Evidence of a role for CutRS and actinorhodin in the secretion stress response in Streptomyces coelicolor M145

CutRS was the first two-component system to be identified in Streptomyces species and is highly conserved in this genus. It was reported >25 years ago that deletion of cutRS increases the production of the antibiotic actinorhodin in Streptomyces coelicolor but despite this early work the function of CutRS has remained enigmatic until now. Here we show that deletion of cutRS upregulates the production of the actinorhodin biosynthetic enzymes up to 260-fold in the cutRS mutant, explaining the increase in actinorhodin production. However, while ChIP-seq identified 86 CutR binding sites in S. coelicolor none of these are in the actinorhodin BGC, so the effect is indirect. The directly regulated CutR targets identified in this study are implicated in extracellular protein folding, including two of the four highly conserved the HtrA-family foldases, HtrA3 and HtrB and a putative VKOR enzyme which is predicted to recycle DsbA following its catalysis of disulphide bond formation in secreted proteins. We thus tentatively propose a role for CutRS in sensing and responding to protein misfolding outside the cell. Since actinorhodin can oxidise cysteine residues and induce disulphide bond formation in proteins its over production in the ?cutRS mutant may be a response to protein misfolding on the extracellular face of the membrane. Overall design: ChIP-seq was carried out to determine the DNA binding sites of the response regulator CutR, part of the two component system CutRS, in Streptomyces coelicolor. This is a conserved two component system found in all member of the genus. CutRS knock out mutants show a different phenotype in the presence of glucose compared to the wild type - and so to understand this further these expirments were carried out on samples grown on DNA (no glucose) and DNAD (with glucose). Being analysed in this experiment is DNA binding of the response regulator CutR in S. coelicolor. S. coelicolor ?cutRS containing pSS170-Cterm3xFLAG-CutRS (CutS and FLAG-tagged CutR under the native promotor) and S. coelicolor ?cutRS containing pSS170-Cterm3x_vnz_FLAG-CutRS (CutS and FLAG-tagged CutR from S. venezuelae under the native promotor) grown in the presence and absence of glucose. This was compared to S. coelicolor WT (no FLAG) grown in the same way and each was carried out and analysed in duplicate.