FIG 3.
ErmBLEF nonsense mutations convert the susceptible WT into ERY-resistant cells. (A) Bacterial growth in the presence of various concentrations of erythromycin (ERY). Overnight E. coli LB cultures were diluted (1/100, normalized to an OD600 of 0.002) into fresh medium supplemented with ampicillin at 100 μg/ml to maintain the ermBL-ermB bearing plasmid. The cell density was recorded at 4, 8, and 24 h after inoculation. Only the 8-h dataset is shown. Error bars indicate standard deviations obtained from three independent experiments. (B) Detection of basal ErmB levels by immunoblotting after 8 h of growth in the absence of erythromycin (ERY). Total soluble proteins were extracted from the same plasmid-borne ermBL-ermB backgrounds shown in panel A. Each lane corresponds to 40 μg of total soluble proteins. Y103A is a catalytically inactive ErmB mutant. The alpha-subunit of RNA polymerase served as the loading control. A 1/1,000 dilution of anti-ErmB and a 1 1/10,000 dilution of anti-RNAPα were used for immunoblotting. (C) Results from a primer extension analysis showing the basal methylation of ribosomes (without ERY) after 8 h of growth. Total RNA was isolated from the same strain backgrounds shown in panels A and B. Each lane corresponds to 250 ng of RNA input.