The major long-term objective is to understand how energy stored as transmembrane ion gradients is used to power the rotation of bacterial flagella. Transmembrane ion gradients and motile processes are fundamental properties of the living state. The molecular details of how ion fluxes are coupled to work and information transfer in biomembranes, and how biomolecules couple chemical energy to microscopic movement remain elusive. Proposed work will contribute to understanding of these basic issues. It is not designed to address specific medical problems, but medical applications of the understanding obtained will doubtless result. The major thrust of our work has been to draw structural and energetic constrains to possible energy coupling mechanisms. Planned work will extend the advances made. Structural work will focus on the bacteria Escherichia coli and Salmonella typhimurium. Rapidly frozen freeze-fractured whole cells; freeze-substituted and freeze-etched cell envelope preparations; and freshly isolated flagella have revealed elaborate intramembrane and cytoplasmic basal flagellar structures. The morphology of these structures will be further characterized using replica and immuno electron microscopy; and single particle image averaging methods. Reversible crosslinkers and immunoprecipitation will be exploited for purification of the additional structures associated with the basal bodies of freshly isolated flagella and determination of their polypeptide composition. The spatial organization of the proteins implicated in energization and switching of flagellar rotation will be investigated by antibody labelling: in the new structures as well as in the plane of cytoplasmic membrane and in cell lysates. Motor dynamics will be related to ion transport chemistry. A biochemical cycle for analysis of the free energy changes occurring during energy transduction in the flagellar motor has been developed based on analysis of swimming cell speed. Temporally resolved, optical measurements of forced and spontaneous changes of motor speed will be made under conditions where ions transfers limit flagellar rotation. Changes in both tethered cell and filament rotation speed induced by photorelease of caged protons will be studied in Escherichia coli. Differences evident in responses between wild type and mutant MotA cells containing defective, plasmid encoded MotA protein, a proton conducting component of the flagellar motor, will be examined. The torque frequency relation of the sodium powered flagella of the bacterium Vibrio alginolyticus will be determined, and speed fluctuations characterized, as a function of limiting cation concentration.
Showing the most recent 10 out of 18 publications
