Features of Rhodobacter sphaeroides ChrR Required for Stimuli to Promote the Dissociation of σE/ChrR Complexes

In the photosynthetic bacterium Rhodobacter sphaeroides, a transcriptional response to the reactive oxygen species singlet oxygen (1O2) is mediated by ChrR, a zinc metalloprotein that binds to and inhibits the activity of the alternative σ factor σE. We provide evidence that 1O2 promotes the dissociation of σE from ChrR to activate transcription in vivo. To identify what is required for 1O2 to promote the dissociation of σE/ChrR complexes, we analyzed the in vivo properties of variant ChrR proteins with amino acid changes in conserved residues of the C-terminal cupin-like domain (ChrR-CLD). We found that 1O2 was unable to promote the detectable dissociation of σE/ChrR complexes when the ChrR-CLD zinc ligands (His141, His143, Glu147, and His177) were substituted with alanine, even though individual substitutions caused a 2-fold to 10-fold decrease in zinc affinity for this domain relative to that for wild-type ChrR (Kd ∼ 4.6 × 10− 10 M). We conclude that the side chains of these invariant residues play a crucial role in the response to 1O2. Additionally, we found that cells containing variant ChrR proteins with single amino acid substitutions at Cys187 or Cys189 exhibited σE activity similar to those containing wild-type ChrR when exposed to 1O2, suggesting that these thiol side chains are not required for 1O2 to induce σE activity in vivo. Finally, we found that the same aspects of R. sphaeroides ChrR needed for a response to 1O2 are required for the dissociation of σE/ChrR complexes in the presence of the organic hydroperoxide t-butyl hydroperoxide.