FIG 2.

Levels of aminoacylated tRNA^Phe correlate with antibiotic persistence. (A) Representative Northern blots with a ³²P-5′-end-labeled tRNA^Phe probe in which 10 μg total tRNA was separated by aminoacylated and deacylated tRNA species on an acid urea gel. For each strain, the left lane was grown in medium A and the right lane was grown in medium B. Left panel is tRNA purified from the E. coli MG1655 relA⁺ strain, and right panel is tRNA purified from the E. coli ΔrelA strain. (B) Percent aminoacylated tRNA^Phe levels in vivo in wild-type pheS/pheT, pheT G318W, pheS A294G, and pheS A294S in both the relA⁺ strain (left panel) and a ΔrelA strain (right panel). Cultures were grown to late log phase in either medium A (solid bars), which is a supplemented M9 minimal medium that contains 40 μg/ml of all 20 proteogenic amino acids, or medium B (striped bars), which is a supplemented M9 minimal medium with 40 μg/ml of 18 proteogenic amino acids, 10 μg/ml Tyr, 40 μg/ml m-Tyr, and no Phe. Error bars represent standard deviations from 3 biological replicates. *, P value < 0.04; statistical analysis was performed using multiple t tests.