Effector binding by Bacillus subtilit CodY

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DESCRIPTION (provided by applicant): Bacillus subtilis employs several adaptive strategies when faced with nutrient limitation. Expression of many of these adaptive mechanisms is under regulation by CodY, a highly conserved protein in Gram-positive bacteria. CodY senses nutrient availability by interaction with its effectors, GTP and the branched-chain amino acids (BCAAs). Upon effector binding, CodY is able to bind to and repress its DNA targets. It is uncertain which amino acids in CodY are necessary for interaction with these effectors, and it is unclear how effector binding alters CodY such that it interacts with DNA. The goal of this project is to address both of these underlying questions. As CodY has been shown to regulate expression of virulence factors in several bacterial pathogens, it is an attractive target for novel antimicrobial therapy. However, development of this line of therapy will require examination of these basic issues concerning CodY activity. The first aim of this application will identify amino acids essential for effector binding. Homology searches and x-ray crystallography studies have yielded some clues about which CodY residues may be important for GTP and BCAA binding, respectively. These residues will be mutagenized, and their impact on effector binding will be determined. The second aim of this application will investigate how effector binding increases CodY's affinity for DNA. Partial proteolysis has indicated that a conformational change occurs in CodY upon binding to BCAAs, but no change was observed upon CodY binding to GTP. Near-UV circular dichroism, spectrofluorometry, and Fourier transform infrared spectroscopy will be used to identify whether GTP induces a subtle alteration in the tertiary structure of CodY. Experiments in the third aim will assess the physiological impact of the loss of CodY effector binding on gene expression. These codY mutations will be introduced into the B. subtilis chromosome, and their effects will be assayed by reporter fusion experiments and microarray analysis. The results of this work will allow us to understand how bacteria regulate gene expression in response to nutrient starvation. Disruption of this regulation may result in a novel means of treatment of certain bacterial infections.