Electrostatic interactions between rotor and stator in the bacterial flagellar motor

Abstract

Bacterial flagellar motors rotate, obtaining power from the membrane gradient of protons or, in some species, sodium ions. Torque generation in the flagellar motor must involve interactions between components of the rotor and components of the stator. Sites of interaction between the rotor and stator have not been identified. Mutational studies of the rotor protein FliG and the stator protein MotA showed that both proteins contain charged residues essential for motor rotation. This suggests that functionally important electrostatic interactions might occur between the rotor and stator. To test this proposal, we examined double mutants with charged-residue substitutions in both the rotor protein FliG and the stator protein MotA. Several combinations of FliG mutations with MotA mutations exhibited strong synergism, whereas others showed strong suppression, in a pattern that indicates that the functionally important charged residues of FliG interact with those of MotA. These results identify a functionally important site of interaction between the rotor and stator and suggest a hypothesis for electrostatic interactions at the rotor-stator interface.

Figure 1

Complementation of the fliG motA double-mutant strain by wild-type fliG and motA genes on plasmids. Shown are swarms in soft-agar plates of (A) strain DFB245, a fliG motA double mutant; (B) strain DFB245 transformed with plasmid pSL27, which encodes FliG; (C) strain DFB245 transformed with plasmid pJZ19, which encodes MotA; and (D) strain DFB245 transformed with both pSL27 and PJZ19. Flagellar staining showed that strains A and C were nonflagellate, whereas strains B and D had 2.4 and 4.5 flagella per cell, respectively (averages for 50 cells).

Figure 2

Examples of swarming phenotypes of fliG motA double mutants, illustrating cases of synergism and suppression. FliG and MotA proteins were expressed from two compatible plasmids in a fliG motA double-mutant strain. (A) Synergistic effect of combining the MotA mutation R90A with the FliG mutation R281A. (B) Synergistic effect of combining the MotA mutation E98Q with the FliG mutation D289A. (C) Mutual suppression of the mutations R90E (MotA) and D289K (FliG). (D) Mutual suppression of the mutations E98K (MotA) and R281V (FliG).

Figure 3

Effects of KCl on swarming motility of wild-type E. coli and strains with mutations in key charged residues. (A) Swarming rates of wild-type and mutant strains in soft-agar plates containing KCl at the concentrations indicated. Data are the average of 10 determinations for the wild type and 2 determinations for each of the mutants. (B) Swarming rates of the charged-residue mutants, measured relative to wild-type controls present on the same plates at various concentrations of KCl (mean ± SD; n = 3).

Figure 4

(A) Hypothesis for charged-residue interactions at the rotor–stator interface. The functionally important charged residues of wild-type FliG and MotA, and hypothesized electrostatic interactions between them, are shown. Solid lines indicate interactions of primary importance for motor function, and thin dashed lines indicate interactions of secondary importance. The interactions are drawn as occurring simultaneously, but might actually occur sequentially, as the rotor and stator move relative to one another or as one of the proteins undergoes a conformational change. (B) An example of strong suppression and its rationalization in the framework of the model. Mutually suppressing mutations of FliG and MotA, and their hypothesized effects on electrostatic interactions at the rotor–stator interface, are shown. Double-headed arrows indicate repulsive interactions in the mutants that take the place of attractive interactions in the wild type. (C) An example of strong synergism.