Utilization of Aspartate as a Nitrogen Source in Escherichia coli: ANALYSIS OF NITROGEN FLOW AND CHARACTERIZATION OF THE PRODUCTS OF ASPARTATE CATABOLISM

Abstract

15N NMR, reverse-phase high performance liquid chromatography and gas chromatography/mass spectrometry have been used to follow nitrogen metabolism in Escherichia coli labeled on medium containing L-[15N]aspartate. The flow of 15N through various nitrogen-containing metabolites was followed over the course of the labeling period. For wild-type E. coli labeled on L-[15N]aspartate as sole source of nitrogen, significant 15N labeling was detected only in the intracellular L-glutamate, L-alanine, L-aspartate, and putrescine pools. Intracellular concentrations of L-aspartate and L-glutamate differed significantly in extracts of an arginine auxotroph (argG-), which is deficient in a potential aspartate-assimilating reaction. When the L-[15N]aspartate containing labeling medium was supplemented with unlabeled arginine, extracts of wild-type E. coli were shown to contain significant amounts of unlabeled ammonia and putrescine. There was substantial dilution of 15N in the glutamate pool. The observation that glutamate, aspartate, and alanine are 15N-labeled, but ammonia is not, suggests that these amino acids are not the immediate source of ammonia. Our results suggest that arginine may be an intermediate for the degradation of some of the aspartate and that arginine may be an intermediate for ammonia production during nitrogen-limited growth. Our results also strongly suggest the presence of a previously uncharacterized pathway of arginine degradation.