Jennifer A Taylor1,2, Cintia C Santiago3, Joe Gray4, Kimberly A Wodzanowski3, Kristen E DeMeester3, Jacob Biboy4, Waldemar Vollmer4, Catherine L Grimes3,5, Nina R Salama1,2. 1. Department of Microbiology, University of Washington, Seattle, Washington. 2. Human Biology Division, Fred Hutchinson Cancer Research Center, Seattle, Washington. 3. Department of Chemistry and Biochemistry, University of Delaware, Newark, Delaware. 4. Centre for Bacterial Cell Biology, Biosciences Institute, Newcastle University, Newcastle upon Tyne, United Kingdom. 5. Department of Biological Sciences, University of Delaware, Newark, Delaware.
Abstract
The bacterial cell wall, composed of peptidoglycan (PG), provides structural integrity for the cell and is responsible for cell shape in most bacteria. Here we present tools to study the cell wall using a clickable PG-specific sugar, 2-alkyne muramic acid (MurNAc-alk), as a metabolic probe. Here we present a new reaction pathway for generating MurNAc-alk. We also include protocols for labeling PG synthesis in Helicobacter pylori, determining the identity of the labeled muropeptides using LC-MS/MS, sample preparation of cells labeled for a short fraction of the doubling time, and visualization using 3D structured illumination microscopy.
The bacterial cell wall, composed of peptidoglycan (PG), provides structural integrity for the cell and is responsible for cell shape in most bacteria. Here we present tools to study the cell wall using a clickable PG-specific sugar, 2-alkyne muramic acid (MurNAc-alk), as a metabolic probe. Here we present a new reaction pathway for generating MurNAc-alk. We also include protocols for labeling PG synthesis in Helicobacter pylori, determining the identity of the labeled muropeptides using LC-MS/MS, sample preparation of cells labeled for a short fraction of the doubling time, and visualization using 3D structured illumination microscopy.
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