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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.


Electron micrographs of flagella. MK1 cells harboring plasmids (a: pTY102 (FliG), b: pTY201 (GFP-FliG), or c: pTY202 (FliG-GFP) were grown in VC medium overnight and were inoculated into VPG medium containing 2.5 μg/ml chloramphenicol and 0.1% arabinose, and were then incubated at 30°C for 4 hr. Cells were harvested by centrifugation and were negatively stained with potassium phosphotungstate. Bar, 2 μm.
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f4-7_59: Electron micrographs of flagella. MK1 cells harboring plasmids (a: pTY102 (FliG), b: pTY201 (GFP-FliG), or c: pTY202 (FliG-GFP) were grown in VC medium overnight and were inoculated into VPG medium containing 2.5 μg/ml chloramphenicol and 0.1% arabinose, and were then incubated at 30°C for 4 hr. Cells were harvested by centrifugation and were negatively stained with potassium phosphotungstate. Bar, 2 μm.

Mentions: Next, flagellated Vibrio cells expressing GFP-fused FliG were observed using negative staining and electron microscopy. Profiles of the flagellation were not different between wild-type FliG cells and GFP-fused FliG cells (Fig. 4). These results show that the GFP fusion does not affect the flagellar morphogenesis at all.


Characterization of the flagellar motor composed of functional GFP-fusion derivatives of FliG in the Na + -driven polar flagellum of Vibrio alginolyticus
Electron micrographs of flagella. MK1 cells harboring plasmids (a: pTY102 (FliG), b: pTY201 (GFP-FliG), or c: pTY202 (FliG-GFP) were grown in VC medium overnight and were inoculated into VPG medium containing 2.5 μg/ml chloramphenicol and 0.1% arabinose, and were then incubated at 30°C for 4 hr. Cells were harvested by centrifugation and were negatively stained with potassium phosphotungstate. Bar, 2 μm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC5036772&req=5

f4-7_59: Electron micrographs of flagella. MK1 cells harboring plasmids (a: pTY102 (FliG), b: pTY201 (GFP-FliG), or c: pTY202 (FliG-GFP) were grown in VC medium overnight and were inoculated into VPG medium containing 2.5 μg/ml chloramphenicol and 0.1% arabinose, and were then incubated at 30°C for 4 hr. Cells were harvested by centrifugation and were negatively stained with potassium phosphotungstate. Bar, 2 μm.
Mentions: Next, flagellated Vibrio cells expressing GFP-fused FliG were observed using negative staining and electron microscopy. Profiles of the flagellation were not different between wild-type FliG cells and GFP-fused FliG cells (Fig. 4). These results show that the GFP fusion does not affect the flagellar morphogenesis at all.

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.