Evidence for mutagenesis by nitric oxide during nitrate metabolism in Escherichia coli

Abstract

In Escherichia coli, nitrosative mutagenesis may occur during nitrate or nitrite respiration. The endogenous nitrosating agent N2O3 (dinitrogen trioxide, nitrous anhydride) may be formed either by the condensation of nitrous acid or by the autooxidation of nitric oxide, both of which are metabolic by-products. The purpose of this study was to determine which of these two agents is more responsible for endogenous nitrosative mutagenesis. An nfi (endonuclease V) mutant was grown anaerobically with nitrate or nitrite, conditions under which it has a high frequency of A:T-to-G:C transition mutations because of a defect in the repair of hypoxanthine (nitrosatively deaminated adenine) in DNA. These mutations could be greatly reduced by two means: (i) introduction of an nirB mutation, which affects the inducible cytoplasmic nitrite reductase, the major source of nitric oxide during nitrate or nitrite metabolism, or (ii) flushing the anaerobic culture with argon (which should purge it of nitric oxide) before it was exposed to air. The results suggest that nitrosative mutagenesis occurs during a shift from nitrate/nitrite-dependent respiration under hypoxic conditions to aerobic respiration, when accumulated nitric oxide reacts with oxygen to form endogenous nitrosating agents such as N2O3. In contrast, mutagenesis of nongrowing cells by nitrous acid was unaffected by an nirB mutation, suggesting that this mutagenesis is mediated by N2O3 that is formed directly by the condensation of nitrous acid.

FIG. 1.

Production of the mutagenic nitrosating agent N₂O₃ during nitrate/nitrite metabolism in E. coli. NO· is a minor by-product of nitrite reductase activity.

FIG. 2.

Reduction of nitrate- and nitrite-induced mutagenesis by a ΔnirB (nitrite reductase) mutation. Cultures were grown anaerobically to saturation in medium containing nitrate or nitrite, and from 2 to 3 × 10⁹ cells from each culture were plated. The strains used were CC106 (wild type), BW1506 (nfi), and BW1590 (nfi ΔnirB). Error bars each represent the standard deviation for nine cultures. Concentrations of nitrite greater than 20 mM were not used because they were lethal (28).