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Activation of Bacillus Subtilis Cody and Its Role in Sporulation.

Activation of Bacillus Subtilis Cody and Its Role in Sporulation.


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Bacillus subtilis CodY protein is a DNA-binding global transcriptional regulator that responds to branched-chain amino acids (isoleucine, leucine and valine) (BCAAs) and GTP and represses many stationary phase genes during exponential growth. 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. I found that F71 residue of CodY is necessary for stimulation by GTP and BCAAs and R61 residue is necessary for stimulation by BCAAs only. The F71E mutant form of CodY had no detectable activity in vivo and the mutant protein did not respond to the effectors in vitro. The CodY mutant R61A retained partial repression of target promoters in vivo and was able to respond to GTP but not to BCAAs in vitro. By comparing the ability of branched-chain amino acids and their analogs to stimulate wild-type CodY for DNA binding, I found that the 2-amino group and its chirality, and the 3-methyl group are critical requirements for the interaction of BCAAs with CodY. As CodY has been implicated in negative regulation of sporulation, I employed in vivo and in vitro studies to identify potential sporulation targets of CodY. Using gel shift assays, I identified several phosphorelay genes, including kinA, kinB, kinC, spo0F, spo0B, spo0A and kinC, as potential direct sporulation targets of CodY. Further, by using DNaseI footprinting assay I identified a protected region for kinC but not for spo0A and kinB promoters. My results implicated CodY in the regulation of the master stationary phase regulator spo0A. CodY was found to repress both spo0A Pv-lacZ and Pv+Ps-lacZ fusions in S6C medium and a Pv+Ps-lacZ fusion in enriched DS medium. My results also implicated CodY in the regulation of spoIIQ, an essential gene that functions in stage II-III of sporulation. I found that CodY is involved in the repression of spoIIQ-lacZ expression in DS medium. I demonstrated that CodY binds to the spoIIQ promoter directly and identified a protected region. I also showed that a putative CodY binding box located in the spoIIQ promoter was required for the in vitro binding.


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Product Details
  • ISBN-13: 9781243428745
  • Publisher: Proquest, Umi Dissertation Publishing
  • Publisher Imprint: Proquest, Umi Dissertation Publishing
  • Height: 254 mm
  • Weight: 513 gr
  • ISBN-10: 1243428740
  • Publisher Date: 02 Sep 2011
  • Binding: Paperback
  • Spine Width: 17 mm
  • Width: 203 mm


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