Transport of C4-dicarboxylates by anaerobically grown Escherichia coli. Energetics and mechanism of exchange, uptake and efflux.

Abstract

Transport activities for uptake, efflux and exchange of C4-dicarboxylates were observed in anaerobically grown Escherichia coli. All three transport modes were only present in strains containing the transcriptional activator FNR of anaerobic respiration, and were repressed by nitrate and O2. The kinetic and energetic parameters of C4-dicarboxylate transport and the mechanism of the uptake, efflux and exchange reactions were analyzed in whole cells and in membrane vesicles. Fumarate/succinate exchange could be characterized as homologous or heterologous 1:1 counter-exchange. The external substrate was determined as divalent fumarate2- (or succinate2-) at pH 6-9, whereas monovalent H-fumarate dominated as the substrate at pH 3-4. The exchange was not inhibited by dissipation of delta p or constituents of it (delta psi or delta pH). We conclude that this transport mode functions as an electroneutral exchange of C4-dicarboxylates. The uptake of C4-dicarboxylates did not depend on internal counter-substrate and resulted in an accumulation of the substrate. Similar to antiport, fumarate was accepted in the divalent form at pH values greater than or equal to 6 and in the monovalent form at pH 3.5-6. The uptake was inhibited by dissipation of delta p or delta psi. Artificially imposed delta pH, delta psi or fumarate gradients were able to drive fumarate uptake. An involvement of Na+ could not be detected. Thus the uptake is likely to operate as an electrophoretic H+/fumarate symport. Independent of the presence of an external counter-substrate, the substrates were secreted from cells or membrane vesicles loaded with succinate or fumarate. The efflux was electrogenic. Energizing the cells or membrane vesicles inhibited efflux, maximal efflux rates were obtained only after dissipation of delta p or delta psi. An imposed K(+)-diffusion potential (outside positive) inhibited succinate excretion. The efflux of succinate from de-energized membrane vesicles generated a delta psi of -70 mV. It is thus suggested that succinate efflux functions as a H+/succinate symport.