A transcriptional response to singlet oxygen in Rhodobacter sphaeroides is controlled by the group IV sigma factor sigma(E) and its cognate anti-sigma ChrR. Crystal structures of the sigma(E)/ChrR complex reveal a modular, two-domain architecture for ChrR. The ChrR N-terminal anti-sigma domain (ASD) binds a Zn(2+) ion, contacts sigma(E), and is sufficient to inhibit sigma(E)-dependent transcription. The ChrR C-terminal domain adopts a cupin fold, can coordinate an additional Zn(2+), and is required for the transcriptional response to singlet oxygen. Structure-based sequence analyses predict that the ASD defines a common structural fold among predicted group IV anti-sigmas. These ASDs are fused to diverse C-terminal domains that are likely involved in responding to specific environmental signals that control the activity of their cognate sigma factor.

In the photosynthetic bacterium Rhodobacter sphaeroides, a transcriptional response to the reactive oxygen species singlet oxygen ( 1 O 2 ) is mediated by ChrR, a zinc metalloprotein that binds to and inhibits activity of the alternative sigma factor, σ E . We provide evidence that 1 O 2 promotes dissociation of σ E from ChrR to activate transcription in vivo. To identify what is required for 1 O 2 to promote dissociation of σ E /ChrR complexes, we analyzed the in vivo properties of variant ChrR proteins with amino acid changes in conserved residues of the Cterminal cupin-like domain (ChrR-CLD). We found that 1 O 2 was unable to promote detectable dissociation of σ E /ChrR complexes when the ChrR-CLD zinc ligands (His 141 , His 143 , Glu 147 , and His 177 ) were substituted with alanine, even though individual substitutions caused a 2-to 10-fold decrease in zinc affinity for this domain relative to that of wild-type ChrR (K d ∼4.6 × 10 −10 M). We conclude that the side chains of these invariant residues play a crucial role in the response to 1 O 2 . Additionally, we found that cells containing variant ChrR proteins with single amino acid substitutions at Cys 187 or Cys 189 exhibited σ E activity similar to those containing wild-type ChrR when exposed to 1 O 2 , suggesting that these thiol side chains are not required for 1 O 2 to induce σ E activity in vivo. Finally, we found that the same aspects of R. sphaeroides ChrR needed for a response to 1 O 2 are required for dissociation of σ E /ChrR in the presence of the organic hydroperoxide, tert-butyl hydroperoxide (t-BOOH).

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