Genomic-driven Approaches in E.coli Physiology

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，补充了高通量 用于定义基因功能、调节和新陈代谢的仪器方法 在全基因组范围内。记者基因融合可以用来了解如何以及何时进行 基因的开启或关闭，反过来可以提供一个线索(S)关于他们 功能。正常染色体上基因的系统突变分析 地点可以产生对其角色的洞察力。条件表达式或 基因的过度表达创造了新的表型，这些表型具有信息量。方法 也被开发用于细胞表型的全球分析，其中 可以同时测试成百上千个生长特性，这 也允许人们测试和分析基因功能。 在这里，我们将结合几种新开发的、简便的分子遗传工具 大肠杆菌K12基因组操作的生理学、表型研究 测试和全球基因表达谱，以研究E.co/I 生理组与无机正磷酸盐(PI)代谢有关。我们会 执行这些实验以检验以下一般假设： 基因调控机制在转录和转录水平上协同作用 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。这些步骤包括吸收的途径 胞外PI(PI(Ext))进入细胞并随后掺入 胞内PI(PI(Int))转化为ATP和其他有机磷酸盐，它们起到 细胞新陈代谢中的磷酸供体。 具体目标是：(1)发现受环境PI调控的新基因 水平，以定义它们的属性和功能，并定义基因如何 对PI控制没有反应的PI吸收本身被调节；(2) 确定在将不同步骤结合在一起的重要监管因素 磷代谢和描述磷/磷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；以及(3)确定乙酰磷酸的作用(S) 在细胞生理学中，因为它与磷的代谢有关，通过检测调节 扰乱乙酰磷酸盐代谢的后果。

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.