Dynamics of sodium-driven stator units in bacterial flagellar motors

以作者查詢圖書館館藏

、以作者查詢臺灣博碩士

、以作者查詢全國書目

、勘誤回報

、線上人數：104

、訪客IP：18.118.16.91

姓名

林再順(Tsai-Shun Lin) 查詢紙本館藏

畢業系所

物理學系

論文名稱

(Dynamics of sodium-driven stator units in bacterial flagellar motors)

相關論文

★ 多細菌鞭毛馬達的同步轉動量測	★ Investigating Stators Assembly of Flagellar Motors in Escherichia Coli by PALM
★ 被動粒子在不同的流體型態	★ Lab on the Agar Plates
★ Probing the Physical Environments of Bacterial Swarm Colony	★ Spiral-coil Formation in Semi-flexible Self-propelled Chain System
★ Real-Time Measurement of Vibrio alginolyticus Polar Flagellar Growth	★ Foraging behavior of Caenorhabditis elegans
★ Jamming State of Active Nematics	★ Probing Escherichia coli Energetics under Starvation by Single-Cell Measurements
★ Probing Cell Wall Synthetic Dynamics by Bacterial Flagellar Motor in Escherichia coli	★ 高密度二維群游細菌系統之動力學
★ Deformation Dynamics of Active 2D Tetragonal Pseudo-Crystal	★ Probing Ion-Flux of Bacterial Flagellar Motors by Correlative Microscopy
★ Aliivibrio fischeri in Motion	★ 主動粒子的擴張行為

檔案

[Endnote RIS 格式]

[Bibtex 格式]

[相關文章]

[文章引用]

[完整記錄]

[館藏目錄]

[檢視]

[下載]

本電子論文使用權限為同意立即開放。
已達開放權限電子全文僅授權使用者為學術研究之目的，進行個人非營利性質之檢索、閱讀、列印。
請遵守中華民國著作權法之相關規定，切勿任意重製、散佈、改作、轉貼、播送，以免觸法。

摘要(中)

許多細菌擁有奈米尺度的細菌鞭毛馬達結構，其大小約為50 奈米。透過轉動鞭毛馬達，細菌可以主動地在低雷諾數的環境中活動以取得維生的重要物質。一個鞭毛馬達的基本結構含有一條螺旋狀的鞭毛、一個轉子與為數約一打的定子單元。其中轉子是馬達的旋轉主體與鞭毛連接，帶動鞭毛轉動。而定子單元則是獨立的各自獨立的蛋白複合體，負責推動轉子轉動，其過程是藉由傳遞特定的離子(氫離子或鈉離子)至細胞體內，進而轉換離子驅動勢(Ion-movie force)至機械能。此外，定子單元的狀態在運轉的馬達周圍是動態的，有機率性的與馬達結合或分離。目前已知，與馬達結合的定子單元數目會隨環境浮動並且對於馬達的輸出至關重要。因此了解定子單元組裝的動力學特性對於解釋細菌鞭毛馬達的基本運作原理與特性相當的重要。
這份論文的主要研究主要集中在鈉離子型態的定子單元上，其中包含了溶藻弧菌身上的原生定子，與人工合成表現在大腸桿菌上的嵌合體定子。我們應用了多種光學顯微技術來解析馬達上定子單元的數目與其動力學行為，應用的技術包含了全內反射營光顯微術 (TIRF)、光漂白螢光恢復術 (FRAP)、光啟動定位顯微術 (PALM)、與後焦面干涉術 (BFPI)。這些不同的技術，被整合到了一台商業化的通用顯微鏡上。除此之外，我們更進一步設計使該顯微系統加入了可以即時切換的注流系統，控制細菌的環境條件，用來做鈉離子濃度的切換；也加入了焦面鎖定系統，達到長時間觀測下穩定的影像。
在這本論文中，主要探討三個與定子相關的計畫。第一個執行的計畫主要目標在於利用超解析顯微鏡PALM探討定子在馬達周圍的分布。根據實驗數據估計，在我們系統的PALM最佳的分辨率小於5奈米。當PALM應用在定位定子的實驗中，雙色螢光分子各別標記了轉子結構上的FliN蛋白與定子結構上的PomA蛋白。在經過仔細的量測，這兩個蛋白共定位的比例和化學固定對於實驗造成的影響後，我們發現定子在轉子周圍的密度，大約呈現定值而後隨距離增加而下降。然而，定子的密度分布卻遠低於現有模型的預測，根據該結果，我們提出未來在PALM實驗系統上優化的可能性；第二個計畫主要是分析溶藻弧菌中，馬達力矩輸出相關的FliL蛋白對於定子組裝於馬達上數量的影響。透過在TIRF顯微術下，計算馬達上FliL與定子的數量，其化學劑量在馬達上比值約為1:1。此外，當FliL蛋白被移除時，透過FRAP量測，定子在馬達上交換速率的時間尺度會些微上升。而追蹤蛋白在膜上擴散的速率顯示，FliL與定子在膜上的功能應該是各自獨立。我們的這些結果表明了FliL對於定子在馬達上的功能具有輔助定子的功能，並提出了可能的組裝過程；第三個計畫主要利用在大腸桿菌上的嵌合體馬達，探討環境中鈉離子濃度對於定子在馬達上組裝動力學的相關反應。我們發現鈉驅動的嵌合體馬達上，定子的數目會有所調整以因應鈉離子驅動勢的變化，因此可以在很寬的鈉離子濃度範圍內，保持一個穩定的速度。根據量測，馬達在鈉離子濃度5mM下，定子在馬達上的結合最為穩定，高於其他更高的鈉離子濃度條件。並且，當環境中鈉離子濃度快速的從高濃度切換到低濃度時，馬達上運作的定子數目出現先快速的下降再回升的現象，該結果與現行用來預測定子組裝過程的吸收模型有所不同，這樣的結果暗示著，當鈉離子濃度突然間的下降，定子可能進入了尚未了解的亞穩態。
總結來說，這本論文中應用了複合式顯微術，結合電腦控制的流道系統，量測了鞭毛馬達的轉速與定子螢光實驗，提供了一個前沿的系統從不同角度綜合量測蛋白質的動力學。我們對鈉驅動鞭毛馬達的發現凸顯了生物分子複雜的動態機制，並且為更深入研究鞭毛馬達的運作機制提供了可能性。

