Defining the disulphide stress response in Streptomyces coelicolor A3(2): identification of the σ^R regulon

Abstract

SummaryIn the Gram-positive, antibiotic-producing bacterium Streptomyces coelicolor A3(2), the thiol-disulphide status of the hyphae is controlled by a novel regulatory system consisting of a sigma factor, s R , and its cognate anti-sigma factor, RsrA. Oxidative stress induces intramolecular disulphide bond formation in RsrA, which causes it to lose affinity for s R , thereby releasing s R to activate transcription of the thioredoxin operon, trxBA. Here, we exploit a preliminary consensus sequence for s R target promoters to identify 27 new s R target genes and operons, thereby defining the global response to disulphide stress in this organism. Target genes related to thiol metabolism encode a second thioredoxin (TrxC), a glutaredoxin-like protein and enzymes involved in the biosynthesis of the low-molecular-weight thiol-containing compounds cysteine and molybdopterin. In addition, the level of the major actinomycete thiol buffer, mycothiol, was fourfold lower in a sigR null mutant, although no candidate mycothiol biosynthetic genes were identified among the s R targets. Three s R target genes encode ribosome-associated products (ribosomal subunit L31, ppGpp synthetase and tmRNA), suggesting that the translational machinery is modified by disulphide stress. The product of another s R target gene was found to be a novel RNA polymerase-associated protein, RbpA, suggesting that the transcriptional machinery may also be modified in response to disulphide stress. We present DNA sequence evidence that many of the targets identified in S. coelicolor are also under the control of the s R homologue in the actinomycete pathogen Mycobacterium tuberculosis.

Activation of the Redox-regulated Chaperone Hsp33 by Domain Unfolding

“…Upon exposure to oxidative stress, disulfide bonds of RsrA form, and zinc is released. This is accompanied by major structural rearrangements that lead to the release of R , which is then able to activate transcription of its target genes (18,30). In contrast to Hsp33 where the oxidized conformation unfolds and the reduced zinc-bound conformation is fully structured, disulfidebonded RsrA appears to contain more ␣-helical secondary structure than the reduced, zinc-bound form.…”

“…The switch that regulates the activity of Hsp33 is a novel, very high affinity zinc-binding motif (CXCX [27][28][29][30][31][32] CXXC) that is located in the C terminus of the protein (3). The four absolutely conserved cysteines that constitute this redox switch are kept in the reduced deprotonated thiolate anion state and together coordinate one zinc(II) ion (K D ϳ 10 Ϫ18 M) (3).…”

Cellular Defenses against Superoxide and Hydrogen Peroxide

“…One wonders, then, whether glutaredoxins and thioredoxins are actually needed only when H 2 O 2 stress is of such long duration as to compensate for the sluggish reactivity of protein thiols; whether they repair a small cohort of extremely reactive thiolate enzymes that have so far escaped detection; whether thiolate oxidations are primarily driven by reactions with oxygen or hydroxyl radicals, rather than H 2 O 2 ; or whether disulfide stress arises in natural environments from a different types of stressor, mimicked by thiol agents such as diamide. In fact, many bacteria-including Bacillus, Mycobacteria, Rhodobacter, and Streptomyces-do not include glutaredoxins or thioredoxins in their H 2 O 2 -inducible regulons (123)(124)(125). Instead, for example, Streptomyces controls synthesis of its thioredoxins with an anti-sigma factor protein that is activated when sulfur exchange reactions create a disulfide bond (126); notably, this system is easily triggered by diamide but requires millimolar doses of H 2 O 2 .…”

Regulation of inducible peroxide stress responses

“…Disulphide stress can be specifically elicited in cells by treatment with the thiol-specific oxidant, diamide, whereas peroxide stress has more far-reaching effects on cellular metabolism. Further evidence for the distinction between peroxide stress and disulphide stress has emerged from analysis of the Streptomyces coelicolor s R regulon (Paget et al, 2001a). Induction of the s R regulon with diamide activates transcription of thioredoxin and thioredoxin reductase, thereby acting to restore oxidized thiols in the cell to their appropriate reduced state (Paget et al, 1998).…”