Abstract
Penicillin-binding proteins (PBPs) are membrane-associated proteins involved in the biosynthesis of peptidoglycan (PG), the main component of bacterial cell walls. These proteins were discovered and named for their affinity to bind the β-lactam antibiotic penicillin. The importance of the PBPs has long been appreciated; however, the apparent functional redundancy of the ~5 to 15 proteins that most bacteria possess makes determination of their individual roles difficult. Existing techniques to study PBPs are not ideal because they do not directly visualize protein activity and can suffer from artifacts. Therefore, development of new methods for studying the roles of distinct PBPs in cell wall synthesis was compulsory. Due to penicillin's covalent mode of inhibition, fluorophore-conjugated analogs can be utilized to visualize PBP activity. Herein, we describe a general protocol to label and detect subsets of active PBPs in live, Gram-positive bacteria using fluorescent β-lactams.