摘要(英)

Bacterial flagellar motor (BFM) is a nanometer-size molecular machine (~50 nm) in many bacteria species. By rotating the flagellar motor, bacteria have the motility to search life essentials actively at low Reynold number environment. A flagellar motor composes of a helical flagellum, a rotor and about dozen of stator units. A rotor is a rotary body connecting to a flagellum. Stator units are small independent protein complex that drive the rotor rotation and consume ion-motive force by transporting specific ions (mostly H+ or Na+) into cytoplasmic to drive the rotor rotation. Moreover, stator units dynamically associate/dissociate at the circumference of a functional rotor. The stoichiometry of stator units in a functional motor is highly dynamic and imperative to the power output. Therefore, understanding the assembly kinetics of stator units is crucial to elucidate bacteria flagellar motor′s the primary working mechanism and characteristics.
The work in this thesis focuses mainly on sodium-type stator units, including wild-type stator in Vibrio alginolyticus and chimeric stator in Escherichia coli. We applied multiple techniques of optical microscopy to reveal the stoichiometry and kinetic of stator units, including total internal reflection fluorescence microscopy (TIRF), fluorescence recovery after photobleaching (FRAP), photoactivated localization microscopy (PALM), and back focal plane interferometry (BFPI). These techniques were designed and integrated into a commercial microscope. The apparatus was further devised to perform an on-time perfusion system for ion concentration switching and a focus-lock system for long-term focus stability.
In this thesis, three related projects are presented. The first project aims to investigate the distribution of stator units around a motor by PALM. The estimated spatial resolution is smaller than 5 nm using PALM in our system. We applied two-color labelling on both rotor protein FliN and stator protein PomA for the stator localization experiments. After we carefully examed the colocalization ratio and the fixation effect, we found the stator density is a constant near the rotor and decayed with distance. However, the stator number density is lower than the model. Further investigation is required for the PALM experimental system. The second project aims to analyze the affection of torque association protein, FliL, to stator-units assemble in V. alginolyticus. Using TIRF microscopy to count protein quantity, the stator units and FliL protein stoichiometric ratio is close to 1:1 in a functional motor. Besides, the exchange time of stator units is slightly increased in the absence of FliL from the FRAP experiment. Measurement of the diffusion rate suggests the two proteins are independent on the membrane. Our results suggest that FliL plays a supporting role to the stator in the BFM. The third project investigates the relation between sodium concentration and the kinetic response of chimaeric stator units in E. coli. We found the sodium-driven chimaeric BFMs maintained constant speed over a wide range of sodium concentrations by adjusting stator units in compensation to the sodium-motive force (SMF) changes. The BFM has the maximum number of stator units and is most stable at 5 mM sodium concentration rather than higher sodium concentration. Upon rapid exchange from high to low sodium concentration, the stator number shows a drop and then resurrection that is different from predictions of simple absorption model. This may imply the existence of a metastable hidden state of the stator unit during the sudden loss of sodium ions.
In conclusion, our correlated microscopy combining BFM speed and fluorescence measurements with computer-controlled microfluidic devices provided a cutting-edge system to investigate protein complex dynamics. Our findings of sodium-driven BFMs highlight the complex and dynamical mechanism of biomolecules and pave the way of the complete understanding of BFM working mechanisms.

