Investigation of the Mechanism of the Bacterial Flagellar Motor

该项目的目的是了解细菌鞭毛马达的机制，鞭毛马达是一种直径约50纳米（1/20,000毫米）的旋转分子电动机，最大速度超过100,000转/分。许多细菌通过游泳来导航它们的环境。螺旋桨是约20nm厚的螺旋鞭毛细丝，马达由氢离子（H+）或钠离子（Na+）沿着称为质子动力（pmf）或钠动力（smf）的电化学梯度流动驱动。这些梯度由细胞膜上的电压和浓度差异组成，是生物自由能的主要形式。每个马达的最大输出功率约为十亿分之一瓦特，比其他已知的由ATP水解驱动的分子马达高100到1000倍。转子是细胞质膜上的一组环，直径约45纳米，周围有大约12个独立的扭矩产生单元，这些扭矩产生单元固定在细胞壁上，当离子流过转子时，就会对转子施加推力。我们将使用一系列生物物理技术，一些相对完善，另一些是全新的，来测量单个鞭毛马达的特性，并测量和控制它们所在的单个细菌细胞的smf。为了测量电机的速度，我们将把直径数百纳米的聚苯乙烯珠子附着在鞭毛细丝上，并通过荧光显微镜拍摄高速视频（高达每秒2400帧）或测量聚焦在珠子上的红外激光束的挠度来测量它们的旋转。我们将使用的鞭毛经过基因工程改造，使这些实验成为可能。这些细丝携带了一种突变，使它们能自发地粘在珠子上。产生扭矩的单元是嵌合体，包含来自不同物种的成分，使我们能够利用大肠杆菌（通常有H+驱动的马达）的所有可能的遗传工具来研究Na+驱动的马达。这些嵌合单元将在一种化学诱导剂的控制下在细菌内产生，这将使我们能够控制每个马达的数量。我们将以我们实验室最近的两个实验创新为基础。其中一个允许首次检测到鞭毛马达的基本扭矩产生步骤。当通过降低Na+浓度来降低smf时，在只含有一个单元的嵌合马达中可以看到14度的台阶。我们认为，每一步可能对应于一个或两个离子穿过电机，但需要在一系列不同条件下对许多步骤进行详细和系统的测量，以确保情况确实如此。我们特别需要控制和测量每个细胞中的smf。这将使用我们最近的另一项创新，它允许单细胞测量smf的荧光显微镜指示染料，报告内部Na+浓度。通过扩展这些技术，我们将系统地研究在smf的不同单元数和不同浓度和电压部分的值下鞭毛电机的转矩-速度关系。最后的实验将尝试通过测量单个微型电池内离子的积累来测量通过单个电机的微小离子通量。基本计算表明，用于测量内部Na+浓度的荧光技术足够灵敏，使这成为可能，即使离子电流将比典型的单离子通道电流小数百倍。对鞭毛马达的理解将有助于分子马达领域的发展，并为可能的技术应用奠定基础。它还将有助于实现在分子尺度上设计和制造人工机器的长期目标。

The aim of the project is to understand the mechanism of the bacterial flagellar motor, a rotary molecular electric motor with a diameter of ~50 nm ( 1/20,000th of a mm) and a maximum speed in excess of 100,000 r.p.m. Many species of bacteria navigate their environment by swimming. The propellers are helical flagellar filaments ~20 nm thick and the motor is driven by the flow of ions, either H+ or Na+, down an electrochemical gradient called the protonmotive force (pmf) or sodium-motive force (smf). These gradients consist of a voltage and a concentration difference across the cell membrane and are the primary form of biological free-energy. Each motor has a maximum power output of about one million-billionth of a Watt, 100 to 1000 times higher than other known molecular motors which are powered by ATP hydrolysis. The rotor is a set of rings in the cytoplasmic membrane, about 45 nm in diameter and surrounded by about a dozen independent torque generating units which are anchored to the cell wall and push on the rotor when ions flow through. We will use a range of biophysical techniques, some relatively well established and others brand-new, to measure the properties of single flagellar motors and to measure and control the smf of the single bacterial cells that they are in. To measure the motor speed we will attach polystyrene beads hundreds of nanometres in diameter to flagellar filaments and measure their rotation either by taking high-speed videos (up to 2400 frames per second) with a fluorescence microscope, or by measuring the deflection of an infra-red laser beam focussed onto the beads. The flagella we will use are genetically engineered to make these experiments possible. The filaments carry a mutation that makes them stick spontaneously to beads. The torque-generating units are chimeras containing components from different species, allowing us to study a Na+-driven motor with all the genetic tools that are possible using E. coli (which normally has H+-driven motors). These chimeric units will be produced inside bacteria under the control of a chemical inducer that will allow us to control the number in each motor. We will build on two recent experimental innovations in our lab. One of these allowed the first ever detection of the fundamental torque-generating step in the flagellar motor. Steps of 14 degrees were seen in chimeric motors containing only one unit, when the smf was reduced by lowering the Na+ concentration. We believe that each step may correspond to one or two ions crossing the motor, but will need to make detailed and systematic measurements of many steps under a range of different conditions to be sure that this is the case. In particular we will need to control and measure the smf in each cell. This will use our other recent innovation, which allows single-cell measurements of smf by fluorescence microscopy of indicator dyes that report internal Na+ concentration. By extending these techniques, we will make a systematic survey of the torque-speed relationship of the flagellar motor with different numbers of units and different values of both the concentration and voltage parts of the smf. The final experiment will attempt to measure the tiny ion flux through a single motor by measuring the accumulation of ions inside single mini-cells. Basic calculations indicate that the fluorescence technique for measuring internal Na+ concentration is sensitive enough to make this possible, even though the ionic current will be hundreds of times smaller than a typical single-ion-channel current. Understanding the flagellar motor will contribute to the wider field of molecular motors and will lay the foundations for possible technological applications. It will also contribute towards the long-term goal of designing and building artificial machines at the molecular scale.