臺灣博碩士論文加值系統

水平基因轉移 (horizontal gene transfer, HGT) 是細菌間交換遺傳物質的重要機制，藉由這個機制可以幫助獲得遺傳物質的細菌得到新的能力，像是增加對環境的適應力和增強致病能力等。在一些細菌的基因體中，有些藉由水平基因轉移的基因片段，被稱作是基因體小島 (genomic islands, GIs)，外來的基因小島模組與他所攜帶的生合成/代謝路徑如何融入細菌原有的基因調控網絡是我們感到好奇的。實驗室先前的研究發現，克雷白氏肺炎桿菌1084的染色體 (chromosome) 上存在一個大小為208-kb的基因體小島KPHPI208，由八個基因模組(genomic modules, GMs)所組成 (GM1-GM8)，經過註解發現GM1是攜帶負責合成基因毒性的Colibactin所需的基因。實驗室之前的研究發現在細胞實驗及動物實驗皆會造成感染的細胞有DNA雙股斷裂的現象，另外，國外則有研究指出，在大腸桿菌中，合成Colibactin的酵素也能幫助螯鐵分子 (siderophore) yersiniabactin的合成。另外，GM6則是攜帶合成具有抑菌能力的細菌素Microcin E492 (MccE492)所需的基因。根據前人的研究，MccE492是由兩部分所組成，分別是具有抗菌活性的胜肽及螯鐵分子enterobactin。本研究將觀察在克雷白氏肺炎菌中製造Colibactin和Microcin的基因體小島模組與負責合成螯鐵分子的基因體小島模組之間的交互作用。本研究除了利用先前構築的1084SΔclbA之外，也利用同源重組的技術成功在克雷白氏肺炎桿菌1084S和ΔclbA中，將mceAB基因剔除得到突變菌株ΔmceAB以及ΔmceABΔclbA。實驗結果顯示，相較於野生菌型 (1084S)，剔除mceAB基因後抑菌能力顯著下降，而利用質體將基因補回可以恢復抑菌能力。然而mceAB基因缺損雖然對螯鐵分子分泌量無顯著上的影響，但利用在in vivo細胞感染模式下，藉由Real-time PCR偵測到螯鐵分子及基因體小島模組中目標基因的表現量的改變。我們比較細菌感染細胞後與感染細胞前的基因表現，發現螯鐵分子yersiniabactin和基因體小島模組GM4中基因的表現量有顯著增加的情形，這個結果顯示水平轉移得來的基因模組之間可能有一些調控關係，而且這些外來的基因模組還會去影響原本細菌基因體的其他基因表現。

Horizontal gene transfer between bacteria has been shown to be an important mechanism for exchange of genetic determinants. These confer a selective advantage to the recipient, e.g., adaption to environment and enhance pathogenicity. Bacterial genomes contain clusters of genes that are acquired by horizontal gene transfer, named genomic islands (GIs). How the acquired biosynthesis/metabolic pathways merged into the existing network of the cell remain elusive. In our previous study, we have identified a 208-kb genomic island from newly sequenced Klebsiella pneumonia 1084 genome. This 208-kb genomic island, KPHPI208, is composed of 8 genomic modules (GMs), GM1~GM8. GM1 consists of genes responsible for colibactin production. In our previous study, the colibactin-producing K. pneumonia 1084 was demonstrated to induces DNA double-strand breaks both in vitro and in vivo. In Escherichia coli, the biosynthesis of colibactin required a 4’- phosphopantetheinyl transferase (PPTase). Interestingly, PPTase also contribute to the synthesis of yersiniabactin, which is also contained as a GM in KPHPI208 (GM3). GM6 consists of genes responsible for microcin E492 (MccE492) production. MccE492 is a low molecular weight bacteriocin. It is composed of polypeptide core with C-terminal enterobactin. In this study we aimed to determine the possible regulation of gene expression among colibactin, microcin and siderophore GMs. In addition to previously constructed 1084SΔclbA, we generated mceAB knockout mutant strain ΔmceAB and double mutant strain ΔmceABΔclbA using allelic exchange. The mutant strains we also utilized by the members in our group to perform functional analysis, including antimicrobial test, siderophore secretion, and to establish the biochemical assay procedure for the toxins. We designed primers targeting the genes in the 8 GMs and other virulence genes in the chromosome. Compared to wild type strain, ΔmceAB showed decreased antimicrobial activity. While we did not see the differences of siderophore secretion between the mutant and wild type, using Real-Time PCR we were able to detect the expression of target gene in siderophore modules and other genomic modules in vivo. We also identified increased transcription of yersiniabactin GM and GM4 in cell culture infection model. Our result suggests a possible regulation among horizontally acquired genomic modules carried in the HPI.

