Prsentation PowerPoint

Can bacterial filaments regrow?Namba Protonic NanoMachine ProjectOsaka Univsersity, Japan.

Guillaume Paradis

Ismal Duchesne

Simon Rainville

www.ulaval.caElectron Microscopy E.coli

Why move?

1Why study bacterial motility? Toxicity and virulence increases with motility

Antibiotics crisis

Nanotechnology2www.ulaval.caE.coli near a surface3

www.ulaval.caWhat does it look like under the microscope?33D biased random walk4

JM Berg, JL Tymoczko, and L Stryer. Biochemistrywww.ulaval.ca5How do bacteria move?Complex assemby of over 20 types of proteins45nm-diameter rotative motorRotation in both direction up 18000 rpmProton powered

www.ulaval.ca18000 rpm -> like Formula 1

Marvel of nanotechnology

Not powered directly by ATP5Filament structure

6

DavidE. Tanner and al. Theoretical and computational investigation of flagellin translocation and bacterial flagellum growth.Biophysical Journalwww.ulaval.caHookUniversal jointCan have the filament to rotate in another axis than the motor

HAP 1,3

10-20 m rigid filament with 2 nm central channel

Cap at the end (HAP2)6MethodologyCells are grown to the exponential phase of growthA first Fluorescent labelling is doneFilaments are cut using a femtosecond laserCoordinates of the bacteria are notedA second labelling is done during a second growth period of 2h (37C)Cells are revisited7

www.ulaval.caSeems simple, but in practice very hard7Experimental setup

8Pulsed laser (1 khz)80 fs, Near infraredFluorescent microscopy at 546nm and 488nmCustom-made flow-cellBacteria with 1 or 2 filaments

www.ulaval.caPulsed laser 250 kHz using a pockels cell with crossed polarizers

Fs = 10-15

Salmonella enterica

8Cutting the filamentLaser is opened

Filament cut itself on the laser beam

Acceleration of the rotation shows cutting9

www.ulaval.caRed arrow points position of the laser9Results after 2h growth period44 filaments were cut and revisited

No regrowth was observed

10www.ulaval.caWhat we knowWhen 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 seen11www.ulaval.caLitterature not sure if cap can replace.

11Overexpression 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 observed12www.ulaval.caControlsBacteria in the same flow-cell left untouched were used as controls

90-95% of filaments still turning were bicolor13

www.ulaval.caControls (2)In one instance, a second filament had grown on a cell with a cut filament14

www.ulaval.caPrevious study15Turner and Berg (2012)

Cells were sheared mechanically

Let overnight at 7C

Tends to show regrowth

Turner L et al. J. Bacteriol. 2012;194:2437-2442www.ulaval.caSheared mechanically : what is it?15ConclusionsNo 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

16www.ulaval.caFuture and ongoing experiments

17A chain mechanism for flagellum growth, Nature, 2013Rate of growth vs length of the filament

Study of a proposed chain mechanism modelwww.ulaval.caThank youDr Simon RainvillephD studentsGuillaume ParadisIsmael Duchesne

Dr Kelly HuguesUniversity of UtahDr Marc ErhardtHelmholtz Centre for infection research, Germany18

www.ulaval.ca