Substantial DNA damage from submicromolar intracellular hydrogen peroxide detected in Hpx ^- mutants of Escherichia coli

Abstract

Since the discovery of catalase, it has been postulated that aerobic organisms generate enough oxidants to threaten their own fitness and, in particular, their genetic stability. An alternative is that these enzymes exist to defend the cell against more-abundant oxidants imposed by external sources. These hypotheses were tested directly through study of Hpx ؊ (katG katE ahpCF) mutants of Escherichia coli, which lack enzymes to scavenge hydrogen peroxide (H 2O2). These strains grew well in anaerobic medium but poorly when they were aerated. The Hpx ؊ bacteria formed filaments and exhibited high rates of mutagenesis, both indicators of DNA damage. An additional recA mutation caused Hpx ؊ cells to die rapidly upon aeration, even though the intracellular H 2O2 was <1 M. Spin-trap experiments detected substantial hydroxyl radicals, and cell-permeable iron chelators eliminated both the phenotypic defects and hydroxyl-radical formation, confirming that the Fenton reaction was responsible. An Hpx ؊ oxyR strain exhibited even more DNA lesions than did the Hpx ؊ mutant, indicating that the OxyR stress response induced protein(s) that suppressed DNA damage. One critical protein was Dps, an iron-sequestration protein, because Hpx ؊ dps mutants exhibited sensitivity similar to that of the Hpx ؊ oxyR mutant. These results reveal that aerobic E. coli generates sufficient H 2O2 to create toxic levels of DNA damage. Scavenging enzymes and controls on free iron are required to avoid that fate. The rate constant of the Fenton reaction measured at physiological pH was much higher than under the acidic conditions that were used to determine the commonly cited value.Fenton ͉ oxidative DNA damage ͉ Dps

Quantitative proteomics of intracellular Porphyromonas gingivalis

“…A reduction in free iron within P. gingivalis, as would result from under-expression of iron acquisition systems and over-expression of ferritin, could also be indicative of oxidative stress. Lower levels of iron in bacteria will limit the production of OH − from H 2 O 2 via Fenton chemistry [56]. Consistent with the concept that intracellular P. gingivalis are under a degree of oxidative stress is the over-expression of two alkyl hydroperoxide reductases, a thiol peroxidase and rubrerythrin, all of which can be involved in protection against oxidative stress [57,58].…”

Catalase (KatA) and Alkyl Hydroperoxide Reductase (AhpC) Have Compensatory Roles in Peroxide Stress Resistance and Are Required for Survival, Persistence, and Nasal Colonization inStaphylococcus aureus

“…Nucleic acid damage was observed in the ahpC katA strain via loss of PCR product and an increase in spontaneous rifampin-resistant mutants. Park et al also reported an increase in the number of DNA lesions in the ahpC katG mutant of E. coli (42).…”

“…Supplementation of the media with an iron chelator or catalase enhanced the growth of Hpx Ϫ cells due to a reduction in the Fenton reaction. Furthermore, overproduction of Dps, an iron sequestration protein, complemented the growth defect of the Hpx Ϫ strain, probably due to its iron-binding activity (42).…”

Cellular Defenses against Superoxide and Hydrogen Peroxide

“…1) depends upon the coordination environment of the iron atom. Early measurements conducted at acid pH indicated the value was quite low (21), but at physiological pH it has been measured to range from 5000 -20,000 M −1 s −1 (13,22). The hydroxyl radical that is generated reacts at nearly diffusion-limited rates near the site of its formation.…”

“…These strains grew well anaerobically but exhibited a variety of aerobic growth defects that derived from the accumulation of endogenous O 2 − . Similarly, E. coli catalase/ peroxidase mutants are poisoned by the micromolar doses of intracellular H 2 O 2 that accumulate (13,14). A key goal has been to track these defects back to the biochemical lesions that cause them.…”