The gentamicin accumulation assay was performed using the method described previously (43 (link)). Briefly, A. baumannii AB5075_UW wild-type and its dksA::Tn26 mutant were grown to OD = 0.6 in MH broth. Culture aliquots (500 μl) were transferred to 2 ml sterile Eppendorf tubes, and gentamicin-Texas Red conjugate was added to each sample at a final gentamicin concentration of 500 μg/ml. Reactions were protected from light and incubated for 30 min at 37°C with shaking at 200 rpm. Cells were then pelleted by centrifugation at 8000 g for 1 min, washed with 400 μl PBS, and the pellet resuspended in 1 ml DMSO and stored at −20°C prior to measurement.
Photophysical measurements were performed with a FLS980 photoluminescence spectrometer (Edinburgh Instruments) equipped with a Xe1 Xenon Arc Lamp (450 W ozone free, excitation range 230–1000 nm) for steady-state measurements. Excitation (lex) was performed at 550 nm, and emission spectra were recorded in DMSO at 28°C with 1 nm step-size, 0.1s integration time, and slit-width of Δlex = Δlem = 1.5 nm for both strains.
Photophysical measurements were performed with a FLS980 photoluminescence spectrometer (Edinburgh Instruments) equipped with a Xe1 Xenon Arc Lamp (450 W ozone free, excitation range 230–1000 nm) for steady-state measurements. Excitation (lex) was performed at 550 nm, and emission spectra were recorded in DMSO at 28°C with 1 nm step-size, 0.1s integration time, and slit-width of Δlex = Δlem = 1.5 nm for both strains.
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