High Throughput Approaches to Define the Spatial Organization of Proteins in E. coli

在过去的二十年里，一个令人惊讶的发现是发现细菌细胞具有显著水平的细胞组织。了解细菌细胞的结构需要知道蛋白质的位置以及每种蛋白质如何与其特定的结合伙伴相互作用。从历史上看，大多数关于特定蛋白质的空间定位及其伴侣的识别的知识都是通过许多单个实验室的重点研究产生的。细菌DNA序列的解码已经允许高通量的努力来定义细菌细胞的空间关系。此外，有了这些DNA序列在手，几个实验室就可以专门为所有研究人员提供全面的工具。本研究将利用这些现有资源，并以新颖的方式利用它们，以实现两个目标。基于微型细胞的原理，由于它们有缺陷的极性限制形成，富含天然靶向于极点和隔膜的蛋白质，该项目将使用微型细胞来识别定位于细胞极点或隔膜的蛋白质。首先，将使用高通量方法鉴定定位于细胞极点或中间细胞的新蛋白质。类似定位的蛋白质之间的相互关系将被确定，其结合伙伴的身份也将被确定。其次，现有的蛋白质融合文库将以一种新的方式用于识别新的结合伙伴和识别极性蛋白质。该项目的目标是生成一个新的大型微生物细胞组织数据集，并将这些数据提供给科学界。这项研究的结果将影响目前对细胞组织和结构的理解。更广泛的影响这些研究的更广泛的影响是双重的。首先，由于该项目的广泛性和综合性，这项研究的结果将对当前对细菌细胞的理解产生重大而直接的影响，无论是在蛋白质-蛋白质相互作用水平还是在空间组织水平。这将是微生物学界的宝贵资源。第二，这个项目将在一个跨学科的研究中培养几名学生（研究生和本科生）。这些学生将有独特的机会获得分子遗传学，细胞生物学和蛋白质组学方面的经验。在这些研究中，培养具有广泛背景的学生和博士后是重中之重，并支持NSF在教学、培训和推广方面的重点。

Intellectual MeritA surprising finding in the past two decades has been the discovery that bacterial cells possess a remarkable level of cellular organization. Understanding how a bacterial cell is structured requires knowing where proteins are positioned and how every protein interacts with its specific binding partner(s). Historically, most knowledge on the spatial localization of specific proteins and the identification of their partners has occurred through focused studies by many individual laboratories. The decoding of the DNA sequences of bacteria has allowed for high throughput efforts to define the spatial relationships in bacterial cells. Moreover, having these DNA sequences in hand has allowed dedicated efforts by several labs to generate comprehensive tools available to all researchers. This study will take advantage of these existing resources and will utilize them in novel ways to achieve two goals. Based upon the principle that mini cells, because of their defective polar restriction formation, are enriched for proteins that are naturally targeted to the poles and the septum, this project will use mini cells to identify proteins that localize to the poles or the septum of cells. First, new proteins that localize to cell poles or to midcells will be identified by using high throughput methods. The interrelationships between proteins that are similarly localized will be determined as will the identity of their binding partners. Second, an existing protein fusion library will be used in a novel manner to identify new binding partners and to identify polar proteins. The goals of this project are to generate a large new microbial cellular organization dataset and to make these data available to the scientific community. Results from this study will impact current understanding of cellular organization and structure.Broader ImpactThe broader impact of these studies is twofold. First, because of the broad and comprehensive nature of this project, results from this research will have significant and immediate impact on current understanding of the bacterial cell, both at the level of protein-protein interactions and at the level of spatial organization. This will be an invaluable resource to the microbiology community. Second, this project will result in the training of several students (graduate and undergraduate) in an interdisciplinary study. These students will have the unique opportunity to gain experiences in molecular genetics, cell biology and proteomics. Training of students and postdoctoral fellows from a wide range of backgrounds is a high priority during these studies and supports NSF emphases on teaching, training, and outreach.