Engineering ROS Metabolism through a Context-Independent Futile Cycle

Abstract

In the face of a weakening antibiotic arsenal, attention has shifted towards the identification of novel antimicrobial mechanisms and the development of new antibiotic classes. We seek to explore the effects of metabolic futile cycles on reactive oxygen species production in E. coli, and in particular to evaluate the performance of a single-step futile cycle utilizing the F1 subunit of ATP synthase in increasing endogenous ROS production. We hypothesize that the F1 subunit of ATP synthase can, when overexpressed and solubilized in the cytosol, act as a general ATPase, hydrolyzing ATP in vivo. According to futile cycle modeling done previously in the Brynildsen lab, this will increase endogenous ROS production and accordingly, sensitivity to H2O2 attack. As peroxide is used by the immune system as well as various antibiotics as a killing strategy for eliminating microbial infection, this effect could lead to the development of novel antibiotics. This hypothesis was confirmed—induction of a single-step F1 ATPase futile cycle confers a hundredfold reduction in survival fraction of E. coli exposed to a final H2O2 concentration of 2 mM, compared to a strain expressing a catalytically inactive version of the F1 ATP synthase subunit.