This project will study the mechanism by which microorganisms actively accumulate metals, against their concentration gradient, through the cell envelope. The ability of microbes to acquire iron is an important aspect of microbial biochemistry and physiology, and TonB is a Gram-negative bacterial cell envelope protein that is needed for the function of iron transporter proteins. It is proposed to transfer energy from the cytoplasmic membrane of the cell through the periplasm to the outer membrane. These studies will use genetic engineering, chemical modification, fluorescence spectroscopy, and microscopy to elucidate the biochemical functions of TonB. We will determine if TonB normally bridges between the inner and outer membranes of bacterial cells, by interaction with the peptidoglycan layer underlying the outer membrane, and we will assess the importance of this association to iron acquisition by Escherichia coli. Comparative measurements of the strength of interactions that occur between TonB and peptidoglycan, and between TonB and FepA (an iron transporter), will illuminate the nature of their functional interaction during the uptake of solutes. Secondly, the experiments will evaluate a proposed rotatory mechanism of TonB action, using fluorescence spectroscopic studies, and characterize the rate of lateral diffusion of TonB in the bacterial membranes. These experiments will test existing theories of TonB- and energy-dependent outer membrane transport, and provide biophysical data that allows insight into long-sought mechanisms of bacterial cell envelope physiology.
Broader Impacts:
The understanding of the mechanism of this prototypic outer membrane transport system has wide ranging implications, because transporters of this class function in all Gram-negative bacteria. Furthermore, the membrane system under investigation is itself unprecedented, because it involves active transport across a membrane that cannot sustain an ion gradient. The project involves and develops methodologies that permit direct observation of membrane transport reactions in living cells, and these procedures advance the study of membrane biochemistry. It will accomplish its goals with new biochemical and spectroscopic techniques, while integrating the teaching and training of student researchers within its overall scientific objectives. This project will train undergraduate students, graduate students, and a postdoctoral researcher in membrane biochemistry, including novel theories and new biophysical methodologies associated with analysis of bacterial membrane transport. As an integral feature of this project intended to promote scientific education and training, students will receive instruction in molecular biological and biochemical techniques, membrane biochemistry and fluorescence spectroscopy, that they will then use in their assigned independent research. The participants include individuals at the University of Oklahoma and Purdue University, from a broad spectrum of ethnic and cultural backgrounds. The PI is committed to broadening research opportunities for under-represented groups, and he will make efforts to communicate the fundamental research findings to audiences outside the field of membrane biochemistry, including the medical community, and high school students and teachers throughout Oklahoma.
