Skip to main content
. 2020 Aug 4;9:e58706. doi: 10.7554/eLife.58706

Figure 1. Voltage is not necessary for aminoglycoside uptake or inner membrane pore formation in E. coli but is required for bactericidal activity.

(A) Colony forming units (CFUs) of untreated cells (blue) over four time points compared to cells treated with 50 µM CCCP (yellow), 100 µg/mL kanamycin (orange), and 50 µM CCCP + 100 µg/mL kanamycin (purple). Each curve averages three biological replicates, with mean and standard deviation plotted for each time point. (B) Ribosomal sucrose gradient depth plotted against 254 nm absorbance from LB grown E. coli treated with vehicle (blue), 100 µg/mL kanamycin (orange). The 30S, 50S, and 70S peaks are labeled. (C) Ratio of the area under the curve for the 30S + 50S to 70S peaks from E. coli in PMM pH 7.5, +50 μM CCCP, or pH 6 in the presence or absence of kanamycin. (D) Propidium iodide (3.75 µM in PMM) fluorescence in cells that were untreated (blue), 50 µM CCCP (yellow), 100 µg/mL kanamycin (orange), and 50 µM CCCP + 100 µg/mL kanamycin (purple) treated. The curve is the mean (solid) and standard deviation (shaded) for three biological replicates.

Figure 1.

Figure 1—figure supplement 1. Aminoglycosides enter cells and induce ribosomal dissociation in the abscence of membrane voltage.

Figure 1—figure supplement 1.

(A) Ribosomal sucrose gradient depth plotted against 254 nm absorbance from E. coli in treatment conditions from Figure 1C. (B) Ratio of the area under the curve for the 30S + 50S to 70S peaks from nuoA::kanR and nuoH::kanR E. coli strains in the absence and presence of gentamicin. (C) The uptake of 3.75 µM propidium iodide (PI) was measured by microscopy in cells that were 100 μg/mL kanamycin (orange), 100 μg/mL kanamycin + 50 µM CCCP (yellow), and 100 μg/mL chloramphenicol (green) treated. The mean (line) and standard deviation (shaded) are plotted over time. (D) Texas red fluorescence of a population of cells treated with 10 μg/mL gentamicin Texas red (GTTR) in cells pretreated with 0 μM CCCP (orange) or 50 μM CCCP (purple). The solid line indicates the mean, and the shaded error bars indicate the standard deviation at each time point.