Visualizing Bacterial Penicillin-Binding Proteins

Bacterial membranes harboring penicillin-binding proteins (PBPs) were prepared from LB – carbenicillin (50 μg/ml) cultures grown for 12 h. Uninhibited PBPs were labeled with a fluorescent penicillin (Bocillin FL; Invitrogen, Carlsbad, CA, United States) and quantified by measuring fluorescence intensity as described previously (Zhao et al., 1999 (link)). To observe PBP localization in the bacterial membrane, Bocillin FL-labeled cells were further subjected to a membrane dye, FM-6-64 (Invitrogen; Kocaoglu et al., 2012 (link)) and then observed using a confocal laser scanning microscope (SP8X; Leica) at excitation/emission wavelengths of 488/500–535 and 515/621–678 nm for Bocillin FL and FM-6-64, respectively.