INTRODUCTION

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Molecular oxygen chemically oxidizes redox enzymes in all aerobic organisms, generating a flux of H₂O₂ that can potentially damage the cell. The propensity of enzymes for autooxidation depends on their electronic and physical structures, and so the rate of H₂O₂ production presumably varies among organisms, depending on the types and abundance of these enzymes (12). Escherichia coli generates about 14 µM H₂O₂ per s when it grows aerobically on glucose (16).

This H₂O₂ can potentially damage enzymes by oxidizing sulfhydryl and iron-sulfur moieties, and on conversion to a hydroxyl radical it can produce mutagenic and lethal lesions (18). Therefore, microbes typically contain multiple catalases and/or peroxidases. Alkyl hydroperoxide reductase is the primary scavenger of endogenous H₂O₂ in E. coli (16). Catalase contributes little when H₂O₂ levels are low, but it becomes the more effective scavenger when H₂O₂ levels are high or, presumably, when the absence of a carbon source depletes the cell of the NADH necessary for Ahp activity. Mutants that lacked both of these scavengers accumulated 2 µM H₂O₂ and grew poorly. This, then, represents a toxic dose of H₂O₂, and it is clear that in the absence of scavengers, sufficient H₂O₂ is generated by metabolic sources to achieve it.

The concentration of H₂O₂ inside any cellular compartment depends on the rates of its influx and/or endogenous formation, balanced against the rates of scavenging and efflux. In some discussions the transit of H₂O₂ across membranes is stipulated to be so rapid that endogenous H₂O₂ effluxes from the cell before Ahp or catalase can scavenge it. If so, the only role of these enzymes would be to detoxify the environment once H₂O₂ had accumulated in it and reentered cells. For this reason it has been proposed that H₂O₂ scavenging is a communal activity (11).

A troubling consequence of such a situation would be that very dilute bacteria, having little collective scavenging activity, would be unable to detoxify an H₂O₂-containing environment enough to make it habitable. This situation could arise when bacteria enter new environments. It may also occur when pathogens are challenged with H₂O₂ inside a phagolysosome; the fact that catalase is not required for full virulence has been explained in this way (4).

However, there are problems with the idea that the intracellular H₂O₂ concentration is equivalent to that outside the cell. First, isolated cells can clearly survive the micromolar amount of H₂O₂ that exists in growth media (16), even though this is a toxic dose that inhibits scavengerless mutants. This result argues that endogenous scavengers are sufficiently active and permeability is sufficiently poor that they can lower the H₂O₂ concentration in the cell well below that of the environment. Second, catalase is compartmentalized in the lysosomes of eukaryotic cells, a site of H₂O₂ production. It seems reasonable to assume that this localization ensures that H₂O₂ is scavenged before it can exit and toxify the cell; the amount of catalase in the lysosome would be inadequate for this effect if H₂O₂ efflux were truly unlimited. More recently, it has been shown that some bacteria also compartmentalize catalases in their periplasms (2, 3, 9, 17), an effort that would be wasted if H₂O₂ rapidly equilibrated between that compartment and the cytoplasm.

This study was undertaken to measure the activities of E. coli Ahp and catalase and the permeability coefficient of H₂O₂. With this information in hand, intracellular concentrations of H₂O₂ can be predicted, and the steepness and physiological significance of transmembrane concentration gradients can be appraised. We find that scavenging processes are more rapid than the flow of H₂O₂ across membranes. As a consequence, E. coli reduces endogenous H₂O₂ to submicromolar levels, and these cells can continue to grow when environmental H₂O₂ concentrations exceed what can be tolerated internally.

	MATERIALS AND METHODS

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Chemicals, enzymes, and growth conditions are described in the accompanying report (16). When indicated, minimal glucose medium included Casamino Acids (CAA) at 0.2% (minimal glucose CAA medium) or L-amino acids at 0.5 mM (minimal glucose 20 AA medium). Tryptophan was added when CAA was used. The strains used in this study were derived from E. coli K-12 (Table 1); isogenic strains were used in all experiments.

