Defining the OrrA regulon and its role in development and antibiotic production in Streptomyces venezuelae NRRL B-65442

Streptomyces bacteria have complex life cycles involving hyphal growth, sporulation and the production of diverse specialised metabolites, including antibiotics. In this study, we investigated the role of the highly conserved orphan response regulator OrrA in Streptomyces venezuelae NRRL B-65442. We show that S. venezuelae ∆ orrA mutants are defective in sporulation and used chromatin immunoprecipitation followed by sequencing to identify five OrrA binding sites in vivo . Tandem-mass-tag proteomics revealed that OrrA directly activates two of these putative target genes, wblA and vnz_04640 , a finding consistent with previous work on OrrA in the distantly related Streptomyces coelicolor . We also demonstrate that deleting wblA blocks sporulation and that overexpressing wblA restores sporulation in the ∆ orrA mutant. Additionally, chloramphenicol biosynthesis is upregulated in both the ∆ orrA and ∆ wblA mutants compared with the wild-type strain. Taken together, these results indicate that the primary function of OrrA is to regulate WblA production and that reduced intracellular WblA levels underlie the phenotypes observed in the ∆ orrA mutant.