Contribution of Many Charged Residues at the Stator-Rotor Interface of the Na ⁺ -Driven Flagellar Motor to Torque Generation in Vibrio alginolyticus

Abstract

In torque generation by the bacterial flagellar motor, it has been suggested that electrostatic interactions between charged residues of MotA and FliG at the rotor-stator interface are important. However, the actual role(s) of those charged residues has not yet been clarified. In this study, we systematically made mutants of Vibrio alginolyticus whose charged residues of PomA (MotA homologue) and FliG were replaced by uncharged or charge-reversed residues and characterized the motilities of those mutants. We found that the members of a group of charged residues, 7 in PomA and 6 in FliG, collectively participate in torque generation of the Na ؉ -driven flagellar motor in Vibrio. An additional specific interaction between PomA-E97 and FliG-K284 is critical for proper performance of the Vibrio motor. Our results also reveal that more charged residues are involved in the PomA-FliG interactions in the Vibrio Na ؉ -driven motor than in the MotA-FliG interactions in the H ؉ -driven one. This suggests that a larger number of conserved charged residues at the PomA-FliG interface contributes to the robustness of the Vibrio motor against mutations. The interaction surfaces of the stator and rotor of the Na ؉ -driven motor seem to be more complex than those previously proposed in the H ؉ -driven motor.

Load- and polysaccharide-dependent activation of the Na+-type MotPS stator in the Bacillus subtilis flagellar motor

“…More than ten MotAB and PomAB stators can assemble around a rotor through the MotA-FliG and PomA-FliG interactions, respectively67891011. The stator is anchored to the peptidoglycan (PG) layer through a well-conserved PG binding motif within the C-terminal periplasmic domain of MotB or PomB121314.…”

Domain-based biophysical characterization of the structural and thermal stability of FliG, an essential rotor component of the Na⁺-driven flagellar motor

“…The basal body, which contains the rotor, comprises several ring structures, the L, P, MS, and C rings. The stator, which is an ion-conducting energy-converting complex, surrounds the rotor and generates torque through interaction with components of the rotor [1,2]. The flagellar motor uses mainly two kinds of ions, H + or Na + , as the energy source and the stator is composed of two kinds of membrane proteins.…”

“…On the other hand, the Na + -driven flagellar motor of marine Vibrio contains such conserved charged residues in PomA and FliG, but single mutations of these conserved residues do not strongly affect the motility [23,24]. Compared with E. coli , the number of conserved charged residues of the flagellar motor in Vibrio is greater, and the contribution of each residue for torque generation may be smaller in Vibrio motor [2,25]. Furthermore, we found that a specific interaction between the charged residues is critical for the correct assembly of the stators around the rotor and is important for torque generation [2].…”

“…Compared with E. coli , the number of conserved charged residues of the flagellar motor in Vibrio is greater, and the contribution of each residue for torque generation may be smaller in Vibrio motor [2,25]. Furthermore, we found that a specific interaction between the charged residues is critical for the correct assembly of the stators around the rotor and is important for torque generation [2]. …”

Loose coupling in the bacterial flagellar motor

“…MotA has four TM segments, two relatively short segments in the periplasm and two larger segments in the cytoplasm (18). The segment of MotA between TM2 and TM3 contains well-conserved charged residues that engage in electrostatic interactions with charged residues of FliG (19)(20)(21). The Asp-32 regulated conformational change has been shown to affect this rotor-interacting region of MotA (17).…”