關鍵字(中)

★ 細菌鞭毛馬達

關鍵字(英)

★ bacterial flagellar motor
★ sodium-motive force
★ stator exchange
★ membrane protein
★ FliL

論文目次

摘要 I
Abstract III
致謝 V
Contents VI
Abbreviations VIII
1 Introduction 1
1.1 Molecular motors 1
1.2 Bacteria flagellar motor 2
1.2.1 Bacteria in motion 2
1.2.2 The structure of bacterial flagellar motors 5
1.2.3 Torque generation of flagellar motors 6
1.2.4 Motor speed 9
1.2.5 Dynamical adaption of stator units 11
2 Materials and Methods 15
2.1 Bacteria strains and cultruing 15
2.2 Channel slide construction 17
2.2.1 Cleaning microscope slides 17
2.2.2 Open-ended sample slides 18
2.2.3 Slide with antibody coating 18
2.3 Motility measurements 22
2.3.1 Bead assay 22
2.3.2 Tethered cell 23
2.3.3 Bead assay with flow 24
2.4 Fluorescence measurements 26
2.4.1 Live static sample 26
2.4.2 Dead-fixed sample (Bacterial proteins in situ fixation) 26
2.5 Microscopy and implement 28
2.5.1 Bright field microscope with high speed image recording 28
2.5.2 Back-focal-plane interferometry 28
2.5.3 Total internal reflection fluorescence microscopy 31
2.5.4 Fluorescence recovery after photobleaching 32
2.5.5 Photoactivated localization microscopy 34
2.5.6 Focus-lock system 35
3 Stator Units Localization by Super-Resolution Microscopy 38
3.1 Aims and experiment design 38
3.2 Fluorescence intensity to the localization resolution. 38
3.3 Simulation of stator localization in high resolution. 40
3.4 Colocalization of rotor and stator units 42
3.5 Effect of cell fixation 45
3.6 The density of stator units around a rotor 46
4 Protein FliL Association to the Stator Units 49
4.1 Aims and experiment design 49
4.2 Effect of FliL on stator abundance 49
4.3 Stator units and FliL exchange dynamics 50
4.4 Membrane diffusivity 52
5 Ion-Concentration-Dependent Stator Dynamics 56
5.1 Aims and experiment design 56
5.2 Sodium dependent assembly of stator units at steady state 56
5.3 Inferring stator number under dynamical sodium motive force 59
5.4 Sodium dependent stator kinetic 60
5.5 Stator response to sodium concentration shift. 66
6 Conclusions 70
7 Bibliography 72
8 List of Figures 82
9 List of Tables 88

