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理，这是一种直径约50 nm(1/20000毫米)、最高速度超过100,000转/分的旋转分子电动马达。许多种类的细菌通过游泳来适应它们的环境。推进器是约20纳米厚的螺旋鞭毛细丝，发动机由H+或Na+离子沿着称为质子动力(PMF)或钠动力(SMF)的电化学梯度流动驱动。这些梯度由跨细胞膜的电压和浓度差组成，是生物自由能的主要形式。每台马达的最大功率输出约为十亿分之一瓦，比其他已知的由三磷酸腺苷水解提供动力的分子马达高出100到1000倍。转子是细胞质膜上的一组环，直径约45 nm，周围环绕着大约12个独立的扭矩产生单元，这些单元固定在细胞壁上，当离子通过时推动转子。我们将使用一系列生物物理技术，一些相对成熟，另一些是全新的，来测量单鞭毛马达的性质，并测量和控制它们所在的单个细菌细胞的SMF。为了测量马达速度，我们将把直径数百纳米的聚苯乙烯珠子固定在鞭毛细丝上，并通过用荧光显微镜拍摄高速视频(高达2400帧/秒)或通过测量聚焦在珠子上的红外线激光的偏转来测量它们的旋转。我们将使用的鞭毛是经过基因工程改造的，以使这些实验成为可能。这些细丝携带着一种突变，使它们自发地粘在珠子上。扭矩产生单元是嵌合体，包含来自不同物种的成分，使我们能够研究由Na+驱动的马达，以及使用大肠杆菌(通常有H+驱动的马达)可能使用的所有遗传工具。这些嵌合单位将在一种化学诱导剂的控制下在细菌内产生，这种化学诱导剂将允许我们控制每个马达中的数量。我们将在实验室最新的两项实验创新的基础上再接再厉。其中之一使得有史以来第一次检测到鞭毛马达中产生扭矩的基本步骤。在只含一个单元的嵌合电机中，当降低Na+浓度时，SMF降低，出现14度台阶。我们认为，每一步都可能对应于一到两个离子穿过发动机，但需要在一系列不同的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。特别是，我们需要控制和测量每个单元中的SMF。这将使用我们最近的另一项创新，允许通过荧光显微镜对报告内部Na+浓度的指示染料进行单细胞SMF测量。通过推广这些技术，我们将对不同单元数、不同SMF浓度和电压部分的值的鞭毛电机的扭矩-转速关系进行系统的研究。最后的实验将尝试通过测量单个微型细胞内离子的积累来测量通过单个马达的微小离子通量。基本计算表明，尽管离子电流将比典型的单离子通道电流小数百倍，但用于测量内部Na+浓度的荧光技术足够灵敏，足以使这一点成为可能。了解鞭毛马达将有助于分子马达的更广泛领域，并将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造机器的长期目标。

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.