Respiration of Escherichia coli can be fully uncoupled via the nonelectrogenic terminal cytochrome bd-II oxidase

Abstract

The respiratory chain of Escherichia coli is usually considered a device to conserve energy via the generation of a proton motive force, which subsequently may drive ATP synthesis by the ATP synthetase. It is known that in this system a fixed amount of ATP per oxygen molecule reduced (P/O ratio) is not synthesized due to alternative NADH dehydrogenases and terminal oxidases with different proton pumping stoichiometries. Here we show that P/O ratios can vary much more than previously thought. First, we show that in wild-type E. coli cytochrome bo, cytochrome bd-I, and cytochrome bd-II are the major terminal oxidases; deletion of all of the genes encoding these enzymes results in a fermentative phenotype in the presence of oxygen. Second, we provide evidence that the electron flux through cytochrome bd-II oxidase is significant but does not contribute to the generation of a proton motive force. The kinetics support the view that this system is as an energy-independent system gives the cell metabolic flexibility by uncoupling catabolism from ATP synthesis under non-steady-state conditions. The nonelectrogenic nature of cytochrome bd-II oxidase implies that the respiratory chain can function in a fully uncoupled mode such that ATP synthesis occurs solely by substrate level phosphorylation. As a consequence, the yield with a carbon and energy source can vary five- to sevenfold depending on the electron flux distribution in the respiratory chain. A full understanding and control of this distribution open new avenues for optimization of biotechnological processes.

FIG. 1.

Diagram of all NADH:quinone oxidoreductases and quinol:oxygen oxidoreductases in E. coli and their proton translocation properties. Cyt, cytochrome; Q, quinone.

FIG. 2.

Biomass-specific ubiquinone-8 content (A) and redox state (B) during exponential growth in minimal medium on glucose in a shake flask culture for different respiratory deletion mutants: ubiquinone pool during aerobic batch growth in minimal media containing 50 mM glucose for strains BW25113 (wild type), MB21 (ΔappB), MB28 (ΔcydB), and MB20 (ΔcyoB). The error bars indicate standard deviations (n = 3). UQ8, ubiquinone-8.

FIG. 3.

Analysis of the ubiquinone pool during continuous growth in glucose-limited chemostat conditions for different respiratory deletion mutants: redox state (A) and content (B) of the ubiquinone pool of MB30, MB37, and MB34 during aerobic glucose-limited chemostat growth in minimal media containing 50 mM glucose. The error bars indicate standard deviations (n = 3).