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Published bySara Aubrie Terry Modified over 8 years ago
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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
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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.
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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.
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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.
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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.
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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 B T 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.
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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.
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