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

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

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Bacterial Proteins*
  • Binding Sites
  • Cysteine / genetics
  • Cysteine / physiology
  • Disulfides / metabolism*
  • Metalloproteins / genetics
  • Metalloproteins / metabolism
  • Metalloproteins / physiology*
  • Molecular Sequence Data
  • Mutagenesis
  • Oxidation-Reduction
  • Sequence Homology, Amino Acid
  • Sigma Factor / metabolism*
  • Spores, Bacterial
  • Streptomyces / genetics
  • Streptomyces / metabolism
  • Streptomyces / physiology
  • Sulfhydryl Compounds / metabolism*
  • Thioredoxins / metabolism
  • Transcription Factors / genetics
  • Transcription Factors / metabolism
  • Transcription Factors / physiology*
  • Zinc / metabolism*

Substances

  • Bacterial Proteins
  • Disulfides
  • Metalloproteins
  • RsrA protein, Streptomyces coelicolor
  • Sigma Factor
  • Sulfhydryl Compounds
  • Transcription Factors
  • Thioredoxins
  • Zinc
  • Cysteine