Structural and functional insights into StnY, a ribbon-helix-helix (RHH) family transcription factor regulating antibiotic resistance in Streptomyces flocculus CGMCC4.1223

Deciphering how bacteria respond to antibiotic stress is essential for developing strategies to combat the increasing global antibiotic resistance gene (ARG) crisis. Here, we identified an unprecedented antibiotic resistance operon characterized by a single-domain transcription factor (TF) StnY, which responds to streptonigrin (STN) antibiotic and controls the activation of resistance genes stnK4 and stnG4 in Streptomyces flocculus CGMCC4.1223. To the best of our knowledge, StnY represents the first RHH family TF regulating ARG and it helically wraps around the promoter of the resistance operon in an octameric form. Unlike conventional TFs with distinct effector-binding domains, StnY utilizes its DNA-binding domain to bind the STN effector, facilitating the dissociation of StnY-DNA complex. Consequently, the vicinal oxygen chelates (VOC) family protein StnK4 sequesters STN to prevent cellular damage, while the major facilitator superfamily (MFS) protein StnG4 effluxes STN out of the cell. Furthermore, genome analysis reveals the widespread distribution of RHH-VOC-MFS gene cassettes in actinomycetes, the primary source of antibiotics. This study elucidates function mode of a resistance operon governed by a TF lacking an effector-binding domain, offering new insights into ARG regulation and the potential of ARG-guided antibiotics discovery, highlighting TFs as promising targets for addressing ARG.