綠膿桿菌為一種機會性致病菌，可引發急性和慢性感染。PsrA是綠膿桿菌的轉錄調節因子，能控制此菌多種毒力相關基因的表現。在野生型PAO1菌株過表現psrA會在許多與群體感應調節相關之表型中顯示出差異，例如綠膿菌素產量的增加、自體誘導物N-acyl homoserine lactone之減少、酪蛋白酶活性降低以及泳動運動和群體移動能力減低。雖然我們發現過表現psrA能活化與綠膿菌素生物合成相關之基因表現，但凝膠電泳遷移分析（EMSA）顯示PsrA不與phzM、phzD2和phzS之啟動子結合，表示PsrA應該不是直接對這些基因進行調控。由於psrA過表達改變了多種與群體感應相關之表現型，且轉錄組分析結果顯示PsrA顯著下調群體感應相關基因lasR，藉由EMSA確認了PsrA可直接與lasR啟動子結合，表示PsrA直接抑制lasR的表現。利用lasR啟動子區域之連續的小片段DNA序列和特定點位突變之lasR啟動子區域DNA片段，找出確切的PsrA結合位點位於5'-TAAACGTTTGCTTA-3'。此外，長鏈脂肪酸－oleate能阻斷PsrA與lasR啟動子之結合，並且恢復psrA過表現菌株的表現型。本實驗證明了PsrA直接調節抑制lasR表現而間接調節了群體感應。而長鏈脂肪酸－oleate能夠阻斷PsrA與lasR啟動子的結合。

Pseudomonas aeruginosa is an opportunistic pathogen that can cause both acute and chronic infections. PsrA is a transcriptional regulator that regulates gene expression of several virulence-related genes in P. aeruginosa. Compared with the wild-type PAO1 strain, the psrA overexpression strain shows difference in several quorum sensing-regulated phenotypes including higher production of pyocyanin, reduced N-acyl homoserine lactone autoinducer, decreased caseinase activity, reduced swimming and swarming motility. Although pyocycanin biosynthetic genes were activated in the psrA overexpressing strain, electrophoretic mobility shift assay (EMSA) showed that PsrA did not bind to the promoter of phzM, phzD2, and phzS. Since psrA overexpression changes several quorum sensing-regulated phenotypes and transcriptome analysis showed that PsrA significantly down regulates quorum sensing related gene lasR, we used EMSA to check whether PsrA binds to the lasR promoter. The results proved that PsrA directly binds to the promoter of lasR. Several truncated and site-specific mutated DNA fragments of the promoter region of lasR were used to find out the exact PsrA binding site. The PsrA binding site was found to be located at 5’-TAAACGTTTGCTTA-3’. In addition, this study demonstrated that long-chain fatty acid oleate blocked the PsrA binding to the lasR promoter and restores several phenotypes of the psrA overexpression strain. This study proves that PsrA down regulates lasR expression and indirectly regulates quorum sensing. Also, long-chain fatty acid oleate can inhibit PsrA binding to lasR promoter.

摘要．．．．．．．i
ABSTRACT．．．．ii
誌謝．．．．．．．iii
縮寫字對照表．．．iv
目錄．．．．．．．vi
表目錄．．．．．．viii
圖目錄．．．．．．ix
壹、前言．．．．10
1.1綠膿桿菌．．．．10
1.2綠膿菌素．．．．11
1.3群體感應．．．．12
1.4 轉錄因子PsrA．．．13
1.5 研究目的．．．．14
貳、材料與方法．．．15
2.1 菌株與培養條件細胞培養液製備．．．．15
2.2 細胞培養條件．．．．．15
2.3 綠膿菌素之定量．．．．15
2.4質體 DNA 製備 ．．．．．16
2.5聚合酶連鎖反應．．．．．16
2.6 DNA純化．．．．．．．．17
2.7 PsrA 蛋白表現與純化．．17
2.7.1 IPTG 誘導基因表現．．17
2.7.2 His-tag 親和性管柱純化蛋白質．．．18
2.8聚丙烯醯胺膠體製備與電泳分離．．．19
2.8.1聚丙烯醯胺膠體製備．．．．．．．19
2.8.2聚丙烯醯胺膠體電泳分離．．．．．19
2.8.3考馬斯亮藍染色．．．．．．．．．．．20
2.9凝膠電泳遷移試驗．．．．．．．．．．．20
2.10 N-acyl homoserine lactones 訊號分子測試．．．21
2.11蛋白酶測試．．．．．．．．．．．．．．．．．．．21
2.12生物膜定量分析．．．．．．．22
2.13泳動運動能力分析．．．．．．22
2.14群體移動能力分析．．．．．．23
參、結果 ．．．．．．．．24
3.1 psrA過表現菌株產生大量綠膿菌素．．．．．24
3.2 PsrA蛋白之表現與純化．．．．．．．．．．24
3.3 PsrA不會與phz啟動子結合．．．．．．．．25
3.4 PsrA直接與lasR啟動子結合．．．．．．．．25
3.5 Oleate抑制PsrA與lasR啟動子的結合．．．．26
3.6過表現psrA基因使AHL之產生下降．．．．．．26
3.7過表現psrA基因使群體移動能力減低．．．．．27
3.8過表現psrA使酪蛋白酶產量減少．．．．．．．28
3.9過表現psrA使生物膜形成減慢與減少．．．．．28
肆、討論．．．．．．．．．30
伍、參考文獻．．．．．．．34
附錄．．．．．59

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