Testable in silico Hypotheses for E. coli Growth

描述（由申请人提供）：概述：细菌在人类疾病，生物恐怖主义和处理环境中很重要。我们现在有了关键细菌基因组的完整DNA序列。我们现在需要了解控制整个基因组功能的机制。阐明这些机制将产生广泛的科学影响，以及我们如何理解病原菌中抗生素耐药性的产生。具体说明：该R 01计划使用基因组规模的实验方法来研究大肠杆菌的转录调控网络及其适应性进化过程中的最佳生长表型。迄今为止，该计划已经取得了几个重要的里程碑：1）它已经使用表型相平面分析来预测和测量最佳生长状态; 2）它已经表明，在迄今为止检查的超过100个案例中，约有70%的实验室适应性进化的终点与野生型和敲除（KO）菌株的基因组规模计算机模型的先验计算一致; 3）在适应性进化之前、期间和之后，它导致了表达谱分析，表征了E.大肠杆菌转录组，并表明有多种用途的基因组，以产生一个特定的生长表型; 4）它已经研究了野生型和转录因子（TF）KO菌株中的氧转移，并通过使用表达谱确定了大量的新的调控相互作用，在大肠杆菌。5）将myc标签放在这些TF上，开始确定其结合位点的基因组位置的过程。基于这些结果，该R 01计划能够回答关于E.大肠杆菌基因组及其在基因组尺度上的进化可塑性。因此，我们提出了以下两个具体的目标，重点是确定转录调控网络在E。I）通过使用已建立的环境和遗传转换实验，广泛和系统地阐明了测序的K-12 MG 1655菌株中的网络结构，和II）在适应性进化到在甘油和乳酸盐上的最佳生长后，以及在选择的转录因子敲除的适应性进化后。这些特定的目标是理解原核生物基因组如何对其环境做出反应，以及这些反应如何在适应性进化过程中被修改以更好地适应给定环境的核心。

DESCRIPTION (provided by applicant): GENERAL: Bacteria are important in human disease, bioterrorism, and dealing with the environment. We now have full DMA sequences for the genomes for key bacteria. We now need to understand the mechanism that govern whole genome functions. Elucidating these mechanisms will have a broad scientific influence, and how we understand the generation of antibiotic resistance in pathogenic bacteria. SPECIFIC: This R01 program has used genome-scale experimental methods to study the transcriptional regulatory network in Escherichia coli and its optimal growth phenotypes during adaptive evolution. The program has to date achieved several important milestones; 1) it has used phenotypic phase plane analysis to predict and measure optimal growth states; 2) it has shown that in about 70% of the >100 cases examined to date, that the endpoint of laboratory adaptive evolution is consistent with the a priori computations of the genome-scale in silico model for wild-type and knock-out (KO) strains; 3) it has led to expression profiling before, during and after adaptive evolutions characterizing the extensive change in the E. coli transcriptome and shown that there are multiple uses of the genome to produce a particular growth phenotype; 4) it has studied the oxygen shift in wild-type and transcription factor (TF) KO strains, and by using expression profiling determined a large number of new regulatory interactions in E. coli; and 5) it has put myc-tags on these TFs to begin the process of determining the genome location of their binding sites. Based on these results, this R01 program is in a position to answer broad and fundamental questions about the use and regulation of the E. coli genome and its evolutionary plasticity on a genome-scale. We thus put forth the following two specific aims that focus on determining the transcriptional regulatory network in E. coli I) a broad and systematic elucidation of the structure of the network in the sequenced K-12 MG1655 strain through the use of established environmental and genetic shift experiments and II) after adaptive evolution to optimal growth on glycerol and lactate, and after adaptive evolution of selected transcription factor knock- outs. These specific aims lie at the heart of understanding how prokaryotic genomes respond to their environments and how such responses are modified during adaptive evolution to better fitness in a given environment.