecha and Demple, 1994). The rates of HO production were expressed as µM/s by assuming a cellular volume of 3.2 10 liter (Imlay and Fridovich, 1991a). Superoxide anion production was measured in isolated membranes prepared by a standard procedure (Imlay and Fridovich, 1991a). The rate of O production was measured by following the superoxide dismutase-sensitive rate of cytochrome c reduction at 550 nm ( - = 21 mM cm) (Boveris, 1984). The reaction mixture consisted of 50 mM potassium phosphate buffer (pH 7.4), 20 µM cytochrome c, 100 µM NADH, and 0.2 mg/ml of membrane protein, with or without 50 units of bovine copper-zinc superoxide dismutase.

Enzymatic Activities

-Galactosidase activity in sodium dodecyl sulfate-CHCl-treated cells was determined as described by Miller(1992). Two different approaches were used to assay the total catalase concentration of the cells. Catalase activity in cell lysates was determined as described previously (González-Flecha and Demple, 1994) and normalized to either the protein content of the lysate or the number of cells extracted. Protein concentration in the extracts was measured by the method of Lowry (Lowry et al., 1951) using bovine serum albumin as standard. All the measurements were carried out in a Perkin-Elmer Lambda 3A UV/Vis spectrophotometer. In the second approach, catalase activity was measured by following the rate of elimination of HO by cells suspended in PBS containing 2 mM HO. The HO concentrations in the supernatants were determined by the horseradish peroxidase-scopoletin method. NADH-cytochrome c reductase activity was measured in bacterial membrane preparations (0.3-0.5 mg protein/ml) by following the superoxide dismutase-insensitive cytochrome c reduction as described above but using 50 µM cytochrome c in the presence of 50 units of bovine copper-zinc superoxide dismutase (Trumpower and Simmons, 1979).

Hydrogen Peroxide and Glucose/Glucose Oxidase Treatments

Overnight cultures of BGF931 and BGF933 were diluted 1:100 in fresh LB and grown for 1, 3, and 7 h. For the pulse-type treatment with HO, cultures at the indicated times were treated with different concentrations of HO, and the -galactosidase activity directed by katG`::lacZ was followed for 30 min after addition of HO. In the case of treatment with glucose/glucose oxidase to generate a continuous flux of HO, cultures at the indicated times were supplemented with 10 mM glucose, and various amounts of glucose oxidase, and -galactosidase activity were followed for 60 min after addition of glucose oxidase.

Statistics

Results are indicated as the mean value of four independent experiments ± S.E. Statistical significance of differences was analyzed by ANOVA, followed by Dunnett's test for other comparisons (Winer, 1971).


RESULTS

Hydrogen Peroxide Generation and Elimination in E. coli

The metabolic production of hydrogen peroxide in intact bacteria was evaluated initially in exponentially growing AB1157 after suspension of the cells in fresh PBS free of HO. The HO concentration measured in the extracellular medium increased with time, reaching a plateau at 0.15 ± 0.01 µM after about 5 min for a cell density of 10 cells ml (Fig. 1A). Bacteria suspended at 10- or 100-fold lower densities approached the same plateau, but with slower kinetics (Fig. 1A). Cells resuspended in PBS initially containing 1.5 µM HO rapidly destroyed the extracellular HO and again reached a plateau concentration of 0.15 µM (Fig. 1B). The combined results correspond to the equilibration between intra- and extracellular HO and indicate an intracellular steady-state concentration of HO of 0.15 µM in exponentially growing E. coli.


Figure 1: Hydrogen peroxide production and elimination by E. coli. HO concentration was measured in the extracellular medium of AB1157 incubated in PBS supplemented with 0 (A) and 1.5 µM (B) HO. A, cell densities after resuspension were 10 cells ml (opentriangles), 10 cells ml (opencircles), 10 cells ml (filledtriangles), and no cells (filledcircles). B, cell density after resuspension was 10 cells ml.



