Structural parameters of flagellar rod and filament assembly in Bacillus subtilis

项目摘要 细菌组装称为纳米机器的大型细胞外复合物，以与它们的 环境纳米机器发挥特定的功能，包括底物分泌，细胞运动， 发病机制细菌蛋白纳米机器的调控和结构组成本质上是 复杂.一种这样的纳米机器，鞭毛装置，对细菌运动至关重要，它的组装是 高度有序。许多调节系统是适当的，以确保正确的组装鞭毛发生， 在空间和时间上。许多研究调查了鞭毛结构的组成，以及如何 宿主调节其各种组分的转录和翻译。然而，关于这一点知之甚少。 控制鞭毛精确组装的系统。 鞭毛的主要成分之一是胞外丝，它负责 产生推动力来移动细胞。大多数专注于细丝组装的研究使用革兰氏染色法， 阴性微生物鼠伤寒沙门氏菌和大肠杆菌。调查显示，这两个 相关的生物体保持调节细丝长度以及修复的独特机制。是否 对丝长度的精确调节对于在所有物种中的有效运动是必要的，这是未知的。使用 遗传上易处理的革兰氏阳性模式菌枯草芽孢杆菌，我们建议确定机制 这种生物体用来控制纤维生长和长度，以及纤维修复是否发生。 此外，调节鞭毛杆从膜结合的细胞跨越的长度的机制， 在B中鞭毛运动到胞外丝。subtilis未知。B由四种蛋白质组成。 枯草杆菌棒，但其组装顺序目前未知。此外，杆必须精确地穿过大的 在革兰氏阴性（周质）和革兰氏阳性（肽聚糖）生物体中启动组装的距离 鞭毛丝因此，细胞必须调节杆长度以准确地跨越这些深度。虽然 革兰氏阴性S.鼠伤寒沙门氏菌显示杆长度是通过与外部的相互作用确定的 膜，革兰氏阳性生物体中缺乏外膜表明存在不同的机制。 我们将确定棒的结构组成，并确定控制棒长度的组件。 这个提议和实验的目的是集中在鞭毛的结构组织上 杆和丝，具体地说：（i）评估控制鞭毛丝长度的细胞机制， 伸长，和（ii）限定组成杆的部件并确定其长度的调节，和 组装件.我们将使用遗传学、生物化学和细胞生物学技术来解决所提出的实验。 总的来说，这些目标旨在促进我们对细菌纳米机器调控及其 对细胞生理和健康的贡献。

PROJECT SUMMARY Bacteria assemble large extracellular complexes called nanomachines to differentially interact with their environment. Nanomachines enact specific functions, including substrate secretion, cell motility, and pathogenesis. The regulation and structural composition of bacterial protein nanomachines is inherently complex. One such nanomachine, the flagellar apparatus, is critical for bacterial motility, and its assembly is highly ordered. Many regulatory systems are in place to ensure that proper assembly of the flagella occurs both spatially and temporally. Many studies investigate the composition of the flagellar structure as well as how the host regulates transcription and translation of its various components. However, less is known regarding the systems in place controlling accurate assembly of the flagellum. One of the major components of the flagellum is the extracellular filament, which is responsible for generating thrust to mobilize the cell. The majority of studies focusing on filament assembly use the Gram- negative organisms Salmonella typhimurium and Escherichia coli. Investigations suggest that these two closely related organisms maintain distinct mechanisms regulating filament length, as well as repair. Whether the precise regulation of filament length is necessary for efficient motility across all species is unknown. Using the genetically tractable, Gram-positive model bacterium Bacillus subtilis, we propose to determine the mechanisms employed by this organism to control filament growth and length, as well as whether filament repair occurs. Additionally, mechanisms regulating the length of the flagellar rod spanning from the membrane-bound flagellar motor to the extracellular filament in B. subtilis is unknown. Four proteins putatively make up the B. subtilis rod, but their order of assembly is currently unknown. Further, the rod must precisely traverse a large distance in both Gram-negative (periplasm) and Gram-positive (peptidoglycan) organisms to initiate assembly of the flagellar filament. Thus, the cell must regulate rod length to accurately span these depths. Although studies in the Gram-negative S. typhimurium show rod length is determined via interaction with the outer membrane, a lack of an outer membrane in Gram-positive organisms indicates a different mechanism is in place. We will identify the structural composition of the rod and determine the components controlling rod length. The aims of this proposal and experiments suggested focus on the structural organization of the flagellar rod and filament, specifically to: (i) assess the cell mechanisms controlling flagellar filament length and elongation, and (ii) define the components that make up the rod and determine the regulation of its length and assembly. We will address the proposed experiments using genetic, biochemical, and cell biology techniques. Overall, these aims intend to promote our understanding of bacterial nanomachine regulation and their contributions to cell physiology and fitness.