Investigating the Rotation of Stator Units of the Bacterial Flagellar Motor

最引人注目的生物机器之一是使细菌能够游泳的旋转马达。这台机器正在对大肠杆菌进行研究，它比任何其他自由生活的东西都更为人所知。大肠杆菌生活在你的肠道里。它的细胞直径约为1微米，长约2微米--1万个细胞可以并排放在你手指的宽度上。每个电池由大约4根细长的螺旋丝推动，每个螺旋丝在其底部由旋转马达驱动。这些细丝沿着细胞的长轴在细胞后面聚集成一束，并以每秒近20个身体长度的速度向前推进。电子显微镜(由Cryo EM)最近的研究表明，驱动鞭毛旋转的力产生单元(定子单元)本身就是旋转发动机，其直径大约是鞭毛电机转子的7倍，因此旋转速度应该是7倍左右。这一假设将通过脉冲荧光漂白来验证，如下所述。众所周知，运动性可以增强细菌的毒力。然而，这项工作的主旨不是探索它的医学相关性，而是理解基础科学，它有可能为生物灵感设计提供蓝图，并激励本科生进入这个快速增长的领域。定子单元由跨越细胞膜的5个拷贝的MOTA蛋白和2个拷贝的MOTB蛋白组装而成。MoTA五聚体被认为是围绕MOTB二聚体旋转的，由从细胞外部流向内部的质子提供动力。MOTB的一端附着在细胞壁的刚性框架(肽聚糖层)上，而MOTA的另一端在鞭毛转子的表面与蛋白质FliG结合。在这个项目中，研究人员将通过将刚性荧光探针融合到MoTA的阿尔法螺旋链上，并用短而强烈的偏振光脉冲对其进行漂白来测试这一命题。随后这些标记的旋转将跟随着较弱的偏振探测光束。这种方法的有效性已经在实验中得到证明，实验表明鞭毛马达的内部和外部组件作为一个单位旋转。这项工作的结果应该提供对为旗舰发动机提供动力的定子单元的力产生机制的基本了解。该奖项反映了NSF的法定使命，并通过使用基金会的智力优势和更广泛的影响审查标准进行评估，被认为值得支持。

One of the most remarkable biological machines is the rotary motor that enables bacteria to swim. This machine is being studied in the bacterium Escherichia coli, about which more is known than any other free-living thing. E.coli lives in your gut. Its cells are about 1 micrometer in diameter by 2 micrometers long -- 10,000 will fit side by side across the width of your finger. Each cell is propelled by about 4 long thin helical filaments, each driven at its base by a rotary motor. The filaments coalesce into a bundle behind the cell along its long axis and push it forward, nearly 20 body lengths per second. Recent work by electron microscopy (by cryo EM) suggests that the force-generating units (stator units) that drive flagellar rotation are themselves rotary engines about 7 times smaller in diameter than the rotor of the flagellar motor, and thus should spin about 7 times faster. This hypothesis will be tested by pulsed fluorescence bleaching, as explained below. Motility is known to enhance bacterial virulence. However, the main thrust of this work is not to explore its medical relevance but rather to understand the fundamental science which has the potential to provide a blueprint for bio-inspired design and inspire undergraduate students to enter this rapidly growing field. Stator units are assembled from 5 copies of the protein MotA and 2 copies of the protein MotB, which span the cell membrane. A MotA pentamer is thought to surround and rotate about a MotB dimer, powered by protons flowing from the outside to the inside of the cell. One end of MotB is attached to the rigid framework of the cell wall (the peptidoglycan layer) while the opposite end of MotA engages the protein FliG at the surface of the flagellar rotor. In this project, investigators will test this proposition by fusing a rigid fluorescent probe to the alpha-helical chains of MotA and bleaching it with a short but intense pulse of polarized light. Subsequent rotation of such markers will be followed by a weaker polarized probe beam. The effectiveness of this method has already been shown in experiments that demonstrated that the inner and outer components of the flagellar motor rotate as a unit. The results of this work should provide fundamental understanding of the mechanism of force generation of the stator units that power the flagellar motor.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.