Improved measurements of scant hydrogen peroxide enable experiments that define its threshold of toxicity for Escherichia coli.

Escherichia coli is a model organism that has been exploited to reveal key details of hydrogen peroxide stress: the biomolecules that H2O2 most rapidly damages and the defensive tactics that organisms use to fend it off. Much less clear is the amount of exogenous H2O2 that is sufficient to injure the bacterium and/or to trigger its stress response. To fill this gap, we need to study the behavior of cells when they are exposed to defined amounts of H2O2 on an hours-long time scale. Such experiments are difficult because bacteria rapidly consume H2O2 that is added to test cultures. Further, lab media itself can generate H2O2, and media components interfere with the quantification of H2O2 levels. In this study we describe mechanisms by which media components interfere with H2O2 determinations, and we identify simple ways to minimize and correct for this interference. Using these techniques, it was shown that standard media generate so much H2O2 that most intracellular H2O2 derives from the medium rather than from endogenous metabolism. Indeed, bacteria spread on plates must induce their stress response or else perish. Finally, two straightforward methods were used to sustain low-micromolar steady-state concentrations of H2O2. In this way we determined that > 2 μM extracellular H2O2 is sufficient to trigger the intracellular OxyR stress response, and > 5 μM begins to impair cell growth in a minimal medium. These concentrations are orders of magnitude lower than the doses that have typically been used in lab experiments. The new approaches should enable workers to study how various organisms cope with natural levels of H2O2 stress.