Alternative respiratory electron transport pathways drive differential aminoglycoside susceptibility

Bacterial bioenergetics is increasingly recognized as a central determinant of antibiotic tolerance and resistance. Here, we define how architectural variation within the aerobic electron transport system (ETS) modulates proton motive force (PMF) and governs antibiotic susceptibility. We constructed four Escherichia coli strains with unbranched respiratory pathways differing in proton-translocation efficiencies. A low-PMF ETS variant exhibited selective tolerance to aminoglycosides, while susceptibility to β-lactams and tetracycline remained unchanged. Using fluorescent gentamicin, we demonstrate that reduced PMF directly limits drug uptake. Interestingly, wild-type E. coli was as susceptible as the high-PMF variant. These results establish respiratory energy transduction as a mechanistic bottleneck for aminoglycoside efficacy and position ETS remodeling as a bioenergetic route to antibiotic tolerance. IMPORTANCE Infectious diseases remain a major global health challenge, and the failure of antibiotics to effectively treat them is compounding the threat. This study shows that the way bacteria produce energy can strongly influence how well certain antibiotics work. By engineering bacteria with different respiratory pathways, we demonstrate that bacteria in a low-energy state became specifically more tolerant to aminoglycoside antibiotics such as gentamicin and tobramycin. Importantly, this tolerance was linked to reduced drug entry into the cell rather than resistance gained due to acquisition of adaptive genetic change. The findings reveal that bacterial energy metabolism is not simply a housekeeping process, but a major factor determining antibiotic effectiveness. This work helps explain why some bacteria survive antibiotic treatment despite lacking resistance mutations and highlights bacterial respiration as a promising target for developing strategies to restore antibiotic sensitivity and combat persistent infections.