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

Regents Of The University Of Michigan - Ann Arbor, Ann Arbor MI

Investigators

Abstract

Intellectual Merit A 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 Impact The 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.