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

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