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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.


Cellular localization of GFP-fused FliG in multiple polar flagella mutants. MK1 cells harboring plasmids (a: pTY200 (GFP), b: pTY201 (GFP-FliG), or c: pTY202 (FliG-GFP) ) were grown in VPG medium containing 2.5 μg/ml chloramphenicol and 0.1% arabinose at 30°C for 4 hr. Cells were observed by fluorescence microscopy as described in the Materials and Methods.
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f3-7_59: Cellular localization of GFP-fused FliG in multiple polar flagella mutants. MK1 cells harboring plasmids (a: pTY200 (GFP), b: pTY201 (GFP-FliG), or c: pTY202 (FliG-GFP) ) were grown in VPG medium containing 2.5 μg/ml chloramphenicol and 0.1% arabinose at 30°C for 4 hr. Cells were observed by fluorescence microscopy as described in the Materials and Methods.

Mentions: The subcellular localizations of GFP-fused FliG proteins were observed by fluorescent microscopy in fliG flhG cells. GFP was distributed throughout the cells and was not localized at the cell poles. Intense fluorescent dots of both GFP-fused FliG were detected at cell poles as reported previously (Fig. 3). However, the intensity of those dots was much stronger than the fluorescent dots of fliG cells without the flhG mutation. We could not observe the difference of the fluorescent dot profiles between GFP-FliG and FliG-GFP.


Characterization of the flagellar motor composed of functional GFP-fusion derivatives of FliG in the Na + -driven polar flagellum of Vibrio alginolyticus
Cellular localization of GFP-fused FliG in multiple polar flagella mutants. MK1 cells harboring plasmids (a: pTY200 (GFP), b: pTY201 (GFP-FliG), or c: pTY202 (FliG-GFP) ) were grown in VPG medium containing 2.5 μg/ml chloramphenicol and 0.1% arabinose at 30°C for 4 hr. Cells were observed by fluorescence microscopy as described in the Materials and Methods.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC5036772&req=5

f3-7_59: Cellular localization of GFP-fused FliG in multiple polar flagella mutants. MK1 cells harboring plasmids (a: pTY200 (GFP), b: pTY201 (GFP-FliG), or c: pTY202 (FliG-GFP) ) were grown in VPG medium containing 2.5 μg/ml chloramphenicol and 0.1% arabinose at 30°C for 4 hr. Cells were observed by fluorescence microscopy as described in the Materials and Methods.
Mentions: The subcellular localizations of GFP-fused FliG proteins were observed by fluorescent microscopy in fliG flhG cells. GFP was distributed throughout the cells and was not localized at the cell poles. Intense fluorescent dots of both GFP-fused FliG were detected at cell poles as reported previously (Fig. 3). However, the intensity of those dots was much stronger than the fluorescent dots of fliG cells without the flhG mutation. We could not observe the difference of the fluorescent dot profiles between GFP-FliG and FliG-GFP.

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.