RECEPTOR MEDIATED UPTAKE OF IRON COMPLEXES IN BACTERIA

本研究的长期目标是阐明 大肠杆菌和相关生物主动 运输铁络合铁载体或铁结合蛋白， 满足他们对铁的营养需求。 细菌在体内获得铁 从不溶性铁络合物中产生三价铁 螯合剂或它们直接利用宿主铁结合蛋白， 转铁蛋白或氯化血红素。 特异性外膜受体识别铁- 结合分子以允许它们的能量依赖性摄取。 的能量 跨外膜的转运是通过质子 从细胞质膜到这些配体结合受体的动力 通过TonB及其辅助蛋白ExbB和ExbD的活性。 TonB依赖性是细菌铁摄取系统中的常见主题， 包括铁载体和宿主铁结合蛋白， 已知的细菌病原体。 高选择性受体的功能 与同源转运渗透酶协同作用，使微生物 吸收足够的铁以在有氧环境中生存和生长， 在动物宿主中。 本研究以E.大肠杆菌肠杆菌素转运系统， 研究TonB受体分子细节的原型， 依赖铁的吸收。 遗传选择策略和生化 技术将被用来定义拓扑和功能的特点， FepA受体，以探测其与TonB的相互作用，并确定 Fep通透酶的基本结构和功能特征。 这些研究将涉及以下具体目标：（1）表位 标记和缺失突变以改进FepA的拓扑图; (2)选择定义FepA功能结构域的突变;（3）遗传学上的 和FepA-TonB相互作用的生化交联分析;（4） 结合能力和突变体选择用于功能表征 的膜蛋白的Fep通透酶复合物;和（5）描绘 酶促条件下从肠杆菌素中除去铁 酯酶Fes. 由于铁的可用性的限制是一个关键 微生物种群生长和传播的控制因素， 它被认为是毒力的重要组成部分， 细菌病原体 了解细菌铁吸收过程 因此，在分子水平上设计有效的策略是很重要的。 以保留这种重要的营养素，供潜在的有毒生物体使用。

The long-term objective of this study is to elucidate the molecular processes by which Escherichia coli and related organisms actively transport iron complexed to siderophores or iron-binding proteins to satisfy their nutritional iron requirements. Bacteria obtain iron in an aerobic environment from insoluble ferric complexes by producing ferric chelators or they directly utilize host iron-binding proteins like transferrin or hemin. Specific outer membrane receptors recognize iron- binding molecules to allow their energy-dependent uptake. The energy for transport across the outer membrane is derived by coupling the proton motive force from the cytoplasmic membrane to these ligand-bound receptors through the activity of TonB and its accessory proteins ExbB and ExbD. TonB dependence is a common theme in bacterial iron uptake systems, including those for siderophores and host iron-binding proteins in recognized bacterial pathogens. The highly selective receptors function in concert with cognate transport permeases to enable microbes to assimilate sufficient iron to survive and grow in aerobic environments or in an animal host. This study focuses on the E. coli enterobactin transport system as a prototype for investigating the molecular details of TonB-receptor- dependent iron uptake. Genetic selection strategies and biochemical techniques will be used to define topological and functional features of the FepA receptor, to probe its interactions with TonB, and to define basis structural and functional characteristics of the Fep permease. These studies will involve the following specific aims: (1) epitope tagging and deletion mutagenesis to refine the topological map of FepA; (2) selection of mutations defining FepA functional domains; (3) genetic and biochemical cross-linking analysis of the FepA-TonB interaction; (4) binding capabilities and mutant selection for functional characterization of membrane proteins of the Fep permease complex; and (5) delineation of enzymatic requirements for iron removal from enterobactin catalyzed by the esterase Fes. Since the limitation of iron availability is a critical control factor in the growth and dissemination of microbial populations, it is recognized as a significant component of virulence capacity for bacterial pathogens. Understanding the bacterial iron uptake process at a molecular level is therefore important to designing effective strategies to withhold this vital nutrient for potentially virulent organisms.