Glycerol-Driven Energy and Proteostasis Underpin Antibiotic Tolerance in Escherichia coli

Bacterial persisters are frequently described as metabolically dormant, yet the endogenous metabolic programs that sustain survival during prolonged nutrient limitation remain poorly understood. Here, using stationary-phase Escherichia coli as a model of antibiotic tolerance, we combine proteomics, genetics, metabolic phenotyping, and single-cell imaging to define a metabolic framework underlying persistence. Perturbation of tricarboxylic acid cycle function broadly reprogrammed stationary-phase physiology, suppressing lipid and glycerol metabolism, altering energy homeostasis and proteostasis, and reducing antibiotic tolerance. Systems-level analyses identified phospholipid-derived glycerol catabolism as a central metabolic node linking endogenous carbon recycling to persistence. Genetic disruption of glycerol utilization impaired proton motive force homeostasis, reduced formation of large polar protein aggregates, altered division-associated remodeling, and sensitized cells to antibiotic-induced lysis. Functional metabolic assays further revealed that persisters retain a selective capacity to utilize glycerol for rapid proton motive force restoration without growth resumption. Together, our findings support a model in which stationary-phase persisters are not metabolically inert but sustained through endogenous metabolic rewiring that coordinates energy maintenance, proteostasis, and antibiotic tolerance.