Stress-induced evolution of Escherichia coli points to original concepts in respiratory cofactor selectivity

Abstract

Bacterial metabolism is characterized by a remarkable capacity to rapidly adapt to environmental changes. We restructured the central metabolic network in Escherichia coli to force a higher production of NADPH, and then grew this strain in conditions favoring adaptive evolution. A six-fold increase in growth capacity was attained that could be attributed in multiple clones, after whole genome mutation mapping, to a specific single mutation. Each clone had an evolved NuoF*(E183A) enzyme in the respiratory complex I that can now oxidize both NADH and NADPH. When a further strain was constructed with an even higher degree of NADPH stress such that growth was impossible on glucose mineral medium, a solid-state screening for mutations restoring growth, led to two different types of NuoF mutations in strains having recovered growth capacity. In addition to the previously seen E183A mutation other clones showed a E183G mutation, both having NADH and NADPH oxidizing ability. These results demonstrate the unique solution used by E. coli to overcome the NADPH stress problem. This solution creates a new function for NADPH that is no longer restricted to anabolic synthesis reactions but can now be also used to directly produce catabolic energy.

Fig. 1.

(A) and (B) Generation of ΔΨ in at the respiratory chain ISO vesicles. The quenching of oxonol V fluorescence was used to monitor potential membrane generation in ISO vesicles prepared from NA23 (A) and NA23E04 (B) strains. Solid and dash-dot lines represent quenching upon NADH and NADPH, respectively.

Fig. 2.

Putative structure of the NuoF subunit from E. coli deduced from the T. Thermophilus Nqo1 structure. The two prosthetic [4Fe-4S] and [2Fe-2S] clusters from N3 and N1a, and the FMN cofactor are in black.

Fig. 3.

(A) and (B) Comparison of the FMN and NAD(P)H binding sites in the NuoF and NuoF* subunits. NADH and NADPH are superimposed and docked in front of FMN. Distances from the negatively charged NADPH phosphate to FMN and residue 183 are in angstroms. Sterically hindrance between the NADPH phosphate and glutamate 183 is seen. NADH is not obstructed and can be placed in front of FMN.