參考文獻

1. Feynman, R. P. There’s plenty of room at the bottom. J. Microelectromechanical Syst. 1, 60–66 (1992).
2. Purcell, E. M. The efficiency of propulsion by a rotating flagellum. Proc. Natl. Acad. Sci. U. S. A. 94, 11307–11 (1997).
3. Schuhmacher, J. S., Thormann, K. M. & Bange, G. How bacteria maintain location and number of flagella? FEMS Microbiol. Rev. 39, 812–822 (2015).
4. Kearns, D. B. A field guide to bacterial swarming motility. Nat. Rev. Microbiol. 8, 634–644 (2010).
5. McCarter, L. L. Dual flagellar systems enable motility under different circumstances. J. Mol. Microbiol. Biotechnol. 7, 18–29 (2004).
6. Ulitzur, S. & Kessel, M. Giant flagellar bundles of Vibrio alginolyticus (NCMB 1803). Arch. Mikrobiol. 94, 331–339 (1973).
7. Berg, H. C. E. coli in Motion.(Springer New York, 2004). doi:10.1007/b97370
8. Berg, H. C. & Brown, D. A. Chemotaxis in Escherichia coli analysed by Three-dimensional Tracking. Nature 239, 500–504 (1972).
9. Taylor, B. L. & Koshland, D. E. Reversal of flagellar rotation in monotrichous and peritrichous bacteria: generation of changes in direction. J. Bacteriol. 119, 640–642 (1974).
10. Darnton, N. C., Turner, L., Rojevsky, S. & Berg, H. C. On torque and tumbling in swimming Escherichia coli. J. Bacteriol. 189, 1756–1764 (2007).
11. Son, K., Guasto, J. S. & Stocker, R. Bacteria can exploit a flagellar buckling instability to change direction. Nat. Phys. 9, 494–498 (2013).
12. Yorimitsu, T. & Homma, M. Na+-driven fagellar motor of Vibrio. Components 1505, 82–93 (2001).
13. Sowa, Y., Hotta, H., Homma, M. & Ishijima, A. Torque-speed relationship of the Na+-driven flagellar motor of Vibrio alginolyticus. J. Mol. Biol. 327, 1043–1051 (2003).
14. Muramoto, K. et al. High-speed rotation and speed stability of the sodium-driven flagellar motor in Vibrio alginolyticus. J. Mol. Biol. 251, 50–58 (1995).
15. Magariyama, Y. et al. Simultaneous measurement of bacterial flagellar rotation rate and swimming speed. Biophys. J. 69, 2154–2162 (1995).
16. Ogawa, R., Abe-Yoshizumi, R., Kishi, T., Homma, M. & Kojima, S. Interaction of the C-terminal tail of Flif with Flig from the Na+-driven flagellar motor of Vibrio alginolyticus. J. Bacteriol. 197, 63–72 (2015).
17. Lynch, M. J. et al. Co-folding of a FliF-FliG split domain forms the basis of the MS:C ring interface within the bacterial flagellar motor. Structure 25, 317–328 (2017).
18. Xue, C. et al. Crystal structure of the FliF-FliG complex from Helicobacter pylori yields insight into the assembly of the motor MS-C ring in the bacterial flagellum. J. Biol. Chem. 293, 2066–2078 (2018).
19. Lloyd, S. A. & Blair, D. F. Charged residues of the rotor protein FliG essential for torque generation in the flagellar motor of Escherichia coli. J. Mol. Biol. 266, 733–744 (1997).
20. Yakushi, T., Yang, J., Fukuoka, H., Homma, M. & Blair, D. F. Roles of charged residues of rotor and stator in flagellar rotation: Comparative study using H+-driven and Na+-driven motors in Escherichia coli. J. Bacteriol. 188, 1466–1472 (2006).
21. Takekawa, N., Kojima, S. & Homma, M. Contribution of many charged residues at the stator-rotor interface of the Na+-driven flagellar motor to torque generation in Vibrio alginolyticus. J. Bacteriol. 196, 1377–1385 (2014).
22. Brown, P. N., Hill, C. P. & Blair, D. F. Crystal structure of the middle and C-terminal domains of the flagellar rotor protein FliG. EMBO J. 21, 3225–3234 (2002).
23. Brown, P. N., Terrazas, M., Paul, K. & Blair, D. F. Mutational analysis of the flagellar protein FliG: Sites of interaction with FliM and implications for organization of the switch complex. J. Bacteriol. 189, 305–312 (2007).
24. Paul, K., Brunstetter, D., Titen, S. & Blair, D. F. A molecular mechanism of direction switching in the flagellar motor of Escherichia coli. Proc. Natl. Acad. Sci. U. S. A. 108, 17171–17176 (2011).
25. Carroll, B. L. et al. The flagellar motor of Vibrio alginolyticus undergoes major structural remodeling during rotational switching. Elife 9, 1–19 (2020).
26. Vartanian, A. S., Pazs, A., Fortgang, E. A., Abramsons, J. & Dahlquist, F. W. Structure of flagellar motor proteins in complex allows for insights into motor structure and switching. J. Biol. Chem. 287, 35779–35783 (2012).
27. Minamino, T. et al. Structural insight into the rotational switching mechanism of the bacterial flagellar motor. PLoS Biol. 9, (2011).
28. dosSantos, R. N., Khan, S. & Morcos, F. Characterization of C-ring component assembly in flagellar motors from amino acid coevolution. R. Soc. Open Sci. 5, (2018).
29. Brown, P. N., Mathews, M. A. A., Joss, L. A., Hill, C. P. & Blair, D. F. Crystal structure of the flagellar rotor protein FliN from Thermotoga maritima. J. Bacteriol. 187, 2890–2902 (2005).
