Probing Ion-Flux of Bacterial Flagellar Motors by Correlative Microscopy

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：27

、訪客IP：18.226.98.128

姓名

曾昭凱(Chao-Kai Tseng) 查詢紙本館藏

畢業系所

物理學系

論文名稱

(Probing Ion-Flux of Bacterial Flagellar Motors by Correlative Microscopy)

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	★ Dynamics of sodium-driven stator units in bacterial flagellar motors
★ 高密度二維群游細菌系統之動力學	★ Deformation Dynamics of Active 2D Tetragonal Pseudo-Crystal
★ 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)

細菌可以藉由旋轉鞭毛來游向營養物質或避免毒素，其中鞭毛由稱為細菌鞭毛馬達的蛋白質複合物所驅動。細菌鞭毛馬達是一種分子機器，它在黏性環境中為游泳運動提供推進力。如同許多旋轉馬達一樣，細菌鞭毛馬達的結構也可分為轉子和定子。細菌鞭毛馬達上的定子利用儲存橫跨在細胞膜上的電化學梯度中的能量與轉子交互作用藉以產生機械扭矩。然而，鞭毛運動能量轉換的機制仍然被了解甚少。我們對離子流入與鞭毛馬達旋轉之間的關係很感興趣。有許多實驗結果提出細菌鞭毛馬達的旋轉可能與通過鞭毛馬達的離子通量緊密耦合。然而，沒有任何證據已被直接觀測到。
為了驗證細菌鞭毛馬達的緊密耦合模型，我們設計了一種新的相關顯微鏡來研究細菌鞭毛馬達的機制。相關顯微鏡結合了單細胞測量的三個重要功能，即活體單細胞鈉離子濃度的測量、即時細菌鞭毛馬達轉速的測量和馬達運動的控制。我們藉由附著在馬達萬象軸上的磁珠子來使用磁力鑷子操縱細菌鞭毛馬達的運動。背焦面偵測器可以在磁鑷施加外部扭矩時同時測量鞭毛馬達的旋轉速度。最後，細胞體內的鈉離子濃度可以通過鈉離子螢光指示劑和螢光顯微鏡觀察。
細胞內鈉離子濃度是鈉離子流進與流出的平衡。大腸桿菌具有部份鈉離子調控的能力。我們測量了當磁力鑷子加快或減慢鞭毛馬達旋轉時細胞體內鈉離子濃度的變化。結果表明在加速操作中鈉離子濃度增加了大約 20%。但是，減速操作中並未顯示鈉離子濃度發生顯著變化。我們推測鈉離子的變化可能被固定子單元的組裝所抵消了。在我們的實驗中觀察到的證據揭示了鈉離子流入與鞭毛馬達旋轉之間的緊密耦合關係。未來還需要利用相關顯微鏡來研究，用以完成理解細菌鞭毛馬達的能量使用。

摘要(英)

Bacteria can swim to the nutrient or avoid toxin by rotating their flagella, where the flagella are driven by a protein complexes called bacterial flagellar motor. Bacterial flagellar motor is a molecular machine that produces propulsive force for swimming motility in the viscous environments. Like many rotary motors, the structure of flagellar motor can also be divided into rotor and stator. The stator of flagellar motor utilizes the energy stored in the electrochemical gradient across the cytoplasmic membrane interacting with rotor to generate mechanical torque. However, the mechanism of flagellar motor energy conversion remains poorly understood. We are interested in the relationship between the ion influx to the motor rotation. There are experimental evidences supporting that the motor rotation tightly couples with the ion flux through the motor. However, there is no direct test has been made.
To verify the tight-coupling model of bacterial flagellar motors, we designed a new correlative microscope to investigate the mechanism of bacterial flagellar motor. The correlative microscope combines three important functions for single-cell measurements, which are the live single-cell sodium-ion concentration measurement, instant bacterial flagellar motor rotational speed measurement, and the motion control of motor. We used the magnetic tweezers to manipulate the motion of flagellar motor via a magnetic bead attached to its hook. The back-focal-plane detection can simultaneously measure the rotational speed of motor when an external torque is applied by the magnetic tweezers. At the same time, the intracellular sodium-ion concentration is monitored via ion fluorescence indicator using fluorescence microscope.
The intracellular sodium-ion concentration is the balance of sodium influx and efflux. Escherichia coli has partial homeostasis of internal sodium-ion concentration. We measured the change of sodium-ion concentration inside a cell when the magnetic tweezers speed up or slow down the rotation of flagellar motor. The results showed an increase of approximately 20% of the sodium concentration in the accelerated operations. However, there was no significant change in intracellular sodium-ion concentration during the deceleration operations. We speculated that the change might be offset by the assembly of extra stator-units. The evidences observed in our experiment reveal the tight-coupling relationship between influx of ion to the rotation of flagellar motor. Further experiments using the correlative microscope are required for the complete understanding of bacterial flagellar motor energy transduction.

