Role for the oxyS Gene in Regulation of Intracellular Hydrogen Peroxide in Escherichia coli

Bacterial strains and experimental procedures.The strains ofE. coli used in this study are listed in Table1. Strains BGF416 and BGF420 were constructed by transduction (18) of the ΔoxyS2::Cm allele from strain GS035 into strains AB1157 and BGF611, respectively. Plasmid poxyS has the oxySgene under the control of the tac promoter in a multicopy vector; even without induction by isopropyl-β-d-thiogalactoside, the level of theoxyS transcript expressed from poxyS is similar to the level seen in H₂O₂-treated wild-type E. coli (1). Plasmid psyxO has the gene in the reverse orientation. Cells were inoculated into Luria-Bertani (LB) broth (18) containing the appropriate antibiotic and incubated overnight at 37°C with gentle shaking (100 rpm). For experimental measurements, the saturated cultures were diluted 100-fold into fresh LB broth and incubated at 37°C for 3 h (optical density at 600 nm, ∼1). Antibiotics were used at the following concentrations (in micrograms per milliliter): tetracycline, 12.5; streptomycin, 50; chloramphenicol, 25; and ampicillin, 100. The intracellular concentration of H₂O₂ was assessed with peroxidase-mediated scopoletin oxidation as previously described (9). Total catalase activity was assayed by monitoring the disappearance of H₂O₂ at 240 nm in cell homogenates as described previously (8, 23) and normalized to the protein concentration determined with bovine serum albumin as the standard (15). The rate of H₂O₂production was calculated from the experimental values for H₂O₂ and catalase concentrations as previously described (3, 8). The rate of O₂⁻production was measured in membrane preparations by monitoring the superoxide dismutase (SOD)-sensitive rate of cytochrome creduction at 550 nm (ɛ_reduced − ɛ_oxidized = 21 mM⁻¹ cm⁻¹) (2, 12). The reaction mixtures consisted of 50 mM potassium phosphate buffer (pH 7.4), 20 μM cytochrome c, 100 μM NADH, and membrane protein (∼0.2 mg/ml), with or without 50 U of bovine CuZn SOD. The rate of respiration by cell cultures was measured with a Clark-type electrode (5). Three-hour cultures were placed in the chamber of an oxygraph, and O₂ concentration was monitored for 5 to 10 min. Values are expressed in nanoatoms of O₂ per minute per 10⁶ cells. The temperature was 37°C. The cellular sensitivity to H₂O₂was assessed on plates. Three-hour cultures were plated on LB plates, and a filter disk (10-mm diameter, Whatman no. 1) was soaked with 150 mmol of H₂O₂ in water and placed in the center of the plate. After 12 to 16 h, the diameter of growth inhibition was measured (11). The frequency of spontaneous mutations to rifampin resistance was measured by plating on LB plates containing 125 μg of rifampin per ml; Rif^r colonies were scored after 24 h of incubation at 37°C (11) and normalized to the number of cells plated (8).

Regulation of intracellular concentrations of hydrogen peroxide.The level of H₂O₂ in theoxyS-deficient strain BGF416 (in exponential phase) was ∼2-fold higher than the level measured for the parentaloxyS⁺ strain (Fig.1A), consistent with regulation mediated by oxyS. This increase was similar to that observed for akatG strain and less than that for a ΔoxyRSstrain (Fig. 1A). We therefore tested whether mutations in bothoxyS and katG would act synergistically to elevate the intracellular H₂O₂ concentration. A ΔoxyS katG double mutant (BGF420) had an ∼3-fold increase in H₂O₂ concentration (Fig. 1A), which shows that katG and oxyS play independent roles in the OxyR-dependent regulation of H₂O₂.

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Fig. 1.

