Biophysical Characterization of Flagellar Motor Functions

Summary

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.

Abstract

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.

Introduction

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 CW_bias (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.

Protocol

1. Cell Preparation

1. Grow overnight cultures of the desired strain carrying the sticky fliC allele ^15,22 in Tryptone Broth (TB, 1% Peptone, 0.5% NaCl) followed by inoculation at 1:100 dilution in 10 mL fresh TB. Grow the culture at 33 °C in a shaker incubator until OD₆₀₀ = 0.5.
2. Pellet the cells at 1,500 x g for 5 - 7 min and re-disperse the pellet vigorously in 10 mL of filter sterilized motility buffer (MB; 10 mM phosphate buffer: 0.05-0.06 M NaCl, 10^-4 M EDTA, 1 µM methionine, pH 7.0).
3. Repeat step 1.2 two more times and re-disperse the final pellet in 1 mL MB.
4. Shear the suspension by passing back and forth ~ 75 times between two syringes with 21 to 23 gauge adapters connected by polyethylene tubing (7 - 12 cm long, 0.58 mm inner diameter). Limit the total time for shearing to 30 - 45 s.
5. Centrifuge the sheared cells at 1,500 x g for 5-7 min and re-disperse the pellet in 100 - 500 µL of MB.

2. Slide Preparation

1. Prepare an imaging chamber by sandwiching two double-sided adhesive tapes between a cover-slip and a microscope slide. For chemotaxis assays, employ any microfluidic chamber that enables the exchange of MB and chemical stimulants.
2. Add 0.01% poly-L-lysine solution in the chamber and after 5 min gently rinse the surfaces with MB (80 - 100 µL).
3. Add 40 µL of the cell suspension into the chamber and allow sufficient time for attachment to the glass surface (7 - 8 min). Flow out unstuck cells by adding 100 µL MB on one side of the chamber, while wicking the solution with a filter paper from the other side.
4. Add 10 - 15 µL of latex beads into the chamber and allow the beads adequate time to settle and attach to the cells (7 - 8 min). Gently rinse with 100 µL of MB, as described in step 2.3, to remove unstuck beads. Use a range of bead-sizes for the experiments so long as a good contrast is available.

3. Bead Tracking

1. Place the sample on a microscope stage and scan the surface for beads attached to motors. Use a 40X phase objective to make observations although phase microscopy is not necessary. Alternatively, employ bright-field imaging so long as sufficient contrast is maintained to clearly distinguish a bright bead on a dark background.
2. Once a bead has been selected, move the stage laterally to position the bead in a pre-determined corner as shown in Figure 1B. Position beads at the same corner to ensure that the direction of rotation of the bead is correctly known. The ideal bead trajectory is approximately circular but elliptical trajectories are admissible.
3. Maintain the sampling frequency higher than twice the rotational frequency of the motor to avoid errors associated with aliasing. In this work, use a motor that was rotating at 50 Hz and sample at frequencies that were 10 times higher (500 Hz), to obtain a smooth signal.

4. Data Analysis

1. Center and scale the PMT output voltages and correct ellipticity in the trajectories with affine transformations if needed ²³. Use a power-spectrum analysis to determine rotation rates ¹⁷.
2. Determine polar angles, θ(t) = atan (y(t)/x(t)). Determine the variations in motor speeds and switching over time by calculating ω ¹⁴.
3. Employ a median filter to smooth the motor speed data. A filter window over two full rotations is recommended ^23,24.

Results

The photomultiplier setup is shown in Figure 1A. It is important that the PMTs have high sensitivities over the range of wavelengths scattered by the beads of interest. The PMTs employed here operate in the visible and near-infrared ranges, and were able to detect light scattered by beads illuminated by a halogen light source. The optimum lighting conditions and supply voltages will vary from one setup to another. For the setup used in this work, a PMT gain ~ 10⁴

Discussion

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 (&#...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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 x 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