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

Abstract

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).

Mechanistic Implications of the Unique Structural Features and Dimerization of the Cytoplasmic Domain of the Pseudomonas Sigma Regulator, PupR

“…Despite sharing only 7–16% identity (31), the PupR-ASD and other known ASDs share a common structural fold comprising a three-helix core bundle (Figure 3). PupR-ASD helices 1–3 superimpose (Figure 3) with a root-mean-square deviations (rmsd) of 2.52–3.18 Å over 40–48 residues of the ChrR-ASD from R. sphaeroides ((30); PDB ID 2Z2S), RseA from E. coli ((34); PDB ID 1OR7), RskA from M. tuberculosis ((37); PDB ID 4NQW), and RslA from M. tuberculosis ((36); PDB ID 3HUG).…”

A constitutively expressed pair of rpoE2–chrR2 in Azospirillum brasilense Sp7 is required for survival under antibiotic and oxidative stress

“…The conserved His141, His143, Glu147 and His177 residues that are involved in the binding of a Zn (Greenwell et al, 2011;Lourenço et al, 2011). The two cysteine residues (Cys186 and Cys188) located at the extreme end of the CLD in the ChrR of C. crescentus and R. sphaeroides are also present in the two ChrR paralogues of A. brasilense.…”

“…The two cysteine residues (Cys186 and Cys188) located at the extreme end of the CLD in the ChrR of C. crescentus and R. sphaeroides are also present in the two ChrR paralogues of A. brasilense. Although these cysteine residues of ChrR in C. crescentus have been shown to be critical for responding to cadmium stress (Lourenço et al, 2011), they were not involved in promoting dissociation of the s E -ChrR complex of R. sphaeroides in response to cadmium stress (Greenwell et al, 2011).…”

“…The lack of autoregulation and regulation by stress agents at the level of transcription of the rpoE2-chrR2 operon suggests that the constitutively expressed RpoE2 and ChrR2 might regulate expression of their target genes by stress-mediated disruption of the RpoE2-ChrR2 complex (Greenwell et al, 2011). The differences in the mechanisms of regulation of rpoE1-chrR1 and rpoE2-chrR2 suggest that the former may be responsible for long-term tolerance to stress whereas the latter might be important in a rapid response to stress.…”

“…In the absence of singlet oxygen ( 1 O 2 ), ChrR forms a 1 : 1 complex with s E , preventing the s factor from binding to the core RNA polymerase and initiating transcription of the target genes. In the presence of 1 O 2 , s E is released from ChrR and triggers the expression of genes needed for protection against photooxidative stress (Glaeser et al, 2011;Greenwell et al, 2011). The genes controlled by s E and their target promoters in R. sphaeroides revealed that the members of the s E regulon and their promoter consensus in several a-proteobacteria might be common (Dufour et al, 2008;Ziegelhoffer & Donohue, 2009).…”