Transport of Succinate in Escherichia coli: I. BIOCHEMICAL AND GENETIC STUDIES OF TRANSPORT IN WHOLE CELLS

Abstract

Abstract A mutant of Escherichia coli unable to metabolize succinate has been used to study the nature of succinate transport in whole cells. It is demonstrated that cells accumulate succinate against a concentration gradient. The uptake of succinate is competitively inhibited by fumarate and malate. The Michaelis constants for the dicarboxylic acids are all in the range of 15 to 30 µm. The accumulation of succinate is prevented by various energy poisons and sulfhydryl reagents. Inhibitors of mitochondrial dicarboxylate transport are without any effect on the succinate uptake system of E. coli. The influx and efflux of succinate in whole cells is temperature dependent. The influx increases rapidly between 15 and 36° and efflux between 36–50°. The transport system is induced by succinate and repressed by glucose. Mutants lacking adenyl cyclase and some lacking enzyme I of phosphotransferase system acquire succinate transport activity when grown in the presence of succinate and cyclic 3',5'-AMP. Mutants of E. coli unable to transport succinate fall into two broad categories which are referred to as dct (dicarboxylate transport) and ct (carboxylate transport). The dct mutants fail to grow on dicarboxylic acids (fumarate, malate, and succinate) but grow normally on the monocarboxylic acid, lactate. The ct mutants do not grow on either the dicarboxylic acids or on lactate. Both classes of mutants grow well on acetate. The dct class of mutants can be genotypically separated into two categories. The dct A mutants map at around 69 min, and the dct B mutant maps at about 17 min (linked to galactose locus) on the genetic map. The ct mutants are also linked to galactose. It is inferred from these experiments that there are at least two components involved in the transport of succinate in E. coli. It is suggested that the ct gene possibly specifies a carboxylate binding protein. This suggestion is based on the evidence that osmotically shocked cells of E.coli lose the capacity for uptake of succinate.