Abstract
We aim to investigate the timing and localization of penicillin‐binding proteins (PBPs) during peptidoglycan (PG) biosynthesis using a combination of fluorescence microscopy, gel electrophoresis, and liquid chromatography‐mass spectrometry (LC‐MS) techniques facilitated by the use of a β‐lactam‐based probe. β‐lactam‐containing compounds are the most widely used class of antibiotics that inhibit PG biosynthesis by binding PBPs. We designed and synthesized fluorescent and biotin derivatives of the antibiotic cephalosporin to selectively label high molecular weight (HMW) PBPs in B. subtilis PY79 in vivo. Our microscopy results indicate that the probe facilitated specific labeling of active HMW PBPs that are localized at newly synthesized septa. The MS analysis identified the labeled proteins as PBP 1a and b, PBP 2a, PBP 2b and PBP 4. Both microscopy and gel‐based analysis indicated that our probe gives cleaner staining and is more selective than BOCILLIN‐FL, a commercially available penicillin analog, which labels both high and low molecular PBPs. Our cephalosporin probe is currently being applied to time‐course studies to visualize the roles of HMW PBPs in B. subtilis.
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