Western Blot analysis of PBPs was performed as described previously with minor modifications. 10 ml of log phase bacteria (OD660 nm =0.5) were exposed to Bocillin FL (25 μg) for 30 min at 37°C. Bacteria were then harvested by centrifugation, washed four times with phosphate-buffered saline, and lysed by freeze-thawing process. Proteins were separated by 1D SDS-PAGE, and the acyl-enzyme complex was detected (excitation: 488nm, emission: 520nm) with a fluorescent scanner (Typhoon 9410, GE Healthcare). Mutagenesis results in a loss of bands of approximately 75 and 78 KDa which correspond to the molecular weight of PBP4 and Lmo0441 proteins respectively.