DNA breaks-mediated cost reveals RNase HI as a new target for selectively eliminating antibiotic resistance

Roberto Balbontín; Nelson Frazão; Isabel Gordo

Early exponential cultures were diluted into pre-warmed medium containing the inducer of the Gam-GFP construction (anhydrotetracycline, 25 ng/ml) and incubated at 37°C with shaking (240 r.p.m.) for 3h, prior to imaging. Bacterial solutions were then placed onto 1% agarose (in 1X PBS) pads mounted in adhesive frames between the microscope slide and a coverglass. Images were acquired on an Applied Precision DeltavisionCORE system, mounted on an Olympus IX71 inverted microscope, coupled to a Cascade II 1024x1024 EM-CCD camera, using an Olympus 100x 1.4NA Uplan SAPO Oil immersion objective, where GFP and mCherry were imaged with FITC (Ex: 475/28, EM: 528/38) and TRITC (Ex: 542/28, Em: 617/73) fluorescence filter sets, respectively, and DIC optics. Images were deconvoluted with Applied Precision's softWorx software, and prepared for presentation (cropping smaller fields to facilitate visualization, and false-coloring green and red fluorescent signals) using Fiji/ImageJ. All microscopy experiments were performed at the Imaging Facility of the IGC.