Azurin為與銅鍵結而能發生電子轉移並進行氧化還原作用的蛋白質，參與細菌去硝化作用的電子傳遞路徑。有關Azurin在電化學、物理特性及腫瘤治療等方面已被廣泛研究，例如能進入癌細胞，抑制其生長及產生細胞毒性。但是Azurin的生化特性對於綠膿桿菌的生物意義目前仍不清楚。在本篇研究中利用同源重組的方式建構azu (PA4922) 剔除株，藉由與PAO1的表現型差異揭露azu的生理功能。結果顯示Δazu的泳動及抽動運動能力、綠膿菌素、螢光菌素、螯鐵分子、胞外多醣產量及對戀臭假單孢菌的競爭能力皆低於PAO1；與PAO1相比Δazu之群體移動能力、酪蛋白酶、溶血能力、AHL訊號分子、生物膜形成及細胞毒性等皆增加。但酚紅指示劑之氧化還原試驗卻未觀察到Δazu與PAO1有明顯差異。為探討azu基因之轉錄調控，本研究利用GUS報導基因及EMSA試驗，證實轉錄活化因子PsrA對於azu的轉錄調控為間接性，而靜止期轉錄調控因子RpoS能鍵結於azu啟動子區域直接影響azu的表現。因先前的研究已知PsrA調控rpoS，因此結合本篇研究我們推論PsrA藉由RpoS調控azu的表現。ΔrpoS及Δazu之抽動性運動及AHL訊號分子的表現型相似。由免疫轉漬法分析得知Azurin可以分泌至胞外，ΔpsrA分泌的Azurin低於PAO1，但ΔrpoS分泌的Azurin高於PAO1。綜合本研究結果，發現azu可能涉及綠膿桿菌PAO1多種生理活性之調控，且RpoS可以鍵結在azu的上游調控azu的表現。

Azurin is a copper-containing redox protein. Previous studies have demonstrated that Azurin is involved in denitrification pathway. Recently, it was discovered to be capable of entering mammalian cells causing cell death. Nevertheless,how the biochemical properties correlates with the physiological properties of Azurin in Pseudomonas aeruginosa PAO1 is still unknown. The purpose of this study is to investigate the biological role of Azurin and its gene regulation. This study constructed an azu deletion mutant and examined its biological properteis. Significant effects on pyocyanin, pyoverdin, and biofilm production, and cytotoxicity to Int407 cells could be observed on the mutant. However, resazurin and phenol red redox assay did not reveal significant difference between PAO1 and Δazu mutant. Using β-glucuronidase reporter gene assay and electrophoretic mobility shift assay, it was revealed that transcriptional regulator PsrA does not regulate azu directly. On the other hand, RpoS, a stationary-phase sigma factor, could bind the azu promoter specifically.Based on a previous study reporting that PsrA regulates rpoS gene expression, we propose that PsrA regulates azu gene expression through RpoS. This notion was supported by the fact that the phenotypic trait of twitching motility and AHL production are consistent in ΔrpoS and Δazu. Western blot analysis illustrated that Azurin could be secreted to extracellular and ΔpsrA decreased Azurin production but not ΔrpoS. Though the function of Azurin has not been clarified, this study has established a strong basis for future analysis of the Azurin and azu regulation.

中文摘要........................................................................................................................ I Abstract........................................................................................................................II 誌謝............................................................................................................................. III
縮寫字對照表 ............................................................................................................ IV 目錄............................................................................................................................ VII 表目錄........................................................................................................................... X 圖目錄......................................................................................................................... XI 壹、 前言...................................................................................................................... 1
1.1 綠膿桿菌........................................................................................................... 1
1.2 Azurin................................................................................................................. 3
1.3 轉錄調控因子 PsrA.......................................................................................... 7
1.4 轉錄調控因子 RpoS......................................................................................... 9
1.5 本篇研究目的................................................................................................. 10

貳、 材料與方法 ....................................................................................................... 11

2.1 菌株、質體與生長環境................................................................................. 11
2.2 細胞與培養條件............................................................................................. 11
2.3 染色體 DNA 萃取.......................................................................................... 12
2.4 質體 DNA 製備.............................................................................................. 12
2.5 聚合酶連鎖反應............................................................................................. 13
2.6 限制酵素處理與接合作用............................................................................. 14
2.7 製備勝任細胞................................................................................................. 14
2.8 熱休克轉型法................................................................................................. 15
2.9 構築突變株..................................................................................................... 15
2.9.1 構築質體................................................................................................ 15
2.9.2 第一次同源重組.................................................................................... 16
2.9.3 第二次同源重組.................................................................................... 16
2.10 接合生殖....................................................................................................... 16

