Torque generation in the bacterial flagellar motor

Lead Research Organisation: University of Oxford
Department Name: Oxford Physics

Abstract

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 energy. Each motor has a maximum power output of about one million-billionth of a Watt, about 100 times higher than other known molecular motors which are powered by the universal energy currency molecule of the cell, ATP. 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 rotation of single flagellar motors far better than has ever been possible in the past. The motor is too small and too similar to everything else that surrounds it to see in a light microscope. Electron microscopes only work on frozen samples, so are no good for seeing the motors when they are working. We get around this by attaching tiny gold particles, which scatter a lot of light so that we can see them, to the bits of the motor that stick outside the cell. We then measure their rotation either by taking high-speed videos (up to 109,500 frames per second!), or by projecting the same image onto a fast position-sensitive detector. We will build on recent experimental innovations in our lab which allowed the first ever detection of the fundamental torque-generating step in the flagellar motor. Steps of 14 degrees were seen in motors containing only one unit, when the SMF was reduced by lowering the Na+ concentration. The torque-generating units were 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). Using Na+-driven motors made sure that we could slow them down enough to see the steps with our old microscope, which was 50 times slower than the new one we have just developed. We are still improving the new microscope, and we hope it will be so good that we can measure steps in the H+-driven motor without doing anything unusual to slow the motor down. 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. To make sure we are interpreting what we see correctly, we will develop and use advanced statistical tools, and mathematical models of the motor mechanism. So far, we can only watch the motor spin while varying the driving force - the 'fuel' if you like. We'd really also like to be able to hold it still, and see how hard it can push. We will do this with tiny cobalt magnets replacing the gold particles as handles. We can twist these with a magnetic field, and see how the motor responds. This will allow all sorts of new probes of the motor, as we hold it still at different angles, push it backwards, and let it run forwards at different speeds. 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.

Technical Summary

1. LDF microscope. Gold particles (80-200 nm diameter) scatter very strongly (plasmon resonance: up to 0.001 of total incident intensity at 632 nm). Calculate particle position by Gaussian Mask Centroid algorithm (RMS noise ~1 nm, bandwidth 1-55 kHz). Optimize the optics and vibration isolation to make the system robust to normal daytime environmental vibrations. Improve the data-transfer capability of high-speed camera. 128x16-pixel 10-bit images at 109 kHz = 270 MB/s: fills camera memory in 8s, 40 min to download. Overcome this limitation on rate of data acquisition with new camera and direct-disc-write technology. 2. Improve yield of spinning particles Protocol: cells incubated with anti-hook IgG, gold particles coated with secondary antibody, blocked with PEG. Mix cells and gold. Cells attached to microscope colverslip. a) Optimize the quantities and order in this protocol. b) if necessary: biotinylate the anti-hook serum (all immunoglobulins), replace secondary antibody on gold with streptavidin. c) if necessary: genetically engineer the hook protein to contain an in-vivo biotinylation signal and proceed as b). 3. Torque, speed and stepping measurements. Torque and speed will be measured with our camera or quadrant photodiode at 5-10 kHz. For stepping rotation we will use the camera and rates up to 110 kHz, selecting only the most stable circular particle trajectories-identified by computer algorithm. Analysis: partition into episodes with different stator number, non-linear filtering and periodicity analysis to determine the step number and step pattern. Further analysis of stepping will be developed according to what the data reveals. 4. Magnetic tweezers. A 4-pole electromagnet wound on a soft iron core and driven by audio amplifiers will deliver fields up to ~0.1 T, with magnitude and direction under computer control, to exert torque on ferromagnetic cobalt nanoparticles ('Turbobeads') attached to flagellar hooks or filaments