中文摘要 i
Abstract ii
目錄 iii
表目錄 vi
圖目錄 vii
第一章、 前言 1
第一節、 克雷白氏肺炎桿菌 1
第二節、 水平基因轉移 2
第三節、 KPHPI208 基因體模組 2
第四節、 克雷白氏肺炎桿菌造成的肝膿瘍與癌症的關係 3
第五節、 Colibactin 4
第六節、 Microcin 6
第七節、 研究動機 7
第二章、 材料與方法 8
第一節、 菌株、質體及生長環境 8
第二節、 利用同源基因互換 (homologous recombination) 的方式建構克雷白氏肺炎桿菌mceAB基因缺損的突變菌株 8
一、 利用聚合酶鏈鎖反應 (Polymerase Chain Reaction, PCR)增幅K.pneumoniae 1084菌株的mceAB基因上下游DNA片段 8
二、 瓊脂膠體電泳(Agarose Gel Electrophoreisis) 9
三、 TA cloning (yT&A® cloning vector kit) 10
四、 細胞轉型(transformation)實驗與藍白篩選(blue and white screening) 11
五、 快速確認插入片段方法 (Cracking) 11
六、 抽細菌的質體DNA (QIAprep® Spin Miniprep kit) 12
七、 利用限制酶 (restriction enzyme) 確認結果 12
八、 利用限制酶確認方向性 13
九、 將確認帶有mceAB基因上下游片段的yT&A vector切限制酶並利用膠體電泳萃取純化 14
十、 DNA接合作用 (Ligation) 15
十一、將yT&A vector上的mceAB基因上下游的DNA片段轉移至自殺性質體pKAS46 16
十二、製作勝任細胞 (competent cell) 18
十三、電穿孔(electroporation)實驗 18
十四、接合生殖 (Conjugation)與挑選突變株 19
第三節、 建構克雷白氏肺炎桿菌mceAB基因缺損突變株的互補質體 21
第四節、 建構克雷白氏肺炎桿菌mceAB和clbA雙重基因缺損的突變菌株 21
第五節、 抑制圈實驗 21
第六節、 偵測螯鐵分子 (SideroTec AssayTM kit)(購於EmergenBio) 22
第七節、 細胞感染實驗 22
第八節、 抽取total RNA (Roche High Pure RNA Isolation Kit) 23
第九節、 total RNA轉cDNA 24
第十節、 Real-time PCR 24
第三章、 研究結果 26
第一節、 在克雷白氏肺炎桿菌1084S中建構突變株 ΔmceAB和ΔmceABΔclbA 26
第二節、 mceAB基因缺損會降低克雷白氏肺炎桿菌的抗菌能力 27
第三節、 在體外（in vitro) 模式中，無法藉由SideroTecTM Assay Kit觀察到mceAB基因缺損對螯鐵分子分泌量的影響 27
第四節、 Colibactin (GM1) 與Microcin E492 (GM6) 基因模組之間的交互作用 28
第五節、 在細胞感染模式中，利用Real-Time PCR來偵測螯鐵分子及KPHPI208的目標基因表現量 29
第六節、 clbA與mceAB基因缺損對KPHPI208其他基因模組之影響 30
第四章、 討論 31
第一節、 克雷白氏肺炎桿菌1084的突變菌株 31
第二節、 mceAB基因缺損對克雷白氏肺炎桿菌1084 之抗菌活性分析 31
第三節、 在體外模式下，mceAB或clbA基因缺損對克雷白氏肺炎桿菌1084之螯鐵分子分泌量分析 32
第四節、 Colibactin與Microcin基因模組之間可能不存在調控關係 32
第五節、 在細胞感染模式下，mceAB或 clbA基因缺損對螯鐵分子及KPHPI208基因模組之影響 33
第五章、 結論 36
第六章、 參考文獻 52

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