The enzyme aspartokinase plays a central role in bacterial amino acid biosynthesis, and its activity is attuned to the physiological needs of the bacterium by complex control mechanisms. Bacillus subtilis has two aspartokinases, one of which (aspartokinase I) is regulated by the cell wall constituent diaminopimelate, while the other (aspartokinase II), the major focus of this research, is controlled by lysine and is unusual in being composed of two different types of subunits which are independently translated from in-phase overlapping genes. These and their control regions have been cloned and their nucleotide sequence has been determined. This proposal aims to apply the sequence information to the study of the mechanisms that control aspartokinase synthesis and of the relationship between structure and function of the enzyme itself. The proposed research will use oligonucleotide directed mutagenesis to alter specific sites in the control regions as well as in the genes encoding the protein structure, in the expectation that the study of these structural perturbations will reveal the mechanisms that underlie control and function. Another aim is the sequencing of the genes that surround the genetic locus of aspartokinase II and the elucidation of their regulatory and metabolic relationship. Bacillus subtilis, the species of bacterium under study in this proposal, has two modes of growth, vegetative under normal environmental conditions, and sporulation under conditions of starvation or other types of stress. The latter process results in the formation of highly durable spores which can be dispersed, and can subsequently germinate when and where conditions permit vegetative growth. Vegetatively growing bacteria, upon receiving specific stimuli to begin sporulation, must alter their entire internal physiology to meet the requirements of the new mode of growth. Certain key enzymes, which play different physiological roles under the two different modes of growth, are thus under a very complex series of controls, which permit the transition of the bacterium from vegetative growth to sporulation. This is a proposal to elucidate the mechanisms of these controls; it could possibly reveal previously unknown mechanisms of control of gene expression. The transition of Bacillus cells from vegetative growth to sporulation is a developmental switch which can serve as a conceptual model for the differentiation of embryonic tissues in higher organisms. The work in this proposal may thus shed indirect light on the latter process.