Investigation of the Mechanism of the Bacterial Flagellar Motor

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.

1. Control and measure electrical (Vm) and chemical (DpNa) components of the smf in single cells with chimeric flagellar motors, simultaneously measure the speed of a motor in the same cell. Vm : control with external pH or the proton uncoupler CCCP, measure by fluorescence microscopy of a membrane-permeable cationic indicator dye. DpNa: control and measure using a fluorescence technique developed recently in our lab. Speed: measure by the well-established back-focal-plane (bfp) method, using sub-micron polystyrene beads attached to flagellar filaments. 2. Improve the time-resolution and lifetime of experimental assays to measure flagellar motor stepping. The bfp method is limited by the slow relaxation in response to a step of 500 nm beads attached via the compliant hook: explore methods to decrease hook compliance or to obtain stable flagellar rotation at slower speeds. The fluorescence microscopy method uses smaller beads and is limited by photodamage and camera speed: explore photo-protective chemicals, less damaging wavelengths, faster cameras. 3. Measure torque-speed relationships for the chimeric flagellar motor with different numbers of torque-generating units and different values of each component of the smf. Vary speed via viscous load on motors with different sized beads . Control number of units by variable expression of chimeric stator proteins. 4. Measure step-sizes and interval length distributions for the chimeric flagellar motor with different numbers of torque-generating units and different values of each component of the smf. Look for: sub-steps, free energy driving each step, rate-constants for mechanochemical transitions, interactions between units. 5. Measure flux through single flagellar motors by monitoring changes in sodium concentration inside mini-cells. The motor flux is estimated to correspond to concentration changes on the order of 0.1 mM /s, which would be measurable using the fluorescence technique.