Present and Potential Functions of Bacterial Transporters

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This award is funded under the American Recovery and Reinvestment Act of 2009 (Public Law 111-5).Intellectual merit. The project will test the hypothesis that nutrient transporters possess functional versatility and genetic malleability that allow them to serve as wedges in the evolutionary divergence of bacteria. Individual transporter genes will be studied in bacteria in which all transporters with overlapping or potentially overlapping activities have been deleted. The properties conferred by each transporter will then be determined. Mutant genes that confer altered growth phenotypes will be selected, and these genes will be sequenced to determine amino acid sequence substitutions that caused the change in transporter activity. This work will be conducted with Acinetobacter baylyi, a bacterium that offers singular advantages for genetic investigation because of its nutritional breadth and its extraordinarily high competence for natural transformation. In the future, genetic constructs in A. baylyi will allow the procedures to be adapted to determine the functions of transporters from other bacteria, including those with complex genomes or those that are difficult to grow.A central question in bacteriology is what makes an organism stand apart? Obvious sources of differences are in genomes where gene rearrangements mark discontinuities in the evolutionary divergence of cell lines. Less obvious, but perhaps as significant, are differences in activities, such as transmembrane transport, that determine how a cell adapts to an often shifting environment. Transmembrane transporters are well suited for accommodating evolutionary divergence because they are poised to have direct contact with the environment, they are abundant (frequently encoded by genes accounting for 10% or more of a genome), and they possess a redundancy that may allow one transporter to maintain an activity while another transporter with overlapping function adapts to a new challenge. To understand the extent to which transporters contribute to evolutionary divergence depends upon knowledge about both their functions and the genetic simplicity with which they can shift from one activity to another. Present information, based upon an overwhelming amount of genome sequence data, provides only a glimpse of the necessary knowledge because sequence similarities are poor predictors of similarities in transporter function. This project is designed to develop a system for learning how a major subset of transporters contributes to physiology and potential genetic adaptation in A. baylyi. A merit of the system is that it will provide a foundation for study of genes easily cloned from other bacteria.Broader impacts. The project will have a broad impact on scientific training and research. Yale graduate students on rotation through the lab and undergraduates will participate in the research. The A. baylyi genetic system is the basis for teaching laboratories around the world because natural transformation provides a swift demonstration of what a gene is and what it does. Support for this research program will facilitate the continued supply of bacterial strains for introductory college teaching and to create upper level teaching laboratories on the genetic analysis of transport. Key research findings will be integrated into classes at local high schools as part of the Yale Outreach Program. Funding will allow further participation in the Yale SURF summer research program, which introduces minority undergraduate students to investigative science. Results of the research will be disseminated through presentations at national meetings and the scientific literature. Bacterial strains will assist other laboratories in cloning and characterizing other genes for bacterial transporters and will thereby provide a useful resource for further research in the scientific community. Results from the project are likely not only to give insight into a basic question of transporter evolution, that is how substrate specificity evolves, but also to provide a knowledge base that will contribute to bioremediation and biotechnological applications, both of which depend upon transport and processing of metabolites.