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

journal contribution
posted on 2023-06-07, 20:07 authored by Mark PagetMark Paget, Jae-Bum Bae, Mi-Young Hahn, Wei Li, Colin Kleanthous, Jung-Hye Roe, Mark J Buttner
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

History

Publication status

  • Published

Journal

Molecular Microbiology

ISSN

0950-382X

Issue

4

Volume

39

Page range

1036-1047

Pages

12.0

Department affiliated with

  • Biochemistry Publications

Notes

Paget was a post-doctoral fellow and recognised researcher on the BBSRC grant that funded the project, and is corresponding author. Paget performed most experiments and wrote up the paper. It defines for the first time the zinc-binding anti-sigma factor family, now recognised as one of the largest in bacteria.

Full text available

  • No

Peer reviewed?

  • Yes

Legacy Posted Date

2012-02-06

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