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 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.
Department of Molecular Microbiology, John Innes Centre, Colney, Norwich NR4 7UH, UK. M.Paget@sussex.ac.uk
M S
MS
Paget
J B
JB
Bae
M Y
MY
Hahn
W
W
Li
C
C
Kleanthous
J H
JH
Roe
M J
MJ
Buttner
D000595:Amino Acid Sequence; D001426:Bacterial Proteins; D001665:Binding Sites; D003545:Cysteine / Q000235:genetics / Q000502:physiology; D004220:Disulfides / Q000378:metabolism*; D008667:Metalloproteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D008969:Molecular Sequence Data; D016296:Mutagenesis; D010084:Oxidation-Reduction; D017386:Sequence Homology, Amino Acid; D012808:Sigma Factor / Q000378:metabolism*; D013171:Spores, Bacterial; D013302:Streptomyces / Q000235:genetics / Q000378:metabolism / Q000502:physiology; D013438:Sulfhydryl Compounds / Q000378:metabolism*; D013879:Thioredoxins / Q000378:metabolism; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D015032:Zinc / Q000378:metabolism*
D001426:Bacterial Proteins; D004220:Disulfides; D008667:Metalloproteins; C121147:RsrA protein, Streptomyces coelicolor; D012808:Sigma Factor; D013438:Sulfhydryl Compounds; D014157:Transcription Factors; D013879:Thioredoxins; D015032:Zinc; D003545:Cysteine