Genomic-driven Approaches in E.coli Physiology

描述（由申请人提供）：有许多微生物模型系统， 分子遗传学提供了强有力的工具， 仪器方法，用于定义基因功能、调节和代谢 在全基因组范围内。报告基因融合可以用来了解如何以及何时 基因是开启或关闭的，这反过来又可以提供关于他们的基因的线索。 功能正常染色体上基因的系统突变分析 位置可以产生对其角色的洞察。条件表达式或 基因的过表达产生了提供信息的新表型。方法 也被开发用于细胞表型的全球分析，其中 可以同时测试数百或数千种生长特性， 也允许人们测试和分析基因功能。 在这里，我们将混合几个新开发的和简易的分子遗传工具， 用于大肠杆菌K12的基因组操作， 测试和全球基因表达谱，以研究E.公司/机构 无机正磷酸盐（Pi）代谢方面的生理组。我们将 进行这些实验以检验以下一般假设： 遗传调节机制在转录和转录水平上协同作用， 为了协调Pi的不同步骤， 代谢与细胞生长。这些步骤包括摄取 细胞外Pi（Pi（ext））进入细胞，随后掺入 细胞内的Pi（Pi（int））转化为ATP和其他有机磷酸盐， 磷酰基供体在细胞代谢中的作用。 具体目的是：（1）鉴定受环境Pi调控的新基因 水平，定义它们的属性和功能，并定义基因如何 对Pi控制无反应的Pi摄取本身受到调节;（2） 确定在耦合不同步骤中重要的调节因素， Pi代谢和描述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，因为它涉及到Pi代谢，通过检查调节 乙酰磷酸代谢紊乱的后果。

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.