The bacterial flagellar motor (BFM) is a rotary molecular machine that drives critical bacterial processes including motility, chemotaxis, biofilm formation, and infection. For over two decades, the bead assay, which measures the rotation of a microparticle attached to the flagellum of a surface-attached bacterium, has been instrumental in deciphering the motor's biophysical mechanisms. This technique has not only quantified the rotational speed and frequency of directional switching as a function of the viscous load on the flagellum but has also revealed the BFM's capacity for mechanosensitive speed modulation, adapting to environmental conditions. Yet, many fundamental mechanistic details of the BFM remain to be discovered, and recent breakthroughs in structural biology, providing atomic-level structures of many motor components, further fuel this active area of biophysical research. This chapter presents an in-depth protocol for the bead assay to measure BFM dynamics, emphasizing advanced methodologies for quantifying the intricate mechanics and rich behavior of this molecular machine.
Keywords: Bacterial flagellar motor; Bead assay; Bending stiffness; Magnetic tweezers; Molecular machine; Protein exchange; Proteorhodopsin; Proton motive force; Single-molecule biophysics; Stator units; Torque.
© 2025. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.