Cloning and characterization of afsR homologue regulatory gene from Streptomyces acidiscabies ATCC 49003

Abstract

Global regulator for secondary metabolism, AfsR, is phosphorylated by the serine/threonine kinase AfsK. Phosphorylation of AfsR activates the transcription of afsS, resulting in the increased production of secondary metabolites in Streptomyces strains. We isolated an afsR homologue regulatory gene from Streptomyces acidiscabies ATCC 49003, which produces thaxtomin A and WS5995B as secondary metabolites. To examine the function of afsR in the production of secondary metabolites in S. acidiscabies, an intact 2,976 bp open reading frame of afsR was identified and characterized. In S. lividans TK 24 strain, the exconjugant harboring afsR high expression vector, began to generate actinorhodin at 36 h of culture, and the amount of accumulated actinorhodin became 10-fold higher than that of the exconjugant harboring the vector lacking the afsR gene. To clarify the in vivo function of afsR, an afsR-disrupted mutant was constructed and analyzed. No morphological difference was observed between the wild-type strain and the afsR disruptant, but production of thaxtomin A and WS5995B of the afsR disruptant were significantly decreased compared to those of the wild-type strains. Specially, WS5995B production was almost abolished by the disruption of only the afsR gene. These changes were restored to the original wild-type phenotype by the introduction of the intact afsR gene into the afsR disruptant, suggesting that the afsR gene participates in the production of secondary metabolites of S. acidiscabies.