30. Reid, S. W. et al. The maximum number of torque-generating units in the flagellar motor of Escherichia coli is at least 11. Proc. Natl. Acad. Sci. U. S. A. 103, 8066–71 (2006).
31. Chen, S. et al. Structural diversity of bacterial flagellar motors. EMBO J. 30, 2972–81 (2011).
32. Beeby, M. et al. Diverse high-torque bacterial flagellar motors assemble wider stator rings using a conserved protein scaffold. Proc. Natl. Acad. Sci. 113, E1917–E1926 (2016).
33. Santiveri, M. et al. Structure and function of stator units of the bacterial flagellar motor. Cell 183, 244-257.e16 (2020).
34. Deme, J. C. et al. Structures of the stator complex that drives rotation of the bacterial flagellum. Nat. Microbiol. 5, 1553–1564 (2020).
35. Zhu, S. et al. Conformational change in the periplamic region of the flagellar stator coupled with the assembly around the rotor. Proc. Natl. Acad. Sci. U. S. A. 111, 13523–8 (2014).
36. Thormann, K. M. & Paulick, A. Tuning the flagellar motor. Microbiology 156, 1275–1283 (2010).
37. Paulick, A. et al. Dual stator dynamics in the Shewanella oneidensis MR-1 flagellar motor. Mol. Microbiol. 1–9 (2015). doi:10.1111/mmi.12984
38. Ito, M., Terahara, N., Fujinami, S. & Krulwich, T. A. Properties of motility in Bacillus subtilis powered by the H+-coupled MotAB flagellar stator, Na+-coupled MotPS or hybrid stators MotAS or MotPB. J. Mol. Biol. 352, 396–408 (2005).
39. Asai, Y., Kawagishi, I., Sockett, R. E. & Homma, M. Hybrid motor with H+- and Na+-driven components can rotate vibrio polar flagella by using sodium ions. J. Bacteriol. 181, 6332–6338 (1999).
40. Yorimitsu, T., Asai, Y., Sato, K. & Homma, M. Intermolecular cross-linking between the periplasmic Loop3-4 regions of PomA, a component of the Na+-driven flagellar motor of Vibrio alginolyticus. J. Biol. Chem. 275, 31387–31391 (2000).
41. Zhou, J. et al. Function of protonatable residues in the flagellar motor of Escherichia coli: A critical role for Asp 32 of MotB. J. Bacteriol. 180, 2729–2735 (1998).
42. Hosking, E. R., Vogt, C., Bakker, E. P. & Manson, M. D. The Escherichia coli MotAB proton channel unplugged. J. Mol. Biol. 364, 921–937 (2006).
43. Li, N., Kojima, S. &Homma, M. Characterization of the periplasmic region of PomB, a Na+-driven flagellar stator protein in Vibrio alginolyticus. J. Bacteriol. 193, 3773–3784 (2011).
44. Takekawa, N. et al. Na+ conductivity of the Na+-driven flagellar motor complex composed of unplugged wild-type or mutant PomB with PomA. J. Biochem. 153, 441–451 (2013).
45. Kojima, S. et al. Stator assembly and activation mechanism of the flagellar motor by the periplasms region of MotB. Mol. Microbiol. 73, 710–718 (2009).
46. Kojima, S. et al. The helix rearrangement in the periplasmic domain of the flagellar stator B subunit activates peptidoglycan binding and ion influx. Structure 26, 590-598.e5 (2018).
47. Mandadapu, K. K., Nirody, J. a, Berry, R. M. & Oster, G. Mechanics of torque generation in the bacterial flagellar motor. Proc. Natl. Acad. Sci. U. S. A. 1–18 (2015). doi:10.1073/pnas.1501734112
48. Zhou, J., Lloyd, S. A. & Blair, D. F. Electrostatic interactions between rotor and stator in the bacterial flagellar motor. Proc. Natl. Acad. Sci. U. S. A. 95, 6436–41 (1998).
49. Michael Silverman & Melvin Simon. Flagellar rotation and the mechanism of bacterial motility. Nature 249, 73–74 (1974).
50. Berg, H. C. & Tedesco, P. M. Transient response to chemotactic stimuli in Escherichia coli. Proc. Natl. Acad. Sci. 72, 3235–3239 (1975).
51. Inoue, Y. Rotation measurements of tethered cells. in The Bacterial Flagellum 163–174 (2017). doi:10.1007/978-1-4939-6927-2_12
52. Ryu, W. S., Berry, R. M. & Berg, H. C. Torque-generating units of the flagellar motor of Escherichia coli have a high duty ratio. Nature 403, 444–7 (2000).
53. Yuan, J. & Berg, H. C. Resurrection of the flagellar rotary motor near zero load. Proc. Natl. Acad. Sci. U. S. A. 105, 1182–1185 (2008).
54. Nord, A. L. et al. Catch bond drives stator mechanosensitivity in the bacterial flagellar motor. Proc. Natl. Acad. Sci. 0, 201716002 (2017).
55. Kasai, T. & Sowa, Y. Measurements of the rotation of the flagellar motor by bead assay. in 185–192 (2017). doi:10.1007/978-1-4939-6927-2_14
56. Berg, H. C. & Turner, L. Torque Generated by the Flagellar Motor of Escherichia coil. 65, (1993).
57. Chen, X. & Berg, H. C. Torque-speed relationship of the flagellar rotary motor of Escherichia coli. Biophys. J. 78, 1036–1041 (2000).
58. Inoue, Y. et al. Torque–speed relationships of Na+-driven chimeric flagellar motors in Escherichia coli. J. Mol. Biol. 376, 1251–1259 (2008).
59. Blair, D. & Berg, H. Restoration of torque in defective flagellar motors. Science (80-. ). 242, 1678–1681 (1988).
60. Manson, M. D., Tedesco, P., Berg, H. C., Harold, F. M. & Van derDrift, C. A protonmotive force drives bacterial flagella. Proc. Natl. Acad. Sci. U. S. A. 74, 3060–4 (1977).
