Hydrogen peroxide fluxes and compartmentalization inside growing Escherichia coli

Escherichia coli generates about 14 μM hydrogen peroxide (H₂O₂) per s when it grows exponentially in glucose medium. The steady-state intracellular concentration of H₂O₂ depends on the rates at which this H₂O₂ is dissipated by scavenging enzymes and by efflux from the cell. The rates of H₂O₂ 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 H₂O₂ was determined. The coefficient is 1.6 × 10^-3 cm/s, indicating that permeability is substantial but not unlimited. These data allowed internal H₂O₂ fluxes and concentrations to be calculated. Under these growth conditions, Ahp scavenges the majority of the endogenous H₂O₂, 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 H₂O₂ concentration inside glucose-grown cells at <10^-7 M, substantially below the level (10^-6 M) at which toxicity is evident. When extracellular H₂O₂ 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 H₂O₂ rationalizes the compartmentalization of scavenging systems and predicts that bacteria that excrete redox-cycling drugs do not experience the same H₂O₂ dose that they impose on their competitors.