Fig. 1: Epitranscriptomic profiling of 23 S and 16 S rRNA and tRNA of Enterococcus faecalis V583 grown in the presence of growth-permissive concentrations of erythromycin.

a Epitranscriptome profiling workflow. Log-phase cultures of V583 were diluted with growth medium containing erythromycin below its MIC (10–200 µg/mL) and allowed to grow for 5-6 doublings to mid-log phase, after which RNA was isolated and RNA modifications quantified by LC-MS/MS. b–d Changes in the levels of RNA modifications in V583 at varying doses of erythromycin (key at bottom of d) compared to untreated cells. Modification levels are shown as fold-change relative to untreated control for 23 S rRNA (b), 16 S rRNA (c), and tRNA (d). Modifications are arranged from left to right in ascending retention time. See Supplementary Table 1 for names, retention times, and precursor and product-ion masses for the RNA modifications. Error bars represent mean ± SD for fold-change values calculated from 3 independent measurements of RNA modification normalized signal intensity in the mutant strain divided by the average signal intensity for three wild-type samples. e, f Effect of erythromycin dose on the ratio of m²A to adenosine. Ribonucleosides were quantified using LC-MS calibration curves, as illustrated for tRNA in the inset. All data are derived from 3 experiments (mean ± SD, n = 3). Statistical analysis by one-way analysis of variance (ANOVA) with Dunnett’s test versus untreated controls; *P < 0.05; **P < 0.005. e Exact p values: 30 μg/ml, 0.0291; 100 μg/ml, 0.0220; 200 μg/ml 0.0235. f Exact p values: 10 μg/ml, 0.0146; 30 μg/ml, 0.0062; 100 μg/ml, 0.0047; 200 μg/ml, 0.0055.