61. Felle, H., Porter, J. S., Slayman, C. L. & Kaback, H. R. Quantitative measurements of membrane potential in Escherichia coli. Biochemistry 19, 3585–3590 (1980).
62. Zilberstein, D., Agmon, V., Schuldiner, S. & Padan, E. Escherichia coli intracellular pH, membrane potential, and cell growth. J. Bacteriol. 158, 246–252 (1984).
63. Bot, C. T. & Prodan, C. Quantifying the membrane potential during E. coli growth stages. Biophys. Chem. 146, 133–137 (2010).
64. Fung, D. C. & Berg, H. C. Powering the flagellar motor of Escherichia coli with an external voltage source. Nature 375, 809–812 (1995).
65. Gabel, C.V. & Berg, H. C. The speed of the flagellar rotary motor of Escherichia coli varies linearly with protonmotive force. Proc. Natl. Acad. Sci. U. S. A. 100, 8748–8751 (2003).
66. Lo, C.-J., Leake, M. C., Pilizota, T. & Berry, R. M. Nonequivalence of membrane voltage and ion-gradient as driving forces for the bacterial flagellar motor at low load. Biophys. J. 93, 294–302 (2007).
67. Minamino, T., Imae, Y., Oosawa, F., Kobayashi, Y. & Oosawa, K. Effect of intracellular pH on rotational speed of bacterial flagellar motorst. J. Bacteriol. 185, 1190–1194 (2003).
68. Meister, M., Lowe, G. & Berg, H. C. The proton flux through the bacterial flagellar motor. Cell 49, 643–650 (1987).
69. Asai, Y., Yakushi, T., Kawagishi, I. & Homma, M. Ion-coupling determinants of Na+-driven and H+-driven flagellar motors. J. Mol. Biol. 327, 453–463 (2003).
70. Lo, C.-J., Leake, M. C. & Berry, R. M. Fluorescence measurement of intracellular sodium concentration in single Escherichia coli cells. Biophys. J. 90, 357–365 (2006).
71. Nord, A. L., Sowa, Y., Steel, B. C., Lo, C.-J. & Berry, R. M. Speed of the bacterial flagellar motor near zero load depends on the number of stator units. Proc. Natl. Acad. Sci. 114, 11603–11608 (2017).
72. Leake, M. C. et al. Stoichiometry and turnover in single, functioning membrane protein complexes. Nature 443, 355–358 (2006).
73. Delalez, N. J. et al. Signal-dependent turnover of the bacterial flagellar switch protein FliM. Proc. Natl. Acad. Sci. U. S. A. 107, 11347–51 (2010).
74. Delalez, N. J., Berry, R. M. & Armitage, J. P. Stoichiometry and Turnover of the Bacterial Flagellar Switch Protein. (2014). doi:10.1128/mBio.01216-14.Updated
75. Lele, P. P., Hosu, B. G. & Berg, H. C. Dynamics of mechanosensing in the bacterial flagellar motor. Proc. Natl. Acad. Sci. U. S. A. 110, 11839–44 (2013).
76. Tipping, M. J., Delalez, N. J., Lim, R., Berry, R. M. & Armitage, J. P. Load-dependent assembly of the bacterial flagellar motor. MBio 4, 1–6 (2013).
77. Wadhwa, N., Phillips, R. & Berg, H. C. Torque-dependent remodeling of the bacterial flagellar motor. Proc. Natl. Acad. Sci. 116, 201904577 (2019).
78. Schoenhals, G. J. & Macnab, R. M. FliL is a membrane-associated component of the flagellar basal body of Salmonella. Microbiology 145, 1769–1775 (1999).
79. Motaleb, M. A., Pitzer, J. E., Sultan, S. Z. & Liu, J. A novel gene inactivation system reveals altered periplasmic flagellar orientation in a Borrelia burgdorferi flil mutant. J. Bacteriol. 193, 3324–3331 (2011).
80. Partridge, J. D., Nieto, V. & Harshey, R. M. A new player at the flagellar Motor: FliL controls both motor output and bias. MBio 6, e02367-14 (2015).
81. Rajagopala, S.V. et al. The protein network of bacterial motility. Mol. Syst. Biol. 3, 128 (2007).
82. Suaste-Olmos, F. et al. The flagellar protein FliL is essential for swimming in Rhodobacter sphaeroides. J. Bacteriol. 192, 6230–6239 (2010).
83. Li, H. & Sourjik, V. Assembly and stability of flagellar motor in Escherichia coli. Mol. Microbiol. 80, 886–899 (2011).
84. Zhu, S., Kumar, A., Kojima, S. & Homma, M. FliL associates with the stator to support torque generation of the sodium-driven polar flagellar motor of Vibrio. Mol. Microbiol. 98, 101–110 (2015).
85. Attmannspacher, U., Scharf, B. E. & Harshey, R. M. FliL is essential for swarming: Motor rotation in absence of FliL fractures the flagellar rod in swarmer cells of Salmonella enterica. Mol. Microbiol. 68, 328–341 (2008).
86. Belas, R. & Suvanasuthi, R. The Ability of proteus mirabilis to sense surfaces and regulate virulence gene expression Involves FliL , a Flagellar Basal Body Protein. J. Bacteriol. 187, 6789–6803 (2005).
87. Fukuoka, H., Wada, T., Kojima, S., Ishijima, A. & Homma, M. Sodium-dependent dynamic assembly of membrane complexes in sodium-driven flagellar motors. Mol. Microbiol. 71, 825–835 (2009).
88. Tipping, M. J., Steel, B. C., Delalez, N. J., Berry, R. M. & Armitage, J. P. Quantification of flagellar motor stator dynamics through in vivo proton-motive force control. Mol. Microbiol. 87, 338–347 (2013).
89. Suzuki, Y. et al. Effect of the MotA(M206I) mutation on torque generation and stator Assembly in the Salmonella H+-driven fagellar motor. J. Bacteriol. 201, 1–14 (2019).
