The σF-specific anti-sigma factor RsfA is one of the protein kinases that phosphorylates the pleiotropic anti-anti-sigma factor BldG in Streptomyces coelicolor A3(2)

• SigF-specific anti-sigma factor RsfA interacts with the anti-anti-sigma factor BldG.
• BldG is specifically phosphorylated by the kinase activity of RsfA.
• RsfA interacts with the anti-anti-sigma factor SCO0869.
• rsfA is expressed in early differentiation stages and repressed by osmotic stress.

The anti-anti-sigma factor BldG has a role in the morphological differentiation and antibiotic production of Streptomyces coelicolor A3(2). Together with the anti-sigma factor UshX it is involved in the “partner-switching”-like activation of the sigma factor σH that has a dual role in the osmotic stress response and morphological differentiation in S. coelicolor A3(2). Although BldG is phosphorylated in vivo in S. coelicolor, neither of the interacting anti-sigma factors UshX and ApgA is found to phosphorylate it. By using a combination of several approaches, we demonstrated a direct interaction between BldG and the anti-sigma factor RsfA, which has been previously shown to regulate antibiotic production and morphological differentiation in S. coelicolor and to specifically interact with the sporulation-specific sigma factor σF. RsfA phosphorylates BldG in vitro, demonstrating that RsfA is a specific kinase for BldG and negatively regulates its activity. However, another interacting anti-anti-sigma factor homolog, SCO0869, was not phosphorylated by RsfA. Transcriptional analyses of rsfA revealed a single promoter, the activity of which was repressed by osmotic stress and decreased during differentiation. These data suggested that BldG has a pleiotropic role in the regulation of at least two sigma factors, σH and σF, in S. coelicolor.