The overall research objective is determination of the mechanism of bacterial flagellar rotation. The research will enhance understanding of the molecular basis of motility and its regulation, as well as of chemosmotic energy transduction. These fundamental issues are key to deciphering how living cells work. The understanding gained will be relevant for diagnosing the cellular basis of metabolism-related diseases. Models for force-generation in the bacterial flagellar motor may now be proposed based on initial assignment of the proteins involved in motility to the recently-discovered basal body structures. Intramembrane particles that ring the flagellum are thought to conduct protons and exert or enable torque on the flagellum by contacts made with the belled cytoplasmic extension of the basal body. Further work is planned to establish these assignments, to map spatial distributions and to characterize functional interactions of these modules with themselves and CheY protein. Salmonella typhimurium extended flagellar basal bodies have been purified. The major proteins of the extended structure are FliG, FliM and FliN, proposed earlier by geneticists to form a structural complex, the """"""""switch complex"""""""". Current protocols will be improved to identify minor components which may form a distinct protein export apparatus within the extended basal body and/or be involved in motility. The arrangement of the FliG, FliM and FliN proteins will be determined by immuno and mass-mapping electron microscopy of wild-type and partial extended basal bodies isolated from mutant strains that are either blocked in assembly or assemble defective structures. Bacterial chemotaxis is mediated by CheY- motor interactions. Binding of CheY protein to extended basal bodies will be characterized using biochemical column and spectroscopic methods. Mass analysis will seek to directly measure the number and spatial distribution of CheY molecules bound to extended basal bodies, once conditions to stabilize such binding have been identified. The involvement of the flagellar intramembrane ring particles in motility is based on correlation of their presence with expression of the MotA and MotB proteins. Fusion constructs of the MotB protein with peptides that fluoresce or that may be biotinylated will be made in order to establish that the MotB protein localizes to the particle rings and, if so, to examine the dynamic equilibrium between assembled and unassembled protein. Work will also aim to extend preliminary experiments that indicate that the organization of the particle rings depends upon contacts made with cytoplasmic structure and the energized-state of the cell membrane; based on utilization of time-resolved freeze-fracture electron and video microscopies and caged compounds.
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