Clarification of energy transduction of bacterial flagellar motor using hybrid bacteria

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Bacterial flagellar motor is known as a molecular machine of bacteria, of which diameter is about 40 nm. The driving force is a difference of ion concentration, proton and sodium, between the inside and outside of the cell. We have made the chimera cell using these cells. This cell is suitable for a rotational measurement, plays an important role for elucidation of this mechanism. We succeeded in a measurement of step-size motion of rotation for the first time (Nature, 2005). However, it was not yet confirmed about a basic property of this chimera cell. Therefore we tried to clarify a basic property of this chimera cell. As a result, the torque-speed relations of a chimera cell had the similar characteristics of two motors. Furthermore, stepwise increase of the rotational speed with an incorporation of stator complex was observed, and the torque-speed relationship by one stator complex could be measured.In addition, imaging technology in a cell is necessary to clarify the information in bacteria cell, the construction situation of a motor complex and a rotational motion a motor. There are several problems about the imaging technology using bacteria cell compared to the normal method, by small and shape of cells. We optimized evanescent illumination and tried to observe of GFP fused protein in bacteria cell. As a result, we succeeded in observing the diffusional motion of GFP fused FliG protein which is one of the motor proteins. Furthermore, we succeeded in estimating the diffusion constant of the fluorescent spots. In addition, we were able to make clear that the position of the GFP fused FliG protein coincided with a pivot point of a cell using a tethered cell assay method.