Asymmetric division as a morphological checkpoint for B. subtilis sporulation

不对称分裂作为枯草芽孢杆菌孢子形成的形态检查点

• 批准号: 7495534
• 负责人: tinya C fleming
• 金额: $ 5.13万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-09-24 至 2010-09-23
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7495534
• 关键词: Bacillus anthracis; Bacillus subtilis; Bacteria; Biological Assay; Cell Differentiation process; Cell division; Cells; Dependence; Development; Developmental Process; Disease; Endopeptidases; Endospore-Forming Bacteria; Ensure; Escherichia coli; Event; Face; Fluorescence Recovery After Photobleaching; Gene Expression; Genetic; Goals; Green Fluorescent Proteins; Investigation; Lead; Link; Localized; Membrane; Methods; Microscopy; Modeling; Mothers; Mutation; Organism; Pathway interactions; Peptide Hydrolases; Peptidoglycan; Play; Process; Production; Protein Dynamics; Proteins; Regulation; Reporting; Reproduction spores; Research; Rest; Role; Side; Signal Transduction; Soil; Staging; Techniques; Testing; Time; base; design; genetic regulatory protein; intracellular protein transport; macromolecule; mutant; promoter; protein localization location; protein protein interaction; reconstitution; research study; response; sigma-E factor; transcription factor

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.

描述（由申请人提供）：我研究的长期目标是发现将不对称细胞分裂与细胞分化耦合的机制。不对称分裂是许多发育过程中的关键步骤，包括枯草芽孢杆菌的孢子形成。活孢子的产生需要细胞特异性转录因子 sigma F (CTF) 和 sigma E (CTE) 的分裂依赖性激活。在这项研究中，我将评估不对称分裂在 CTF 和 aE 活动的时间和空间调节中的作用。 aF 激活蛋白 SpollE 在分裂过程中定位于隔膜，因此代表了感测隔膜完成的理想候选者，这是激活 aF 之前所必需的。此外，pro-oE（CTE 的非活性形式）和 SpollGA（将 pro-aE 加工成活性 aE）可能定位于隔膜，也受到分裂的调节。细胞特异性定位提供了一种机制，通过这种机制，分隔可以确保前孢子中的 CTF 和母细胞中的 aE 适当的时间和空间激活。我将开发一种新方法来测试 SpollE、SpollGA 和 pro-aE 是否定位于隔膜的前孢子侧或母细胞侧。通过利用分裂 GFP 技术，我将能够可视化这些蛋白质是否仅存在于前孢子隔膜、仅存在于母细胞隔膜或隔膜的两侧。隔膜处的蛋白质-蛋白质相互作用也可能调节 aF 和 aE 活性。使用光漂白后荧光恢复 (FRAP) 将识别蛋白质迁移率的变化，这种变化因蛋白质相互作用的变化而变化。我将在分隔的不同阶段、在隔膜的相对侧以及在隔膜处与膜的其他区域进行比较，对 GFP 与 SpollE、SpollGA 和 pro-aE 的融合进行 FRAP。根据这些结果，我将开发 SpollE、SpollGA 和 pro-aE 如何响应分隔而发挥作用的机制模型。分隔可能作为 CTE 激活的直接形态学检查点。我将进行基因研究来测试这种可能性。此外，我将在大肠杆菌中表达 pro-aE 及其已知的调节蛋白，以测定 aE 激活所需的成分。这项研究将揭示发育过程中的重要机制，并可能揭示发育过程中基因表达异常导致疾病的基础。此外，这项研究将进一步了解常见土壤细菌枯草芽孢杆菌和其他内生孢子形成细菌（例如炭疽芽孢杆菌）的孢子形成。