Function of proline residues of MotA in torque generation by the flagellar motor of Escherichia coli

Abstract

Bacterial flagellar motors obtain energy for rotation from the membrane gradient of protons or, in some species, sodium ions. The molecular mechanism of flagellar rotation is not understood. MotA and MotB are integral membrane proteins that function in proton conduction and are believed to form the stator of the motor. Previous mutational studies identified two conserved proline residues in MotA (Pro 173 and Pro 222 in the protein from Escherichia coli) and a conserved aspartic acid residue in MotB (Asp 32) that are important for function. Asp 32 of MotB probably forms part of the proton path through the motor. To learn more about the roles of the conserved proline residues of MotA, we examined motor function in Pro 173 and Pro 222 mutants, making measurements of torque at high load, speed at low and intermediate loads, and solvent-isotope effects (D2O versus H2O). Proton conduction by wild-type and mutant MotA-MotB channels was also assayed, by a growth defect that occurs upon overexpression. Several different mutations of Pro 173 reduced the torque of the motor under high load, and a few prevented motor rotation but still allowed proton flow through the MotA-MotB channels. These and other properties of the mutants suggest that Pro 173 has a pivotal role in coupling proton flow to motor rotation and is positioned in the channel near Asp 32 of MotB. Replacements of Pro 222 abolished function in all assays and were strongly dominant. Certain Pro 222 mutant proteins prevented swimming almost completely when expressed at moderate levels in wild-type cells. This dominance might be caused by rotor-stator jamming, because it was weaker when FliG carried a mutation believed to increase rotor-stator clearance. We propose a mechanism for torque generation, in which specific functions are suggested for the proline residues of MotA and Asp32 of MotB.

FIG. 1

Membrane topologies of MotA and MotB. Functionally important residues identified in previous mutational studies (40, 42) are indicated. Residue numbers are for the MotA and MotB proteins from E. coli.

FIG. 2

Examples of swarming phenotypes of Pro 173 and Pro 222 mutants. The motA-defective strain MS5037 was transformed with plasmids (derivatives of pRF4) expressing the mutant MotA proteins indicated. The plate contained tryptone broth and 0.28% agar. It was inoculated with 1 μl of a saturated culture of each strain and photographed after 8 h at 32°C.

FIG. 3

Immunoblot showing cellular levels of representative mutant MotA proteins. Cell membranes were prepared from mid-log cultures and electrophoresed and immunoblotted as described in Materials and Methods. w.t., wild type.

FIG. 4

Effects of viscous load on the swimming speeds of wild-type cells and the MotA mutants P173S and P173T. Swimming speeds and medium viscosity were measured at 32°C in media that contained Ficoll (average molecular weight, 400,000) at concentrations ranging from 0 to 16%, as described in Materials and Methods. The reciprocals of swimming speeds are plotted against the relative viscous load, which was obtained as the product of viscosity and swimming speed. Results are shown for three independent experiments with the wild type and two independent experiments with each mutant. Values are the means ± standard errors of the mean for at least 50 cells. Practically all of the uncertainty in the estimates of load comes from the measurements of swimming speed, because measurements of viscosity are much more precise. Errors along the two axes are therefore expected to be correlated, as indicated. Best-fit straight lines are drawn to guide the eye.

FIG. 5

Motor torques in tethered cells of Pro 173 mutants. Flagellar filaments were sheared and tethered to coverslips on a microscope stage thermostated at 32°C, and cell rotation rate, size, and radius of gyration were measured. Torques were computed as described in Materials and Methods. Values are the means ± standard errors of the mean for the sample sizes indicated.

FIG. 6

Proton conductance, as assayed by growth impairments, of MotA-MotB channels with mutations in Pro 173 and Pro 222. Wild-type cells were transformed with derivatives of plasmid pLW3 (38), which overexpresses MotA or its mutant variants and also a MotB-Rop fusion protein, both from the inducible trp promoter. (Left) Representative growth curves. Open symbols, uninduced cultures; filled symbols, cultures induced at t = 0 with indoleacrylic acid (final concentration, 100 μg/ml). Circles and triangles represent results of two independent experiments, which are fitted to solid and dashed lines, respectively. (Right) Summary of growth rates of the Pro 173 and Pro 222 mutants, with and without induction by indoleacrylic acid (mean ± standard deviation; n = 3).

FIG. 7

Proton conduction, as assayed by growth impairments, by MotA-MotB channels with mutations in both proteins. The mutations present in MotA and MotB are indicated. Q4Ter signifies a stop codon in place of codon 4 of motB (allele 33 in reference 6). Black bars, uninduced; grey bars, induced with indoleacrylic acid. Values are the means ± standard deviations (n = 5 to 9). w.t., wild type.

FIG. 8

Dominance of Pro 222 mutations of MotA and modulation of this dominance by mutations in fliG. The plates were inoculated with 1 μl of saturated cultures of the indicated strains and incubated at 32°C. Strains that contained only wild-type MotA were incubated for 6 h (top) or 7.5 h (bottom); others were incubated for 7.5 to 8 h. Three isolates of each strain are shown. w.t., wild type.

FIG. 9

Alignment of selected MotA sequences in the segments around Pro 173 and Pro 222. M3 and M4 indicate parts of membrane segments 3 and 4. Coils below the sequences indicate segments predicted to be α-helical by a neural-net algorithm (27).

FIG. 10

Proposed mechanism for coupling proton flow to motor rotation at the Asp 32-Pro 173-Pro 222 site. (A) Mechanism of motor rotation. The diagram shows a single stator complex and a small section of the rotor. A series of projections on the edge of the rotor, drawn as white triangles, interact with points projecting from the stator so that relative movement of rotor and stator is restricted. Other sites on the rotor and stator, colored gray and termed trigger sites, interact so that when the rotor and stator are properly aligned, a conformation change is triggered in the stator. The trigger sites might be formed from the charged residues of MotA and FliG that have been shown to interact at the rotor-stator interface (41). Other elements pictured are the functionally important residues Asp 32 of MotB and Pro 173 and Pro 222 of MotA. In an initial state (panel i), Asp 32 is unprotonated, and the trigger sites on the rotor and stator are close to each other but not yet aligned. A small movement of the rotor, which might be accelerated by electrostatic interactions between the rotor and stator, brings the trigger sites into alignment so that they can interact to promote a conformational change in the stator (panel ii). This conformational change, which is suggested to involve Pro 173, opens a gate in a gate in a proton channel to the periplasm. A proton enters and binds to Asp 32 (the filled oval signifies the protonated site), triggering a further conformational change in the stator (panel iii). This conformational change affects contacts at the rotor-stator interface to drive movement of the rotor, toward the right for the geometry pictured. Next, the proton is released from Asp 32 to the cytoplasm, and the stator returns to its starting conformation. The rotor is driven farther to the right at this step because the stator engages the next projecting site on the rotor (panel iv). The net result is transfer of one proton across the membrane and movement of the rotor to the right by one rotor subunit. (B) A mechanism for switching the direction of rotation. Switching could occur by a coordinated movement of the projecting sites on the rotor relative to the trigger sites. The events in the stator would be the same as those pictured in part A, but for the geometry shown at the bottom, the rotor would move to the left.