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Temporal and Spatial Organization of Peptidoglycan Biosynthesis |
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| Bacterial cells are surrounded by layers of peptidoglycan, a crosslinked carbohydrate polymer that functions as a protective exoskeleton. These PG layers enable bacteria to withstand high internal osmotic pressures and also play an important role in maintaining cell shape. Peptidoglycan is assembled on the bacterial cell surface from a simple disaccharide building block, which is made inside the cell. We know a great deal about the biosynthesis of this disaccharide building block, but we do not understand how it is converted into a complex, three-dimensional polymer on the cell surface. What proteins are involved in this process? How are their activities coordinated with cell division? When and where is new peptidoglycan made during cell growth and division? These are some of the questions we are trying to answer.
We are developing chemical probes to visualize new sites of peptidoglycan synthesis on bacterial cell surfaces. Unlike proteins, which can be visualized using genetically encoded fluorescent tags or fluorescent antibodies, there are still no general methods to visualize carbohydrate polymers on cell surfaces with good temporal and spatial resolution. Our strategy to visualize new sites of PG synthesis relies on the use of fluorescently labeled substrate-binding antibiotics. Microorganisms produce a diverse array of natural products that bind to intermediates along the pathway to mature peptidoglycan, and we have developed a set of tools that enable us to dissect the mechanisms of these types of antibiotics. Using our knowledge of the recognition chemistry and mechanism of action of one such antibiotic, ramoplanin, we have prepared a set of fluorescent probes that bind to the initiation sites of PG synthesis on bacterial cell surfaces. These probes stain the new division sites, old poles, and side walls of Bacillus subtilis cells. The side walls are stained in a helical pattern, which is presumed to reflect the underlying biosynthetic machinery involved in PG synthesis.
With a probe to monitor new sites of PG synthesis in hand, we can begin to answer many questions about the genes that control this helicoid pattern of PG synthesis. We may also be able to learn more about the structure of PG itself from the patterns in which it is laid down.
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Selected Publications: |
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"Imaging Peptidoglycan Biosynthesis in Baccillus subtilis with Fluorescent Antibiotics" Tiyanont K, Doan T, Lazarus MB, Fang X, Rudner DZ, and Walker S, Proc. Natl. Acad. Sci. USA, 2006 (in press).
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