Transport of Succinate in Escherichia coli II. CHARACTERISTICS OF UPTAKE AND ENERGY COUPLING WITH TRANSPORT IN MEMBRANE

Membrane vesicles prepared from a strain of Escherichia coli which lacks membrane-bound succinate dehydrogenase and fumarate reductase activity are capable of accumulating succinate in the presence of D( -)lactate or reduced phenazine methosulfate as electron donors. The steady state, intramembranal concentration of succinate reaches 30to 50-fold of the external concentration. Both the initial and the steady state rates are higher with ascorbate-phenaz:ne methosulfate than with D( -)lactate. ATP, ADP, and various other metabolic intermediates do not stimulate succinate transport. Magnesium ions are required for maximum uptake. Monovalent cations, such as Na+, I(+, or Li+ also stimulate transport to varying degrees. With ascorbate-phenazine methosulfate as electron donor the Michaelis constant for succinate is about 10 PM, and the inhibition constants for fumarate and L-malate are 12 PM and 22 PM, respectively. Both the ascorbate-phenazine methosulfate-dependent and D( -)lactate-dependent uptake of succinate are inhibited by energy poisons, uncouplers, and various other respiratory chain inhibitors; Compounds known to interfere with phosphorolytic and transphosphorylation reactions are without significant effect on transport. Amongst the thiol reagents, N-ethylmaleimide and showdomycin do not inhibit succinate uptake even at very high concentrations, but pchloromercuribenzoate, Hg++ and Ag++ are very potent inhibitors. Both N-ethylmaleimide and p-chloromercuribenzoate, however, strongly inhibit D-lactate oxidation. From these observations, and from oxamate inhibition data, it is concluded that electrons are coupled to succinate transport directly at the initial step of lactate dehydrogenation. It is proposed that succinate uptake is mediated by alternate reduction and oxidation of a succinate carrier in membranes. Membranes prepared from strains carrying genes dct A or dct B are defective in the uptake of succinate. It thus appears that at least two genetic loci are involved in the formation of the succinate transport system in the membranes.