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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.


Protein expression profiles. MK1 cells harboring plasmids (1: pTY102 (FliG), 2: pTY200 (GFP), 3: pTY202 (FliG-GFP), or 4: pTY201 (GFP-FliG)) were grown at 30°C for 4 hr in VPG medium containing 2.5 μg/ml chloramphenicol and 0.1% arabinose. Whole cell extracts were subjected to SDS-PAGE, followed by immunoblotting using anti-FliG (a), anti-GFP (b), anti-flagellin (c), and anti-PomA (d) antibodies.
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f1-7_59: Protein expression profiles. MK1 cells harboring plasmids (1: pTY102 (FliG), 2: pTY200 (GFP), 3: pTY202 (FliG-GFP), or 4: pTY201 (GFP-FliG)) were grown at 30°C for 4 hr in VPG medium containing 2.5 μg/ml chloramphenicol and 0.1% arabinose. Whole cell extracts were subjected to SDS-PAGE, followed by immunoblotting using anti-FliG (a), anti-GFP (b), anti-flagellin (c), and anti-PomA (d) antibodies.

Mentions: To analyze GFP-fused FliG proteins (N terminal and C terminal FliG are termed GFP-FliG and FliG-GFP, respectively), we used the multiple polar flagellar V. alginolyticus strain, KK148, which is a flhG mutant that expresses the flagellar genes at levels higher than wild-type34. GFP-FliG and FliG-GFP were produced from plasmid vectors using arabinose as an inducer in a fliG deleted strain (MK1, hereafter described as the fliG flhG strain) which was constructed from KK148. The GFP-fused FliG proteins in the Vibrio cells were detected by western blotting using anti-FliG and anti-GFP antibodies (Fig. 1). The molecular weights of GFP and FliG are 27 kDa and 39 kDa, respectively, and thus the fusion protein is estimated as 66 kDa. From whole cell lysates, the 62 kDa band was strongly detected by the anti-FliG antibody and corresponded to the size of the fusion protein (Fig. 1a and 1b). Several bands smaller than 62 kDa were detected in GFP-FliG as well as FliG-GFP. Those smaller bands might represent degradation or truncated products of the fusion proteins, suggesting that there are some differences in stability between GFP-FliG and FliG-GFP, and that the stability of GFP-FliG is relatively lower than FliG-GFP. No effect on flagellar protein expression was detected by western blotting using anti-flagellin or anti-PomA antibodies (Fig. 1c and 1d). The expressions of flagellin and the motor protein, PomA, were repressed by the fliG mutation according to the transcriptional hierarchy of the flagellar regulon.


Characterization of the flagellar motor composed of functional GFP-fusion derivatives of FliG in the Na + -driven polar flagellum of Vibrio alginolyticus
Protein expression profiles. MK1 cells harboring plasmids (1: pTY102 (FliG), 2: pTY200 (GFP), 3: pTY202 (FliG-GFP), or 4: pTY201 (GFP-FliG)) were grown at 30°C for 4 hr in VPG medium containing 2.5 μg/ml chloramphenicol and 0.1% arabinose. Whole cell extracts were subjected to SDS-PAGE, followed by immunoblotting using anti-FliG (a), anti-GFP (b), anti-flagellin (c), and anti-PomA (d) antibodies.
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Related In: Results  -  Collection

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f1-7_59: Protein expression profiles. MK1 cells harboring plasmids (1: pTY102 (FliG), 2: pTY200 (GFP), 3: pTY202 (FliG-GFP), or 4: pTY201 (GFP-FliG)) were grown at 30°C for 4 hr in VPG medium containing 2.5 μg/ml chloramphenicol and 0.1% arabinose. Whole cell extracts were subjected to SDS-PAGE, followed by immunoblotting using anti-FliG (a), anti-GFP (b), anti-flagellin (c), and anti-PomA (d) antibodies.
Mentions: To analyze GFP-fused FliG proteins (N terminal and C terminal FliG are termed GFP-FliG and FliG-GFP, respectively), we used the multiple polar flagellar V. alginolyticus strain, KK148, which is a flhG mutant that expresses the flagellar genes at levels higher than wild-type34. GFP-FliG and FliG-GFP were produced from plasmid vectors using arabinose as an inducer in a fliG deleted strain (MK1, hereafter described as the fliG flhG strain) which was constructed from KK148. The GFP-fused FliG proteins in the Vibrio cells were detected by western blotting using anti-FliG and anti-GFP antibodies (Fig. 1). The molecular weights of GFP and FliG are 27 kDa and 39 kDa, respectively, and thus the fusion protein is estimated as 66 kDa. From whole cell lysates, the 62 kDa band was strongly detected by the anti-FliG antibody and corresponded to the size of the fusion protein (Fig. 1a and 1b). Several bands smaller than 62 kDa were detected in GFP-FliG as well as FliG-GFP. Those smaller bands might represent degradation or truncated products of the fusion proteins, suggesting that there are some differences in stability between GFP-FliG and FliG-GFP, and that the stability of GFP-FliG is relatively lower than FliG-GFP. No effect on flagellar protein expression was detected by western blotting using anti-flagellin or anti-PomA antibodies (Fig. 1c and 1d). The expressions of flagellin and the motor protein, PomA, were repressed by the fliG mutation according to the transcriptional hierarchy of the flagellar regulon.

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.