Avermectins produced by Streptomyces avermitilis are potent anti-parasitic agents that are useful in animal health care, agriculture, and the treatment of human infections. In a search for novel regulators that affect avermectin biosynthesis, comparative transcriptome analysis was performed between wild-type strain ATCC31267 and avermectin overproducing strain 76-02-e, revealing some differentially expressed genes. SAV576, which is downregulated in 76-02-e and encodes a TetR family transcriptional regulator (TFR), was shown to inhibit avermectin production by indirectly affecting the expression of ave genes. SAV576 directly repressed the transcription of its gene SAV576 and of adjacent genes SAV575 (encodes cytochrome P450/NADPH-ferrihemoprotein reductase) and SAV574. The SAV576-binding sites within the bidirectional SAV575-SAV576 promoter region were determined by DNase I footprinting assays. A consensus 15-bp palindromic sequence CCRTACRVYGTATGS was found in these binding sites and shown to be important for SAV576-binding activity. SAV575, an important target gene of SAV576, was shown to exert a positive effect on avermectin production. The study findings extend our limited knowledge of the complex regulation of avermectin biosynthesis and provide a basis for rational genetic manipulation of S. avermitilis to improve avermectin production through control of SAV576 and its target gene.

Streptomyces avermitilis is an important bacterial species used for industrial production of avermectins, a family of broad-spectrum anthelmintic agents. We previously identified the protein SAV576, a TetR family transcriptional regulator (TFR), as a downregulator of avermectin biosynthesis that acts by controlling transcription of its major target gene SAV575 (which encodes cytochrome P450/NADPH-ferrihemoprotein reductase) and ave genes. SAV577, another TFR gene, encodes a SAV577 protein that displays high amino acid homology with SAV576. In this study, we examined the effect of SAV577 on avermectin production and the relationships between SAV576 and SAV577. SAV577 downregulated avermectin biosynthesis indirectly, similarly to SAV576. SAV576 and SAV577 both directly repressed SAV575 transcription, and reciprocally repressed each other's expression. SAV575 transcription levels in various S. avermitilis strains were correlated with avermectin production levels. DNase I footprinting and electrophoretic mobility shift assays indicated that SAV576 and SAV577 compete for the same binding regions, and that DNA-binding affinity of SAV576 is much stronger than that of SAV577. GST pull-down assays revealed no direct interaction between the two proteins. Taken together, these findings suggest that SAV577 regulates avermectin production in S. avermitilis by a mechanism similar to that of SAV576, and that the role of SAV576 is dominant over that of SAV577. This is the first report of two adjacent and similar TFR genes that co-regulate antibiotic production in Streptomyces.