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. 2001 Feb;39(4):1036-47.
doi: 10.1046/j.1365-2958.2001.02298.x.

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

M S Paget  1 , J B Bae, M Y Hahn, W Li, C Kleanthous, J H Roe, M J Buttner
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Free article

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

M S Paget et al. Mol Microbiol. 2001 Feb.
Free article

Abstract

In the Gram-positive bacterium, Streptomyces coelicolor A3(2), expression of the thioredoxin system is modulated by a sigma factor called sigmaR in response to changes in the cytoplasmic thiol-disulphide status, and the activity of sigmaR is controlled post-translationally by an anti-sigma factor, RsrA. In vitro, the anti-sigma 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 sigmaR-dependent transcription, confirming that RsrA is a negative regulator of sigmaR 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 sigmaR.

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