Biophysical mechanisms of the Bacterial Fagellar Motor

In our custom made Magnetic Tweezers setup, we study the dynamical behavior of individual bacterial flagellar motors (BFMs) looking at the rotation of microscopic magnetic beads tethered to them via either the hook or a sheared flagellum. A magnetic field is produced by permanent magnets, mounted on a motorized stage, and can be used to apply external force and torque to the bead.

A modulated diode-laser illumination [1] allows to image the hologram of the bead on a fast CMOS (20 KHz) with 1 us exposure time. Our tracking software can then retrieve the xyz trajectory of the bead with few nm resolution. As the bead rotates off-axis, its position gives informations about the angle of the motor.

Fluorescence can be excited both in epi- and tirf- mode. We have labeled the stators of the flagellar motor (the active torque generating units of the BFM) by different fluorophores, and we can observe their assembly at the anchoring point of rotating cells tethered to the glass slide via the flagellum. Interestingly, we have found that BFMs with labeled stators change behavior when switching their direction of rotation [2].

The stators are mechano-sensors, and assemble in the BFM when the resistance to rotation increases. By manipulation of individual motors tethered to beads of different diameter, we have quantified the binding and unbinding rates of stator recruitment, and we have proposed that a catch-bond (a bond strengthened instead of weakened by tension) keeps each stator assembled within the BFM [3].

[1] D.Dulin, X.Hachair, S.Barland, F.Pedaci. Efficient illumination for microsecond tracking microscopy. PLoS ONE 9(9): e107335. (Sep 2014). [PDF]

[2] M.Heo*, A.L.Nord*, D.Chamousset, E. van Rijn, HJE Beaumont, F.Pedaci. Impact of fluorescent protein fusions on the bacterial flagellar motor. Sci. Rep. 7, 12583 (Oct. 2017). [PDF][SI]

[3] A.L.Nord, E.Gachon, R.Perez-Carrasco, J.A.Nirody, A.Barducci, R.M.Berry, F.Pedaci.A catch-bond drives stator mechanosensitivity in the Bacterial Flagellar Motor. PNAS doi:10.1073/pnas.1716002114 (Nov. 2017). [PDF+SI]