Effect of ionophores on denitrification inFlexibacter canadensis

Abstract

Denitrification by Flexibacter canadensis was investigated by measuring the production and (or) consumption of nitrite, nitric oxide (NO), and nitrous oxide (N2O) under anaerobic conditions. Carbonyl cyanide m-chlorophenylhydrazone (CCCP), carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP), 2,4-dinitrophenol, and nigericin, but not valinomycin-K+inhibited the production of nitrite and N2O from nitrate by intact cells. However, CCCP, FCCP, 2,4-dinitrophenol, nigericin, and valinomycin-K+did not affect nitrite production from nitrate by cell-free extracts. These results suggest that nitrate transport was dependent on the transmembrane pH gradient but not on the membrane potential. CCCP, FCCP, and nigericin but not 2,4-dinitrophenol and valinomycin-K+caused NO accumulation during the reduction of nitrite, and also inhibited NO consumption and N2O production from nitrite by intact cells. These results preclude an explanation for NO accumulation based on the collapse of the proton motive force by ionophores, and imply that CCCP, FCCP, and nigericin perhaps dissociated a nitrite reductase–nitric oxide reductase complex, and (or) inhibited nitric oxide reductase specifically. 2,4-Dinitrophenol and CCCP did not inhibit the reduction of N2O to dinitrogen. Addition of ≤ 1.16 μM dissolved NO did not affect the production of nitrite from nitrate, or the disappearance of nitrite or N2O. The rate of NO consumption was linear with concentrations of dissolved NO up to 67 nM. Above 67 nM NO, NO consumption was inhibited, suggesting that NO is toxic to nitric oxide reductase.Key words: ionophores, denitrification, nitric oxide, Flexibacter canadensis.