關鍵字(中)

★ 細菌鞭毛馬達
★ 相關顯微鏡
★ 鈉離子通量
★ 大腸桿菌

關鍵字(英)

★ bacterial flagellar motor
★ correlative microscopy
★ sodium-ion flux
★ E. coli strain MTB24 mini cell

論文目次

摘要 i
Abstract iii
Acknowledgement v
Content vi
1. Introduction 1
1-1 Bacterial flagellar motor 1
1-2 Stator – the torque-generating unit 3
1-2-1 Structure and function 3
1-2-2 Energy transduction 5
1-3 Mechanism of torque generation 7
1-3-1 Torque-speed relationships 7
1-3-2 Stator assembly 9
1-3-3 Stator remodeling 12
1-4 Models of rotary motor 14
1-4-1 Turnstile model 14
1-4-2 Proton turbine model 17
1-4-3 Inchworm model 18
1-5 External torque application 21
1-5-1 Electrorotation 21
1-5-2 Optical tweezer and wrench 22
1-5-3 Magnetic tweezer 23
2. Experimental Methods 26
2-1 Bacterial strain 26
2-2 Sample preparation 27
2-2-1 Bead assay 27
2-2-2 Protocol 29
2-3 Correlative microscopy 31
2-3-1 Applying external torque on BFM 31
2-3-2 Motion measurement of BFM 34
2-3-3 Sodium ion fluorescence indicator 36
3. Results 39
3-1 Motor numbers of mini cell 39
3-2 The loading efficiency of sodium indicator 40
3-3 Calibration of sodium indicator 42
3-4 Ion flux under external torque 44
4. Discussion 48
5. Reference 50

