Insight into the assembly mechanism in the supramolecular rings of the sodium-driven Vibrio flagellar motor from the structure of FlgT

Abstract

Flagellar motility is a key factor for bacterial survival and growth in fluctuating environments. The polar flagellum of a marine bacterium, Vibrio alginolyticus, is driven by sodium ion influx and rotates approximately six times faster than the proton-driven motor of Escherichia coli. The basal body of the sodium motor has two unique ring structures, the T ring and the H ring. These structures are essential for proper assembly of the stator unit into the basal body and to stabilize the motor. FlgT, which is a flagellar protein specific for Vibrio sp., is required to form and stabilize both ring structures. Here, we report the crystal structure of FlgT at 2.0-Å resolution. FlgT is composed of three domains, the N-terminal domain (FlgT-N), the middle domain (FlgT-M), and the C-terminal domain (FlgT-C). FlgT-M is similar to the N-terminal domain of TolB, and FlgT-C resembles the N-terminal domain of FliI and the α/β subunits of F1-ATPase. To elucidate the role of each domain, we prepared domain deletion mutants of FlgT and analyzed their effects on the basal-body ring formation. The results suggest that FlgT-N contributes to the construction of the H-ring structure, and FlgT-M mediates the T-ring association on the LP ring. FlgT-C is not essential but stabilizes the H-ring structure. On the basis of these results, we propose an assembly mechanism for the basal-body rings and the stator units of the sodium-driven flagellar motor.

Fig. 1.

Schematic diagram of the bacterial flagellar motor. The left half shows the Na⁺-driven motor (Vibrio sp.), and the right half the H⁺-driven motor (E. coli and Salmonella). The name of the component protein is shown in parentheses. IM, inner membrane; OM, outer membrane; PG, peptidoglycan layer.

Fig. 2.

Structure of FlgT. (A) Cα ribbon drawing of FlgT. The FlgT-N, FlgT-M, and FlgT-C domains are shown in cyan, green, and red, respectively. (B) Cα backbone trace of FlgT shown in rainbow colors from the N terminus (blue) to the C terminus (red). The Cys-115 and Cys-287 residues, which form an intramolecular disulfide bond, are displayed in ball model. (C) Comparison of the FlgT-M domain (green) with the N-terminal domain of TolB (orange) (PDB ID code: 1CRZ). (D) Comparison of the FlgT-C domain (cyan) with the N-terminal domain of the F₁-β subunit (magenta) (PDB ID code: 1SKY, chain E).

Fig. 3.

Motilities of cells expressing the domain deletion mutant variants of FlgT. (A) Primary structure of FlgT and its mutant variants used in this study. The region shown in blue (Met-1 to Ala-23) is the secretion signal sequence. FlgT-N (Ser-24 to Tyr-109), FlgT-M (Lys-121 to Cys-287), and FlgT-C (Pro-292 to Leu-377) are shown in cyan, green, and pink, respectively. (B) Motility assay of cells expressing the mutant variants of FlgT. Fresh colonies of ΔflgT (TH6) cells harboring the plasmids [1, pSU41; 2, pTH104 (FlgT-NMC); 3, pTH105 (FlgT-NMC+His₆); 4, pLSK9 (FlgT-NM); 5, pLSK9-his (FlgT-NM+His₆); 6, pLSK10 (FlgT-MC); 7, pLSK10-his (FlgT-MC+His₆); 8, pLSK11 (FlgT-NC); 9, pLSK11-his (FlgT-NC+His₆); the asterisk represents the attached hexahistidine tag] were inoculated onto 0.25% agar VPG500 plates containing kanamycin and were then incubated at 30 °C for 4 h. (C) Protein expression profiles. Whole-cell extracts and periplasmic fractions of ΔflgT cells harboring the plasmids noted above were prepared as reported (40) and were subjected to SDS-PAGE, followed by immunoblotting using an anti-FlgT antibody.

Fig. 4.

Electron microscopic images of hook-basal bodies purified from V. alginolyticus. (A–E) Hook-basal bodies were isolated from TH7 (ΔflgT flhG) cells harboring the plasmids, pTH105 (FlgT-NMC+His₆, positive control) (A), pLSK9-his (FlgT-NM+His₆) (B), pLSK10-his (FlgT-MC+His₆) (C), pLSK11-his (FlgT-NC+His₆) (D), and pSU41 (vector control) (E). The asterisk represents the attached hexahistidine tag. The hook-basal bodies were negatively stained with 2% phosphotungstic acid (pH 7.4). (Scale bar: 50 nm.) (F) Schematic representation of the hook-basal body of V. alginolyticus.

Fig. 5.

Characterization of domain deletion mutant variants of FlgT. TH7(ΔflgT flhG) cells harboring the plasmids [1, pTH105 (FlgT-NMC+His₆); 2, pLSK9-his (FlgT-NM+His₆); 3, pLSK10-his (FlgT-MC+His₆); or 4, pLSK11-his (FlgT-NC+His₆)] were grown, and their hook-basal bodies (HBB) were isolated according to Materials and Methods. The protein samples were subjected to SDS/PAGE, followed by immunoblotting using (A) anti-FlgT, (B) anti-FliF, (C) anti-MotX, and (D) anti-MotY antibodies. The asterisk represents the attached hexahistidine tag.

Fig. 6.

A plausible model for the basal body ring formation of the Na⁺-driven motor. The H ring is constructed by FlgT and/or other components, then MotX and MotY assemble to the basal body with the help of FlgT-M and form the T ring. The stator units assemble around the rotor through the T ring, resulting in the formation of a functional motor. Without FlgT, no H ring is constructed. MotX and MotY may occasionally attach to the basal body to form the T ring; however, the ring is unstable and easy to break up. As a result, the stator unit is not properly incorporated into the motor. FlgT and MotY are shown as space-filling models colored cyan for FlgT-N, green for FlgT-M, magenta for FlgT-C, violet for MotY-N, and yellow for MotY-C.