Intracellular Sources of Hydrogen Peroxide

The contribution of cytosolic enzymes to the total production of HO in intact E. coli was estimated by using the uncoupler of electron transport FCCP, which reduces to negligible levels the rate of HO production associated with mitochondrial respiratory chain (Boveris and Chance, 1973). Addition of FCCP to E. coli AB1157 decreased the HO steady-state concentration and production rate to about one-seventh the value of untreated cells (). Thus, the production of oxygen free radicals by the respiratory chain in intact bacteria accounts for most of the HO generation.

To identify specific sites of the respiratory chain at which single electrons might leak to form O, inhibitors of electron transport were used. The maximal rate of O production by autoxidation of the flavin-semiquinone of NADH-dehydrogenase (FMNH) was measured by supplementing E. coli membrane preparations with NADH and the site I inhibitor rotenone (Boveris and Chance, 1973). Similarly, the maximal production of O by ubisemiquinone (UQH) autoxidation was measured by supplementing E. coli membranes with NADH and the site II inhibitor antimycin (Boveris and Chance, 1973). The latter rate was assumed to represent the sum of the rates of O production at UQH plus FMNH. The effect of these inhibitors on O and HO production was also tested for intact bacteria by following the generation of HO by the cells (). As described previously for mitochondria (Turrens and Boveris, 1980), both rotenone and antimycin showed a biphasic effect on HO production peaking at a concentration of 0.5 µM for rotenone and 1 µM for antimycin, when each compound blocked 50% of the electron flux through the respiratory chain, as evaluated by the NADH-cytochrome c reductase activity (data not shown). Using these concentrations, both the rate of O production in E. coli membranes and the rate of HO production in intact bacteria were increased 1.5-2-fold after rotenone treatment, and 2-3-fold after antimycin treatment (). These results indicate that both NADH dehydrogenase and ubiquinone have significant potential to leak electrons to form O in E. coli.

Hydrogen Peroxide and Superoxide Production during Aerobic Growth

The rate of HO production during aerobic growth in rich medium showed a biphasic profile with a progressive increase during exponential growth, followed by a decrease after cessation of cell growth (Fig. 2). To estimate the contribution of oxygen free radicals produced at the respiratory chain level to the total oxygen free radical production, we studied the relationship between the rate of O production and the number of active respiratory chains. The O production by isolated membranes was linearly related to the number of respiratory chain units/cell, estimated by the NADH-cytochrome c reductase activity (Fig. 3).


Figure 2: Hydrogen peroxide production during aerobic growth. Overnight cultures of E. coli AB1157 were diluted 1:100 in fresh LB and incubated at 37 °C. At different time points samples were taken to measure OD (opencircles) and assay HO production rate (filledcircles).




Figure 3: Superoxide anion production as a function of the number of respiratory chain units in isolated membranes.



Effect of Respiratory Chain Mutations on Intracellular HOGeneration

It has been reported that mutants defective in the biosynthesis of ubiquinone or menaquinone are also defective in the ability to induce the synthesis of catalases during aerobic growth (Hassan and Fridovich, 1978). To test the consequences of specific defects in NADH dehydrogenase or cytochrome oxidase on the production of HO by E. coli, a series of isogenic strains with mutations affecting these components was assayed for HO steady-state and catalase concentrations. The rate of HO production by GO103 (deficient in cytochrome oxidase d) was significantly lower than the rate of HO production by either GO104 (deficient in cytochrome oxidase o) or the parental strain GR70N (I). The production of HO was also significantly lower in strains MWC215 (deficient only in NADH dehydrogenase 2) and MWC232 (deficient in NADH dehydrogenase 1 and 2), and significantly higher in strain MWC190 (deficient in NADH dehydrogenase 1), compared with the control strain (I). In all the strains studied, the catalase concentration varied directly with the rate of HO production. This balance kept the steady-state concentration of HO within a 2-fold range for all the strains (I) and reflects a kind of homeostasis for reactive oxygen.

