Abstract
Abstract Escherichia coli K-12 has been shown to possess two transport systems for aspartate. One system specified by the genotype dct is nonspecific toward a wide variety of C4-dicarboxylic acids whereas another system specified by the genotype ast is more specific for aspartate and transports this substrate with a much higher affinity. The existence of these two systems was unequivocally shown from a comparison of the kinetics of transport, patterns of competitive inhibition, and by isolation and characterization of mutants devoid of each system or both. Aspartate was accumulated by strains with a tricarboxylic acid cycle metabolic block (citrate synthase), however, cells without this lesion were unable to do so due to rapid catabolism. C4-Dicarboxylic acids which enter the cell via the dct system were not accumulated. Membrane vesicles actively transported and accumulated aspartate and were stimulated by d(-)-lactate, however, membrane vesicles from ast strains did not accumulate aspartate and were not stimulated by d(-)lactate. C4-Dicarboxylic acids, although transported, were not accumulated and no stimulation by d(-)-lactate was observed. The low affinity dct system was absolutely necessary for dicarboxylic acid catabolism, whereas the ast high affinity system was not. The high affinity system, however, could be shown to be adequate to serve as a transport system for aspartate as a nitrogen source and as a supply route for the C4 acids required for anaplerosis.
Highlights
The existence of these two systems was unequivocally shown from a comparison of the kinetics of transport, patterns of competitive inhibition, and by isolation and characterization of mutants devoid of each system or both
The findings described in this publication support our preliminary observat,ion that more than one aspartate transport system is present in E. co& and, for the first time, clearly shows the unique existence of two transport systems having distinct metabolic roles yet facilitating the transport of a common metabolite, aspartic acid, across the bacterial membrane
Effect of Aspartic Acid as Nitrogen Source-In a medium with aspartic acid as a nitrogen source, E. coli bearing the dct lesion were found to grow as well as the parent strain indicating that aspartic acid most hkely enters the cell via a route other than that available for the other dicarboxylic acids
Summary
The existence of these two systems was unequivocally shown from a comparison of the kinetics of transport, patterns of competitive inhibition, and by isolation and characterization of mutants devoid of each system or both. In asl strains, the kinetics of aspartate uptake was resolved into a single component; that is, a low affinity dicarboxylic acid transport system (V,,, of 25 nmoles per min per mg of cells, dry weight, and a K, of 30 pM) (Fig. 2B).
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