Mutational analysis of RsrA, a zinc‐binding anti‐sigma factor with a thiol–disulphide redox switch

Abstract

SummaryIn the Gram-positive bacterium, Streptomyces coelicolor A3(2), expression of the thioredoxin system is modulated by a sigma factor called s R in response to changes in the cytoplasmic thiol±disulphide status, and the activity of s R is controlled post-translationally by an anti-sigma factor, RsrA. In vitro, the antisigma factor activity of RsrA, which contains seven cysteines, correlates with its thiol±disulphide redox status. Here, we investigate the function of RsrA in vivo. A constructed rsrA null mutant had very high constitutive levels of disulphide reductase activity and s R -dependent transcription, confirming that RsrA is a negative regulator of s R and a key sensor of thiol±disulphide status. Targeted mutagenesis revealed that three of the seven cysteines in RsrA (C11, C41 and C44) were essential for anti-sigma factor activity and that a mutant RsrA protein containing only these three cysteines was active and still redox sensitive in vivo. We also show that RsrA is a metalloprotein, containing near-stoichiometric amounts of zinc. On the basis of these data, we propose that a thiol±disulphide redox switch is formed between two of C11, C41 and C44, and that all three residues play an essential role in anti-sigma factor activity in their reduced state, perhaps by acting as ligands for zinc. Unexpectedly, rsrA null mutants were blocked in sporulation, probably as a consequence of an increase in the level of free s R .

Reduced immunopathology and mortality despite tissue persistence in aMycobacterium tuberculosismutant lacking alternative σ factor, SigH

“…The arrangement of genes at the M. tuberculosis sigH locus resembles that of the Streptomyces coelicolor A3(2) sigR locus (38,39), and it has been shown that the gene adjacent to sigR (rsrA) inhibits SigR activity by functioning as SigR-sequestering anti-factor (40). Because MT3318 transcriptional levels parallel those of sigH, it appears likely that the SigH and the MT3318 gene product are produced in equimolar quantities.…”

Regulation of inducible peroxide stress responses

“…Oxidation of RsrA leads to the formation of disulphide bonds and the concomitant release of Zn(II). Thus, it is likely that the role of RsrA as a sensor of oxidative stress involves one or more reactive cysteine residues co-ordinated to Zn(II) (Paget et al, 2001b). A. OxyR is a tetrameric DNA-binding protein that can be reversibly oxidized to form a disulphide linking Cys-199 and Cys-208 (E. coli numbering).…”

OxyR is a key regulator in response to oxidative stress in Streptomyces avermitilis

“…In S. coelicolor, the activity of s R is regulated by anti-s factor RsrA, which is inactivated by formation of disulphide bonds and releases s R to transcribe its regulon under oxidative stress (Bae et al, 2004;Kang et al, 1999;Paget et al, 2001a, b). An unstable larger isoform of s R with a 55 aa N-terminal extension is induced by diamide treatment; this extension is transcribed from the s R -dependent promoter and translated from an upstream start codon (Kim et al, 2009).…”