Growth Phase-dependent Triggering of a Response to HO

The above results suggested an almost continuous response of E. coli to different physiological rates of HO generation. We therefore used a strain (BGF931) carrying a katG`::lacZ operon fusion to examine whether such a continuous response could be observed at the level of transcription of the catalase gene. We first examined the response of the reporter fusion to acute ``pulse-type'' exposures. The initial HO concentrations required for maximal induction of oxyR-dependent katG transcription varied with the growth phase and were, respectively, 15, 25, and 100 µM for 1-h, 2-h, and 3-h cultures (Fig. 4). The 7-h culture had a somewhat higher initial expression of katG`::lacZ but displayed a < 2-fold induction by HO over the range 2 µM to 1 mM (Fig. 4D). These HO concentrations provided about the same HO:catalase initial ratio for the 1-3-h cultures (), so that the HO concentration remained above the physiological value for 15 min (data not shown).


Figure 4: Induction of katG`::lacZ by acute HO exposure. Overnight cultures of BGF931 in LB were diluted 1:100 in fresh LB and incubated at 37 °C for 1, 2, 3, or 7 h. At the indicated times, samples were treated with the indicated concentration of HO and incubated for a further 30 min. At 5, 10, 20, and 30 min, samples were taken and assayed for -galactosidase activity. No increase in -galactosidase activity was observed in the oxyR strain BGF933 (not shown). Values in the figure correspond to maximal induction, obtained 10 min after HO addition.



To evaluate regulation of katG expression in cells subjected to a ``ramp-type'' stimulus, we subjected E. coli BGF931 to a constant and defined external flux of HO provided by glucose/glucose oxidase. The rates of HO production were chosen from Fig. 2to yield 2-fold increases in both the rate of production and the intracellular concentration of HO at any given time. A 2-fold increase was chosen to mimic the elevated HO generation observed between hours 2 and 3 of the growth curve (Fig. 2), which is associated with the induction of katG during exponential growth. As in the case of the pulse-type stimuli, the effective concentration of HO depended on the catalase concentration in the cells, and on the growth phase. Catalase activity in lag-phase cells destroyed only 18% of the HO produced by glucose/glucose oxidase (Fig. 5A). In exponentially growing cells 90% of the external flux was destroyed by intracellular catalase (Fig. 5B). Despite this difference, the 2-fold increases in HO production significantly induced katG`:lacZ expression during both the lag and the exponential phase (Fig. 6). In contrast, katG`:lacZ was hardly induced during early stationary phase by the 2-fold increase in the HO production rate (Fig. 6).


Figure 5: Time course of HO production by the glucose/glucose oxidase system. Growth conditions were as in Fig. 4. At 1 h (A) and 3 h (B), glucose (Glc) was added to a final concentration of 10 mM and followed by 0.4 mg/ml (A) or 10 mg/ml (B) of glucose oxidase (GO) to generate HO at a flux equal to the intracellular rate (Fig. 2). The amount of HO in the culture medium was measured at the indicated times.




Figure 6: Induction of katG expression by extracellular fluxes of HO.Experimental conditions were as described in Fig. 5. Values in the figure correspond to maximal induction, obtained at 15, 45, and 60 min after glucose oxidase (GO) addition to the 1-h (0.4 mg/ml), 3-h (10 mg/ml), and 7-h (60 mg/ml) cultures, respectively. No increase in -galactosidase activity was observed in BGF933 (not shown).