H₂O₂ scavenging by whole cells and cell extracts. H₂O₂ was detected throughout this study using the Amplex red/horseradish peroxidase detection method (16). To compare the ability of whole cells and cell extracts to scavenge 1.5 µM H₂O₂, cultures were grown overnight in Luria broth (LB), diluted to 0.01 optical density unit (OD) at 600 nm in fresh LB, and grown for ca. six generations. Cells were then pelleted and washed twice with phosphate-buffered saline (PBS). Half the cells were resuspended in PBS (pH 7.3) at 1/20 the original culture volume, and half the cells were resuspended at 1/20 the original volume in 50 mM potassium phosphate (KPi, pH 7.8). The whole cells were then diluted to an OD of 0.030 in 5 ml of PBS, and H₂O₂ was then added to a final concentration of 1.5 µM. At various time intervals, 0.45 ml was removed, and residual H₂O₂ was quantitated by the Amplex red/horseradish peroxidase assay. The cells that had been suspended in 50 mM KPi were lysed by sonication, and cell debris was removed by centrifugation at 13,000 × g for 20 min. Cell extract was diluted, by the same ratio as the whole cells, into 5 ml of PBS, producing a cell extract that represented 0.030 OD cells. H₂O₂ was then added, and at timed intervals the remaining H₂O₂ was measured.

View larger version (19K): [in this window] [in a new window]   FIG. 1.   Decomposition of H2O2 by HPI in vitro and in vivo. (A) First-order decomposition of H2O2 by extracts from wild-type (MG1655) and HPI cells (JI364). (B) Kinetics of decomposition of H2O2 by whole Ahp HPII HPI+ (JI372) cells suspended at 0.1 OD.

Mixed-culture experiments. To monitor katG::lacZ expression in cells grown in mixed cultures, pure cultures were first grown overnight anaerobically in LB. Cultures were then diluted to 0.01 OD and grown to 0.1 OD anaerobically. These exponentially growing cultures were mixed at a 9:1 ratio of Ahp⁺ to Ahp (MC4100:LC70 and GS022:MC4100 ahp, respectively), where only one strain contained katG::lacZ. Both pure and mixed cultures were then aerated by vigorous shaking in room air. Once cultures reached 0.3 to 0.4 OD, an aliquot was removed from the mixed cultures, diluted, and plated on LB and LB plus kanamycin plates in order to determine the precise ratio of the two strains. The cultures were chilled on ice for 10 min, centrifuged, washed, and resuspended in 1/15 the culture volume in cold 50 mM KPi buffer (pH 7.0). Cells were lysed by sonication, and cell debris was removed by centrifugation at 13,000 × g for 20 min. Extracts were then assayed for -galactosidase activity (16). To calculate the specific activity of the katG::lacZ strain within the mixed culture, the protein concentration of the extract was multiplied by the fraction of cells that contained the katG::lacZ allele. Cultures were grown and assayed in duplicate.

	RESULTS

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Rationale. The concentration of hydrogen peroxide inside bacterial cells is established by the balance between the processes that generate it and those that dissipate it.

(1)

In this equation H_in denotes the intracellular concentration of H₂O₂. The fluxes (in moles per second) refer to influx into the cell from the external medium (J_influx), endogenous H₂O₂ production (J_prod), efflux out of the cell (J_efflux), and H₂O₂ decomposition by catalase (J_Kat) and Ahp (J_Ahp). In this report J will refer to the flux per single cell.

HPI activity in vivo. To obtain a term for J_Kat, catalase activity was assayed in vitro and extrapolated to the enzyme density found in vivo. The activity was primarily due to HPI, the OxyR-regulated enzyme that is encoded by katG, rather than HPII, the katE-encoded enzyme that is induced in stationary phase (13). Ahp was not active in these in vitro assays because NADH was not provided. Many catalases exhibit nonlinear kinetics when they are assayed with high (millimolar) concentrations of H₂O₂; the enzymes convert from a highly active form to a less active form during the first few minutes of the assay (15). HPI also gradually diminished in activity during exposure to millimolar H₂O₂ concentrations. However, it exhibited first-order behavior throughout the period of the assay when its activity was measured with micromolar concentrations of H₂O₂ (Fig. 1A). The inhibition by high concentrations of H₂O₂ is not an in vitro artifact, as high concentrations also inhibited in vivo in the Ahp HPII strain JI372. Larger amounts were needed to cause inhibition in vivo, because the intracellular concentrations are 10-fold less than the external concentrations (see Discussion).

