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.
| Original language | English |
|---|---|
| Pages (from-to) | 1036-47 |
| Number of pages | 12 |
| Journal | Molecular Microbiology |
| Volume | 39 |
| Issue number | 4 |
| Publication status | Published - 2001 |
Keywords
- Amino Acid Sequence
- Bacterial Proteins
- Binding Sites
- Cysteine
- Disulfides
- Metalloproteins
- Molecular Sequence Data
- Mutagenesis
- Oxidation-Reduction
- Sequence Homology, Amino Acid
- Sigma Factor
- Spores, Bacterial
- Streptomyces
- Sulfhydryl Compounds
- Thioredoxins
- Transcription Factors
- Zinc