DISCUSSION

The experiments presented here indicate that the major source of HO in intact E. coli is probably the respiratory chain, which can account for as much as 87% of the total HO production. Similar to eukaryotic mitochondria, the leakage of single electrons from the bacterial respiratory chain was observed at the NADH dehydrogenase and ubiquinone sites. Accordingly, changes both in the number of respiratory chain units/cell and in the composition of the electron transport chain affected the rate of hydrogen peroxide production. Our results showed that the rate of HO production changes dramatically during aerobic growth and was linearly related to the number of respiratory chain units/cell estimated by the specific activity of NADH-cytochrome c reductase. Bacteria seem to cope with this changing generation of oxygen radicals by elevating the expression of antioxidant functions, represented here by the katG-encoded catalase. Indeed, mutational suppression of catalase-hydroperoxidase I, but not of catalase-hydroperoxidase II, increased intracellular HO concentrations to 0.3 µM, which demonstrates a role for catalase-hydroperoxidase I as a defensive enzyme.()

Interestingly, the calculated rate of HO production in exponentially growing E. coli approached values similar to the rate of production in mammalian cells (4 µM s) (González-Flecha et al., 1993). This production followed the theoretical 2:1 stoichiometry for superoxide:hydrogen peroxide (2O + 2H HO + O), which indicated that most of the HO generation in E. coli arises as a by-product of O generation.

Mutational inactivation of different components of the respiratory chain also affected the rate of HO production. Elimination of either or both NADH dehydrogenases (in strains MWC190, MWC215, or MWC232) decreased the rate of HO production. NADH dehydrogenase 1 is a multisubunit complex homologous in structure and function to the eukaryotic complex I (Meinhardt et al., 1989). This enzyme contains four iron-sulfur clusters and FMN and couples its reaction to the generation of proton-motive force (Matsushita et al., 1987). NADH dehydrogenase 2, in contrast, contains FAD and no iron (Hayashi et al., 1989), and its function is not related to proton translocation (Matsushita et al., 1987). Both dehydrogenases would be likely to react with O to generate superoxide. The results presented here show that the inactivation of either NADH dehydrogenase 1 or NADH dehydrogenase 2 decreased the rate of HO production by only 25-30%. These results contrast with those of Imlay and Fridovich (1991b), in which the residual O production in membranes isolated from a strain lacking NADH dehydrogenase 2 was only 8% of that for the parental strain. However, in intact bacteria lacking the NADH dehydrogenases, reducing equivalents can and evidently do still enter the respiratory pathway at the ubiquinone level via alternative dehydrogenases, such as succinate dehydrogenase or lactate dehydrogenase (Ingledew and Poole, 1984).

HO production was also affected by changing the energy status of E. coli by directing the electron flux through components with higher or lower energetic efficiency. In isolated mitochondria HO production strongly depends upon the energy status; an ``energized'' condition (state 4, with slow O consumption and ADP phosphorylation) corresponds to a highly reduced steady state for the respiratory carriers and to a relatively high HO generation; a ``de-energized'' condition (state 3, with fast O consumption and ATP production) corresponds to a highly oxidized steady state for the respiratory chain components and to a relatively low HO production (Boveris and Chance, 1973). In our experiments, mutants utilizing the so-called ``coupled'' components of the respiratory chain (NADH dehydrogenase 1 in strain MWC215 and cytochrome bo in strain GO103) (Calhoun et al., 1993) appear to be in a de-energized condition, with the respiratory carriers largely in the oxidized state and generating HO at a relatively low rate. In contrast, the mutant strain GO104 utilizing the ``uncoupled'' cytochrome d (Calhoun et al., 1993) seems to be in an energized state with highly reduced electron transport carriers. This observation could extend to the growth of bacteria on different carbon sources or other conditions that determine different patterns of ``coupled-uncoupled'' components.

