Visualizing Localization of Fluorescent Fusion Proteins in MRSA

MRSA COL strains with green fluorescent protein (GFP)-labeled PBP2, yellow fluorescent protein (YFP)-labeled PBP4, or cyan fluorescent protein (CFP)-labeled FtsZ were obtained as referenced.^{28 (link),36 (link),37} Overnight cultures of bacteria were inoculated 0.5% into MHB and incubated at 37 °C until an OD₆₀₀ of 0.6. BPEI was added (64 μg/mL, the MIC of MRSA COL), and the cells were incubated for 60 min. After growth, 1 mL of culture was harvested by centrifugation, washed once in PBS, and resuspended in PBS. Next, 1 μL of resuspended cells was placed on a thin layer of 1.2% agarose in PBS. Samples were observed using a Zeiss Axio Observer microscope equipped with a Photometrics CoolSNAP HQ2 camera (Roper Scientific) and Metamorph 7.5 software (Molecular Devices). Images were analyzed using ImageJ software. To quantitatively assess the localization of the fusion proteins, the fluorescence signal at the septum and at the peripheral cell wall was determined for >100 cells with fully formed septa. Automated calculations of fluorescent ratios between septal and membrane signals were performed by eHooke software as previously described.^{38 (link)} Statistical analyses were performed using GraphPad Prism 7 (GraphPad Software). Unpaired Student’s t tests were used to compare fluorescence ratios between peripheral and septal wall signal intensity.