參考文獻

Ashkin, A., Dziedzic, J. M., Bjorkholm, J. E., and Chu, S. “Observation of a single-beam gradient force optical trap for dielectric particles.” Opt. Lett. Vol 11, pp. 288-290, 1986.
Berg, H. C. and Anderson, R. A. “Bacteria swim by rotating their flagellar filaments.” Nature. Vol 245, pp. 380-382, 1973.
Berg, H. C., and Turner, L. “Torque generated by the flagellar motor of Escherichia coli.” Biophys J. Vol 65, pp. 2201-2216, 1993.
Berg, H. C. “The rotary motor of bacterial flagella.” Annu Rev Biochem. Vol 72, pp. 19-54, 2003.
Berry, R. M. “Torque and switching in the bacterial flagellar motor. an electrostatic model.” Biophys J. Vol 64, pp. 961-973, 1993.
Berry, R. M. “Theories of rotary motors.” Philos Trans R Soc Lond B Biol Sci. Vol 355, pp. 503-509, 2000.
Blair, D. F., and Berg, H. C. “Mutations in the MotA protein of Escherichia coli reveal domains critical for proton conduction.” J Mol Biol. Vol 221, pp. 1433-1442, 1991.
Brown, M. T., Steel, B. C., Silvestrin, C., Wilkinson, D. A., Delalez, N. J., Lumb, C. N., Obara, B., Armitage, J. P., and Berry, R. M. “Flagellar hook flexibility is essential for bundle formation in swimming Escherichia coli cells.” J Bacteriol. Vol 194, pp. 3495-3501, 2012.
Chang, Y., Moon, K. H., Zhao, X., Norris, S. J., Motaleb, M. A., and Liu, J. “Structural insights into flagellar stator- rotor interactions.” Elife. Vol 8, 2019.
Chang, Y., Zhang, K., Carroll, B. L., Zhao, X., Charon, N. W., Norris, S. J., Motaleb, M. A., Li, C., and Liu, J. “Molecular mechanism for rotational switching of the bacterial flagellar motor.” Nat Struct Mol Biol. Vol 27, pp. 1041-1047, 2020.
Chen, X. and Berg, H. C. “Torque-Speed Relationship of the Flagellar Rotary Motor of Escherichia coli.” Biophys J. Vol 78, pp. 1036-1041, 2000.
Elston, T. C., and Oster, G. “Protein turbines. I: The bacterial flagellar motor.” Biophys J. Vol 73, pp. 703-721, 1997.
Farley, M. M., Hu, B., Margolin, W., and Liu, J. “Minicells, Back in Fashion.” J Bacteriol. Vol 198, pp. 1186-1195, 2016.
Farré, A., Marsà, F., and Montes-Usategui, M. “Optimized back-focal-plane interferometry directly measures forces of optically trapped particles.” Opt Express. Vol 20, pp. 12270-12291, 2012.
Gabel, C. V. and Berg, H. C. “The speed of the flagellar rotary motor of Escherichia coli varies linearly with protonmotive force.” PNAS. Vol 100, pp. 8748-8751, 2003.
Gittes, F., and Schmidt, C. F. “Interference model for back-focal-plane displacement detection in optical tweezers.” Opt Lett. Vol 23, pp. 7-9, 1998.
Inoue, Y., Lo, C. J., Fukuoka, H., Takahashi, H., Sowa, Y., Pilizota, T., Wadhams, G. H., Homma, M., Berry, R. M., and Ishijima, A. “Torque-Speed Relationships of Na^+-driven Chimeric Flagellar Motors in Escherichia coli.” J Mol Biol. Vol 376, pp. 1251-1259, 2008.
Khan, S. and Berg, H. C. “Isotope and thermal effects in chemiosmotic coupling to the flagellar motor of Streptococcus.” Cell. Vol 32, pp. 913-919, 1983.
Khan, S., Ivey, D.M., Krulwich, T. A. “Membrane ultrastructure of alkaliphilic Bacillus species studied by rapid-freeze electron microscopy.” J Bacteriol. Vol 174, pp. 5123-5126, 1992.
Kihara, M. and Macnab, R. M. “Cytoplasmic pH mediates pH taxis and weak-acid repellent taxis of bacteria.” J Bacteriol. Vol 145, pp. 1209-1221, 1981.
Kojima, S., and Blair, D. F. “Conformational change in the stator of the bacterial flagellar motor.” Biochemistry. Vol 40, pp. 13041-13050, 2001.
Kojima, S., Takao, M., Almira, G., Kawahara, I., Sakuma, M., Homma, M., Kojima, C., and Imada, K. “The helix rearrangement in the periplasmic domain of the flagellar stator B subunit activates peptidoglycan binding and ion influx.” Structure. Vol 26, pp. 590-598, 2018.
La Porta, A., Wang, M. D. “Optical torque wrench: angular trapping, rotation, and torque detection of quartz microparticles.” Phys Rev Lett. Vol 92, 2004.
Larsen, S. H., Adler, J., Gargus, J. J., and Hogg, R. W. “Chemomechanical coupling without ATP: the source of energy for motility and chemotaxis in bacteria.” PNAS. Vol 71, pp. 1239-1243, 1974.
