A fundamental question in cellular physiology is how cells recognize and respond to changes in their environment. The applicant's overall goal is to understand the network of regulatory systems controlling the utilization of nitrogen compounds in the model low G+C Gram-positive bacterium, Bacillus subtilis. These studies will provide insight into how nitrogen metabolism is controlled in the agriculturally and commercially important Bacillus spp. as well as in important low G+C Gram-positive pathogens such as Staphylococcus, Streptococcus and Enterococcus. ? ? Two very similar global transcription factors, GInR and TnrA, are responsible for the activation of gene expression during nitrogen-limited growth in B. subtilis. The activity of TnrA is regulated by a novel signal transduction system where the feedback-inhibited form of GS binds TnrA, blocking its DNA binding activity. One goal of this project is to understand the molecular mechanism responsible for the protein-protein interaction between TnrA and GS. Generalized and site-directed mutagenesis will be performed to identify amino acid residues required for feedback inhibition of GS and for the interaction between TnrA and GS. The stoichiometry and equilibrium binding constant of the interaction between GS and TnrA will be determined. The TnrA binding site will be defined by protein footprinting. All the available genetic evidence indicates that the feedback-inhibited form of GS also regulates the activity of GInR. It will be determined whether GS directly binds to GInR, activating its DNA binding activity. Alternatively feedback inhibited GS may indirectly regulate GlnR by a partner switching mechanism. Biochemical and genetic approaches will be used to search for factors required for the regulation of GInR activity by glutamine synthetase. ? ?
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