90. Morimoto, Y.V., Nakamura, S., Kami-Ike, N., Namba, K. & Minamino, T. Charged residues in the cytoplasmic loop of MotA are required for stator assembly into the bacterial flagellar motor. Mol. Microbiol. 78, 1117–1129 (2010).
91. Sowa, Y. et al. Direct observation of steps in rotation of the bacterial flagellar motor. Nature 437, 916–919 (2005).
92. Hata, H. et al. High pressure inhibits signaling protein binding to the flagellar motor and bacterial chemotaxis through enhanced hydration. Sci. Rep. 10, 2351 (2020).
93. Okunishi, I., Kawagishi, I. & Homma, M. Cloning and characterization of motY, a gene coding for a component of the sodium-driven flagellar motor in Vibrio alginolyticus. J. Bacteriol. 178, 2409–2415 (1996).
94. Kitaoka, M. et al. A novel dnaJ family gene, sfLA, encodes an inhibitor of flagellation in marine vibrio species. J. Bacteriol. 195, 816–822 (2013).
95. Lin, T.-S., Zhu, S., Kojima, S., Homma, M. & Lo, C.-J. FliL association with flagellar stator in the sodium-driven Vibrio motor characterized by the fluorescent microscopy. Sci. Rep. 8, 11172 (2018).
96. Pilizota, T. & Shaevitz, J. W. Fast, multiphase volume adaptation to hyperosmotic shock by Escherichia coli. PLoS One 7, e35205 (2012).
97. Fukuoka, H., Yakushi, T., Kusumoto, A. & Homma, M. Assembly of motor proteins, PomA and PomB, in the Na+-driven stator of the flagellar motor. J. Mol. Biol. 351, 707–17 (2005).
98. Cras, J. J., Rowe-Taitt, C. A., Nivens, D. A. & Ligler, F. S. Comparison of chemical cleaning methods of glass in preparation for silanization. Biosens. Bioelectron. 14, 683–688 (1999).
99. Nishiyama, M. & Kojima, S. Bacterial motility measured by a miniature chamber for high-pressure microscopy. Int. J. Mol. Sci. 13, 9225–39 (2012).
100. Kiernan, J. A. Formaldehyde, Formalin, Paraformaldehyde And Glutaraldehyde: What They Are And What They Do. Micros. Today 8, 8–13 (2000).
101. Visscher, K., Gross, S. P. & Block, S. M. Construction of multiple-beam optical traps with nanometer-resolution position sensing. IEEE J. Sel. Top. Quantum Electron. 2, 1066–1076 (1996).
102. Gittes, F. & Schmidt, C. F. Interference model for back-focal-plane displacement detection in optical tweezers. Opt. Lett. 23, 7 (1998).
103. Farré, A., Marsà, F. & Montes-Usategui, M. Optimized back-focal-plane interferometry directly measures forces of optically trapped particles. Opt. Express 20, 12270 (2012).
104. Axelrod, D., Thompson, N. L. & Burghardt, T. P. Total internal reflection fluorescent microscopy. J. Microsc. 129, 19–28 (1983).
105. Axelrod, D. Chapter 9 Total internal reflection fluorescence microscopy. in Encyclopedia of Cell Biology 2, 245–270 (Elsevier, 1989).
106. Tokunaga, M., Kitamura, K., Saito, K., Iwane, A. H. &Yanagida, T. Single molecule imaging of fluorophores and enzymatic reactions achieved by objective-type total internal reflection fluorescence microscopy. Biochem. Biophys. Res. Commun. 235, 47–53 (1997).
107. Funatsu, T., Harada, Y., Tokunaga, M., Saito, K. &Yanagida, T. Imaging of single fluorescent molecules and individual ATP turnovers by single myosin molecules in aqueous solution. Nature 374, 555–559 (1995).
108. Tokunaga, M., Imamoto, N. & Sakata-Sogawa, K. Highly inclined thin illumination enables clear single-molecule imaging in cells. Nat. Methods 5, 159–161 (2008).
109. Diaspro, A. Optical Fluorescence Microscopy. Advancement Of Science 138, (Springer Berlin Heidelberg, 2011).
110. Bulinski, J. C., Odde, D. J., Howell, B. J., Salmon, T. D. & Waterman-Storer, C. M. Rapid dynamics of the microtubule binding of ensconsin in vivo. J. Cell Sci. 114, 3885–3897 (2001).
111. Betzig, E. Proposed method for molecular optical imaging. Opt. Lett. 20, 237 (1995).
112. Yildiz, A. Myosin V walks hand-over-hand: Single fluorophore imaging with 1.5-nm localization. Science (80-. ). 300, 2061–2065 (2003).
113. Dickson, R. M., Cubitt, A. B. & Tsien, R. Y. On / off blinking and switching behaviour of single molecules of green fluorescent protein. Nature 388, 355–358 (1997).
114. Patterson, G. H. & Lippincott-Schwartz, J. A photoactivatable GFP for selective photolabeling of proteins and cells. Science 297, 1873–7 (2002).
115. Betzig, E. et al. Imaging intracellular fluorescent proteins at nanometer resolution. Science 313, 1642–5 (2006).
116. Thompson, R. E., Larson, D. R. & Webb, W. W. Precise nanometer localization analysis for individual fluorescent probes. Biophys. J. 82, 2775–83 (2002).
117. Lacoste, T. D. et al. Ultrahigh-resolution multicolor colocalization of single fluorescent probes. Proc. Natl. Acad. Sci. U. S. A. 97, 9461–9466 (2000).
118. Tang, H. & Blair, D. F. Regulated underexpression of the FliM protein of Escherichia coli and evidence for a location in the flagellar motor distinct from the MotA/MotB torque generators. J. Bacteriol. 177, 3485–3495 (1995).
