Imaging bacterial peptidoglycan biosynthesis with fluorescent antibiotics

The peptidoglycan (PG) layer surrounding bacterial cells is essential for cell survival; therefore, its biosynthetic pathway has long been a target of antibiotic development. Although PG biosynthesis has been studied for over four decades, the machinery controlling when and where new PG is made is still not well understood. A prerequisite for understanding PG assembly is an ability to visualize the sites where nascent PG is made. Most techniques for doing so are either indirect or involve harsh treatment of the cells, which can introduce artifacts.; It has been suggested that labeled antibiotics that bind to PG precursors may be useful for imaging PG to help determine the genes that control the biosynthesis of this polymer. This thesis describes the preparation and evaluation of fluorescent derivatives of two PG-binding antibiotics, vancomycin and ramoplanin, for imaging new PG biosynthesis in bacterial cells. Conditions for visualizing normal patterns of PG synthesis are described, and it is shown that ramoplanin probes are better imaging agents than vancomycin probes.; Studies with the fluorescent antibiotics suggest that new PG is incorporated in a helical pattern along the cylindrical walls of Bacillus subtilis cells. Both left- and right-handed helices are observed, but only one handedness is detected in a given cell. This helical pattern of PG synthesis is not determined by Mbl, as previously suggested. The utility of fluorescent derivatives of ramoplanin for imaging outer membrane permeable Escherichia coli cells is also demonstrated.; These studies provide a starting point for utilizing substrate-binding antibiotics to identify the machinery responsible for directing PG synthesis in both Gram-positive and Gram-negative bacteria.