Pathway-specific regulation revisited: cross-regulation of multiple disparate gene clusters by PAS-LuxR transcriptional regulators.

PAS-LuxR regulators are highly conserved proteins devoted to the control of antifungal production by binding to operators located in given promoters of polyene biosynthetic genes. The canonical operator of PimM, archetype of this class of regulators, has been used here to search for putative targets of orthologous protein PteF in the genome of Streptomyces avermitilis, finding 97 putative operators outside the pentaene filipin gene cluster (pte). The processes putatively affected included genetic information processing; energy, carbohydrate, and lipid metabolism; DNA replication and repair; morphological differentiation; secondary metabolite biosynthesis; and transcriptional regulation, among others. Seventeen of these operators were selected, and their binding to PimM DNA-binding domain was assessed by electrophoretic mobility shift assays. Strikingly, the protein bound all predicted operators suggesting a direct control over targeted processes. As a proof of concept, we studied the biosynthesis of the ATP-synthase inhibitor oligomycin whose gene cluster included two operators. Regulator mutants showed a severe loss of oligomycin production, whereas gene complementation of the mutant restored phenotype, and gene duplication in the wild-type strain boosted oligomycin production. Comparative gene expression analyses in parental and mutant strains by reverse transcription-quantitative polymerase chain reaction of selected olm genes corroborated production results. These results demonstrate that PteF is able to cross-regulate the biosynthesis of two related secondary metabolites, filipin and oligomycin, but might be extended to all the processes indicated above. This study highlights the complexity of the network of interactions in which PAS-LuxR regulators are involved and opens new possibilities for the manipulation of metabolite production in Streptomycetes.