Probing Cell Wall Synthetic Dynamics by Bacterial Flagellar Motor in Escherichia coli

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孫翊仁(Yi-Jen Sun) 查詢紙本館藏

畢業系所

物理學系

論文名稱

(Probing Cell Wall Synthetic Dynamics by Bacterial Flagellar Motor in Escherichia coli)

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摘要(中)

細菌需要不斷分裂來確保種族的延續，由於技術上的困難，在這個動態的生長與分裂過程中，我們對於細菌DNA複製的了解比細胞壁生合成的過程來的詳盡許多。因此在細菌分裂的過程中，有個簡單卻基本的問題我們尚未能解答：「母細菌如何在自身插入新的細胞壁來生成兩個子細胞的細胞壁結構？」
細菌鞭毛馬達的整個結構從細胞內膜一路貫穿到細胞體外，穩固的鑲嵌在細胞壁之中。因此當細菌的細胞壁在生長時，馬達位置的移動只會與細菌插入細胞壁的方式有關。利用這個特性，我們發展了一套新方法，利用細菌鞭毛馬達作為細胞壁上的標記來了解細胞壁插入的機制。
我們將細菌鞭毛馬達標上螢光，並觀察在生長時馬達是如何位移的。利用馬達的位移，我們確認了細菌體中在生長時沒有細胞壁插入的部位以及其範圍。而當細菌在延長自身身長時，馬達在有細胞壁插入的部分會維持在同樣的相對位置，我們由此推論細菌身上任何部位細胞壁插入的速度都是等值的。在細菌進入分裂階段時，位於細菌中心的馬達會逐漸遠離中心，意味著新的細胞隔膜是完全由新的細胞壁材料所構成的。利用以上我們對細胞壁插入機制的了解，我們能利用Bernoulli-shift map來推測馬達在每一代細菌身上的位置。
Bernoulli-shift map平滑化的函數特性告訴我們馬達在細菌表面上的分布是由新生成的馬達所決定的。我們用不同顏色的螢光染劑標記了不同時間點出生的馬達，證明了馬達分布所呈現的凹函數(Concave function)分布是由於馬達傾向於生成在細菌中心所造成的。
有了螢光標記細菌鞭毛馬達這項技術讓我們追蹤馬達的動態，我們對細菌細胞壁生合成機制與穿膜蛋白的分布機制的探討將能有更多的發展性。

摘要(英)

Bacterial reproduction is a critical and dynamic life process. Here, we raise a simple yet fundamental question that how does the mother cell remodels the cell wall into two daughter cells? Compare to the DNA replication, we have far less understanding in the mechanism of cell wall remodeling due to the technical difficulties.
In this thesis, we develop a new approach using membrane anchored protein as landmarks to study the cell wall synthesis dynamics. Bacterial flagellar motors (BFM) are membrane protein complexes anchored firmly on the cell wall. Thus BFMs position changes along with cell growth depend solely on the spatiotemporal coordinate of the cell wall insertion.
By tracking fluorophore labeled BFMs in Escherichia coli while cell reproduce, we confirm the existence and determine the size of the cell wall growth inert zone. The normalized axial position of the motor remains constant while cell elongate, indicate a uniform axial cell wall insertion rate. During division, the mid-cell motors will be moved away from cell center indicating that the septum is completely formed by new cell wall material. With the understanding of the cell wall insertion, we built a modified Bernoulli-shift map to predict the position of the motors in each generation once it was formed.
The smoothing property of the Bernoulli-shift map also indicate the motor distribution only determine by the newly born motor. With sequentially labeling motors with different color fluorophore, we are able to distinguish the newly born motors and confirm the concave distribution of the BFM is contributed by the preference of the motors to synthesize in the cell center.
With this new experimental method, we open a new door to study the cell wall dynamics and membrane anchored proteins dynamics.

關鍵字(中)

★ 細菌細胞壁
★ 大腸桿菌
★ 細菌鞭毛馬達
★ 螢光顯微鏡

關鍵字(英)

★ Bacterial cell wall
★ Escherichia coli
★ Bacterial flagellar motor
★ Fluorescence microscopy

論文目次

摘要 i
Abstract ii
Acknowledgments iii
Glossary vi
Acronym vi
1 Introduction 1
1.1 Bacterial Cell Wall 1
1.1.1 Function of Bacteria Cell Wall 1
1.1.2 Types of Bacteria Cell Wall 1
1.1.3 Structure of Bacteria Cell Wall 2
1.2 Cell Wall Remodeling 5
1.2.1 Cell Wall Synthetic Complexes 5
1.2.2 Cell Wall Insertion 8
1.3 Bacterial Flagellar Motor (BFM) 11
1.3.1 Function and Structure 11
1.3.2 Flagellar Distribution 12
1.4 Aim of This Work 13
2 Material and Method 14
2.1 Bacterial Strain 14
2.1.1 Biotinylation 15
2.2 Fluorescent Dye 17
2.2.1 Streptavidin-Biotin Interaction 17
2.3 Growth Condition 19
2.3.1 Medium/Buffer 19
2.3.2 Growth Condition 19
2.4 Technique 20
2.4.1 Labelling Process 20
2.4.2 Poly-L-Lysine Slide 20
2.4.3 Gel Pad 21
2.5 Apparatus 21
2.5.1 Microscopy 21
2.5.2 Phase Contrast 22
2.5.3 Perfect Focus System (PFS) 23
2.5.4 Image Acquisition 24
2.6 Image Analysis 25
2.6.1 Positioning Center of the Motor 25
2.6.2 Cell Contour and Midline 25
2.6.3 Tracking Motor Position on the Cell 26
2.6.4 BFM Stability on the Cell Envelop 26
2.6.5 Inert Zone Detection 26
2.6.6 Cell Wall Insertion in Elongation 27
2.6.7 Cell Wall Insertion in Division 27
2.6.8 Predictable BFM Position between generations 27
2.6.9 Non-uniform BFM Distribution 28
3 Results 29
3.1 BFM Stability on the Cell Envelop 29
3.2 Inert Zone Detection 30
3.3 Cell Wall Insertion in Elongation 31
3.3.1 In Relative Axial Coordinate 31
3.3.2 Hubble’s Law in cell wall insertion 31
3.3.3 In Normalized Axial Coordinate 32
3.4 Cell Wall Insertion in Division 34
3.4.1 In Normalized Axial Coordinate 34
3.4.2 Direct Tracking of Newly Born Pole 35
3.5 Predictable BFM Position between generations 35
3.5.1 Cell growth and BFM positioning model 35
3.5.2 Predict BFM Position with Bernoulli Shift Map 36
3.6 Non-uniform BFM Distribution 38
3.6.1 Mono-Color Labelling 38
3.6.2 Simulation 38
3.6.3 Dual-Color Labelling 40
4 Discussion 41
Bibliographies 43
Appendix A 46
Filter Spectrum 46

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指導教授

羅健榮(Chien-Jung Lo)

審核日期

2019-8-24

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