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.
