Asymmetric division as a morphological checkpoint for B. subtilis sporulation

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DESCRIPTION (provided by applicant): The long-term goals of my research are to discover the mechanisms that couple asymmetric cell division to cell differentiation. Asymmetric division is a key step in many developmental processes, including sporulation of the bacterium Bacillus subtilis. The production of viable spores requires the division-dependent activation of the cell-specific transcription factors, sigma F (CTF) and sigma E (CTE). In this study, I will assess the role of asymmetric division in the temporal and spatial regulation of CTF and aE activity. The aF activating protein SpollE localizes to the septum during division and therefore represents an ideal candidate for sensing completion of septation, which is required before activation of aF. In addition, pro-oE, the inactive form of CTE, and SpollGA, which processes pro-aE into active aE, may localize to the septum and also be regulated by division. Cell-specific localization offers one mechanism by which septation may ensure proper temporal and spatial activation of CTF in the forespore and aE in the mother cell. I will develop a new method to test if SpollE, SpollGA and pro-aE localize to the forespore side or mother cell side of the septum. By utilizing split GFP techniques I will be able to visualize if these proteins are present only at the forespore septum, only at the mother cell septum or on both sides of the septum. Protein-protein interactions at the septum may also regulate aF and aE activity. The use of Fluorescence Recovery After Photobleaching (FRAP) will identify changes in protein mobility, which varies as a result of changes in protein interactions. I will perform FRAP on GFP fusions to SpollE, SpollGA and pro-aE at different stages of septation, on opposing sides of the septum and at the septum in comparison to other regions of the membrane. From these results I will develop mechanistic models for how SpollE, SpollGA and pro-aE function in response to septation. Septation may function as a direct morphological checkpoint for activation of CTE. I will perform genetic investigations to test that possibility. In addition, I will express pro-aE and its known regulatory proteins in E coli to assay the necessary components for aE activation. This research will reveal important mechanisms in development and possibly uncover the basis of diseases that result from aberrant gene expression during development. In addition, this study will further our understanding of sporulation by the common soil bacterium Bacillus subtilis and other endospore forming bacteria, such as B. anthracis.