Supplementary Material for: Mutation in <b><i>afsR</i></b> Leads to A-Factor Deficiency in <b><i>Streptomyces griseus</i></b> B2682

<b><i>Background/Aims:</i></b> A-factor, a γ-butyrolactone autoregulator, in <i>Streptomyces griseus</i> is involved in the regulation of differentiation and antibiotic production. Here we studied the <i>S. griseus</i> B2682-AFN (A-factor negative) bald mutant that harbors a nonsense mutation in the <i>afsR</i> gene encoding a pleiotropic regulator. Our aim was to prove that this mutation is the cause of the A-factor deficiency in AFN. We also studied whether AfsR regulates A-factor production by AfsA, which is supposed to be the only specific key enzyme in A-factor biosynthesis. <b><i>Methods:</i></b> Wild <i>afsR</i> was cloned to the pHJL401 shuttle vector and was transformed to the <i>S. griseus</i> AFN and B2682 strains. During phenotypic characterization, sporulation, antibiotic, protease, A-factor, and AfsA protein production were studied. <b><i>Results:</i></b> Transformation of AFN by a wild <i>afsR</i> restored its phenotype including sporulation, antibiotic, extracellular protease, and A-factor production. Introduction of <i>afsR</i> to the B2682 wild-type strain resulted in antibiotic and extracellular protease overproduction that was accompanied with an elevated A-factor level. AfsA was detected both in AFN and B2682. <b><i>Conclusions:</i></b> AfsR has an effect on the regulation of A-factor production in <i>S. griseus</i>. The presence of AfsA is not sufficient for normal A-factor production. AfsR regulates A-factor biosynthesis independently of AfsA.