Clarification of energy transduction of bacterial flagellar motor using hybrid bacteria

细菌鞭毛马达是细菌的分子机器，直径约40 nm。驱动力是电池内外的离子浓度之差，质子和钠。我们用这些细胞制造了嵌合体细胞。该池适用于转动测量，对阐明这一机理起着重要作用。我们首次成功地测量了旋转的步长运动（Nature，2005）。然而，关于这种嵌合体细胞的基本性质尚未得到证实。因此，我们试图阐明这种嵌合体细胞的基本性质。因此，嵌合体细胞的转矩-速度关系具有两个电机的相似特性。此外，还可以观察到定子复合体的加入使转速逐步增加，可以测定一个定子复合体的转矩-速度关系。此外，为了弄清细菌细胞内的信息、马达复合体的构造情况和马达的旋转运动，需要细胞内的成像技术。细菌细胞成像技术与常规方法相比存在一些问题，如细胞体积小、形状不规则等。我们优化了倏逝光照明条件，并尝试在细菌细胞中观察GFP融合蛋白。结果，我们成功地观察到GFP融合的FliG蛋白的扩散运动，FliG蛋白是动力蛋白之一。此外，我们成功地估计了荧光点的扩散常数。此外，我们能够使用系留细胞测定方法明确GFP融合的FliG蛋白的位置与细胞的枢轴点一致。

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.