Hydrogen peroxide fluxes and compartmentalization inside growing Escherichia coli

Abstract

Escherichia coli generates about 14 microM hydrogen peroxide (H(2)O(2)) per s when it grows exponentially in glucose medium. The steady-state intracellular concentration of H(2)O(2) depends on the rates at which this H(2)O(2) is dissipated by scavenging enzymes and by efflux from the cell. The rates of H(2)O(2) degradation by the two major scavenging enzymes, alkyl hydroperoxide reductase and catalase, were quantified. In order to estimate the rate of efflux, the permeability coefficient of membranes for H(2)O(2) was determined. The coefficient is 1.6 x 10(-3) cm/s, indicating that permeability is substantial but not unlimited. These data allowed internal H(2)O(2) fluxes and concentrations to be calculated. Under these growth conditions, Ahp scavenges the majority of the endogenous H(2)O(2), with a small fraction degraded by catalase and virtually none persisting long enough to penetrate the membrane and exit the cell. The robust scavenging activity maintains the H(2)O(2) concentration inside glucose-grown cells at <10(-7) M, substantially below the level (10(-6) M) at which toxicity is evident. When extracellular H(2)O(2) is present, its flux into the cell can be rapid, but the internal concentration may still be an order of magnitude lower than that outside. The presence of such gradients was confirmed in experiments that revealed different degrees of oxidative stress in cocultured scavenger-deficient mutants. The limited permeability of membranes to H(2)O(2) rationalizes the compartmentalization of scavenging systems and predicts that bacteria that excrete redox-cycling drugs do not experience the same H(2)O(2) dose that they impose on their competitors.

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.

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.

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 J_Ahp = 2.1 × 10⁻¹⁸ mol/s (H_in) (H_in + 1.2 × 10⁻⁶ 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.

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.