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.). ? ? ?
