Institute of Multidisciplinary Research for Advanced Materials, Tohoku University, Aoba-ku, Sendai 980-8577, Japan.
Biochem Biophys Res Commun. 2010 Mar 26;394(1):130-5. doi: 10.1016/j.bbrc.2010.02.129. Epub 2010 Feb 23.
The bacterial flagellar motor is a rotary motor driven by the electrochemical potential of a coupling ion. The interaction between a rotor and stator units is thought to generate torque. The overall structure of flagellar motor has been thought to be static, however, it was recently proved that stators are exchanged in a rotating motor. Understanding the dynamics of rotor components in functioning motor is important for the clarifying of working mechanism of bacterial flagellar motor. In this study, we focused on the dynamics and the turnover of rotor components in a functioning flagellar motor. Expression systems for GFP-FliN, FliM-GFP, and GFP-FliG were constructed, and each GFP-fusion was functionally incorporated into the flagellar motor. To investigate whether the rotor components are exchanged in a rotating motor, we performed fluorescence recovery after photobleaching experiments using total internal reflection fluorescence microscopy. After photobleaching, in a tethered cell producing GFP-FliN or FliM-GFP, the recovery of fluorescence at the rotational center was observed. However, in a cell producing GFP-FliG, no recovery of fluorescence was observed. The transition phase of fluorescence intensity after full or partially photobleaching allowed the turnover of FliN subunits to be calculated as 0.0007s(-1), meaning that FliN would be exchanged in tens of minutes. These novel findings indicate that a bacterial flagellar motor is not a static structure even in functioning state. This is the first report for the exchange of rotor components in a functioning bacterial flagellar motor.
细菌鞭毛马达是一种由耦合离子的电化学势驱动的旋转马达。转子和定子单元之间的相互作用被认为会产生扭矩。尽管人们一直认为鞭毛马达的整体结构是静态的，但最近的研究证明定子在旋转马达中是可以交换的。了解工作中的旋转马达中转子组件的动力学对于阐明细菌鞭毛马达的工作机制非常重要。在本研究中，我们专注于功能齐全的鞭毛马达中转子组件的动力学和周转率。构建了 GFP-FliN、FliM-GFP 和 GFP-FliG 的表达系统，并且每个 GFP 融合蛋白都能功能性地整合到鞭毛马达中。为了研究在旋转马达中是否会发生转子组件的交换，我们使用全内反射荧光显微镜进行了光漂白后荧光恢复实验。在产生 GFP-FliN 或 FliM-GFP 的拴系细胞中，在旋转中心观察到荧光的恢复。然而，在产生 GFP-FliG 的细胞中，未观察到荧光的恢复。完全或部分光漂白后荧光强度的过渡阶段允许计算 FliN 亚基的周转率为 0.0007s(-1)，这意味着 FliN 会在几十分钟内被交换。这些新发现表明，即使在工作状态下，细菌鞭毛马达也不是静态结构。这是关于在功能齐全的细菌鞭毛马达中转子组件交换的首次报道。
