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

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.

Keywords: C-ring; FliG; GFP; Na+-driven; Vibrio alginolyticus; basal body; flagellar motor.

Figure 1

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.

Figure 2

Swarming abilities of multiple polar flagella mutants. Fresh colonies of NMB198 (a) or MK1 (b and c) cells harboring plasmids (1: pTY102 (FliG), 2: pTY200 (GFP), 3: pTY201 (GFP-FliG), or 4: pTY202 (FliG-GFP)) were inoculated on 0.25% agar VPG plates containing 2.5 μg/ml chloramphenicol and 0.1% arabinose, and were then incubated at 30°C for 6 hr (a and b) or 24 hr (c).

Figure 3

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.

Figure 4

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.

Figure 5

Fractionation by sucrose density gradient centrifugation. The basal bodies were prepared from MK1 cells harboring plasmids pTY102 (FliG) (a), pTY201(GFP-FliG) (b), or pTY202 (FliG-GFP)(c). The basal bodies (lane 0) were applied to a 20–60% (w/w) stepwise sucrose gradient in TEC. After centrifugation at 72,000×g for 90 min at 4°C, the gradient was divided into 20 fractions from the top to the bottom. Proteins in each fraction were separated by SDS-PAGE and were detected by immunoblotting using anti-MotY (upper panel) or anti-FliG antibodies (lower panel).

Figure 6

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 previously. 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.