2.11 生長曲線測試............................................................................................... 17
2.12 泳動性測試................................................................................................... 17
2.13 群體移動能力測試....................................................................................... 18
2.14 抽動性運動測試........................................................................................... 18
2.15 螢光色素鑒定............................................................................................... 18
2.16 螢光色素定量測試....................................................................................... 19
2.17 綠膿菌素鑒定............................................................................................... 19
2.18 綠膿菌素定量測定....................................................................................... 19
2.19 螯鐵分子測試............................................................................................... 20
2.20 溶血性測試................................................................................................... 20
2.21 酪蛋白酶測試............................................................................................... 21
2.22 菌落形態測試............................................................................................... 21
2.23 生物膜定量分析........................................................................................... 21
2.24 N-acyl homoserine lactones 訊號分子測試................................................. 22
2.25 細胞毒殺測試............................................................................................... 22
2.25.1 處理細菌上清液.................................................................................. 22
2.25.2 處理細菌.............................................................................................. 23
2.26 細菌侵入性試驗........................................................................................... 24
2.27 細菌競爭試驗............................................................................................... 25
2.28 Resazurin 氧化還原指示劑測試................................................................... 25
2.29 酚紅指示劑測試........................................................................................... 26
2.30-葡萄糖苷酸酶報導基因試驗之標準曲線配置........................................ 26
2.31-葡萄糖苷酸酶報導基因試驗.................................................................... 26
2.32 IPTG 誘導基因表現...................................................................................... 27
2.33 His-tag 親和性管柱純化蛋白質................................................................... 28
2.34 聚丙烯醯胺膠體電泳................................................................................... 29
2.35 考馬斯亮藍染色........................................................................................... 29
2.36 蛋白質的透析............................................................................................... 30
2.37 凝膠電泳遷移試驗....................................................................................... 30
2.37.1 生物素標定 DNA 建立....................................................................... 30
2.37.2 製備凝膠電泳遷移試驗膠體.............................................................. 30
2.37.3 凝膠電泳遷移試驗.............................................................................. 31
2.38 抗體製備....................................................................................................... 32
2.39 免疫轉漬分析............................................................................................... 32

參、 結果.................................................................................................................... 34

3.1 Azurin 蛋白質功能區域分析.......................................................................... 34
3.2 構築 GUS 報導基因質體............................................................................... 34

3.3 綠膿桿菌 PAO1 與 psrA 剔除株對 azu 啟動子序列的調控能力................ 35
3.4 綠膿桿菌 PAO1 與 rpoS 剔除株對 azu 啟動子序列的調控能力................ 35
3.5 綠膿桿菌 PAO1 與 anr 剔除株中對 azu 啟動子序列的調控能力.............. 36
3.6 綠膿桿菌 PAO1 與 dnr 剔除株中對 azu 啟動子序列的調控能力.............. 37
3.7 RpoS 與 azu 啟動子區域結合調控 azu 基因表現......................................... 37
3.8 構築重組蛋白................................................................................................. 38
3.9 Azurin 蛋白質的表現與純化.......................................................................... 38
3.10 免疫轉漬分析綠膿桿菌 azu 過度表現株中 Azurin 分泌情形.................. 38
3.11 構築 azu 剔除株 ........................................................................................... 39
3.12 構築 azu 過度表現株與回補株................................................................... 40
3.13 綠膿桿菌 PAO1 野生株與 azu 剔除株 (Δazu) 的生長曲線測試............. 40
3.14 剔除 azu 基因對於綠膿桿菌 PAO1 泳動能力的影響 ............................... 40
3.15 剔除 azu 對於綠膿桿菌 PAO1 群體移動能力的影響 ............................... 41
3.16 剔除 azu 對於綠膿桿菌 PAO1 抽動性運動能力的影響 ........................... 42
3.17 剔除 azu 對於螢光菌素分泌的影響........................................................... 42
3.18 剔除 azu 對於綠膿菌素分泌的影響........................................................... 43
3.19 剔除 azu 對於綠膿桿菌 PAO1 螯鐵分子的影響 ....................................... 44
3.20 剔除 azu 對於綠膿桿菌 PAO1 溶血能力的影響 ....................................... 44
3.21 剔除 azu 對於綠膿桿菌 PAO1 酪蛋白酶活性的影響 ............................... 45
3.22 剔除 azu 對於綠膿桿菌 PAO1 菌落型態的影響 ....................................... 46
3.23 剔除 azu 對於綠膿桿菌 PAO1 生物膜形成影響 ....................................... 46
3.24 剔除 azu 對於綠膿桿菌 PAO1 產生自體誘導物 AHL 的影響 ................. 47
3.25 剔除 azu 對於綠膿桿菌 PAO1 細胞毒性的影響 ....................................... 48
3.26 剔除 azu 對於綠膿桿菌 PAO1 被巨噬細胞 RAW 264.7 吞噬能力的影響
................................................................................................................................ 49
3.27 剔除 azu 對於綠膿桿菌 PAO1 侵入人類腸道細胞 Int-407 之分析.......... 49
3.28 剔除 azu 對戀臭假單孢菌競爭能力的影響............................................... 50
3.29 剔除 azu 對於綠膿桿菌 PAO1 氧化還原能力的影響 ............................... 50
3.30 剔除 azu 對於綠膿桿菌 PAO1 進行反硝化作用的影響 ........................... 51

肆、 討論.................................................................................................................... 53


伍、 參考文獻 ........................................................................................................... 61

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