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Characterization of the flagellar motor composed of functional GFP-fusion derivatives of FliG in the Na + -driven polar flagellum of Vibrio alginolyticus

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ABSTRACT

The polar flagellum of Vibrio alginolyticus is driven by sodium ion flux via a stator complex, composed of PomA and PomB, across the cell membrane. The interaction between PomA and the rotor component FliG is believed to generate torque required for flagellar rotation. Previous research reported that a GFP-fused FliG retained function in the Vibrio flagellar motor. In this study, we found that N-terminal or C-terminal fusion of GFP has different effects on both torque generation and the switching frequency of the direction of flagellar motor rotation. We could detect the GFP-fused FliG in the basal-body (rotor) fraction although its association with the basal body was less stable than that of intact FliG. Furthermore, the fusion of GFP to the C-terminus of FliG, which is believed to be directly involved in torque generation, resulted in very slow motility and prohibited the directional change of motor rotation. On the other hand, the fusion of GFP to the N-terminus of FliG conferred almost the same swimming speed as intact FliG. These results are consistent with the premise that the C-terminal domain of FliG is directly involved in torque generation and the GFP fusions are useful to analyze the functions of various domains of FliG.

No MeSH data available.


Schematic location of GFP and FliG in the basal body. The stator complex and FliG are superimposed onto EM micrographs of the S. typhimurium flagellar basal body reported previously5. The structural model of intact FliG and C-terminal MotB was drawn by a pdb viewer of MolFeat (FiatLux Co., Tokyo, Japan) using pdb data of 3HJL and 2ZVZ, respectively.
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f6-7_59: Schematic location of GFP and FliG in the basal body. The stator complex and FliG are superimposed onto EM micrographs of the S. typhimurium flagellar basal body reported previously5. The structural model of intact FliG and C-terminal MotB was drawn by a pdb viewer of MolFeat (FiatLux Co., Tokyo, Japan) using pdb data of 3HJL and 2ZVZ, respectively.

Mentions: GFP-FliG (N terminal fusion) retained the function for swimming and switching and that phenotype was similar to the wild-type FliG except for the decrease in swarming ability. The N terminal helices of FliG are believed to interact with FliF (MS ring), thus the fused GFP is expected to be located in the inside of the C ring, and therefore the bulky effect of GFP on flagellar function might be very small (Fig. 6). On the other hand, FliG-GFP (C terminal fusion) also worked in the flagellar motor, and both the swimming speed and the switching frequency were significantly reduced. Phenol, which is a repellent and induces a tumbling movement, did not work for the FliG-GFP motor. The swarming ability of FliG-GFP is comparable to that of GFP-FliG which conferd the better ability of swimming and switching. We do not know this reason and the slow motility may escape from the trap of cells by agar matrix. The C terminal region of FliG is believed to interact with a cytoplasmic domain of a stator protein and the interaction between conserved charged residues of FliG and the stator protein of MotA is essential to generate torque according to studies of E. coli19. However, these conserved residues are not essential for the Vibrio flagellar motor20,21, and the C terminal regions in the species are exchangeable20. The C-terminal GFP is predicted to be present around the outer region of the C ring from a docking image of the crystal structure of FliG into the 3D EM reconstruction (Fig. 6)25,42.


Characterization of the flagellar motor composed of functional GFP-fusion derivatives of FliG in the Na + -driven polar flagellum of Vibrio alginolyticus
Schematic location of GFP and FliG in the basal body. The stator complex and FliG are superimposed onto EM micrographs of the S. typhimurium flagellar basal body reported previously5. The structural model of intact FliG and C-terminal MotB was drawn by a pdb viewer of MolFeat (FiatLux Co., Tokyo, Japan) using pdb data of 3HJL and 2ZVZ, respectively.
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Related In: Results  -  Collection

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f6-7_59: Schematic location of GFP and FliG in the basal body. The stator complex and FliG are superimposed onto EM micrographs of the S. typhimurium flagellar basal body reported previously5. The structural model of intact FliG and C-terminal MotB was drawn by a pdb viewer of MolFeat (FiatLux Co., Tokyo, Japan) using pdb data of 3HJL and 2ZVZ, respectively.
Mentions: GFP-FliG (N terminal fusion) retained the function for swimming and switching and that phenotype was similar to the wild-type FliG except for the decrease in swarming ability. The N terminal helices of FliG are believed to interact with FliF (MS ring), thus the fused GFP is expected to be located in the inside of the C ring, and therefore the bulky effect of GFP on flagellar function might be very small (Fig. 6). On the other hand, FliG-GFP (C terminal fusion) also worked in the flagellar motor, and both the swimming speed and the switching frequency were significantly reduced. Phenol, which is a repellent and induces a tumbling movement, did not work for the FliG-GFP motor. The swarming ability of FliG-GFP is comparable to that of GFP-FliG which conferd the better ability of swimming and switching. We do not know this reason and the slow motility may escape from the trap of cells by agar matrix. The C terminal region of FliG is believed to interact with a cytoplasmic domain of a stator protein and the interaction between conserved charged residues of FliG and the stator protein of MotA is essential to generate torque according to studies of E. coli19. However, these conserved residues are not essential for the Vibrio flagellar motor20,21, and the C terminal regions in the species are exchangeable20. The C-terminal GFP is predicted to be present around the outer region of the C ring from a docking image of the crystal structure of FliG into the 3D EM reconstruction (Fig. 6)25,42.

View Article: PubMed Central - PubMed

ABSTRACT

The polar flagellum of Vibrio alginolyticus is driven by sodium ion flux via a stator complex, composed of PomA and PomB, across the cell membrane. The interaction between PomA and the rotor component FliG is believed to generate torque required for flagellar rotation. Previous research reported that a GFP-fused FliG retained function in the Vibrio flagellar motor. In this study, we found that N-terminal or C-terminal fusion of GFP has different effects on both torque generation and the switching frequency of the direction of flagellar motor rotation. We could detect the GFP-fused FliG in the basal-body (rotor) fraction although its association with the basal body was less stable than that of intact FliG. Furthermore, the fusion of GFP to the C-terminus of FliG, which is believed to be directly involved in torque generation, resulted in very slow motility and prohibited the directional change of motor rotation. On the other hand, the fusion of GFP to the N-terminus of FliG conferred almost the same swimming speed as intact FliG. These results are consistent with the premise that the C-terminal domain of FliG is directly involved in torque generation and the GFP fusions are useful to analyze the functions of various domains of FliG.

No MeSH data available.