| dc.description.abstract | Bacterial flagellar motor (BFM) is a nanometer-size molecular machine (~50 nm) in many bacteria species. By rotating the flagellar motor, bacteria have the motility to search life essentials actively at low Reynold number environment. A flagellar motor composes of a helical flagellum, a rotor and about dozen of stator units. A rotor is a rotary body connecting to a flagellum. Stator units are small independent protein complex that drive the rotor rotation and consume ion-motive force by transporting specific ions (mostly H+ or Na+) into cytoplasmic to drive the rotor rotation. Moreover, stator units dynamically associate/dissociate at the circumference of a functional rotor. The stoichiometry of stator units in a functional motor is highly dynamic and imperative to the power output. Therefore, understanding the assembly kinetics of stator units is crucial to elucidate bacteria flagellar motor′s the primary working mechanism and characteristics.
The work in this thesis focuses mainly on sodium-type stator units, including wild-type stator in Vibrio alginolyticus and chimeric stator in Escherichia coli. We applied multiple techniques of optical microscopy to reveal the stoichiometry and kinetic of stator units, including total internal reflection fluorescence microscopy (TIRF), fluorescence recovery after photobleaching (FRAP), photoactivated localization microscopy (PALM), and back focal plane interferometry (BFPI). These techniques were designed and integrated into a commercial microscope. The apparatus was further devised to perform an on-time perfusion system for ion concentration switching and a focus-lock system for long-term focus stability.
In this thesis, three related projects are presented. The first project aims to investigate the distribution of stator units around a motor by PALM. The estimated spatial resolution is smaller than 5 nm using PALM in our system. We applied two-color labelling on both rotor protein FliN and stator protein PomA for the stator localization experiments. After we carefully examed the colocalization ratio and the fixation effect, we found the stator density is a constant near the rotor and decayed with distance. However, the stator number density is lower than the model. Further investigation is required for the PALM experimental system. The second project aims to analyze the affection of torque association protein, FliL, to stator-units assemble in V. alginolyticus. Using TIRF microscopy to count protein quantity, the stator units and FliL protein stoichiometric ratio is close to 1:1 in a functional motor. Besides, the exchange time of stator units is slightly increased in the absence of FliL from the FRAP experiment. Measurement of the diffusion rate suggests the two proteins are independent on the membrane. Our results suggest that FliL plays a supporting role to the stator in the BFM. The third project investigates the relation between sodium concentration and the kinetic response of chimaeric stator units in E. coli. We found the sodium-driven chimaeric BFMs maintained constant speed over a wide range of sodium concentrations by adjusting stator units in compensation to the sodium-motive force (SMF) changes. The BFM has the maximum number of stator units and is most stable at 5 mM sodium concentration rather than higher sodium concentration. Upon rapid exchange from high to low sodium concentration, the stator number shows a drop and then resurrection that is different from predictions of simple absorption model. This may imply the existence of a metastable hidden state of the stator unit during the sudden loss of sodium ions.
In conclusion, our correlated microscopy combining BFM speed and fluorescence measurements with computer-controlled microfluidic devices provided a cutting-edge system to investigate protein complex dynamics. Our findings of sodium-driven BFMs highlight the complex and dynamical mechanism of biomolecules and pave the way of the complete understanding of BFM working mechanisms. | en_US |