第六型分泌系統為革蘭氏陰性菌傳送有毒物質至目標對象的方式之一，在綠膿桿菌PAO1中，共有三套第六型分泌系統基因叢集，分別命名為H1-T6SS、H2-T6SS與H3-T6SS基因叢集。本實驗室先前的研究發現，sfa2 (PA1663) 為RpoN (σ54) 依賴性轉錄活化因子，在sfa2基因缺損突變菌株中，H2-T6SS相關基因表現量降低，因此本研究建構轉錄因子rpoN (PA4462) 基因缺損突變菌株 (ΔrpoN)，探討綠膿桿菌PAO1的H2-T6SS基因叢集是否確實透過Sfa2與RpoN兩種因子調控。利用GUS報導基因系統得知Sfa2與RpoN可以正向轉錄調控H2-T6SS基因叢集，透過凝膠電泳遷移分析得知，Sfa2與RpoN分別會鍵結在H2-T6SS基因叢集之上游序列432-bp至403-bp與672-bp至503-bp的位置。此外，透過GUS報導基因系統與免疫轉漬法釐清可能由H2-T6SS基因叢集調控的三組hcp/vgrG操作子，分別為PA0263/PA0262、PA1512/PA1511與PA5267/PA5266，由結果得知除PA0263/PA0262外，其餘兩組的操作子轉錄與轉譯結果皆會受Sfa2與RpoN影響，其中以PA5267/PA5266所受到的調控最為顯著，利用凝膠電泳遷移分析發現，只有RpoN會與此三組hcp/vgrG操作子之上游序列結合，Sfa2則不會。本研究中發現ΔrpoN的抽動運動性、泳動運動性、螯鐵能力、螢光素與綠膿素產量、生物膜生成皆低於綠膿桿菌野生株PAO1，溶血能力與群體感應能力則優於綠膿桿菌PAO1。綜合本研究的結果得知Sfa2和RpoN可共同正向調控H2-T6SS基因叢集與三組hcp/vgrG操作子，且RpoN調控綠膿桿菌PAO1眾多的生理活性表現。

Type VI secretion system (T6SS) is an apparatus for Gram-negative bacteria to deliver effector proteins directly into target cells. There are three T6SS gene clusters, named H1-, H2-, and H3-T6SS gene cluster, in Pseudomonas aeruginosa PAO1. The aim of this study is to elucidate the role of both sfa2 (PA1663), a putative RpoN transcriptional activator encoding gene located in the H2-T6SS gene cluster and rpoN (PA4462), a transcription factor in regulation of H2-T6SS. In previous studies, the expression of H2-T6SS gene cluster were down-regulated in sfa2 deletion mutant strain. This study constructed an rpoN deletion mutant strain (ΔrpoN) to elucidate the relationship between Sfa2 and RpoN in regulation of H2-T6SS gene expression. Analysis of the upstream region of H2-T6SS with β-glucuronidase (GUS) reporter gene assay confirmed that Sfa2 and RpoN co-regulate the expression of the H2-T6SS gene cluster. Using recombinant Sfa2 and RpoN in an electrophoretic motility shift assay, this study found that the Sfa2 binding site and RpoN binding site are located from nucleotide position -432 to -403 and -672 to -503, respectively in the upstream region of the H2-T6SS gene cluster. This study also showed that the expression of Hcp decreases in both the sfa2 and rpoN deletion mutant strains. By GUS reporter gene assay, PA5267 (hcpB) is the major one of three hcp genes (PA0263, PA1512 and PA5267) regulated by Sfa2 and RpoN. However, only RpoN can bind to the upstream refion of three hcp genes but not Sfa2. Compared to PAO1, twitching motility, swimming motility, iron chelation, the production of pyocyanin and pyoverdin, and biofilm formation reduced in ΔrpoN. Conversely, the hemolysis and quorum sensing ability increased in ΔrpoN. Combined results in this study, we suggest that Sfa2 interacts with RpoN to up-regulate both H2-T6SS gene cluster and hcp genes expression and RpoN plays an important role in regulating phenotypic traits in P. aeruginosa PAO1.