Mutational changes in the composition of the respiratory chain prompted complementary changes in catalase activity. It is worth noting that, even in the strains with a decreased rate of HO production (GO103, MWC215, and MWC232), catalase expression was regulated to keep the intracellular HO steady-state concentration at 0.1-0.2 µM. These results suggest that at least some basal induction of the oxyR regulon occurs even without exogenous HO stress, triggered by values that exceed the 0.1-0.2 µM physiological value. This represents a sensitivity 25-fold greater than previously associated with oxyR-dependent catalase induction, which occurred in response to a pulse-type exposure to 5 µM HO (Demple and Halbrook, 1983). A key difference is likely to be the more constant HO generation by respiration leakage, as in the case of the ramp-type oxidative stress, which might exert a cumulative effect by constantly activating OxyR protein. We have shown here that pulse-type stimuli may provide an increased HO concentration for only 15 min. A critical question for understanding the difference between these two situations is the half-life of activated OxyR protein, which has not been determined. A long half-life would favor more dramatic inducing effects of a modest HO pulse (1-5 µM) than would a short half-life. Important factors that could remove activated OxyR might be proteolysis and direct reversal of the activated form, which is thought to be an oxidized protein (Storz et al., 1990).

It is interesting to note that the magnitude of the HO response not only depends on the magnitude and type of stimulus but also on the growth phase, being maximal at logarithmic phase and almost negligible during early stationary phase. This observation is in good agreement with the reported resistance of bacteria to HO in stationary phase cultures (Jenkins et al., 1988; Hengge-Aronis. 1993) and with the dual regulation of katG (Ivanova et al., 1994) and dps and oxyS (Altuvia et al., 1994) by both oxyR and by the stationary phase regulator rpoS. We do not know if rpoS regulation merely supersedes that by oxyR in stationary phase cells, or whether the activity of OxyR may be less under those conditions.

  
Table: Bacterial strains used in this study


  
Table: Effect of respiratory chain blockers on HO production in intact E. coli and O production in membrane preparations

Exponentially growing E. coli AB1157 cells were incubated with FCCP, rotenone, or antimycin for 10 min, washed, resuspended in PBS, and assayed for HO or O production and catalase activity. ND, not determined.


  
Table: Hydrogen peroxide metabolism in respiratory chain mutants

Exponentially growing bacteria were assayed for HO and catalase concentrations. Cyt d and Cyt bo, deficient in cytochrome oxidases d and o, respectively; NDH-1 and NDH-2, deficient in NADH dehydrogenases 1 and 2, respectively.


  
Table: Elimination of HO by intracellular catalase in pulse-type models of oxidative stress

Overnight cultures of BGF931 in LB were diluted 1:100 in fresh LB and incubated at 37 °C. Cultures grown for 1, 2, and 3 h were treated with the concentration of HO required for maximal induction of the katG`::lacZ fusion, and the concentration of HO in the extracellular medium was followed. [HO], initial concentration of HO; elimination t, half-life of HO in the extracellular medium; [Catalase], catalase concentration before the addition of HO



FOOTNOTES

*
This work was supported by National Institutes of Health Grant CA37831 (to B. D.) and a grant from the Amyotrophic Lateral Sclerosis Association (to B. D.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
This author acknowledges the generous support of a fellowship from the Pew Charitable Trusts.

To whom correspondence should be addressed: Dept. of Molecular and Cellular Toxicology, Harvard School of Public Health, 665 Huntington Ave., Boston, MA 02115. E-mail: demple@mbcrr.harvard.edu.

The abbreviations used are: FCCP, carbonyl cyanide p-trifluoromethoxyphenylhydrazone; LB, Luria-Bertani medium; PBS, phosphate-buffered saline; FMNH, flavin-semiquinone of the NADH dehydrogenase; UQH, ubisemiquinone; GO, glucose oxidase.

B. González-Flecha and B. Demple, manuscript in preparation.


ACKNOWLEDGEMENTS

We are grateful to Dr. Robert Gennis (University of Illinois) for providing us with strains GR70N, GO103, GO104, MWC190, MWC215, and MWC232; Dr. Gisela Storz (National Institute of Child Health and Human Development) for providing plasmid pAQ24; and Dr. Nancy Kleckner (Harvard University) for providing bacteriophage RS45. We also thank Drs. Elena Hidalgo, Rafael Rodriguez Ariza, and Tatsuo Nunoshiba for helpful discussions.


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