The Character Of Stator Protein PomB And H-ring Protein FlgT Of Vibrio Alginolyticus, A Na~+-driven Flagellar Motor

The Na+-driven bacterial flagellar motor is a molecular machine powered by anelectrochemical potential gradient of sodium ions across the cytoplasmic membrane. Themarine bacterium Vibrio alginolyticus has a single polar flagellum that enables it to swim inliquid. The flagellar motor contains a basal body and a stator complex, which are composed ofseveral proteins. PomA, PomB, MotX and MotY are thought to be essential components ofthe stator that are required to generate the torque of the rotation. Several mutations have beeninvestigated to understand the characteristics and function of the ion channel in the stator andthe mechanism of its assembly around the rotor to complete the motor. In this thesis, wesummarize recent results of the Na+-driven motor in the polar flagellum of Vibrio.The stator proteins PomA and PomB form a complex that couples Na+influx to torquegeneration in the polar flagellar motor of Vibrio alginolyticus. This stator complex is anchoredto an appropriate place around the rotor through a putative peptidoglycan-binding (PGB)domain in the periplasmic region of PomB (PomBC). To investigate the function of PomBC, aseries of N-terminally truncated variants and in-frame deletions in the linker region betweenthe transmembrane (TM) segment and the PGB domain of PomB was constructed. A PomBCfragment consisting of residues135to315(PomBC5) formed a stable homodimer andsignificantly inhibited the motility of wild-type cells when over-expressed in the periplasm.In-frame deletion (PomBΔL) of up to80residues retained function and its over-expressionwith PomA impaired cell growth. This inhibitory effect was suppressed by a mutation at thefunctionally critical Asp (D24N) in the TM segment of PomB, suggesting that high Na+influxthrough the mutant stator causes growth impairment. The mutations in putative interacteionsuface helixs of PomB homodimer which is preferred to destroy the interaction ofPomB-PomB in periplasm still formed dimer. Overproduction of the functionalPomA/PomBΔLstators also reduced motile fractions of the cells, and such effect is slightlyrelieved by a mutation (L168P) in the putative N-terminal α-helix connecting to the PGB domain without affecting the growth inhibition, suggesting that the conformational change ofthe region including the PGB domain affects stator assembly. Our results reveal the commonfeatures of the periplasmic region of PomB/MotB, and that flexible linker that contains ‘plug’segment is important for the control of Na+influx through the stator complex as well as thestator assembly. GFP fused PomA/PomBΔLredused the plar localization of stators that meansthe linker region of PomB can affect the motility of motor by reducing the assembly of satatorcomplexes around the rotor.