Effect of genetic deficiency in oxyS orkatG on the intracellular concentration of hydrogen peroxide or catalase activity. (A) Steady-state H₂O₂concentrations in intact cells. (B) Total catalase activity in cell extracts. Values are the means of four to six independent experiments ± SEMs. Strain abbreviations: WT1, AB1157 (oxyRS⁺ katG⁺); oxyS, BGF416 (oxyR⁺ oxyS2::Cm);katG, BGF611 (oxyRS⁺katG17::Tn10); oxyS katG, BGF420 (oxyR⁺ oxyS2::CmkatG17::Tn10); WT2, RK4936 (oxyRS⁺ katG⁺); oxyR, TA4112 [Δ(oxyRS-btuB)3].

Increased steady-state concentrations of H₂O₂can result from decreases in the rate of its decomposition or increases in the rate of H₂O₂ production. The catalase–HP-I is the major H₂O₂-decomposing activity in exponentially growing E. coli (8, 14), and it is possible that oxyS regulateskatG by a posttranscriptional mechanism (1) distinct from the transcriptional activation of katGmediated by OxyR. However, the deletion of oxyS did not alter the total catalase activity in either the wild-type or thekatG background (Fig. 1B). As previously reported, thekatG mutant strain had an ∼70% lower catalase activity due to the lack of a functional catalase–HP-I (the remainder is thekatE-encoded enzyme [8, 14]).

Since oxyS did not seem to affect expression of the primary H₂O₂ scavenging activity, we hypothesized thatoxyS might influence the cellular generation of H₂O₂, most of which arises from the dismutation of O₂⁻ by SOD (10). We therefore tested the ΔoxyS, katG, and ΔoxyRSstrains for changes in the rate of O₂⁻production. The rates of superoxide anion production in membrane preparations (12) increased 6-fold in the ΔoxySstrain and 2.5-fold in the ΔoxyRS strain compared to their wild-type counterparts (Table 2). Expression of oxyS from the multicopy plasmid poxyS (1) complemented the ΔoxyS phenotype by preventing the increased superoxide production (Table 2). In the wild-type strain, the rate of O₂⁻ production was not changed significantly by poxyS, the vector plasmid, or a plasmid with oxyS in the reverse orientation (psyxO [1]) (Table 2 and data not shown).

Unexpectedly, there seemed to be a small decrease in O₂⁻ production in the katG strain BGF611, but this was not statistically significant (Table 2). However, this strain is oxyR proficient, and it may be that there is diminished O₂⁻ production due to OxyR-dependent induction of oxyS as a result of the increased H₂O₂ concentration in this strain (Fig. 1A). Indeed, forcing the increased expression of oxySby itself with poxyS was sufficient to decrease O₂⁻ production in BGF611 as well as in the ΔoxyRS strain TA4112 (Table 2).

The rates of H₂O₂ production were calculated from the experimental values for the steady-state H₂O₂ concentration and the total catalase concentration (3, 8). Deletion of oxyS in strain BGF416 increased H₂O₂ production 1.7-fold, compared to a 6-fold increase in O₂⁻generation in this strain (Table 2). We have not determined whether H₂O₂ production is decreased by poxyS in the ΔoxyS strain. In principle, the 2:1 O₂⁻-H₂O₂ stoichiometry of superoxide dismutation (3) would predict a maximal threefold increase in H₂O₂ generation in the ΔoxyS strain. However, reactions other than SOD can consume O₂⁻ (7), and such reactions may contribute to this difference. The lack of a significant increase in H₂O₂ production in the ΔoxyRSstrain (TA4112) (Table 2) could also be related to non-SOD pathways consuming superoxide.

