This is a proposal to study the biochemical regulation of enzyme activity during metabolic adaptation and differentiation. Two general questions are posed: (1) what biochemical mechanisms catalyze and regulate the selective inactivation and degradation of enzymes in vivo? and (2) how is the synthesis of enzymes developmentally regulated? These phenomena occur in most cell types and are poorly understood. The experimental system is the differentiating bacterium Bacillus subtilis, in which we have characterized in detail two target enzymes of nucleotide biosynthesis, aspartate transcarbamylase and glutamine PRPP amidotransferase, which are selectively degraded in nutrient-starved (sporulating) cells. The synthesis of these two enzymes is also abruptly shut off prior to their degradation. It is proposed to continue extensive study of the developmental regulation of these enzymes by pursuing the following specific aims: 1. To reconstruct the degradation process in vitro from purified components and to explain the regulation of degradation by the stringent response; 2. To isolate and characterize mutants and clones of genes involved in degradation; 3. To test the hypotheses that degradation of the amidotransferase is preceded by oxidative inactivation of an essential Fe-S cluster; 4. To elucidate the structure and transcription in vitro of the cloned aspartate transcarbamylase (pyrB) gene; 5. To construct mutant forms of the pyrB gene and test their functioning in vivo so as to test hypotheses concerning regulation of pyrB expression; and if resources are adequate 6. To study developmental and nutritional regulation of other genes of the pyr cluster by cloning, sequencing and expression studies with mutant clones.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI011121-16
Application #
3124896
Study Section
Microbial Physiology and Genetics Subcommittee 2 (MBC)
Project Start
1976-05-01
Project End
1991-04-30
Budget Start
1988-05-01
Budget End
1989-04-30
Support Year
16
Fiscal Year
1988
Total Cost
Indirect Cost
Name
University of Illinois Urbana-Champaign
Department
Type
Schools of Arts and Sciences
DUNS #
041544081
City
Champaign
State
IL
Country
United States
Zip Code
61820
Bussey, L B; Switzer, R L (1993) The degA gene product accelerates degradation of Bacillus subtilis phosphoribosylpyrophosphate amidotransferase in Escherichia coli. J Bacteriol 175:6348-53
Sheehan, S M; Switzer, R L (1991) Intracellular serine protease-4, a new intracellular serine protease activity from Bacillus subtilis. Arch Microbiol 156:186-91
Quinn, C L; Stephenson, B T; Switzer, R L (1991) Functional organization and nucleotide sequence of the Bacillus subtilis pyrimidine biosynthetic operon. J Biol Chem 266:9113-27
Graves, L M; Switzer, R L (1990) Aspartokinase II from Bacillus subtilis is degraded in response to nutrient limitation. J Biol Chem 265:14947-55
Graves, L M; Switzer, R L (1990) Aspartokinase III, a new isozyme in Bacillus subtilis 168. J Bacteriol 172:218-23
Sheehan, S M; Switzer, R L (1990) Intracellular serine protease 1 of Bacillus subtilis is formed in vivo as an unprocessed, active protease in stationary cells. J Bacteriol 172:473-6
Onate, Y A; Vollmer, S J; Switzer, R L et al. (1989) Spectroscopic characterization of the iron-sulfur cluster in Bacillus subtilis glutamine phosphoribosylpyrophosphate amidotransferase. J Biol Chem 264:18386-91
Grandoni, J A; Switzer, R L; Makaroff, C A et al. (1989) Evidence that the iron-sulfur cluster of Bacillus subtilis glutamine phosphoribosylpyrophosphate amidotransferase determines stability of the enzyme to degradation in vivo. J Biol Chem 264:6058-64
Switzer, R L (1989) Non-redox roles for iron-sulfur clusters in enzymes. Biofactors 2:77-86
Lerner, C G; Stephenson, B T; Switzer, R L (1987) Structure of the Bacillus subtilis pyrimidine biosynthetic (pyr) gene cluster. J Bacteriol 169:2202-6

Showing the most recent 10 out of 13 publications