Recent findings suggest that bacterial flagellar motors sense a variety of environmental signals and remodel in response. The bead-assays discussed here are expected to help explain the role of remodeling in cellular adaptation to environmental stressors.
The role of flagellar motors in bacterial motility and chemotaxis is well-understood. Recent discoveries suggest that flagellar motors are able to remodel in response to a variety of environmental stimuli and are among the triggers for surface colonization and infections. The precise mechanisms by which motors remodel and promote cellular adaptation likely depend on key motor attributes. The photomultiplier-based bead-tracking technique presented here enables accurate biophysical characterization of motor functions, including adaptations in motor speeds and switch-dynamics. This approach offers the advantage of real-time tracking and the ability to probe motor behavior over extended durations. The protocols discussed can be readily extended to study flagellar motors in a variety of bacterial species.
Flagellar motors enable cells to swim by rotating helical extracellular filaments. The amount of torque the motor can generate for a given length of the flagellum (i.e., the viscous load) determines the swimming speeds. On the other hand, its ability to switch the direction of rotation controls cell migration in response to chemicals, a process known as chemotaxis. Chemotaxis and motility being virulence factors 1-3, flagellar motors have been well-characterized over the years 4. Mounting evidence now suggests that the motor acts as a mechanosensor — it mechanically detects the presence of solid substrates 5,6. This ability likely helps in triggering surface colonization and infections 5,7. As a result, the mechanisms whereby the motor senses surfaces and initiates signaling are of significance 8,9.
The flagellar motor can be readily studied by tethering the flagellum to a substrate and observing cell rotation. Such tethering was first achieved by Silverman and Simon, who worked with a polyhook mutant in E. coli and successfully attached hooks to glass substrates with anti-hook antibodies 10. The tethered-cell assay enabled researchers to study the responses of the motor-switch to a variety of chemical stimuli. For example, Segall and co-workers chemically stimulated tethered cells with the aid of iontophoretic pipettes. The corresponding changes in CWbias (the fraction of the time motors spin clockwise, CW) enabled them to measure the kinetics of adaptation in the chemotaxis network 11,12. While the tethered cell assay was effective in studying switch responses, it was only able to offer insights into motor mechanics over a limited range of viscous loads 13. To overcome this problem, Ryu and co-workers tethered spherical, latex beads to filament stubs on cells stuck to surfaces. The beads were then tracked using back-focal interferometry with weak optical traps 14. By working with beads of different sizes, researchers could study the motor over a much wider range of loads. This assay was later improved by Yuan and Berg, who developed a photomultiplier-based bead-tracking technique combined with laser dark-field illumination. Their method enabled tracking of tethered gold nanobeads that were so tiny (~ 60 nm) that the external viscous resistances were lower compared to the internal viscous resistances to rotation 15,16. This led to the measurements of the maximum achievable speeds in E. coli (~ 300 Hz). In V. alginolyticus, similar bead assays enabled measurements of the spinning rates at intermediate viscous loads (~ 700 Hz) 17. By enabling measurements of motor responses over the entire possible range of viscous loads (from zero-load to near-stall), the bead-assays provided an important biophysical tool to understand the torque-generation process 18,19.
Recently, we modified the Yuan-Berg assay to include optical tweezers that enabled us to apply precise mechanical stimuli to individual motors 6. Using this technique, we showed that the force-generators that rotate the motor are dynamic mechanosensors — they remodel in response to changes in viscous loads. It is possible that such load-sensing triggers cell differentiation into swarming bacteria, although the mechanisms remain unclear. It is also likely that the flagellar motors in other species are also mechanosensitive 20, although direct evidence is lacking. Here, we discuss the photomultiplier-based (PMT) approach for tracking the rotation of latex beads tethered to flagellar filaments 15. In comparison to tracking with ultrafast cameras, the photomultiplier-setup is advantageous because it is relatively straightforward to track single beads in real-time and over long durations. It is particularly useful when studying long-time remodeling in flagellar motor complexes due to environmental stimuli 21. Though we detail protocols specifically for E. coli, they can be readily adapted for studying flagellar motors in other species.
In order to facilitate tethered bead-tracking and correct estimation of motor-torques, the following information should be reviewed. When performing these measurements with flagellated cells, shearing is a critical step. Shearing reduces the flagellar filament to a mere stub, thereby ensuring that the viscous load on the motor is predominantly due to the bead and can be estimated within 10% error 16. Shearing also improves the chances of finding circular trajectories with tightly distributed eccentricities (&#…
The authors have nothing to disclose.
The authors acknowledge Howard Berg for the gift of the bead-tracking microscope/photomultipliers and the Texas A&M Engineering Experiment Station for funds.
Poly-L-lysine Solution (0.1%) | Sigma-Aldrich | P8920 | http://www.sigmaaldrich.com/catalog/product/sigma/p8920?lang=en®ion=US |
Polybead Microspheres | Polysciences, Inc. | 7307 | http://www.sigmaaldrich.com/catalog/product/sigma/p8920?lang=en®ion=US |
1 ml Luer Slip Tip Syringe | Exel Int. | 26048 | http://www.exelint.com/tuberculin_syringes.php |
Clay Adams Intramedic Luer-Stub Adapter 23-gauge | Becton, Dickinson and Company | 427565 | http://www.bd.com/ds/productCenter/ES-LuerStubAdaptors.asp |
Polyethylene tubing | Harvard Apparatus | 59-8325 | http://www.harvardapparatus.com/laboratory-polye-polyethylene-non-sterile-tubing.html |
Photomultiplier Tubes | Hamamatsu | R7400U-20 | Spectral response range of 300 to 920 nm, Peak wavelength 630 nm, 0.78 ns response time http://pdf1.alldatasheet.com/datasheet-pdf/view/212308/HAMAMATSU/R7400U-20.html |
3×1 mm precision slits | Edmund Optics | NT39-908 | 2 slits mounted at right angles to one another on photomultiplier tubes |
Oscilloscope | Tektronix | TBS 1032B | Alternative brands are acceptable. Digital Oscilloscope, TBS 1000B Series, 2 Analogue, 30 MHz, 500 MSPS, 2.5 kpts http://www.tek.com/oscilloscope/tbs1000b-digital-storage-oscilloscope |
8 Pole LP/HP Filter | Krohn-Hite | 3384 | Alternative brands are acceptable. A frequency range from 0.1 Hz to 200 kHz is recommended. http://www.krohn-hite.com/htm/filters/PDF/3384Data.pdf |
Optiphot microscope | Nikon | NA | Any upright or inverted phase microscope can be used. https://www.thorlabs.com/newgrouppage9.cfm?objectgroup_id=754 |
50:50 (R:T) Cube Beamsplitter | ThorLabs | BS013 |