The metabolic production of O₂⁻ and H₂O₂ in E. coli depends on the number of active respiratory chain units per cell and on the energetic state of the chains, which can be altered by directing the electron flux through components with higher or lower energetic efficiency (coupled versus uncoupled components) (10, 22). Therefore, changes in growth conditions or in the proportions of coupled and uncoupled components would determine the rate of free radical production at the respiratory chain. The effect of oxyS on the metabolic production of superoxide and hydrogen peroxide reported here could be due to this type of regulation. Three of the eightoxyS-regulated genes reported so far,fhlA, gadB, and uhpT, are involved in energy metabolism, although they have not been related directly to electron transport processes. The fhlA gene encodes a transcriptional activator of the hydrogenase pleiotropic operon (hypABCDE) (16, 17). The gadB gene encodes a glutamic acid decarboxylase (21), anduhpT encodes the sugar phosphate transporter (6, 13). Perhaps oxyS represses some energy pathways that leak more O₂⁻ and are deleterious to cells under oxidative stress.

To test the hypothesis that oxyS deletion affects the energy metabolism of the cells, we measured the rates of respiration in intact wild-type and ΔoxyS cells. Oxygen uptake by exponentially growing cells was significantly increased in the ΔoxySstrain (mean rate of O₂ consumption ± standard error of the mean [SEM] for three independent experiments, 0.77 ± 0.02 nanoatoms of O₂/min/10⁶ cells; wild type, 0.51 ± 0.06 nanoatoms of O₂/min/10⁶cells), and this effect was largely suppressed by introduction of the multicopy plasmid poxyS (0.63 ± 0.07 nanoatoms of O₂/min/10⁶ cells). As seen for the rate of O₂⁻ production (Table 2), expression of additional oxyS from poxyS in the wild-type strain may slightly decrease the rate of respiration (from 0.51 ± 0.06 to 0.44 ± 0.04 nanoatoms of O₂/min/10⁶cells). We therefore conclude that oxyS does affect cellular respiration. More-detailed studies will be required to delineate the mechanism underlying this regulation.

We previously reported that twofold increases in the rate of production of H₂O₂ are enough to trigger a substantial OxyR-dependent transcription of katG (10) and that twofold increases in the steady-state concentration of H₂O₂ significantly increased the frequency of spontaneous mutation (8). We therefore tested whether the lack of oxyS or oxyS and katG resulted in phenotypic changes. We measured the frequency of spontaneous mutation (a sensitive marker of oxidative DNA damage [8]) and the cellular sensitivity to exogenous H₂O₂ in the various strains. Compared to the wild-type strain (AB1157), both parameters were unchanged in the ΔoxyS strain (BGF416), but there were significant increases in spontaneous mutation (2.8-fold) and H₂O₂ sensitivity (1.5-fold) in the ΔoxyS katG strain (BGF420) (Table3). Multicopy oxyS had an antimutagenic effect in the ΔoxyS and katGstrains and possibly in the oxyRS⁺ strain (Table3). Multicopy oxyS in the double mutant strain BGF420 (ΔoxyS katG), decreased the frequency of spontaneous mutation to almost the same value as obtained the katGstrain without poxyS (Table 3). Interestingly, multicopyoxyS did not complement the mutator phenotype of the ΔoxyRS strain (Table 3). This result suggests that, in addition to oxyS, either katG or some other OxyR-dependent activity is critical for limiting mutagenesis by endogenous H₂O₂. Alternatively, oxySin poxyS is not regulated in response to oxidative stress (1), so the level of the RNA does not adjust to changing H₂O₂ production.

Multicopy oxyS did not significantly alter the sensitivity to hydrogen peroxide in any of the strains listed (data not shown), in agreement with the lack of regulation of katG byoxyS (Fig. 1B).

Our results show an oxyS-dependent regulation of the intracellular production of oxygen free radicals. In this way, theoxyS pathway and the OxyR-dependent induction of catalase–HP-I would provide two independent and complementary mechanisms to limit the levels of H₂O₂ during aerobic growth and possibly under oxidative stress. Elimination of either pathway is still compatible with almost normal aerobic growth, but elimination of both oxyS and katG produces a ΔoxyRS-like phenotype with significant increases in the frequency of spontaneous mutations and sensitivity to H₂O₂. Defining the mechanism(s) by whichoxyS limits O₂⁻ production in respiring E. coli may reveal general pathways to avoid oxidative damage.