Planned Impact

Expectations for research of this nature: The primary impact and beneficiaries are expected to be within the UK and international scientific communities. The immediate, measurable impact will be concentrated as publication in leading international journals, presentations at national and international conferences, and the spread of ideas and techniques via formal and informal scientific collaborations with a wide range of colleagues in the UK and internationally. Helping to understand in detail the molecular machinery of life, which is the goal of this proposal, is a necessary complement to the modern explosion in biological information represented by genome sequencing and other advances in molecular biology. Communications and engagement with beneficiaries The beneficiaries of this research are categorized below. Immediate beneficiaries : 1. the UK and international research communities, 2. students and postdocs trained in the course of the research 3. the general public. Medium-term beneficiaries: 4. UK science, education and industry. Long-term, beneficiaries: 5. General public via emerging nanotechnologies and personal medicine. The mechanisms for reaching these beneficiaries are detailed below. 1. UK and international research communities. We are living in the midst of a revolution in the way the life- and physical sciences interact. Developments in genetics and molecular biology in the last few decades have opened the fundamental processes of life to the quantitative scrutiny that previously was the domain of the mathematical and physical sciences. The current research proposal will advance the new paradigm of single-molecule biology, in which fundamental biological processes are investigated and understood in quantitative, mechanistic, microscopic detail. The impact will be in the form of both experimental techniques and theoretical and conceptual exposition. 2. Students and postdocs trained An important impact of the project will be the training of active researchers. 10 people have left the PI's research group since 2005. Of these 3 have permanent academic posts, 5 are postdocs, one a Senior Scientist in a biotechnology company and one a school teacher. The project will create opportunities for training research students within the Oxford DTC at the Life Sciences Interface, of which the PI is a directorate member. 3. the General Public The BFM has been adopted by the 'intelligent design' community as an example of 'irreducible complexity', presumably because it so appealingly resembles a macroscopic man-made machine. The scientific understanding of its mechanism, assembly and evolution is an important example to demonstrate the power of scientific, rational approach to explain the marvels of nature. This approach is crucial if the UK is to lead the world in the modern knowledge-based economy. Communication with the general public will be through several channels: a web-site that includes a description of the research at a level suitable for an educated layman, research forums, articles in national newspapers on nanotechnology and molecular motors, tours of the laboratory as part of the outreach effort of the Oxford Physics department. 4. Science, Education and Industry People trained, techniques developed and ideas tested during the course of the project will spread into science, industry and education, enriching the scientific culture that is vital to the success of these areas of the UK economy. 5. Emerging nanotechnologies and personal medicine The long-range economic impact of this research will be in these fields. These are far enough in the future to be very difficult to predict in detail. Exploitation and Application: The commercial potential of the methods developed will be assessed and managed by the PI, with the assistance of ISIS, Oxford University's technology transfer subsidiary.

Publications

10 25 50
 
Description We have discovered how the bacterial flagellar motor is assembled, that it is constantly exchanging components, and a lot about how it works.
Exploitation Route They will understand better how the molecular machinery of life works.
Sectors Education

 
Description They have been used to improve our understanding of the molecular machinery of life.
First Year Of Impact 2010
Sector Education
Impact Types Cultural

 
Title Application of magnetic torque to bacterial flagellar motors 
Description We have developed and refined a procedure to attach superparamagnetic beads (1 micron in diameter) onto flagella. We use beads that are streptavidin-coated and biotinylated filaments in order to achieve high specificity and yields. We have demonstrated that Helmholtz coils can be used to apply torque to the beads and to align the particles within a magnetic field. 
Type Of Material Physiological assessment or outcome measure 
Provided To Others? No  
Impact N/A 
 
Title Attachment of E. coli to glass surfaces for single molecule experiments 
Description We have performed a systematic investigation of different types of glass preparations, polylysines, and motility buffers in order to reproduce previously published low load motor speeds and to maximize motor stability. 
Type Of Material Physiological assessment or outcome measure 
Provided To Others? No  
Impact N/A 
 
Title E. coli strains with Biotinylated flagellar hooks 
Description Cell lines: MTB31,MTB32,MTB33,MTB34,MTB40,MTB48,MTB49,MTB50. These flagellum-deleted strains are have biotinylated hooks via one of two successful mutations to the hook protein, allowing us to do kinetics experiments via tagging and/or stiffening the biotin hook. 
Type Of Material Cell line 
Provided To Others? No  
Impact Single-molecule experiments on flagellar motors are now much easier. 
 
Title Fast sample temperature control for high-numerical aperture light microscope suitable for single-molecule observation of molecular motors. 
Description We developed and demonstrated and tested a method of controlling the temperature of a sample in a high-numerical aperture light microscope on a timescale of minutes, suitable for single-molecule observation of molecular motors. 
Type Of Material Technology assay or reagent 
Year Produced 2011 
Provided To Others? Yes  
Impact N/A 
 
Title Labelling Biotinylated flagellar filaments 
Description We have developed a technique to reliably biotinylate bacterial flagellar filaments in E. coli. This allows us to attach streptavidin-coated particles to the filaments, in particular magnetic particles for the controlled application of torque to the flagellar motor. 
Type Of Material Technology assay or reagent 
Provided To Others? No  
Impact N/A 
 
Title Labelling biotinylated flagellar hooks 
Description We have developed protocols that allow efficient tagging of flagellar hooks with gold probes via a biotin inserted into the hook and a streptavidin or neutravidin linker, with no noticeable effects on the cells that are tagged. This allows high speed measurements of flagellar rotation. 
Type Of Material Technology assay or reagent 
Provided To Others? No  
Impact N/A 
 
Title Wavelength selector for use with a white-light laser 
Description We developed a wavelengths selector which receives light from a broad spectrum source, generates a dispersed beam, selectively masks all but the desired wavelengths, then recombines the beam into a fiber optic cable. This allows for rapid and precise wavelength selection for microscopy. 
Type Of Material Technology assay or reagent 
Provided To Others? No  
Impact This allowed for tailored excitation of different sizes of plasmonic nano rods attached to the bacterial flagellar motor. These rods were imaged using polarization microscopy. 
 
