Genetic and Biochemical Analysis of the Interaction ofBacillus subtilisCodY with Branched-Chain Amino Acids

Abstract

Bacillus subtilis CodY protein is a DNA-binding global transcriptional regulator that responds to branchedchain amino acids (isoleucine, leucine, and valine) and GTP. Crystal structure studies have shown that the N-terminal region of the protein includes a GAF domain that contains a hydrophobic pocket within which isoleucine and valine bind. This region is well conserved in CodY homologs. Site-directed mutagenesis was employed to understand the roles of some of the residues in the GAF domain and hydrophobic pocket in interaction with isoleucine and GTP. The F40A, F71E, and F98A forms of CodY were inactive in vivo. They were activatable by GTP but to a much lesser extent by branched-chain amino acids in vitro. The CodY mutant R61A retained partial repression of target promoters in vivo and was able to respond to GTP in vitro but also responded poorly to branched-chain amino acids in vitro unless GTP was simultaneously present. Thus, the GAF domain includes residues essential for full activation of CodY by branched-chain amino acids, but these residues are not critical for activation by GTP. Binding studies with branched-chain amino acids and their analogs revealed that an amino group at position 2 and a methyl group at position 3 of valine are critical components of the recognition of the amino acids by CodY.

Bacillus subtilis CodY Operators Contain Overlapping CodY Binding Sites

“…These results demonstrated that CodY DNA-binding activity was enhanced 5-fold in the presence of isoleucine. Although this level of CodY DNA-binding enhancement is similar to that found in previously published reports (44,46), it is rather low compared to that seen for other DNA-binding proteins. For example, the DNA-binding activities of the TrpR and PurR repressors are enhanced 200-fold by their corepressors (9,51).…”

Hierarchical expression of genes controlled by theBacillus subtilisglobal regulatory protein CodY

“…Previous results indicated that CodY binds to different target sites with varying affinity (29,37). Furthermore, significant variation has been observed in the extent to which mutations in CodY that affect its DNA-binding activity alter the regulation of a limited number of its direct targets (17,37,38). These findings suggest that not all genes regulated by CodY respond equally to a given extent of nutritional limitation (27,37).…”

“…To verify that the variable activities of the mutant set used for the experiments reported here was related to the presence of ILV, we measured transcript abundance for bcaP (a direct CodY target) in strains producing WT CodY and the mutant forms F71Y, R61K, and R61H (ref. 38 and this study), all of which have single amino acid substitutions in the ILV-binding pocket (Fig. S1).…”

“…To modify the activity of CodY without changing the medium composition or the codY gene copy number, we used a set of codY mutant strains that have alterations in the GAF domain (18) that is responsible for ILV binding (37,38, and this study). The mutant strains were expected to behave as though ILV levels were limiting even though excess ILV was provided in the medium.…”

Qualitative Reasoning for Reaction Networks with Partial Kinetic Information

“…Protein CcpA is expressed by reaction r 6 and degraded by reaction r 6 . Transcription at the ilv-leu promoter is well known to be inhibited by CodY through a binding of this latter on the promoter [7,9,14,16,17]. To model this action of CodY on the promoter PIlv−Leu, we introduce the reaction r 4 which activates the binding side BS CodY of CodY at the promoter, which in turn slows down reaction r 5 and thus reduces the promoter's activity.…”