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方面很重要。我们现在有了关键细菌基因组的完整DMA序列。我们现在需要了解控制整个基因组功能的机制。阐明这些机制将具有广泛的科学影响，以及我们如何理解致病菌中抗生素耐药性的产生。特异性：该R01项目使用基因组规模的实验方法研究了大肠杆菌的转录调控网络及其在适应进化过程中的最佳生长表型。到目前为止，该计划已经取得了几个重要的里程碑；1)利用表型相平面分析预测和测量最佳生长状态；2)研究表明，在迄今为止研究的100例病例中，约70%的实验室适应进化终点与野生型和敲除型（KO）菌株基因组尺度的计算机模型的先验计算结果一致；3)在适应性进化之前、期间和之后的表达谱分析表征了大肠杆菌转录组的广泛变化，并表明基因组有多种用途来产生特定的生长表型；4)研究了野生型和转录因子（TF） KO菌株的氧转移，并通过表达谱确定了大肠杆菌中大量新的调控相互作用；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。基于这些结果，该R01程序能够回答有关大肠杆菌基因组的使用和调控及其在基因组尺度上的进化可塑性的广泛而基本的问题。因此，我们提出了以下2个具体目标，重点确定大肠杆菌的转录调控网络：1)通过既定的环境和遗传转移实验对测序的K-12 MG1655菌株的网络结构进行广泛和系统的阐明；2)在适应进化到在甘油和乳酸中获得最佳生长后，以及在选择转录因子敲除的适应进化后。这些特定的目标是理解原核生物基因组如何对环境做出反应，以及这些反应如何在适应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 2 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.