Visualization of Functional Rotor Proteins of the Bacterial Flagellar Motor in the Cell Membrane

Abstract

The bacterial flagellar motor is a rotary motor driven by the electrochemical potentials of specific ions across the cell membrane. Direct interactions between the rotor protein FliG and the stator protein MotA are thought to generate the rotational torque. Here, we used total internal reflection fluorescent microscopy to observe the localization of green fluorescent protein (GFP)-fused FliG in Escherichia coli cells. We identified three types of fluorescent punctate signals: immobile dots, mobile dots that exhibited simple diffusion, and mobile dots that exhibited restricted diffusion. When GFP–FliG was expressed in a Δ fliG background, most of the cells were not mobile. When the cells were tethered to a glass side, however, rotating cells were commonly observed and a single fluorescent dot was always observed at the rotational center of the tethered cell. These fluorescent dots were likely positions at which functional GFP–FliG had been incorporated into a flagellar motor. Our results suggest that flagellar basal bodies diffuse in the cytoplasmic membrane until the axial structure and/or other structures assemble.