Beta-Lactam Titration Assays to Evaluate Penicillin-Binding Protein Selectivity

Penicillin-binding proteins (PBP) are integral to the formation and maintenance of the bacterial peptidoglycan (PG) layer. Beta-Lactam antibiotics bind to the PBPs, ultimately killing cells through disruption of the PG. These molecules often do not inhibit all of the PBP isoforms in a given organism, but instead selectively target a subset of these proteins. This property can be exploited to study the function and localization of individual PBPs in live cells by the use of non-lethal concentrations of beta-lactam antibiotics and/or fluorophore-appended derivatives. Here, we describe a titration assay that can be used to examine the PBP specificity of beta-lactams over a range of concentrations. This assay can be utilized in any live gram-positive bacterium or the lysates from gram-negative organisms.