A conserved NLR activation switch governs global transcriptional control of antibiotic biosynthesis in Streptomyces

Abstract

Nucleotide-binding and oligomerization domain-like receptors (NLRs) are conserved molecular switches that regulate innate immunity across diverse domains of life. While best known for their roles in immunity, bacterial NLR-related proteins also govern non-immune processes. In Streptomyces, the global transcriptional regulator AfsR employs an NLR-type architecture to control antibiotic production. Here, we identify a conserved histidine-aspartic acid motif within the nucleotide-binding and oligomerization domain of AfsR that functions as a universal NLR activation switch. Site-directed mutagenesis of this motif decouples AfsR from native upstream signals, locking it into a constitutive, autoactive “ON” state. Using integrated transcriptomics, ChIP-seq, and in vitro DNA-binding assays, we demonstrate that autoactivation expands the regulatory reach of AfsR via enhanced promoter affinity, directly controlling its canonical target afsS and uncovering wblH as an additional member of the AfsR regulon. Untargeted metabolomics further shows that autoactive AfsR drives systemic reprogramming of specialized metabolism, increasing the production of known antibiotics and awakening silent biosynthetic gene clusters in Streptomyces coelicolor and Streptomyces peucetius. Furthermore, phylogenomic analysis reveals that Actinomycetes encode multiple AfsR-like regulators with undefined roles in biosynthetic gene cluster regulation, suggesting a substantial reservoir of unexplored regulatory diversity that could be harnessed to expand natural product discovery. Together, our work provides fundamental insights into the functional diversity of NLRs and a framework for rationally engineering these regulators to unlock untapped microbial chemical diversity. Significance statement NLR proteins are conserved molecular switches that require specific activating signals to control diverse biological processes in plants, mammals and microbes. We show that a conserved regulatory feature of plant NLR proteins is retained in a bacterial NLR-related transcription factor that controls antibiotic production and can be harnessed to generate a constitutively active regulator. This strategy bypasses the unknown signals that normally limit expression of many biosynthetic genes in Streptomyces, the source of most clinically used antibiotics, leading to increased antibiotic production and activation of previously silent biosynthetic pathways. Our work reveals a highly conserved principle of NLR regulation and provides a strategy for rationally engineering bacterial transcriptional programs to unlock microbial metabolic potential.