中文摘要 I
Abstract II
誌謝 III
縮寫字對照表 IV
目錄 VI
圖目錄 X
表目錄 XII
壹、 前言 1
1.1 綠膿桿菌 1
1.2 細菌的分泌系統 1
1.3 第六型分泌系統 2
1.3.1 第六型分泌系統的結構蛋白 3
1.3.2 第六型分泌系統的功能探討 5
1.4 第六型分泌系統的調控機制 8
1.4.1 雙因子調控系統 8
1.4.2 載鐵調控蛋白 8
1.4.3 群體感應 9
1.5 轉錄因子RpoN (σ54) 10
1.6 轉錄活化因子 11
1.7 研究目的 13
貳、 材料與方法 15
2.1 菌株、質體與生長環境 15
2.2 綠膿桿菌PAO1的RpoN轉錄活化因子之胺基酸序列分析 15
2.3 RpoN轉錄活化因子的親緣性分析 15
2.4 細菌染色體DNA萃取 16
2.5 聚合酶連鎖反應 16
2.6 小量質體DNA萃取 17
2.7 限制性酵素處理與接合作用 18
2.8 勝任細胞製備 18
2.9 熱休克轉型法 19
2.10 接合生殖 19
2.11 建構綠膿桿菌PAO1 rpoN基因缺損突變菌株 20
2.11.1 建構質體 20
2.11.2 第一次同源序列重組互換 20
2.11.3 第二次同源序列重組互換 21
2.12 β-葡萄醣苷酸酶報導基因系統建構與分析 21
2.12.1 建構帶有H2-T6SS基因叢集和hcp/vgrG操作子上游序列之GUS報導基因質體 21
2.12.2 β-葡萄醣苷酸酶活性測量 22
2.13 凝膠電泳遷移分析 23
2.13.1 增幅帶有biotin之DNA片段 23
2.13.2 建構蛋白質表現與利用His6-Tag親和性管柱純化 23
2.13.3 凝膠電泳遷移分析 24
2.14 綠膿桿菌PAO1對惡臭假單胞桿菌F1競爭能力分析 26
2.15 聚丙烯醯胺膠體製備與電泳分離 26
2.15.1 聚丙烯醯胺膠體製備 26
2.15.2 聚丙烯醯胺膠體電泳分離 27
2.16 免疫轉漬法分析綠膿桿菌PAO1胞內Hcp表現量 27
2.17 綠膿桿菌PAO1生長曲線分析 28
2.18 綠膿桿菌PAO1抽動運動能力分析 29
2.19 綠膿桿菌PAO1泳動運動能力分析 29
2.20 綠膿桿菌PAO1群體移動能力分析 29
2.21 綠膿桿菌PAO1酪蛋白分解酶效力分析 30
2.22 綠膿桿菌PAO1溶血能力分析 30
2.23 綠膿桿菌PAO1螯鐵能力分析 30
2.24 綠膿桿菌PAO1螢光素產量定性分析 31
2.25 綠膿桿菌PAO1綠膿素產量定性分析 31
2.26 綠膿桿菌PAO1綠膿素與螢光素產量定量分析 32
2.27 綠膿桿菌PAO1菌落形態與胞外多醣產量分析 32
2.28 綠膿桿菌PAO1生物膜形成的定量分析 32
2.29 綠膿桿菌PAO1群體感應的定性分析 33
參、 結果 34
3.1 綠膿桿菌PAO1的第六型分泌系統 34
3.2 建構rpoN基因缺損突變菌株 35
3.3 GUS報導基因系統分析RpoN對H2-T6SS基因叢集的轉錄調控機制 35
3.4 Sfa2與其他RpoN轉錄活化因子之胺基酸序列特性分析 36
3.5 Sfa2與各菌種之RpoN轉錄活化因子的親緣性分析 37
3.6 GUS報導基因系統分析Sfa2對H2-T6SS基因叢集的轉錄調控機制 37
3.7 重組蛋白質RpoN與Sfa2的純化與表現 38
3.8 凝膠電泳遷移分析尋找RpoN與Sfa2鍵結在H2-T6SS基因叢集上游序列的位置 38
3.9 綠膿桿菌PAO1對惡臭假單胞桿菌F1競爭能力分析 40
3.10 綠膿桿菌PAO1與突變菌株ΔrpoN、Δsfa2的Hcp表現量比較 40
3.11 GUS報導基因系統分析hcp/vgrG操作子的轉錄調控機制 42
3.12 凝膠電泳遷移分析尋找RpoN與Sfa2鍵結在hcp/vgrG操作子之上游序列的位置 43
3.13 綠膿桿菌PAO1與ΔrpoN生長曲線分析 44
3.14 剔除rpoN基因對綠膿桿菌PAO1抽動運動性的影響 45
3.15 剔除rpoN基因對綠膿桿菌PAO1泳動運動性的影響 45
3.16 剔除rpoN基因對綠膿桿菌PAO1群體移動性的影響 46
3.17 剔除rpoN基因對綠膿桿菌PAO1酪蛋白分解酶效力的影響 47
3.18 剔除rpoN基因對綠膿桿菌PAO1溶血能力的影響 47
3.19 剔除rpoN基因對綠膿桿菌PAO1螯鐵能力的影響 48
3.20 剔除rpoN基因對綠膿桿菌PAO1螢光素產量的影響 49
3.21 剔除rpoN基因對綠膿桿菌PAO1綠膿素產量的影響 49
3.22 剔除rpoN基因對綠膿桿菌PAO1菌落形態與胞外多醣產量的影響 50
3.23 剔除rpoN基因對綠膿桿菌PAO1生物膜形成的影響 51
3.24 剔除rpoN基因對綠膿桿菌PAO1群體感應的影響 52
肆、 討論 53
伍、 參考文獻 60

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