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Summary: Our previous study using transposon mutagenesis indicated that disruption of the putative response regulator gene orrA impacted antibiotic production in Streptomyces coelicolor. In this study, the role of OrrA was further characterized by comparing the phenotypes and transcriptomic profiles of the wild‐type S. coelicolor strain M145 and ΔorrA, a strain with an inactivated orrA gene. Chromatin immunoprecipitation using a strain expressing OrrA fused with FLAG showed that OrrA binds the promoter of wblA, whose expression was downregulated in ΔorrA. The interaction of OrrA with the wblA promoter was further validated by a pull‐down assay. Similar to ΔorrA, the deletion mutant of wblA (ΔwblA) was defective in development, and developmental genes were expressed at similar levels in ΔorrA and ΔwblA. Although both OrrA and WblA downregulated actinorhodin and undecylprodigiosin, their roles in regulation of the calcium‐dependent antibiotic and yellow‐pigmented type I polyketide differed. sco1375, a gene of unknown function, was identified as another OrrA target, and overexpression of either sco1375 or wblA in ΔorrA partially restored the wild‐type phenotype, indicating that these genes mediate some of the effects of OrrA. This study revealed targets of OrrA and provided more insights into the role of the orphan response regulator OrrA in Streptomyces. [ABSTRACT FROM AUTHOR]

Summary: Phosphate metabolism is known to be regulated by the PhoPR regulatory system in Streptomyces and some other bacteria. In this study, we report that MtrA also regulates phosphate metabolism in Streptomyces. Our data showed that, in Streptomyces coelicolor, MtrA regulates not only phosphate metabolism genes such as phoA but also phoP under different phosphate conditions, including growth on rich complex media without added inorganic phosphate and on defined media with low or high concentrations of inorganic phosphate. Cross‐regulation was also observed among mtrA, phoP and glnR under these conditions. We demonstrated both in vitro and in vivo binding of MtrA to the promoter regions of genes associated with phosphate metabolism and to the intergenic region between phoR and phoU, indicating that these phosphate metabolism genes are targets of MtrA. We further showed that MtrA in S. lividans and S. venezuelae has detectable regulatory effects on expression of phosphate metabolism genes. Additionally, the MtrA homologue from Corynebacterium glutamicum bound predicted MtrA sites of multiple phosphate metabolism genes, implying its potential for regulating phosphate metabolism in this species. Overall, our findings support MtrA as a major regulator for phosphate metabolism in Streptomyces and also potentially in other actinobacteria. [ABSTRACT FROM AUTHOR]

Summary: In Streptomyces, GlnR is an activator protein that activates nitrogen‐assimilation genes under nitrogen‐limiting conditions. However, less is known regarding the regulation of these genes under nitrogen‐rich conditions. We determined that the developmental regulator MtrA represses nitrogen‐assimilation genes in nitrogen‐rich media and that it competes with GlnR for binding to GlnR boxes. The GlnR boxes upstream of multiple nitrogen genes, such as amtB, were confirmed as MtrA binding sites in vitro by electrophoretic mobility shift assays and in vivo by ChIP‐qPCR analysis. Transcriptional analysis indicated that, on nutrient‐rich medium, MtrA profoundly repressed expression of nitrogen‐associated genes, indicating opposing roles for MtrA and GlnR in the control of nitrogen metabolism. Using in vitro and in vivo analysis, we also showed that glnR is itself a direct target of MtrA and that MtrA represses glnR transcription. We further demonstrated functional conservation of MtrA homologues in the recognition of GlnR boxes upstream of nitrogen genes from different actinobacterial species. As mtrA and glnR are widespread among actinomycetes, this mechanism of potential competitive control over nitrogen metabolism genes may be common in this group, adding a major new layer of complexity to the known regulatory network for nitrogen metabolism in Streptomyces and related species. [ABSTRACT FROM AUTHOR]