Investigating the Rotation of Stator Units of the Bacterial Flagellar Motor

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One of the most remarkable biological machines is the rotary motor that enables bacteria to swim. This machine is being studied in the bacterium Escherichia coli, about which more is known than any other free-living thing. E.coli lives in your gut. Its cells are about 1 micrometer in diameter by 2 micrometers long -- 10,000 will fit side by side across the width of your finger. Each cell is propelled by about 4 long thin helical filaments, each driven at its base by a rotary motor. The filaments coalesce into a bundle behind the cell along its long axis and push it forward, nearly 20 body lengths per second. Recent work by electron microscopy (by cryo EM) suggests that the force-generating units (stator units) that drive flagellar rotation are themselves rotary engines about 7 times smaller in diameter than the rotor of the flagellar motor, and thus should spin about 7 times faster. This hypothesis will be tested by pulsed fluorescence bleaching, as explained below. Motility is known to enhance bacterial virulence. However, the main thrust of this work is not to explore its medical relevance but rather to understand the fundamental science which has the potential to provide a blueprint for bio-inspired design and inspire undergraduate students to enter this rapidly growing field. Stator units are assembled from 5 copies of the protein MotA and 2 copies of the protein MotB, which span the cell membrane. A MotA pentamer is thought to surround and rotate about a MotB dimer, powered by protons flowing from the outside to the inside of the cell. One end of MotB is attached to the rigid framework of the cell wall (the peptidoglycan layer) while the opposite end of MotA engages the protein FliG at the surface of the flagellar rotor. In this project, investigators will test this proposition by fusing a rigid fluorescent probe to the alpha-helical chains of MotA and bleaching it with a short but intense pulse of polarized light. Subsequent rotation of such markers will be followed by a weaker polarized probe beam. The effectiveness of this method has already been shown in experiments that demonstrated that the inner and outer components of the flagellar motor rotate as a unit. The results of this work should provide fundamental understanding of the mechanism of force generation of the stator units that power the flagellar motor.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.