Roles of Charged Residues of Rotor and Stator in Flagellar Rotation: Comparative Study using H ⁺ -Driven and Na ⁺ -Driven Motors in Escherichia coli

Abstract

In Escherichia coli, rotation of the flagellar motor has been shown to depend upon electrostatic interactions between charged residues of the stator protein MotA and the rotor protein FliG. These charged residues are conserved in the Na ؉ -driven polar flagellum of Vibrio alginolyticus, but mutational studies in V. alginolyticus suggested that they are relatively unimportant for motor rotation. The electrostatic interactions detected in E. coli therefore might not be a general feature of flagellar motors, or, alternatively, the V. alginolyticus motor might rely on similar interactions but incorporate additional features that make it more robust against mutation. Here, we have carried out a comparative study of chimeric motors that were resident in E. coli but engineered to use V. alginolyticus stator components, rotor components, or both. Charged residues in the V. alginolyticus rotor and stator proteins were found to be essential for motor rotation when the proteins functioned in the setting of the E. coli motor. Patterns of synergism and suppression in rotor/stator double mutants indicate that the V. alginolyticus proteins interact in essentially the same way as their counterparts in E. coli. The robustness of the rotor-stator interface in V. alginolyticus is in part due to the presence of additional charged residues in PomA but appears mainly due to other factors, because an E. coli motor using both rotor and stator components from V. alginolyticus remained sensitive to mutation. Motor function in V. alginolyticus may be enhanced by the proteins MotX and MotY.

Characterization of PomA Mutants Defective in the Functional Assembly of the Na + -Driven Flagellar Motor in Vibrio alginolyticus

“…In the H ϩ -driven motors of E. coli, it is inferred that the conserved charged residues of FliG and MotA interact, which is important for torque generation (26,33). Mutation of the cytoplasmic domain at L131 and T132 in PomA affects the temperature sensitivity of the mutants at the conserved charged residues of PomA (6).…”

“…In the H ϩ -driven motor of E. coli, it has been shown that charged residues of MotA and FliG are important for torque generation (26,33). On the other hand, the Na ϩ -driven motor of V. alginolyticus may not require the corresponding charged residues for torque generation (29,31).…”

Isolation of Basal Bodies with C-Ring Components from the Na + -Driven Flagellar Motor of Vibrio alginolyticus

“…FliG, 26 molecules of which are incorporated into the motor, appears to be the protein that is most directly involved in torque generation (15). Mutational analysis suggests that electrostatic interactions between conserved charged residues in the C-terminal domain of FliG and the cytoplasmic domain of MotA are important in torque generation (14), although this may not be the case for the Na ϩ -type motor of Vibrio alginolyticus (32,35,36). FliM interacts with the chemotactic signaling protein CheY in its phosphorylated form (CheY-P) to regulate rotational direction (30).…”

Roles of Charged Residues in the C-Terminal Region of PomA, a Stator Component of the Na + -Driven Flagellar Motor

“…Using this chimeric motor system, a comparative study was carried out with E. coli (29). Neutral or charge reversal mutations were introduced into conserved charged residues in the cytoplasmic domain of PomA and the C terminus of FliG from V. alginolyticus.…”