119. Chao, Y. & Z hang, T. Optimization of fixation methods for observation of bacterial cell morphology and surface ultrastructures by atomic force microscopy. Appl. Microbiol. Biotechnol. 92, 381–92 (2011).
120. Joosen, L., Hink, M. A., Gadella, T. W. J. & Goedhart, J. Effect of fixation procedures on the fluorescence lifetimes of Aequorea victoria derived fluorescent proteins. J. Microsc. 256, 166–176 (2014).
121. Berezin, M. Y. &Achilefu, S. Fluorescence lifetime measurements and biological imaging. Chem. Rev. 110, 2641–2684 (2010).
122. Patterson, G. H. &Lippincott-Schwartz, J. A photoactivatable GFP for selective photolabeling of proteins and cells. Science 297, 1873–7 (2002).
123. Heo, M. et al. Impact of fluorescent protein fusions on the bacterial flagellar motor. Sci. Rep. 7, 1–10 (2017).
124. Dickson, R. M., Cubittt, A. B., Tsient, R. Y. &Moerner, W. E. On/off blinking and switching behaviour of single molecules of green fluorescent protein. Nature 388, 355–358 (1997).
125. Leake, M. C., Wilson, D., Gautel, M. &Simmons, R. M. The elasticity of single titin molecules using a two-bead optical tweezers assay. Biophys. J. 87, 1112–1135 (2004).
126. Svoboda, K., Schmidt, C. F., Schnapp, B. J. &Block, S. M. Direct observation of kinesin stepping by optical trapping interferometry. Nature 365, 721–727 (1993).
127. Zhu, S. et al. Molecular architecture of the sheathed polar flagellum in Vibrio alginolyticus. Proc. Natl. Acad. Sci. 201712489 (2017). doi:10.1073/pnas.1712489114
128. Wayne Niblack. An Introduction to Digital Image Processing. (Prentice Hall, 1986).
129. Sauvola, J. &Pietikäinen, M. Adaptive document image binarization. Pattern Recognit. 33, 225–236 (2000).
130. Kumar, M., Mommer, M. S. & Sourjik, V. Mobility of cytoplasmic, membrane, and DNA-binding proteins in Escherichia coli. Biophys. J. 98, 552–559 (2010).
131. Oswald, F., Varadarajan, A., Lill, H., Peterman, E. J. G. & Bollen, Y. J. M. MreB-dependent organization of the E. coli cytoplasmic membrane controls membrane protein diffusion. Biophys. J. 110, 1139–1149 (2016).
132. Castle, A. M., Macnab, R. M. & Shulman, R. G. Coupling between the sodium and proton gradients in respiring Escherichia coli cells measured by 23Na and 31P nuclear magnetic resonance. J. Biol. Chem. 261, 7797–7806 (1986).
133. Nirody, J. A., Berry, R. M. & Oster, G. The limiting speed of the bcterial flagellar Motor. Biophys. J. 111, 557–564 (2016).
134. Wang, B., Zhang, R. & Yuan, J. Limiting (zero-load) speed of the rotary motor of Escherichia coli is independent of the number of torque-generating units. Proc. Natl. Acad. Sci. 114, 201713655 (2017).
135. Khan, S., Dapice, M. & Reese, T. S. Effects of mot gene expression on the structure of the flagellar motor. J. Mol. Biol. 202, 575–584 (1988).
136. Samuel, A. D. T. & Berg, H. C. Torque-generating units of the bacterial flagellar motor step independently. Biophys. J. 71, 918–923 (1996).
137. Block, S. M. & Berg, H. C. Successive incorporation of force-generating units in the bacterial rotary motor. Nature 309, 470–472 (1984).
138. Van DenHeuvel, M. G. L. & Dekker, C. Motor proteins at work for nanotechnology. Science (80-. ). 317, 333–336 (2007).
139. Turner, L., Ryu, W. S. & Berg, H. C. Real-time imaging of fluorescent flagellar filaments. J. Bacteriol. 182, 2793–2801 (2000).
140. Xie, L., Altindal, T., Chattopadhyay, S. & Wu, X. L. Bacterial flagellum as a propeller and as a rudder for efficient chemotaxis. Proc. Natl. Acad. Sci. U. S. A. 108, 2246–2251 (2011).
141. Zhou, X. & Roujeinikova, A. The structure, composition, and role of periplasmic stator scaffolds in polar bacterial flagellar motors. Front. Microbiol. 12, (2021).
142. Nishino, Y., Onoue, Y., Kojima, S. & Homma, M. Functional chimeras of flagellar stator proteins between E. coli MotB and Vibrio PomB at the periplasmic region in Vibrio or E. coli. Microbiologyopen 4, 323–331 (2015).
143. Sowa, Y. & Berry, R. M. Bacterial flagellar motor. Q. Rev. Biophys. 41, 103–32 (2008).
144. Armitage, J. P. & Berry, R. M. Assembly and dynamics of the bacterial flagellum. Annu. Rev. Microbiol. 74, 181–200 (2020).
145. Goodwin, J. S. & Kenworthy, A. K. Photobleaching approaches to investigate diffusional mobility and trafficking of Ras in living cells. Methods 37, 154–164 (2005).
146. Prescher, J. Assembly and optimization of a super-resolution STORM microscope for nanoscopic imaging of biological structures. (LMU München, 2016).
147. Chung, S. H. & Kennedy, R. A. Forward-backward non-linear filtering technique for extracting small biological signals from noise. J. Neurosci. Methods 40, 71–86 (1991).
148. Thompson, R. E., Larson, D. R. & Webb, W. W. Precise Nanometer Localization Analysis for Individual Fluorescent Probes. Biophys. J. 82, 2775–2783 (2002).

指導教授

羅健榮(Chien-Jung Lo)

審核日期

2021-10-25

推文