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Journal of Bacteriology, November 2009, p. 6865-6876, Vol. 191, No. 22
0021-9193/09/$08.00+0     doi:10.1128/JB.00818-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Genetic and Biochemical Analysis of the Interaction of Bacillus subtilis CodY with Branched-Chain Amino Acids{triangledown} ,{dagger}

Anuradha C. Villapakkam ,1,§,{ddagger} Luke D. Handke,2,§,¶ Boris R. Belitsky,2 Vladimir M. Levdikov,3 Anthony J. Wilkinson,3 and Abraham L. Sonenshein1,2*

Graduate Program in Molecular Microbiology, Sackler School of Graduate Biomedical Sciences,1 Department of Molecular Biology and Microbiology, School of Medicine, Tufts University, Boston, Massachusetts 02111,2 Structural Biology Laboratory, Department of Chemistry, University of York, York YO10 5YW, United Kingdom3

Received 23 June 2009/ Accepted 4 September 2009

Bacillus subtilis CodY protein is a DNA-binding global transcriptional regulator that responds to branched-chain 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.

* Corresponding author. Mailing address: Department of Molecular Biology and Microbiology, Tufts University School of Medicine, 136 Harrison Avenue, Boston, MA 02111. Phone: (617) 636-6761. Fax: (617) 636-0337. E-mail: linc.sonenshein{at}tufts.edu

{triangledown} Published ahead of print on 11 September 2009.

{dagger} Supplemental material for this article may be found at http://jb.asm.org/.

§ These authors contributed equally to the work presented.

{ddagger} Present address: Bio-X Program, Department of Chemical and Systems Biology, Stanford University School of Medicine, Stanford, CA 94305-5020.

Present address: Wyeth Pharmaceuticals, 401 N. Middletown Road, Pearl River, NY 10965.

Journal of Bacteriology, November 2009, p. 6865-6876, Vol. 191, No. 22
0021-9193/09/$08.00+0     doi:10.1128/JB.00818-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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