Probing Metabolic Complexity Using a Bacterial Model System

描述（由申请人提供）：生物学中的一个基本问题是如何调节代谢途径的整合，以产生具有可持续性生命特征的健壮但响应性生理。我们在这些领域缺乏基本知识，在不同的基因组序列中仍然没有功能注释的多个orf强调了这一点。因为所有活细胞在整合新陈代谢方面都面临着类似的挑战，为了简单和技术可行性，将使用细菌系统（肠沙门氏菌）来完成概述的目标。这个系统将提供解剖网络的培训，在我以后的职业生涯中可以转移到更难对付的生物体上。通过对硫胺素生物合成系统的遗传分析，鉴定出5个铁硫簇代谢缺陷的条件型硫胺素无性营养体。总的来说，这些位点似乎是铁运输和氧化损伤的Fe- s簇修复所必需的。本文概述了一系列的实验，以解决这三种蛋白质（ApbE， ApbC和RseC）的特定生化功能。本文描述的项目分为两个相似且重叠的目标，最终将定义这些与参与Fe-S代谢的其他蛋白质之间的关系。完成这些目标将需要严格应用生物化学、生物物理、遗传和生理技术的体内和体外实验。本提案中提出的研究的长期目标是有助于理解代谢整合，特别是铁稳态。为了达到这个目标，我们必须增加我们对新陈代谢的生化成分和它们之间存在的联系的知识。这些工作不仅有助于基因组的严格注释，而且为进一步研究铁和自由基代谢提供了基础。细胞代谢是在任何给定时间内发生在活细胞中的所有化学反应的总和。就像互联网或经济一样，新陈代谢是一个网络或系统，所以微小的变化，比如营养物质的去除，可能会产生深远的影响。拟议工作的目的是了解这些化学反应之间的联系，以便预测如果系统以某种方式受到干扰（癌症，饥饿，疾病等）将会遇到的影响。

DESCRIPTION (provided by applicant): A fundamental question in biology is how the integration of metabolic pathways is regulated to produce the robust, but responsive physiology that characterizes sustainable life. Our lack of basic knowledge in these areas is emphasized by the multiple ORFs that remain without functional annotation in diverse genome sequences. Because all living cells face similar challenges in integrating their metabolism, a bacterial system (Salmonella enterica) will be used for simplicity and technical feasibility to complete the outlined objectives. This system will provide training in dissecting a network that can be transferred to more refractory organisms later in my career. Genetic analysis of the system integrated with thiamine biosynthesis has identified a set of five conditional thiamine axotrophs which are defective in Fe-S cluster metabolism. Collectively, these loci appear to be necessary for Fe trafficking, and the repair of oxidatively damaged Fe-S clusters. A series of experiments is outlined to address the specific biochemical function of three of these proteins (ApbE, ApbC, and RseC). The project described herein is divided into two similar and overlapping objectives that will ultimately define the relationship between these and other proteins involved in Fe-S metabolism. Completion of these objectives will require in vivo and in vitro experiments with rigorous application of biochemical, biophysical, genetic, and physiological techniques. The long-term goal of the research presented in this proposal is to contribute to the understanding of metabolic integration, specifically Fe homeostasis. To reach this goal we must increase our knowledge of the biochemical components of metabolism and the connections that exist between them. Such work not only contributes to the rigorous annotation of genomes, but provides the basis for continuing studies on Fe and free radical metabolism. Cellular metabolism is the sum of all of the chemical reactions taking place in a living cell at any given time. Like the internet or the economy, metabolism is a network or system, so small changes, such as the removal of a nutrient, can have profound effects. The objective of the proposed work is to understand the connections between these chemical reactions in order to predict the effects which would be encountered if the system is in some way perturbed (cancer, starvation, disease etc.).