Dynamic Conformational Changes of Flagellar Filament Observed by High-Pressure Microscopy

Abstract

The bacterial flagellar motor is a molecular machine that rotates a flagellum in both directions. CCW rotation allows the left-handed helical filaments to form a bundle that propels the cell smoothly, whereas CW rotation of a filament leads to change the shape of filament in right-handed helix and break the bundle, and inhibits smooth swimming of the cell, called a tumble. The switching in the helical structure is thought to be caused by directional mechanical actions arising from abrupt change of exerted torque by the motor rotation. Here, we show that application of pressure can also change the helical structure of flagellar filaments. The flagellar filaments in E. coli cells were fluorescently labeled, and then the images were acquired by using a high-pressure microscope [1, 2] with some modifications. We measured the diameter and pitch of the individual filaments and then classified them into 11 possible waveforms which are predicted from structural data. At 0.1 MPa (ambient pressure), all flagellar filaments formed left-handed helical structure (normal form). At 40 MPa, we found left-handed forms (normal and coiled forms) and right- (curly I (or II)). At 80 MPa, 80% flagellar filaments took curly I (or II) forms. After the pressure was released, most filaments returned to the initial left-handed structures. The application of pressure is thought to enhance the structural fluctuation and/or association of water molecules with the exposed regions of flagellin molecules, and results in switching the helical from left- to right-handed structure.[1] Nishiyama M. and Y. Sowa. 2012. Microscopic Analysis of Bacterial Motility at High Pressure. Biophys. J. 102:1872-1880.[2] Nishiyama M. and S. Kojima. 2012. Bacterial motility measured by a miniature chamber for high-pressure microscopy. Int. J. Mol. Sci. 13:9225-9239.