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標題: 克雷白氏肺炎桿菌1084基因體小島模組間交互作用與功能之探討
Studies on the interactions among genomic island modules in Klebsiella pneumoniae 1084
作者: Yi-Min Hong
關鍵字: genomic island
Microcin E492
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摘要: 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.
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。
其他識別: U0005-2208201520553100
文章公開時間: 2018-08-24
Appears in Collections:基因體暨生物資訊學研究所



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