Lele, P. P., Hosu, B. G., and Berg, H. C. “Dynamics of mechanosensing in the bacterial flagellar motor.” PNAS. Vol 110, pp. 11839-11844, 2013.
Lim, R. C. “Application of External Torque on the Bacterial Flagellar Motor.” St Catherine′s College - University of Oxford, degree of Doctor, 2015.
Lloyd, S. A., Tang, H., Wang, X., Billings, S. and Blair, D. F. “Torque generation in the flagellar motor of Escherichia coli: evidence of a direct role for FliG but not for FliM or FliN.” J Bacteriol. Vol 178, pp. 223-231, 1996.
Lo, C. J., Sowa, Y., Pilizota, T., Berry, R. M. “Mechanism and kinetics of a sodium-driven bacterial flagellar motor.” PNAS. Vol 110, pp. E2544-E2551, 2013.
Manson, M. D., Tedesco, P., Berg, H. C., Harold, F. M. and van der Drift, C. “A protonmotive force drives bacterial flagella.” PNAS. Vol 74, pp. 3060-3064, 1977.
Meister, M., Lowe, G. and Berg, H. C. “The proton flux through the bacterial flagellar motor.” Cell. Vol 49, pp. 643-650, 1987.
Moon, K. H., Zhao, X., Manne, A., Wang, J., Yu, Z., Liu, J., and Motaleb, M. A. “Spirochetes flagellar collar protein FlbB has astounding effects in orientation of periplasmic flagella, bacterial shape, motility, and assembly of motors in Borrelia burgdorferi.” Mol Microbiol. Vol 102, pp. 336-348, 2016.
Moon, K. H., Zhao, X., Xu, H., Liu, J., and Motaleb, M. A. “A tetratricopeptide repeat domain protein has profound effects on assembly of periplasmic flagella, morphology and motility of the Lyme disease spirochete Borrelia burgdorferi.” Mol Microbiol. Vol 110, pp. 634-647, 2018.
Morimoto, Y. V., Che, Y. S., Minamino, T., and Namba, K. “Proton-conductivity assay of plugged and unplugged MotA/B proton channel by cytoplasmic pHluorin expressed in Salmonella.” FEBS Lett. Vol 584, pp. 1268-1272, 2010.
Mosconi, F., Allemand, J. F., Croquette, V. “Soft magnetic tweezers: a proof of principle.” Rev Sci Instrum. Vol 82, 2011.
Nirody, J. A., Nord, A. L., and Berry, R. M. “Load-dependent adaptation near zero load in the bacterial flagellar motor.” J R Soc Interface. Vol 16, 2019.
Nord, A. L., Sowa, Y., Steel, B. C., Lo, C. J., and Berry, R. M. “Speed of the bacterial flagellar motor near zero load depends on the number of stator units.” PNAS. Vol 114, pp. 11603-11608, 2017.
Reid, S. W., Leake, M. C., Chandler, J. H., Lo, C. J., Armitage, J. P., and Berry, R. M. “The maximum number of torque-generating units in the flagellar motor of Escherichia coli is at least 11.” PNAS. Vol 103, pp. 8066-8071, 2006.
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. Vol 403, pp. 444-447, 2000.
Santiveri, M., Roa-Eguiara, A., Kühne, C., Wadhwa, N., Hu, H., Berg, H. C., Erhardt, M. and Taylor, N. M. I. “Structure and Function of Stator Units of the Bacterial Flagellar Motor.” Cell. Vol 183, pp. 244-257, 2020.
Silverman, M. and Simon, M. “Flagellar rotation and the mechanism of bacterial motility.” Nature. Vol 249, pp. 73-74, 1974.
Sowa, Y., Hotta, H., Homma, M., and Ishijima, A. “Torque-speed Relationship of the 〖Na〗^+-driven Flagellar Motor of Vibrio alginolyticus.” J Mol Biol. Vol 327, pp. 1043-1051, 2003.
Sowa, Y., Rowe, A. D., Leake, M. C., Yakushi, T., Homma, M., Ishijima, A., Berry, R. M. “Direct observation of steps in rotation of the bacterial flagellar motor.” Nature. Vol 437, pp. 916-919, 2005.
Sowa, Y., and Berry, R. M. “Bacterial flagellar motor.” Q Rev Biophys. Vol 41, pp. 103-132, 2008.
Suzuki, H., Yonekura, K. and Namba, K. “Structure of the rotor of the bacterial flagellar motor revealed by electron cryomicroscopy and single-particle image analysis.” J Mol Biol. Vol 337, pp. 105-113, 2004.
Van Oene, M. M., Dickinson, L. E., Cross, B., Pedaci, F., Lipfert, J., and Dekker, N. H. “Applying torque to the Escherichia coli flagellar motor using magnetic tweezers.” Sci Rep. Vol 7, 2017.
Wadhwa, N., Phillips, R., and Berg, H. C. “Torque-dependent remodeling of the bacterial flagellar motor.” PNAS. Vol 116, pp. 11764-11769, 2019.
Xue, R., Ma, Q., Baker, M. A. B., and Bai, F. “A Delicate Nanoscale Motor Made by Nature-The Bacterial Flagellar Motor.” Adv Sci (Weinh). Vol 2, 2015.

指導教授

羅健榮(Chien-Jung Lo)

審核日期

2021-6-30

推文