PapR6, a Putative Atypical Response Regulator, Functions as a Pathway-Specific Activator of Pristinamycin II Biosynthesis in Streptomyces pristinaespiralis

Abstract

There are up to seven regulatory genes in the pristinamycin biosynthetic gene cluster ofStreptomyces pristinaespiralis, which infers a complicated regulation mechanism for pristinamycin production. In this study, we revealed that PapR6, a putative atypical response regulator, acts as a pathway-specific activator of pristinamycin II (PII) biosynthesis. Deletion of thepapR6gene resulted in significantly reduced PII production, and its overexpression led to increased PII formation, compared to that of the parental strain HCCB 10218. However, eitherpapR6deletion or overexpression had very little effect on pristinamycin I (PI) biosynthesis. Electrophoretic mobility shift assays (EMSAs) demonstrated that PapR6 bound specifically to the upstream region ofsnaF, the first gene of thesnaFE1E2GHIJKoperon, which is likely responsible for providing the precursor isobutyryl-coenzyme A (isobutyryl-CoA) and the intermediate C11αβ-unsaturated thioester for PII biosynthesis. A signature PapR6-binding motif comprising two 4-nucleotide (nt) inverted repeat sequences (5′-GAGG-4 nt-CCTC-3′) was identified. Transcriptional analysis showed that inactivation of thepapR6gene led to markedly decreased expression ofsnaFE1E2GHIJK. Furthermore, we found that a mutant (snaFmu) with base substitutions in the identified PapR6-binding sequence in the genome exhibited the same phenotype as that of the ΔpapR6strain. Therefore, it may be concluded that pathway-specific regulation of PapR6 in PII biosynthesis is possibly exerted via controlling the provision of isobutyryl-CoA as well as the intermediate C11αβ-unsaturated thioester.