|
The bacterial flagellar motor is a rotary molecular engine powered by the flow of ions across the inner, or cytoplasmic, membrane of a bacterial cell envelope [1]. Each motor drives a protruding helical filament, and the rotating filaments provide the propulsive force for cells to swim. Ion flux is driven by an electrochemical gradient, the protonmotive force (pmf) or sodium-motive force (smf) in motors driven by H+ and Na+ respectively. The electrochemical gradient consists of a voltage component and a concentration component, and is a key intermediate in the metabolism of both bacteria and higher organisms. The inside of a bacterial cell is typically at an electrical potential about 150 mV below the outside, and also has a slightly lower concentration of H+ or Na+ ions.
 |
 |
Filaments rotate at speeds up to 1000 Hz in swimming cells [2,3]. If cells are attached to a surface by a single flagellar filament, or “tethered”, the motor turns the whole cell body at speeds around 10 Hz [4]. The rotating heart of the motor is a set of rings in the cytoplasmic membrane, about 45 nm in diameter, and containing a total of a few hundred molecules of several different proteins [5]. This rotor is surrounded by a ring of independent torque generators which are anchored to the cell wall and consist of the proteins MotA and MotB [6,7]. There is room for at least 11 torque generators in E. coli [8], maybe as many as 16 in other species.
Flagellar Motor Steps: We recently showed that the flagellar motor takes 26 steps per revolution [9]. We used a genetically engineered chimeric flagellar motor [10] that runs on sodium ions in E. coli., made in the lab of Michio Homma in Nagoya, Japan. We slowed the motor down to the point where steps could be resolved using optical tweezers and single-particle fluorescence tracking. We are trying to improve the time resolution significantly so that we can measure steps under a wide range of conditions, for example with different smf and different numbers of torque-generators.
 |
 |
| The movie shows a high-speed video of a 200 nm fluorescent bead
attached to a flagellar motor, taking 26 steps per revolution. 30x slower than real time, 2400 frames per second, position resolution ~5 nm (right-click movie to download.
|
Single-Molecule Fluorescence: We use a custom-made TIRF microscope to look at fluorescent proteins down to the single-molecule level, inside living bacteria. (This work is being continued by Mark Leake, who is now a Royal Society University Research Fellow.) We counted 22 molecules of MotB, one of the torque-generating stator proteins, per motor, consistent with 11 stators each containing 2 MotB molecules. We also found a diffusing pool of ~200 MotB molecules in the cell membrane, and evidence for exchange between the motor and the pool on a ~1 minute timescale [11].
 |
 |
 |
Measurements of Torque vs speed, pmf and smf: The purpose of a biological motor is to convert chemical energy into mechanical energy. In the case of the bacterial flagellar motor, the chemical input power is the product of ion-motive force and ion flux, and the mechanical output power is the product of torque and rotation speed. The torque-speed relationship Escherichia coli (E.coli) has been measured before, the results are summarized below (blue, green and red lines correspond to 11, 16 and 22 degrees Centigrade respectively). We are now looking at how the torque-speed relationship in the chimera motor depends on each component of the smf. We use fluorescence microscopy to measure both components of the sodium-motive force in single bacteria [12,13] , in the motors of several different species of bacteria. It appears that the two components of smf are equivalent at low high load (near thermodynamic equilibrium?) but not at low load. To measure torque-speed relationship we can vary the drag coefficient of a viscous load attached to the motor [15], or we can use electrorotation or optical tweezers to apply external torque.
 |
 |
Modelling: In collaboration with Jianhua Xing (formerly of the group of George Oster in Berkeley), we are modelling the mechanism of the motor [14].
References
1. Macnab, R. M. 1996. Flagella and motility. In Escherichia coli and Salmonella: Cellular and Molecular Biology. F. C. Neidhart, et al., eds., 2nd Ed. American Society for Microbiology, Washington, D.C. 123-145.
2. Berry, R.M. and Armitage, J.P. (1999). The bacterial flagella motor. Adv. Microb. Physiol. 41:291-337.
3. Lowe, G., M. Meister, and H. C. Berg. 1987. Rapid rotation of flagellar bundles in swimming bacteria. Nature. 325: 637-640.
4. Magariyama Y, Sugiyama S, Muramoto K, Maekawa Y, Kawagishi I, Imae Y, Kudo S. 1994 .Very fast flagellar rotation. Nature 371:752
5. Silverman, M., and M. Simon. 1974. Flagellar rotation and the mechanism of bacterial motility. Nature. 249: 73–74.
6. Francis, N. R., Sosinsky, G. E., Thomas, D. and DeRosier, D. J. 1994. Isolation, characterization and structure of bacterial flagellar motors containing the switch complex. J. Mol. Biol. 235:1261-1270
7. Coulton, J. W., and R. G. E. Murray. 1978. Cell envelope associations of Aquaspirillum serpens flagella. J. Bacteriol.
8. Reid S.W., Leake M.C., Chandler J.H., Lo C-J., Armitage J.P., Berry R.M. (2006) The bacterial flagellar motor contains at least 11 torque-generating units. Proc. Natl. Acad. Sci. USA. 103: 8066-8071.
9. Sowa Y, Rowe AD, Leake MC, Yakushi T, Homma M, Ishijima A, Berry RM. (2005) Direct observation of steps in rotation of the bacterial flagellar motor. Nature. 437:916-919.
10. Asai, Y., Yakushi, T., Kawagishi, I. & Homma, M. Ion-coupling determinants of Na+ -driven and H+ -driven flagellar motors. J Mol Biol 327, 453-63 (2003).
11. Leake MC, Chandler JH, Wadhams GH, Bai F, Berry RM, Armitage JP. (2006). Stoichiometry and turnover in single, functioning membrane protein complexes. Nature. 443:355-358.
12. Lo C.-J., Leake M. C., Berry R. M. (2006) Fluorescence measurement of intracellular sodium concentration in single Escherichia coli cells. Biophys. J. 90: 357-365.
13. Lo C-J, Leake MC, Pilizota T, Berry RM. (2007). Non-equivalence of membrane voltage and ion-gradient as driving forces for the bacterial flagellar motor at low load. Biophys. J. 93:294-302.
14. Xing J., Bai F., Berry R.M., Oster G. (2006) The torque-speed relationship of the bacterial flagellar motor. Proc. Natl. Acad. Sci. USA. 103:1260-1265.
15. Inoue Y, Lo C-J, Fukuoka H, Takahashi H, Sowa Y, Pilizota T, Wadhams GH, Homma M, Berry RM, Ishijima A. (2008) Torque-speed relationships of Na+-driven chimeric flagellar motors in Escherichia coli. J. Mol. Biol. 376:1251-1259.
last updated: 21 November 2011