Forespore Engulfment During B. subtilis Sporulation

描述(申请人提供)：芽孢杆菌属和梭状芽胞杆菌属的细菌产生异常持久和长寿的孢子，这些孢子是炭疽和肉毒杆菌中毒的感染源，并聚集在另一个细胞的细胞质中。细胞结构中这种独特的细胞是由吞噬类吞噬过程产生的，在此过程中，较大的母细胞的膜围绕较小的前孔迁移，直到它完全被包围在母细胞细胞质中。吞噬作用为细菌细胞的动态能力提供了一个戏剧性的例子，但其机制尚不清楚。此前，唯一的吞噬突变体阻止隔膜变薄，在此期间，隔膜内的肽聚糖被变薄，为膜迁移做准备。我们开发了研究吞噬的新工具，并鉴定了在膜迁移以及在吞噬的最后一步膜融合方面存在缺陷的突变体。膜融合缺陷突变体影响一种在许多物种中高度保守且必不可少的蛋白质。该蛋白定位于分裂部位，并参与染色体分离的最后阶段，这表明它可能还参与了细胞分裂完成时的膜融合，这一过程鲜为人知。在隔膜细化过程中，需要产孢子特异性的酶来水解肽聚糖，我们将测试营养自溶素是否可以部分取代产孢子特异性的酶。自溶素存在于所有细菌中(据预测，枯草芽孢杆菌基因组编码了30多种这样的酶)，并被认为可以使肽聚糖重塑，促进细胞伸长和分裂。然而，这些酶是潜在的致命的，因为除非它们在空间和时间上都受到严格的调控，否则它们的活性可能会导致细胞裂解。事实上，许多商业抗生素的杀伤力需要自溶素。吞噬为了解细菌如何控制这些潜在的致命酶提供了一个理想的系统，这些酶是新抗生素的诱人靶标。我们将采用细胞生物学、遗传学和生物化学相结合的方法，研究多肽多糖水解物的空间调节、细菌细胞膜融合的机制以及了解细菌如何在细胞内移动和定位大分子。

DESCRIPTION (provided by applicant): 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