Bacteria from the genera Bacillus and Clostridium produce unusually durable and long-lived spores that are the infectious agent of Anthrax and Botulism, and which are assembled in the cytoplasm of another cell. This unique cell within a cell structure is produced by the phagocytosis-like process of engulfment, during which the membrane of the larger mother cell migrates around the smaller forespore, until it is completely enclosed within the mother cell cytoplasm. Engulfment provides a dramatic example of the dynamic capabilities of the bacterial cell, but its mechanism remains unclear. Previously, the only engulfment mutants blocked septal thinning, during which peptidoglycan within the septum is thinned in preparation for membrane migration. We have developed new tools for the study of engulfment, and identified mutants defective in membrane migration, and in the final step of engulfment, membrane fusion. The membrane fusion defective mutants affect a protein that is both highly conserved and essential in many species. This protein localizes to site of division and is involved in the final stages of chromosome segregation, suggesting that it may also be involved in membrane fusion at the completion of cell division, a process about which little is known. Sporulation-specific enzymes are required to hydrolyze peptidoglycan during septal thinning, and we will test if vegetative autolysins can partially substitute for the sporulation specific enzymes. Autolysins are found in all bacteria (the Bacillus subtilis genome is predicted encode more than 30such enzymes), and are thought to allow peptidoglycan remodeling for cell elongation and division. However, these enzymes are potentially lethal, since unless they are tightly regulated both spatially and temporally, their activity can result in cell lysis. Indeed, the lethality of many commercial antibiotics requires autolysins. Engulfment provides an ideal system for understanding how bacteria control these potentially lethal enzymes, which are attractive targets for novel antibiotics. We will take a combined cell biological, genetic and biochemical approach to study the spatial regulation of peptidoglycan hydrolysis, the mechanism of membrane fusion in bacterial cells, as well as to understand how bacteria move and localize macromolecules within their cells
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