Energetics of plasmid-mediated arsenate resistance in Escherichia coli

Abstract

Plasmid R773, which codes for resistances to arsenate, arsenite, and antimony, was introduced into Escherichia coli strain AN120, a mutant deficient in the H+-translocating ATPase of oxidative phosphorylation. Cultures depleted of endogenous energy reserves were loaded with 74AsO3-4, and arsenate efflux was measured after dilution into medium containing various energy sources and inhibitors. Rapid extrusion of arsenate occurred when glucose was added. Arsenate was extruded both against and down a concentration gradient. In this strain glucose allows formation of both ATP via substrate-level phosphorylation and an electrochemical proton gradient (or protonmotive force) via oxidation of the products of glycolysis. When oxidation was inhibited by cyanide, glucose metabolism still produced arsenate efflux. Energy sources such as succinate, which supplies a protonmotive force but not ATP, did not result in efflux. Measurement of intracellular ATP concentration under each set of conditions demonstrated a direct correlation between the rate of efflux and ATP levels. Osmotically shocked cells lost the ability to extrude arsenate; however, no arsenate-binding activity was detected in osmotic shock fluid from induced cells. These results suggest that the arsenate efflux system is coupled to cellular ATP rather than an electrochemical proton gradient, possibly by an arsenate-translocating ATPase.