The overall objective of this research proposal is to determine the genetic and regulatory properties of the enzymes involved in nitrogen metabolism in Bacillus subtilis. I propose to initiate these studies by focusing on the genetics and regulation of glutamate synthase (GOGAT) an amidotransferase involved in nitrogen metabolism. GOGAT is the main enzyme responsible for the synthesis of glutamate (glt) which occupies a central role in this assimilation of inorganic nitrogen into an organic form. This amidotransferase is composed of 2 nonidentical subunits and catalyzes the following reaction: gln plus alpha kg plus NADPH--greater than 2 glutamate plus NADP. I propose to study GOGAT from B. subtilis because: 1) the enzyme aggregate is readily associated and dissociated; 2) the isolated subunits can be reconstituted to form an active enzyme complex (complementation assay); 3) this complementation assay can be utilized to follow the levels of the 2 subunits following genetic and physiological manipulations; 4) mutations at the gltA and gltB loci (code for 2 subunits of GOGAT) affect glutamine synthetase (GS) (glt plus NH3 plus ATP greater than gln plus ADP plus Pi). The specific aims of this proposal are: 1) determine the physical and kinetic properties of GOGAT; 2) determine the regulation of the gltA and gltB loci and their relationship to levels of GS. These goals will be pursued using biochemical assays for enzyme activity, immunological assays for GOGAT and its subunits and isolation and characterization of mutations at the gltA and gltB loci as well as other mutations that effect the expression of these two loci. It is anticipated that these studies will demonstrate what regulatory signals effect GOGAT. Since this enzyme is essential to the corporation of inorganic nitrogen into an organic form (a central reaction for all types of cells), these results may be of general importance in understanding the regulation of this complex metabolic sequence.