This application asks how the flagellum of the bacterium Salmonella is assembled, and how most of its biomass, external to the cell, is exported. What is the composition and location of the export apparatus? How do its substrates cross the plane of the membrane? What does the assembly process entail? Is the export process ordered? If so, to what extent and what is the mechanism? How is the process energized and how is the energy transduced and controlled? The methods proposed include classical and molecular genetics (e.g., intergenic suppression and scanning deletion analysis), enzymatic biochemistry (characterization of the ATPase activity of the apparatus), and physical biochemistry (affinity chromatography, size filtration chromatography, etc.). A major emphasis will be on obtaining high-resolution structures of a number of the export apparatus components. This will initially be done with the thermophilic bacterium Thermotoga maritima, which has a similar export system but whose proteins are more stable and amenable to X-ray crystallography. These structural studies will be conducted in collaboration with Professor Namba of Osaka University, Japan. The application is one of basic microbiological research, but motility is often a significant contributing factor to pathogenicity. Also, Type III secretion pathways extremely similar to the flagellar protein export pathway are utilized by many pathogens (Yersinia pestis, for example) for secretion of virulence factors; information learned about one branch of this superfamily of pathways can only help with understanding of the other. Finally, the assembly of complex biological structures is one of broad general interest. ? ?
Showing the most recent 10 out of 86 publications
