title: An Activity-Based Probe for Studying Crosslinking in Live Bacteria.
abstract: Penicillin-binding proteins (PBPs) catalyze the crosslinking of peptidoglycan (PG), an essential process for bacterial growth and survival, and a common antibiotic target. Yet, despite its importance, little is known about the spatiotemporal aspects of crosslinking—largely because of a lack of experimental tools for studying the reaction in live bacteria. Here we introduce such a tool: an activity-based probe that enables visualization and relative quantitation of crosslinking in vivo. In Staphylococcus aureus, we show that fluorescent mimics of the natural substrate of PBPs (PG stem peptide) are covalently incorporated into the cell wall, installing fluorophores in place of natural crosslinks. These fluorescent stem peptide mimics (FSPMs) are selectively recognized by a single PBP in S. aureus: PBP4. Thus, we were able to use FSPM pulse-labeling to localize PBP4 activity in live cells, showing that it is recruited to the septum in a manner dependent on wall teichoic acid.
authors.affiliation: Department of Cell Biology, Yale School of Medicine, 333 Cedar Street, New Haven, CT 06520 (USA).|Department of Chemistry, Yale University, 225 Prospect Street, New Haven, CT 06511 (USA).
authors.forename: Samir
authors.initials: S
authors.lastname: Gautam
authors.affiliation: Department of Chemistry, Yale University, 225 Prospect Street, New Haven, CT 06511 (USA).
authors.forename: Taehan
authors.initials: T
authors.lastname: Kim
authors.affiliation: Department of Chemistry, Yale University, 225 Prospect Street, New Haven, CT 06511 (USA).|National Institute of Health Sciences, 1-18-1 Kamiyoga, Setagaya-ku, Tokyo, 158-8501 (Japan).
authors.forename: Takuji
authors.initials: T
authors.lastname: Shoda
authors.affiliation: Department of Chemistry, Yale University, 225 Prospect Street, New Haven, CT 06511 (USA).|Department of Medicine, Massachusetts General Hospital and Harvard Medical School, 55 Fruit Street, Boston, MA 02114 (USA).
authors.forename: Sounok
authors.initials: S
authors.lastname: Sen
authors.affiliation: Department of Chemistry, Yale University, 225 Prospect Street, New Haven, CT 06511 (USA).
authors.forename: Deeksha
authors.initials: D
authors.lastname: Deep
authors.affiliation: Department of Chemistry, Yale University, 225 Prospect Street, New Haven, CT 06511 (USA).
authors.forename: Ragini
authors.initials: R
authors.lastname: Luthra
authors.affiliation: Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Av. da República, 2780-157 Oeiras (Portugal).
authors.forename: Maria Teresa
authors.initials: MT
authors.lastname: Ferreira
authors.affiliation: Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Av. da República, 2780-157 Oeiras (Portugal).
authors.forename: Mariana G
authors.initials: MG
authors.lastname: Pinho
authors.affiliation: Department of Chemistry, Yale University, 225 Prospect Street, New Haven, CT 06511 (USA). david.spiegel@yale.edu.|Department of Pharmacology, Yale School of Medicine, 333 Cedar Street, New Haven, CT 06520 (USA). david.spiegel@yale.edu.
authors.forename: David A
authors.initials: DA
authors.lastname: Spiegel
mesh_terms: D015335:Molecular Probes; D013211:Staphylococcus aureus / Q000378:metabolism*
chemical_list: D015335:Molecular Probes
keywords: bacteria; biosensors; crosslinking; fluorescent probes; peptidoglycans