In all experiments, bacterial cells were cultured in 25mL Luria-Bertani broth (LB) for 16 hours at 37°C, 300RPM, and 80% humidity in 250mL flasks. Unless otherwise noted, the following concentrations were used: 10 μg/mL gentamicin, 100 μg/mL ampicillin, 5 μg/mL ofloxacin, 20 μM CCCP, 1 mM KCN. The concentration of all carbon sources added to potentiate aminoglycosides was normalized to deliver 60 mM carbon (e.g., 10 mM glucose, 20 mM pyruvate, etc.). E. coli (K12 EMG2) and S. aureus (ATCC 25923) were the two parent strains used in this study. Knockouts (Supplementary Table 1 and 2) were constructed by P1-phage transduction from the Keio knockout collection. In E. coli, non-persister stationary phase cells were killed by treatment with 5 μg/mL ofloxacin for 4 hours25 (link), 26 (link). Samples were then washed with phosphate buffered saline (PBS) and suspended in M9 salts with carbon source and antibiotic to determine metabolite-enabled killing of persisters. At specified time points, 10 μL aliquots of samples were removed, serially diluted, and spot-plated onto LB agar plates to determine colony forming units/mL (CFU/mL) and survival. Gent-TR was made as previously described27 (link). Aminoglycoside uptake was measured by incubating stationary phase samples with 10 μg/mL Gent-TR for 5 minutes at 37°C, 300RPM, and 80% humidity. 100 μL of each sample was then washed and resuspended in PBS and analyzed on a BD FACS Aria II flow cytometer. Biofilm survival assays were performed as previously described28 (link). Raw microarray data for S. aureus were downloaded from the Gene Expression Omnibus (GEO) series GSE2097329 (link) and processed with RMA express using background adjustment, quantile normalization, and median polish summarization to compute RMA expression values30 (link). Mouse experiments were performed with female Charles River Balb/C mice in collaboration with ViviSource Laboratories and conformed to the ViviSource IACUC policies and Procedural Guidelines.