Direct observation of steps in rotation of the bacterial flagellar motor

Yoshiyuki Sowa, Alexander Rowe, Mark C. Leake, Toshiharu Yakushi, Michio Homma, Akihiko Ishijima & Richard M. Berry

Figure 1: Rotation measurements of chimaeric Na+-driven flagellar motors in E. Coli.

a)●'H+ driven MotA and MotB of wildtype E. Coli are replaced by Na+ driven PomA and PotB b) ●Reducing the sodium motive force (SMF) reversibly decreases the number of active stator units, and the speed per unit.

c) ●Photodamage in fluorescence experiment reduces the SMF. ●The smooth decline is attributed to a decrease in membrane voltage, whereas the discrete steps are reversible on/off switching of stator units.

Figure 2: Stepping rotation.

a)●Selected images of flourenscent bead attached to flagellar..

b)●Stepping rotation at different speeds for BFP and fluorescence measurements,

●Steps could be resolved for speeds below 7 Hz for BFP and 40 Hz for flourescence experiments.

●Backwards steps occurred at at all speeds, and must be coursed by microscopic reversibility, since the bacteria are ΔChiY.

Figure 3: Analysis of step size and periodicity.

a)●Expansion of traces from fig 2, with stepping algorithm fitted. b)●Presents an example of how information is extracted from data.●The time-data is turned into a histrogram of dwell angles, from which a power spectrum is extracted.●The power spectrum shows a large peak at 26 per revolution.

Figure 4: Summary of step analysis.

●Summary experiment results. (Data from episodes of approx. constant speed).

●1400 revolutions, 9 cells & 28,611 steps. a)●Histogram of step sizes, with multiple gaussian fit to data.

●Forwards step size = 13.7 +- 5.2° (1/26)●Backwards step size = -10.9 +- 3.9° (1/35)●Smaller backwards step size might be caused by reorientation of rotation axis or experimental artifacts.

b)●Power spectrum of a).●(Inset) Power spectrum found with the stepping algorithm

●Notice peaks at 26, 11, 16 & 23 steps per rev.

Discussion

●26 steps per rev. is consistent with periodicity of rotor proteins (FliG)

●10-11 steps per revolution matches stator periodicity

●The peaks at 16 & 23 steps per rev. might reflect unknown symmetries.

●Unusual slow episodes show smaller steps than 1/26 rev. (Data excluded).

●Interval length distributions show both single- and non-single-exponential behavior.

●The two above points and the apparent independence of step size on stator number suggests that the model of the stators as independent poisson-stepping stators needs revision.

●From experiment it cannot be concluded whether one step corresponds to a single ion transit.

Energetic considerations:●ΔG = 0...3k

BT depending on speed,

equivalent of a 75mV potential●Wild type E. Coli has a 150mV membrane potential.

●However, from previous data, ~10 stators pass 1,200 H+ per rev., corresponding to a 3° step per ion.

●From energy conservation an upper bound for step size can be estimated as (Free energy/ ion)/(max. torque/stator). This gives a max step of ~5°.

●Smaller, yet unresolved, substeps might exist or one step is coupled to multiple ion transits, requiring accumulation of ions of mechanical strain in the rotor-stator complex.

●Synthesis of ATP is believed to be coupled to 3-4 ion transits.

Conclusions

●Stepping of the flagellar motor does occur.

●Periodicity of stepping matches the periodicity of rotor and stator proteins. (And perhaps other unknown symmetries.

●Stepping is probably coupled to multiple ion transits.

●Experiment does not agree with the poisson-stepping model.