9723003 Reitzer, Lawrence J. The immediate objectives of this research are to characterize the pathways of aspartate and arginine catabolism and their function in Escherichia coli. The regulation of the previously undescribed arginine succinyl transferase (AST) pathway suggested that it not only degrades arginine, but also other amino acids during nitrogen-limited growth. The gene for one AST pathway enzyme was identified, and it appears to be the first of a five-gene ast operon. The products of this operon will be characterized, the genes disrupted and overexpressed, the effects on amino acid catabolism determined, and the promoter characterized. If ast mutants still utilize arginine, the remaining pathway of arginine catabolism will be genetically analyzed. Preliminary results suggest that aspartate is degraded by the AST pathway and a previously undescribed pathway. Mutants blocked in the second pathway have been isolated, the lesion mapped, and complementing DNA isolated. The complementing DNA will be further subcloned, the genes identified, and their regulation analyzed. Analysis of this region's nucleotide sequence suggests that it contains only one (54-dependent operon. This operon codes for a minor citrate synthase isozyme, which appears to be active. This citrate synthase has the potential to coordinate carbon and nitrogen metabolism, therefore, its function will be characterized, regardless of its role in aspartate catabolism. In summary, this research focuses on nitrogen metabolism - specifically, the pathways of arginine and aspartate catabolism, which appear to have multiple functions, possibly including the coordination of carbon and nitrogen metabolism. When considered within the context of the second phase of the E. coli genome project, the analysis of gene function, this research will further an understanding of metabolism in E. coli, which will provide a framework for the genes and metabolism of other organisms with sequenced genomes. Bacteria and other living organisms c onsist primarily of carbon, hydrogen, oxygen, and nitrogen. Nitrogen constitutes about 10% by weight of most organisms, and the rate of its acquisition from the environment frequently limits growth. The availability of the complete sequence of all the genes of some model organisms, such as Escherichia coli, permits the complete analysis of certain aspects of metabolism. This research analyzes the means which E. coli acquires and uses nitrogen from the environment. It also analyzes the specific tasks that are performed when certain sources of nitrogen become available. Such basic understanding of metabolism could suggest methods to harness bacterial biochemistry to perform useful tasks, such as the synthesis of useful chemicals, or to control bacterial growth in some situations.