Title High resolution low load rotation of the BFM 
Description We have collected, to our knowledge, the highest resolution recordings of the rotation of the BFM to date. This was accomplished by attaching 100nm gold nanoparticles to the hook of the BFM. We have achieved sub-nm resolution at 109,500 FPS. This data set is currently being investigated by our group and two separate research groups who have requested the data. 
Type Of Material Database/Collection of data 
Year Produced 2016 
Provided To Others? Yes  
Impact We have published the first paper from this dataset (Nord et al, 2016, New Journal of Physics), demonstrating pauses in the rotation of the BFM at low load. 
 
Description Application of optical torque to molecular motors 
Organisation Delft University of Technology (TU Delft)
Country Netherlands 
Sector Academic/University 
PI Contribution Application of optical torque to molecular motors using optical angular momentum exchange between optical trapping laser beams and birefringent microparticles. Collaboration with Nynke Dekker, TU Delft and Francesco Pedaci, Centre de Biochimie Structurale Montpellier
Start Year 2010
 
Description Application of optical torque to molecular motors 
Organisation University of Montpellier
Department Centre for Structural Biochemistry
Country France 
Sector Academic/University 
PI Contribution Application of optical torque to molecular motors using optical angular momentum exchange between optical trapping laser beams and birefringent microparticles. Collaboration with Nynke Dekker, TU Delft and Francesco Pedaci, Centre de Biochimie Structurale Montpellier
Start Year 2010
 
Title Backscattering Laser Darkfield Microscopy 
Description We have developed a backscattering laser darkfield technique. This builds on forward-scattering laser darkfield techniques which allow high spatial and temporal resolution. By implementing back-scatter we gain: * improved stability and usability for the entire system, with relatively simple optics. * 2-3 orders of magnitude gain in contrast between the scattering of small and large objects, allowing us to pick out small gold probes again the scattering background of cells. * increased light capture allowing better data at a given illumination level. * increased access to the sample, allowing simpler perfusion of buffers. 
Type Of Technology New/Improved Technique/Technology 
Year Produced 2010 
Impact N/A 
 
Title Fast sample temperature control for high-numerical aperture light microscope suitable for single-molecule observation of molecular motors. 
Description We developed and demonstrated and tested a method of controlling the temperature of a sample in a high-numerical aperture light microscope on a timescale of minutes, suitable for single-molecule observation of molecular motors. 
Type Of Technology New/Improved Technique/Technology 
Year Produced 2010 
Impact Use in international research communities leading to publications 
 
Title New Laser Darkfield microscope 
Description We have built a new Laser Dark Field microscope to deliver better performance than that of the prototype described in the grant proposal. Compared to the prototype, we have: * replaced the stage with a sturdy construction with very low inherent noise. * replaced the optical pathways, decoupling upstream noise via optical fibre delivery and sealing the optical pathway inside small diameter tubing. * incorporated a white light laser and monochromator to allow illumination with any selected wavelength band of light. * replaced the QPD with a lower-noise version, and upgrading the software to match. * incorporated a pathway that allows splitting of the light into four polarisation components before imaging, which provides high resolution information for rod experiments. *built the system in a quieter room. * upgrading the optical table to a higher specification. 
Type Of Technology New/Improved Technique/Technology 
Year Produced 2013 
Impact N/A 
 
Description Frontiers of Biological Physics: Molecular Motors 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Public/other audiences
Results and Impact Public Lecture. Saturday 1st October, 2011



This event provides an exciting opportunity to see how new experimental techniques and improved theoretical understanding are opening up the world of biological physics. Topics covered will range from the magnetism and bird migration to natural optics and molecular motors. The event is aimed at the general public and is open to all

no actual impacts realised to date
Year(s) Of Engagement Activity 2011
 
Description Mini motors 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? Yes
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact We were one of three research groups who participated in the Mini Motors stand at the 2012 Royal Society's Summer Science Exhibition. As part of this stand, we explained the difference between swimming in the microbial world to that of everyday experience, and showed off bacterial motility and torque-speed assays. The exhibit was open to the London public for a week and visited by > 10,000 visitors. We also exhibited twice in Oxford.

N/A
Year(s) Of Engagement Activity 2012
 
Description What is Light? 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Schools
Results and Impact Demonstration to primary school children of light and its uses in science.

no actual impacts realised to date
Year(s) Of Engagement Activity 2013