The goal of this proposal is to achieve a detailed understanding of a key step in the sporulation of Bacillus subtilis, engulfment. During engulfment the edges of the sporulation septum migrate around and fully enclose the forespore; ultimately the forespore is fully enclosed within the mother cell cytoplasm and is surrounded by two membranes. Engulfment is essential for both spore morphogenesis and for full activation of late sporulation-specific transcription factors in both cells of the developing sporangium. Although several sporulation-specific proteins have been implicated in engulfment, its mechanism remains obscure. Bacterial cells lack the cytoskeletal proteins associated with similar eukaryotic events, and hence must use a noval mechanism to drive the engulfment of one cell by another. We will use the following specific aims to further our understanding of engulfment: Characterization of the known engulfment proteins to determine their subcellular distribution, to identify proteins with which they interact, to determine their biochemical activities, and to more precisely determine the stage at which they are required for engulfment. Identification of additional proteins required for engulfment, using both genetic and biochemical approaches. The development of a system to observe engulfment in living bacteria, using video microscopy and protein fusions to the green fluorescent protein (GFP) of Aquoria victoria. It is hoped that by identifying a large number of proteins required for engulfment, and by studying their biochemical activities, interactions and subcellular distributions, that we can begin to understand the mechanism by which one bacterial cell can engulf another. Engulfment provides a unique model system for the study of regulated membrane movements in bacteria.
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