Physical-physiological coupling underlies resistance gene transfer driven by environmental subinhibitory tetracyclines.

Antibiotics at subinhibitory concentrations are key drivers of resistance gene dissemination in the environment; however, the mechanisms operating with recipient bacteria remain poorly understood. Here, we propose a Physical-Physiological Coupling hypothesis to explain how tetracyclines promote plasmid transfer. Using a combination of molecular simulations and biological assays, we show that oxytetracycline exerts the strongest effect, increasing conjugation frequency by 4.1-fold relative to controls. Mechanistically, oxytetracycline acts as a specific modulator that binds tightly to the TolC channel (binding energy -7.4 kcal/mol), functioning as a molecular wedge that allosterically stabilizes TolC in an open conformation. Crucially, this conformational rigidity exerts mechanical stress on the surrounding lipid bilayer, inducing interfacial packing defects. Mechanistically, this membrane destabilization is likely to facilitate donor pilus penetration and stabilize the mating pair junction, thereby lowering the physical barrier to conjugative DNA transfer. Physiologically, the recipient strain responds through extensive metabolic reprogramm upregulation of the tricarboxylic acid cycle and a significant surge in intracellular ATP levels. This energy surplus satisfies the heightened demands of the conjugative transfer machinery, while the Tol system and ordered lipid domains further stabilize the mating pair formation. Together, these changes create a high-competence physiological state. Correlation analyses identified intracellular ATP levels and membrane permeability as the primary determinants of enhanced transfer efficiency. Collectively, our findings demonstrate that environmental antibiotics act as signaling molecules that engineer bacterial cells for resistance acquisition by simultaneously lowering physical barriers and boosting physiological capacity.