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Bacillus subtilis CodY operators contain overlapping CodY binding sites.

Lewis V Wray1, Susan H Fisher

  • 1Department of Microbiology, Boston University School of Medicine, 72 East Concord Street, Boston, MA 02118-2526, USA.

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|July 19, 2011
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Summary

Bacillus subtilis CodY protein binds DNA more effectively at pH 6.0 than pH 8.0. This pH-dependent DNA binding influences gene regulation by CodY, a global transcriptional regulator activated by amino acids.

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Area of Science:

  • Microbiology
  • Molecular Biology
  • Genetics

Background:

  • CodY is a global transcriptional regulator in bacteria, activated by branched-chain amino acids.
  • CodY recognizes a specific 15-bp palindromic DNA motif (AATTTTCNGAAAATT).

Purpose of the Study:

  • To investigate the effect of pH on the DNA binding affinity and regulatory function of Bacillus subtilis CodY.
  • To characterize the specific DNA sequences and binding mechanisms of CodY at different pH levels.

Main Methods:

  • Gel mobility shift assays were employed to assess CodY's DNA binding affinity at varying pH.
  • DNase I footprinting was utilized to identify CodY-protected DNA regions within target promoters.
  • Mutational analysis of promoter regions was performed to confirm the role of identified motifs in CodY regulation.

Main Results:

  • Bacillus subtilis CodY exhibits higher DNA binding affinity at pH 6.0 compared to pH 8.0.
  • Isoleucine activation of CodY is enhanced at pH 6.0, with reduced nonspecific DNA binding.
  • CodY binding sites within the hutPp and ureAp(3) promoters contain overlapping copies of the 15-bp motif.

Conclusions:

  • pH significantly influences the DNA binding characteristics and regulatory activity of Bacillus subtilis CodY.
  • Overlapping sequence motifs are crucial for CodY binding to native operator sites, suggesting a complex regulatory mechanism.
  • These findings provide insights into the environmental factors affecting bacterial gene expression regulated by CodY.