Assessment of ClpP activity in vitro.

For the fluorometric peptide assay, the ClpP proteins (at 1 µM monomeric concentration) were added to 500 µM Suc-Luc-Tyr-AMC (Boston Biochem) dissolved in buffer E (50 mM Tris-HCl [pH 8], 200 mM KCl, and 1 mM dithiothreitol [DTT]) or buffer F (with 0.2 M sodium citrate) (42). The final reaction mixture volumes were 50 µl. Reactions were monitored over 6 h at 37°C using a BioTek Synergy HT plate reader set at an excitation of 340/360 and an emission of 440/460, with readings taken at 5-min intervals. For the casein degradation assays, casein (Sigma-Aldrich) was dissolved in buffer E, and 1 µg was used per assay. Samples containing casein and 1 µM the respective ClpP monomer were incubated at 37°C for 3 h with or without the respective ACP compound (500 µM). Reactions were halted by mixing with 2× Laemmli buffer containing β-mercaptoethanol and heating at 90 to 100°C for 5 min. Samples were analyzed for digestion of casein using 12% SDS-PAGE gels, followed by Coomassie blue staining.