1

1

(2)

H₂O₂ scavenging can be limited by its penetration into the cell. In the accompanying report we note that wild-type cells (containing both Ahp and HPI), katG mutants (containing only Ahp), and ahp mutants (containing only HPI) all degraded 1.5 µM extracellular H₂O₂ at similar rates (16). This observation is explicable in part because in the ahp mutant the OxyR protein was activated and induced the synthesis of HPI, to compensate for the lack of Ahp. However, given the likelihood that the kinetic behaviors of these enzymes differ, it seemed coincidental that at this particular H₂O₂ concentration the rates were essentially equivalent. A simplifying explanation might be that the rate-limiting step in scavenging was entry of the H₂O₂ into the cell. If all three strains contained enough scavengers to degrade >80% of the H₂O₂ before it exited, then the rates at which these cells scavenged H₂O₂ would be equivalent within the precision of our measurements. This logic also suggested that the permeability coefficient might be inferred from the rate at which the cells degraded H₂O₂.

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7

16

(3)

19

3

View larger version (18K): [in this window] [in a new window]   FIG. 2.   Decomposition of H2O2 by equivalent amounts of extracellular and intracellular HPI. Extract and whole cells of JI372 (ahpCF katE) were prepared as described in Materials and Methods, and the decomposition of 1.5 µM H2O2 was monitored.

Calculation of permeability coefficient. The value of P can be determined more precisely by comparing the rates at which extracellular and intracellular catalases degrade H₂O₂ (Fig. 2). When a cell extract was used that contained the same amount of HPI as did 0.030 OD bacteria, the rate of H₂O₂ decomposition was 12.2 nM/s, or 9.4 times more rapid than with the equivalent amount of intact cells. Thus, it is immediately apparent that the finite permeability of the membrane limits the rate at which cells scavenge H₂O₂.

View larger version (18K): [in this window] [in a new window]   FIG. 3.   Decomposition of H2O2 by Ahp in vivo. Rates of decomposition were measured in JI367 (Ahp+ HPI HPII, diamonds). Solid line, curve predicted using JAhp = 2.1 × 1018 mol/s (Hin) (Hin + 1.2 × 106 M). Note that the abscissa displays extracellular H2O2 concentrations; intracellular concentrations can be calculated from them by equation 8. Dashed line, the rate of H2O2 diffusion into the suspended cells, predicted from the permeability coefficient. Since cells can degrade H2O2 no faster than it penetrates them, this line represents the maximum possible rate of H2O2 decomposition.

(4)

12

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9

(5)

9

6

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3

3

3

(6)

Ahp activity in vivo. Ahp is the predominant scavenger of the low concentrations of H₂O₂ that are generated by endogenous metabolism. Unfortunately, its activity cannot be reliably assayed in cell extracts, because the AhpC and AhpF subunits dissociate easily (8, 14). Therefore we could quantify only the ability of intact Ahp⁺ HPI cells to scavenge exogenous H₂O₂ (Fig. 3). Scavenging was saturated when the external H₂O₂ concentration exceeded 10 µM. At lower concentrations, the rate of scavenging (data points) was close to the rate at which H₂O₂ entered the cell, as predicted by the permeability coefficient (dashed line).

18

(7)

H₂O₂ homeostasis in E. coli The intracellular steady-state concentration of H₂O₂ in growing cells is established by the relative rates of H₂O₂ influx, efflux, production, and scavenging by HPI and Ahp:

20

(8)

Physiological evidence of transmembrane concentration gradients. Equation 8 predicts that transmembrane flow is limited, so that intracellular and extracellular H₂O₂ concentrations can be substantially different. Two examples will be considered. First, because efflux is slowed by the limited permeability of the membrane, in an Ahp strain the intracellular H₂O₂ should be elevated. Indeed, the OxyR regulon is activated; induction can be observed by use of a katG::lacZ fusion (16). Interestingly, when the Ahp strain is cocultured with a scavenging-proficient strain, the fusion remains highly expressed (0.61 ± 0.04 U/mg) as it is in pure culture (0.69 ± 0.04 U/mg). However, when the converse experiment is performedwhen the fusion is in the wild-type strain of the mixed culturethe fusion is not expressed (0.12 ± 0.02 U/mg compared to 0.05 ± 0.02 U/mg in pure wild-type cultures). The implication is that the H₂O₂ concentration inside the Ahp strain is higher than that inside the wild-type strain, even when these strains are cocultured, due to the efficiency of H₂O₂ scavenging by Ahp inside the latter.

