Can bacterial filaments regrow? Namba Protonic NanoMachine
Project Osaka Univsersity, Japan. Guillaume Paradis Ismal Duchesne
Simon Rainville
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www.ulaval.ca Why study bacterial motility? Toxicity and
virulence increases with motility Antibiotics crisis Nanotechnology
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www.ulaval.ca E.coli near a surface 3
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www.ulaval.ca 3D biased random walk 4 JM Berg, JL Tymoczko, and
L Stryer. Biochemistry
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www.ulaval.ca 5 How do bacteria move? Complex assemby of over
20 types of proteins 45nm-diameter rotative motor Rotation in both
direction up 18000 rpm Proton powered
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www.ulaval.ca Filament structure 6 David E. Tanner and al.
Theoretical and computational investigation of flagellin
translocation and bacterial flagellum growth. Biophysical
Journal
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www.ulaval.ca Methodology Cells are grown to the exponential
phase of growth A first Fluorescent labelling is done Filaments are
cut using a femtosecond laser Coordinates of the bacteria are noted
A second labelling is done during a second growth period of 2h
(37C) Cells are revisited 7
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www.ulaval.ca Experimental setup 8 Pulsed laser (1 khz) 80 fs,
Near infrared Fluorescent microscopy at 546nm and 488nm Custom-made
flow-cell Bacteria with 1 or 2 filaments
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www.ulaval.ca Cutting the filament Laser is opened Filament cut
itself on the laser beam Acceleration of the rotation shows cutting
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www.ulaval.ca Results after 2h growth period 44 filaments were
cut and revisited No regrowth was observed 10
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www.ulaval.ca What we know When the filament is cut, the cap is
lost The cap is needed for filament growth FliD protein is
constantly secreted HAP3 (FlgL) is needed for the cap to form If
there was regrowth, should take minutes to be seen 11
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www.ulaval.ca Overexpression of FliD (HAP2) A genetically
modified strain controlling the expression of the FliD protein was
used The protein was overexpressed (doubled) during the 2h growth
period 18 filaments were cut and revisited No regrowth was observed
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www.ulaval.ca Controls Bacteria in the same flow-cell left
untouched were used as controls 90-95% of filaments still turning
were bicolor 13
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www.ulaval.ca Controls (2) In one instance, a second filament
had grown on a cell with a cut filament 14
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www.ulaval.ca Previous study 15 Turner and Berg (2012) Cells
were sheared mechanically Let overnight at 7C Tends to show
regrowth Turner L et al. J. Bacteriol. 2012;194:2437-2442
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www.ulaval.ca Conclusions No regrowth was observed Bicolor
uncut filaments in the same flowcells were observed Even with
higher levels of cap protein, cut filaments dont regrow In these
conditions, bacterial filaments dont regrow 16
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www.ulaval.ca Future and ongoing experiments 17 A chain
mechanism for flagellum growth, Nature, 2013 Rate of growth vs
length of the filament Study of a proposed chain mechanism
model
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www.ulaval.ca Thank you Dr Simon Rainville phD students
Guillaume Paradis Ismael Duchesne Dr Kelly Hugues University of
Utah Dr Marc Erhardt Helmholtz Centre for infection research,
Germany 18