Genomic-driven Approaches in E.coli Physiology

大肠杆菌生理学中的基因组驱动方法

• 批准号: 6736237
• 负责人: BARRY L WANNER
• 金额: $ 31.31万
• 依托单位: PURDUE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-04-01 至 2006-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6736237
• 关键词: Escherichia coli; biotechnology; functional /structural genomics; gene expression; gene expression profiling; genetic regulation; microarray technology; molecular genetics; phosphates; phosphorus metabolism; phosphorylation; physiology

DESCRIPTION (provided by applicant): With many microbial model systems, molecular genetics provides powerful tools, complementary to high-throughput instrumental approaches, for defining gene function, regulation, and metabolism on a genome-wide scale. Reporter gene fusions can be used to learn how and when genes are turned on or off, which in turn can provide a clue(s) about their function. Systematic mutational analyses of genes in their normal chromosomal locations can generate insights into their roles. Conditional expression or hyperexpression of genes create novel phenotypes that are informative. Methods have also been developed for global analysis of cellular phenotypes in which hundreds or thousands of growth properties can be tested simultaneously, which also allow one to test and assay gene function. Here we will blend several newly developed and facile molecular genetic tools for genome manipulation of Escherichia coli K12 with physiology, phenotypic testing, and global gene expression profiling in order to study the E. co/i physiome with respect to inorganic orthophosphate (Pi) metabolism. We will carry out these experiments to test the following general hypothesis: Genetic regulatory mechanisms act in concert at the transcriptional and posttranscriptional levels in order to coordinate different steps of Pi metabolism with cell growth. These steps include pathways for the uptake of extracellular Pi (Pi (ext))into the cell and subsequent incorporation of intracellular Pi (Pi (int))into ATP and other organophosphates, which act as phosphoryl donors in cell metabolism. Specific Aims are: (1) To identify new genes regulated by environmental Pi levels, to define their properties and functions, and to define how genes for Pi uptake that are unresponsive to Pi control are themselves regulated; (2) To identify regulatory factors that are important in coupling different steps in Pi metabolism and to describe regulatory interactions between the PhoR/PhoB two-component system and other two-component systems that appear to be connected to Pi metabolism; and (3) To identify the role(s) of acetyl phosphate in cell physiology as it relates to Pi metabolism by examining the regulatory consequences of perturbing acetyl phosphate metabolism.

描述（由申请人提供）：对于许多微生物模型系统， 分子遗传学提供了强大的工具，与高通量相辅相成 用于定义基因功能、调控和代谢的工具方法 在全基因组范围内。报告基因融合可用于了解如何以及何时进行 基因被打开或关闭，这反过来又可以提供有关其基因的线索 功能。正常染色体中基因的系统突变分析 地点可以深入了解他们的角色。条件表达式或 基因的过度表达创造了信息丰富的新表型。方法 还被开发用于细胞表型的全局分析，其中 可以同时测试数百或数千种生长特性，这 还可以测试和分析基因功能。 在这里，我们将融合几种新开发的、简便的分子遗传工具 用于大肠杆菌 K12 的生理学、表型基因组操作 测试和全局基因表达谱分析，以研究大肠杆菌 无机正磷酸盐 (Pi) 代谢的生理组学。我们将 进行这些实验来检验以下一般假设： 基因调控机制在转录和转录过程中协同作用 转录后水平以协调 Pi 的不同步骤 新陈代谢与细胞生长有关。这些步骤包括吸收途径 细胞外 Pi (Pi (ext)) 进入细胞并随后掺入 细胞内 Pi (Pi (int)) 转化为 ATP 和其他有机磷酸酯，其作用 细胞代谢中的磷酰基供体。 具体目标是：（1）鉴定受环境Pi调控的新基因 水平，定义它们的特性和功能，并定义基因如何 对 Pi 控制无反应的 Pi 吸收本身受到调节； (2) 至 确定在耦合不同步骤中重要的调节因素 Pi 代谢并描述 PhoR/PhoB 之间的调节相互作用 双组分系统和其他双组分系统 与 Pi 代谢有关； (3) 确定乙酰磷酸的作用 在细胞生理学中，通过检查调节与 Pi 代谢相关 扰乱乙酰磷酸代谢的后果。