Glycerol-driven energy and proteostasis underpin antibiotic tolerance in Escherichia coli

The metabolic pathways that sustain bacterial persistence during nutrient limitation remain poorly understood. Using stationary-phase Escherichia coli as a model of antibiotic tolerance, we used proteomics, genetics, metabolic phenotyping, and single-cell imaging to define a metabolic framework underlying persistence. Perturbation of tricarboxylic acid cycle function 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 critical metabolic pathway linking carbon recycling to persistence. Genetic disruption of key nodes within this pathway impaired proton motive force homeostasis, reduced large polar protein aggregate formation, altered division-associated remodeling, and sensitized cells to antibiotic-induced lysis. Metabolic assays further revealed that persisters retain a selective capacity to utilize glycerol for rapid proton motive force restoration. Together, these findings support a model in which stationary-phase persisters are sustained through metabolic rewiring that coordinates energy maintenance, proteostasis, and antibiotic tolerance.