The long term objectives of the current studies are to determine how L-alanine is synthesized and used by Escherichia coli. Currently, this entails a primarily molecular genetic approach, with utilization of transposable elements as tools for gene cloning, mapping and sequencing. Because solid L- alanine auxotrophs cannot be isolated directly, potential alanine biosynthetic genes are identified by cloning wildtype genes that suppress a leaky alanine mutation. These genes will be mapped and the DNA will be sequenced. L-alanine is the most abundant amino acid in E. coli and is a component of the bacterial cell wall. The synthesis of several cell membrane components, including a cell surface antigen associated with pathogenesis, is regulated by L- alanine, suggesting that this amino acid may be one of the bacterial cell's general regulatory signals. L-alanine, the most abundant amino acid in the bacterium E.coli, is an essential constituent both of proteins and of the cell wall. Because of its involvement in more than one physiological function, it regulates several diverse pathways of gene expression, and is itself synthesized by more than one pathway. Because of these complexities, the biosynthesis of L- alanine and the regulation thereof have proven particularly refractory to genetic analysis for many years. Using some novel methods, Dr. Berg has succeeded in isolating mutations in some of the gene involved in the biosynthesis of L-alanine, and has begun to study the regulation of their expression. It has emerged from her preliminary work that the activity of one of these genes may be regulated in a novel way, viz., by the tightness of physical coiling of the DNA very near the gene. This is a proposal to extend these studies. The proposed work should yield new insights into how a bacterial cell coordinates two rather disparate aspects of its physiology, and may also reveal previously unknown mechanisms of regulation of gene expression.