View larger version (18K): [in this window] [in a new window]   FIG. 4.   Catalase-proficient cells have a growth advantage over catalase-deficient cells in a mixed culture. JI372 (ahpCF katE) and JI377 (ahpCF katE katG) were mixed at a 9:1 ratio of JI372 to JI377 and diluted into aerobic minimal glucose CAA medium containing an additional 2 µM H2O2. The number of viable cells of each strain was then monitored by intermittent dilution and plating on selective plates.

	DISCUSSION

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Quantification of intracellular H₂O₂ stress. To characterize H₂O₂ stress in growing cells, it is necessary to quantify it. We have attempted to do so by measuring the rates at which H₂O₂ is generated and dissipated in wild-type E. coli. The rate of endogenous formation was determined in a connected study (16), and the fluxes through the three routes of dissipation, scavenging by Ahp and catalase and diffusion through the cell membrane, were measured here. The greatest surprise was that E. coli strives to keep H₂O₂ concentrations so low. It synthesizes enough Ahp that, in the absence of exogenous sources, H₂O₂ levels should rise no higher than 20 nM; when levels rise to 100 nM, E. coli increases the rates of Ahp and HPI synthesis by activating the OxyR regulon. Such vigilance may be warranted, since the 100 nM H₂O₂ that accumulates in Ahp mutants is evidently sufficient to accelerate mutagenesis (6), and 2 µM causes substantial growth inhibition (16).

Experimental support for quantitative estimates. The H₂O₂ concentrations that are predicted by equation 6 have been directly measured only in the case of Ahp Kat cultures, which accumulate H₂O₂ in the extracellular medium. However, the predictions that derive from this equation are supported by a number of experimental observations. First, the 100 nM H₂O₂ that is predicted to accumulate in the Ahp strain is within the 50 to 200 nM range, which activated OxyR effectively in vitro (1).

Compartmentalization of H₂O₂ sources and scavenging enzymes. Because virtually no H₂O₂ escapes from E. coli, the stress is effectively contained within the cell. There is no obvious benefit to this, but it proves an expected principle: when highly active scavengers are compartmentalized inside organelles that generate H₂O₂, the H₂O₂ can be effectively contained. Thus, it is plausible that the catalase that is localized in peroxisomes and the peroxidases that are inside mitochondria can eliminate H₂O₂ before it can escape those compartments and threaten cytosolic enzymes and nuclear DNA. Were the permeability coefficient of H₂O₂ as high as, for example, that of molecular oxygen, transmembrane gradients could not be formed at any reasonable activity of the scavenging enzyme, and most H₂O₂ would spill out of these organelles.

Oxidative stress in experimental systems. The speed with which E. coli scavenges H₂O₂ makes it difficult to do certain types of experiments, particularly to monitor the biochemical and physiological effects of the micromolar doses of H₂O₂ that E. coli is likely to confront in the real world. Even uninduced cells scavenge micromolar concentrations of exogenous H₂O₂ very quickly. In a culture of moderate density (0.1 OD), the half-life of H₂O₂ in the medium is only 3.5 min (see, e.g., Fig. 1); at 1.0 OD, it is 20 s. In our hands OxyR-inducing regimens which use micromolar concentrations of H₂O₂ are variably effective because the inducing dose is rapidly scavenged. Millimolar challenge doses of H₂O₂, on the other hand, are slowly degraded in cultures, both because Ahp is saturated and because HPI is less active at high substrate concentrations (Fig. 1). These doses provide more consistent results. Thus, in the past we and other experimenters have reluctantly resorted to challenging cells with higher, nonphysiological concentrations of H₂O₂. We anticipate that the availability of strains that cannot scavenge H₂O₂ will enable us to study the impact of more realistic doses.

1