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In Azospirillum brasilense, an extra-cytoplasmic function σ factor (RpoE10) shows the characteristic 119 amino acid long C-terminal extension found in ECF41-type σ factors, which possesses three conserved motifs (WLPEP, DGGGR, and NPDKV), one in the linker region between the σ 2 and σ 4 , and the other two in the SnoaL_2 domain of the C-terminal extension. Here, we have described the role of the two conserved motifs in the SnoaL_2 domain of RpoE10 in the inhibition and activation of its activity, respectively. Truncation of the distal part of the C-terminal sequence of the RpoE10 (including NPDKV but excluding the DGGGR motif) results in its promoter's activation suggesting autoregulation. Further truncation of the C-terminal sequence up to its proximal part, including NPDKV and DGGGR motif, abolished promoter activation. Replacement of NPDKV motif with NAAAV in RpoE10 increased its ability to activate its promoter, whereas replacement of DGGGR motif led to reduced promoter activation. We have explored the dynamic modulation of σ 2 -σ 4 domains and the relevant molecular interactions mediated by the two conserved motifs of the SnoaL2 domain using molecular dynamics simulation. The analysis enabled us to explain that the NPDKV motif located distally in the C-terminus negatively impacts transcriptional activation. In contrast, the DGGGR motif found proximally of the C-terminal extension is required to activate RpoE10., (© 2022 Wiley Periodicals LLC.)

Aims: Azospirillum brasilense harbors two redox-sensitive Zinc-binding anti-sigma (ZAS) factors (ChrR1 and ChrR2), which negatively regulate the activity of their cognate extra-cytoplasmic function (ECF) σ factors (RpoE1 and RpoE2) by occluding their binding to the core enzyme. Both pairs of RpoE-ChrR control responses to photooxidative stress. The aim of this study was to investigate whether the two RpoE-ChrR pairs cross-talk while responding to the stress., Results: In silico analysis showed a high sequence similarity between ChrR1 and ChrR2 proteins, but differences in redox sensitivity. Using in silico and in vitro methods of protein-protein interaction, we have shown that both ChrR1 and ChrR2 proteins physically bind to their noncognate RpoE proteins. Restoration of the phenotypes of chrR1::Tn5 and chrR2::Km mutants related to carotenoid biosynthesis and photooxidative stress tolerance by expressing chrR1 or chrR2 provided in vivo evidence for the cross-talk. In addition, up- or down-regulation of several identical proteins by expressing chrR1 or chrR2 in the chrR1::Tn5 mutant provided another in vivo evidence for the cross-talk., Innovation: Although multiple redox-sensitive ZAS anti-σ factors occur in some Gram-positive bacteria, no cross-talk is reported among them. We report here, for the first time, that the two ZAS anti-σ factors of A. brasilense also interact with their noncognate σ factors and affect gene expression., Conclusion: The two redox-sensitive ZAS anti-σ factors in A. brasilense may interact with their cognate as well as noncognate ECF σ factors to play an important role in redox homeostasis by facilitating recovery from the oxidative stress.

Aims: Azospirillum brasilense harbors two redox-sensitive Zinc-binding anti-sigma (ZAS) factors (ChrR1 and ChrR2), which negatively regulate the activity of their cognate extra-cytoplasmic function (ECF) σ factors (RpoE1 and RpoE2) by occluding their binding to the core enzyme. Both pairs of RpoE-ChrR control responses to photooxidative stress. The aim of this study was to investigate whether the two RpoE-ChrR pairs cross-talk while responding to the stress. Results: In silico analysis showed a high sequence similarity between ChrR1 and ChrR2 proteins, but differences in redox sensitivity. Using in silico and in vitro methods of protein-protein interaction, we have shown that both ChrR1 and ChrR2 proteins physically bind to their noncognate RpoE proteins. Restoration of the phenotypes of chrR1::Tn5 and chrR2::Km mutants related to carotenoid biosynthesis and photooxidative stress tolerance by expressing chrR1 or chrR2 provided in vivo evidence for the cross-talk. In addition, up- or down-regulation of several identical proteins by expressing chrR1 or chrR2 in the chrR1::Tn5 mutant provided another in vivo evidence for the cross-talk. Innovation: Although multiple redox-sensitive ZAS anti-σ factors occur in some Gram-positive bacteria, no cross-talk is reported among them. We report here, for the first time, that the two ZAS anti-σ factors of A. brasilense also interact with their noncognate σ factors and affect gene expression. Conclusion: The two redox-sensitive ZAS anti-σ factors in A. brasilense may interact with their cognate as well as noncognate ECF σ factors to play an important role in redox homeostasis by facilitating recovery from the oxidative stress. Antioxid. Redox Signal. 20, 42-59. [ABSTRACT FROM AUTHOR]