Coelimycin inside out - negative feedback regulation by its intracellular precursors.

Coelimycin (CPK) producer Streptomyces coelicolor A3(2) is a well-established model for the genetic studies of bacteria from the genus Streptomyces, renowned for their ability to produce a plethora of antibiotics and other secondary metabolites. Expression regulation of natural product biosynthetic gene clusters (BGCs) is highly complex, involving not only regulatory proteins, like transcription factors, but also the products of the biosynthetic pathway that may act as ligands for some regulators and modulate their activity. Here, we present the evidence that intracellular CPK precursor(s) (preCPK) is involved in a negative feedback loop repressing the CPK BGC. Moreover, we provide a characterization of the cluster-encoded efflux pump CpkF. We show that CpkF is essential for the extracellular CPK production. In order to track down which CPK compounds - intra- or extracellular - are the ones responsible for the feedback signal, a luciferase-based reporter system was applied to compare the activity of 13 CPK gene promoters in the wild-type (WT) and two mutated strains. The first strain, lacking the CPK-specific exporter CpkF (ΔcpkF), was unable to produce the extracellular CPK. The second one did not produce any CPK at all, due to the disruption of the CpkC polyketide synthase subunit (ΔcpkC). All tested promoters were strongly upregulated in ΔcpkC strain, while in the ΔcpkF strain, promoter activity resembled the one of WT. These results lead to the conclusion that the CPK polyketide acts as a silencer of its own production. Supposedly this function is exerted via binding of the preCPK by an unidentified regulatory protein. KEY POINTS: •Intracellular coelimycin precursor takes part in a negative cpk cluster regulation •CpkF exporter is essential for the extracellular coelimycin production •Simple method for the analysis of coelimycin P2 production in agar medium.