Natural flavonoids inhibit plasmid conjugation via iron chelation and zinc-responsive envelope stress

• Natural flavonoids inhibit the conjugative transfer of resistance plasmids. • Flavonoids with low log P exhibit superior inhibitory effects on conjugation. • Scutellarein disrupts iron homeostasis and activates envelope stress response. Plasmid-mediated bacterial conjugation is a major driver of horizontal gene transfer (HGT) via direct cell-to-cell contact, significantly accelerating the dissemination of antimicrobial resistance (AMR). Given the limited pipeline of new antibacterial agents, conjugation inhibitors represent a promising alternative strategy to curtail resistance spread. This study aims to identify potent natural compounds that block resistance transmission and elucidate their underlying molecular mechanisms. We conducted conjugation assays to screen plant-derived flavonoids for inhibitory activity against resistant plasmid transfer. Structure–activity relationship (SAR) analysis was employed to delineate the impact of specific substituent groups. Transcriptomic profiling and gene knockout experiments identified and validated critical functional genes. Cell surface hydrophobicity, adhesion and aggregation assays provided mechanistic evidence of gene function in the conjugation process. Herein, we demonstrate that the majority of plant-derived flavonoids potently inhibit the conjugative transfer of two distinct plasmids both in vitro and in vivo . SAR analysis reveals that flavonoids with lower lipophilicity (log P ), particularly those bearing hydroxyl groups, exhibit superior inhibitory efficacy. Conversely, isopentenyl-substituted flavonoids display attenuated activity. Mechanistically, scutellarein, a representative hydroxylated flavonoid, disrupts bacterial iron homeostasis, triggering zinc-responsive envelope stress response (ESR) activation. Zinc influx induces intracellular protective responses mediated by ZraP and glutathione (GSH), whose depletion reduces membrane permeability, reactive oxygen species (ROS) levels and electron transport chain (ETC) activity. Concurrently, GSH oxidation to glutathione disulfide (GSSG) upregulates bhsA expression, altering surface hydrophobicity and flagellar motility, thereby diminishing intercellular adhesion, aggregation and physical encounter frequency. Collectively, our findings uncover the critical roles of iron homeostasis perturbation and ESR activation in controlling plasmid conjugation, underscoring the therapeutic potential of natural flavonoids in mitigating the AMR crisis.