Many essential cellular processes are carried out by complex biological machines located in the cell membrane. The bacterial flagellar motor is a large membrane-spanning protein complex that functions as an ion-driven rotary motor to propel cells through liquid media1,2,3. Within the motor, MotB is a component of the stator that couples ion flow to torque generation and anchors the stator to the cell wall4,5. Here we have investigated the protein stoichiometry, dynamics and turnover of MotB with single-molecule precision in functioning bacterial flagellar motors in Escherichia coli. We monitored motor function by rotation of a tethered cell body6, and simultaneously measured the number and dynamics of MotB molecules labelled with green fluorescent protein (GFP–MotB) in the motor by total internal reflection fluorescence microscopy. Counting fluorophores by the stepwise photobleaching of single GFP molecules showed that each motor contains ∼22 copies of GFP–MotB, consistent with ∼11 stators each containing two MotB molecules. We also observed a membrane pool of ∼200 GFP–MotB molecules diffusing at ∼0.008 µm2 s-1. Fluorescence recovery after photobleaching and fluorescence loss in photobleaching showed turnover of GFP–MotB between the membrane pool and motor with a rate constant of the order of 0.04 s-1: the dwell time of a given stator in the motor is only ∼0.5 min. This is the first direct measurement of the number and rapid turnover of protein subunits within a functioning molecular machine.
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We thank D. Blair for antibodies to flagellin and MotB. The research of M.C.L., J.H.C., R.M.B. and J.P.A. was supported by combined UK research councils via an Interdisciplinary Research Collaboration in Bionanotechnology (IRC), that of G.H.W. by the Biotechnology and Biological Sciences Research Council (BBSRC), and that of F.B. by a Clarendon Scholarship. Author Contributions Fluorescence experiments were carried out by M.C.L. and J.H.C. in the laboratory of R.M.B.; strain construction was done by J.H.C. and G.H.W. in the laboratory of J.P.A.; data analysis was done by M.C.L., R.M.B. and G.H.W.; and simulations were carried out by F.B. and M.C.L. The experiment was designed by M.C.L., R.M.B. and J.P.A.
Reprints and permissions information is available at www.nature.com/reprints. The authors declare no competing financial interests.
This file contains Supplementary Methods and Supplementary Figures 1–13. (PDF 1180 kb)
This file contains text to accompany the below Supplementary Videos. (PDF 10 kb)
Brightfield video for a freely rotating tethered GFP-MotB cell and fixed cell in the same field of view. (MPG 141 kb)
Continuous TIRF bleach of cells in Supplementary Video 1. (MPG 638 kb)
Continuous TIRF illumination on a different pre-bleached fixed GFP-MotB cells showing diffusing GFP-MotB in the membrane. (AVI 212 kb)
Continuous TIRF illumination on a different pre-bleached fixed GFP-MotB cells showing diffusing GFP-MotB in the membrane. (AVI 329 kb)
Brightfield video for a freely rotating tethered GFP-MotB cell. (MPG 232 kb)
Continuous TIRF bleach of cell in Supplementary Video 5. (MPG 143 kb)
Continuous TIRF bleach of cell in Supplementary Video 4, 60 min after the end of the initial bleach. (MPG 143 kb)
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Quantification of very low-abundant proteins in bacteria using the HaloTag and epi-fluorescence microscopy
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