direct observation of steps in rotation of the bacterial flagellar motor yoshiyuki sowa, alexander...

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.