Guillaume Paradis, Ismaël Duchesne and Simon Rainville
Centre for Optics, photonics and lasers (COPL), Department of Physics, Université Laval, Québec, Québec, Canada, G1V 0A6
Contact : simon.rainville@phy.ulaval.ca
Goal
The study of the bacterial flagellar motor using an in vitro system.
Results
 To measure the rotation speed of the fluorescent filaments, we
make a high speed (~500 fps) movie of its rotation. We then select
a small (2 x 2 pixels) region of interest (ROI) in which a filament
passes during each rotation. As the filament comes in and out of
the ROI, the average light intensity goes up and down. We then
take a Fourier transform (FFT) of the signal obtained from the ROI.
Fig. c) shows a typical FFT for a filament freely rotating before we
punch the hole.
Motivation
Although a lot is known about the rotary motor of
bacterial flagella, a complete understanding of how
the motor works is still missing. It is our hope that
the creation of an in vitro system would help the
study of the bacterial flagellar motor as much as it
helped the study of other biological motors like
kinesin, myosin, etc..
c)
d)
 Fig. d) shows the rotation speed of a filament under our control. The
filament’s rotation speed depends linearly on the applied voltage, as
expected. This shows that the hole pierced in the membrane by the
laser remains open for several minutes (between 2-20 minutes).
Concept
We developed an in vitro system that gives us :
1. Direct access to the inside of the cell (allowing us to label cytoplasmic motor
components, to study switching, etc.)
2. Control over the proton motive force (pmf) and thus the rotation speed of the
motor
To do this, we partially trap individual bacteria inside micropipettes in order to achieve a
configuration similar to the whole-cell patch-clamp assay.
 Our system was used in an attempt to determine why ΔFliL mutants of Salmonella enterica lose their
filaments while swarming (but not while swimming). One of the hypothesis is that the pmf alone in those
bacteria is significantly higher when swarming. To test wether a high pmf alone could break the filament
of ΔFliL mutants, we externaly applied a high pmf on them using our in vitro assay. A pmf as high as
-320 mV to filamentous S.enterica using the combination of an applied voltage and a difference of pH
between the exterior and the interior of the pipette.
 We did NOT observe any filaments breaking off the cell. We conclude that a high pmf alone cannot be
the explanation for this mutant’s peculiar behavior. Fig. e) shows that we had control over the motor.
Experimental Setup
e)
1) A single filamentous bacterium is
partly inserted inside a home-made
micropipette having a constriction
closely matching the diameter of
the cell. (Fig a.) The details are
located below.
2) Laser pulses are used to create a
plasma that pierces a hole in the
segment of the bacteria’s
membrane that is inside the
micropipette. This ablation process
can be localized beyond the
diffraction limit (<500 nm).
(Laser : Near IR, 60 fs pulse
duration, 10 kHz repetition rate
~100 pulses, ~5-10 nJ/pulse.)
3) The rotation of the filaments
outside the micropipette is
monitored using high-speed video
microscopy of fluorescently labeled
filaments or attached gold
nanoparticules (Fig. b).
Home-made Microforge
Much efforts were spent to transform the
micropipette fabrication from an ‘art’ to a
robust, precise and flexible process. We
designed a vertical microforge completely
automated using a rotating motor and
platinum filaments. It is versatile enough to
adapt to the small changes in diameter of
different strains of E.coli or other types of
bacteria (eg. Salmonella). Image analysis
of the micropipettes is used instead of
measuring the ‘bubble number’.
Bar : 10µm
 In order to understand what happens to the motors' components
once a hole is made in the bacterium's membrane, we used an E.
coli strain with FliM-YPet construction (JPA-945, kindly provided by
J.P. Armitage). Fig. f) shows how pierced cells (in red) lose
FliM-YPet with time whereas intact cells (in blue) don’t. The data
was corrected for photobleaching.
f)
Conclusion
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We demonstrated control of the bacterium’s pmf
We have a direct physical access to the inside of an isolated bacterium
Our in vitro assay is now operational and produces quantitative data
We invalidated the hypothesis of high pmf causing filaments to fall without FliL.
We have measured a slow irreversible loss of FliM when the bacterium is pierced.
Future Possibilities
 Study switching with direct control over the internal concentration of the related proteins.
 Study the diffusion of torque generating units and other motor proteins in and out of the
motors.
 Slow down the motor to measure steps in the rotation.
Reference
M.Gauthier, D.Truchon and S.Rainville, “Taking control of the flagellar motor,” Photonics North, 2008, vol. 7099