Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/52220
標題: Studies on the Release of Colicin Ib
大腸菌素Colicin Ib 釋出機制之研究
作者: 陳建華
關鍵字: 生物科學類
基礎研究
摘要: Colicin is produced by E. coli or bacteria of Enterobactericeae. It inhibits thegrowth of related bacteria.. Almost all colicin genes are located on plasmid.Downstream of a colicin gene is its corresponding immunity gene coding for theimmunity protein, which would protect the colicin-producing bacteria from the inhibitoryeffect of the colicin. Some colicin genes have lysis genes downstream, which code forthe lysis protein to release the colicin into the environment, but some do not. PlasmidpColIb-P9 is an E. coli plasmid that contains colicin Ib gene (colIb) and its immunitygene (imm), but no lysis gene. E. coli containing this plasmid would produce colicin Iband release it into the cultural medium by an unknown mechanism. Colicin Ib-sensitivebacteria have Cir receptor on the outer membrane. Colicin Ib can enter the sensitivebacteria through Cir receptor and make pores on the inner membrane, leading to ionleakage and bacterial death.We previously obtained a fosmid clone from a Shigella flexneri Taiwan isolate.The insert DNA contains colIb, imm, and ibfA. We have demonstrated that the ibfA genedownstream of imm could enhance release of colicin Ib from the bacteria into culturalmedium. Overproducing the imm and ibfA protein products in E. coli caused the bacteriastop to grow, indicating a damaging effect of the two proteins. Thus, we hypothesize thatthe Imm and IbfA proteins can release colicin Ib from the bacteria but through differentmechanisms. The Imm protein releases colicin Ib from inner membrane to outside of theouter membrane through protein-protein interaction with Imm, translocator protein andreceptor protein, whereas the IbfA protein directly release colicin Ib from cytoplasm intothe cultural medium through activation of a protein secretion pathway. The main purposeof this proposal is to prove this hypothesis. We will purchase knock-out mutants ofcolicin-related membrane protein genes, and generate mutants of colIb, imm, and ibfA towhich different tags will be added. We will determine whether the protein products andthe mutant proteins of the three genes would have interactions with other membraneproteins, release colicin Ib onto the outer membrane or to the cultural medium. Thetopologies colicin Ib in the presence of Imm or IbfA will also be determined. By this way,we may be able to prove or modify the hypothesis.
大腸菌素 (colicin) 由腸內菌產生,抑制種類相近菌之生長。幾乎所有的 colicin基因皆位在質體上,colicin基因下游皆有一immunity 基因,負責做出immunity蛋白,保護產生colicin 的菌不受該colicin 的抑菌作用。一些colicin 基因的下游還有lysis 基因,可將colicin自菌中釋出。大腸菌的pColIb-P9質體,其上有colicin Ib基因 (colIb) 及immunity 基因 (imm),但不具lysis 基因。具有此質體的大腸菌會產生並釋出colicin Ib,但其釋出機制至今仍不清楚。敏感菌的外膜上具Cir receptor蛋白,colicin Ib藉此進入後,再經由 translocator 蛋白將活性 domain 嵌入菌的內膜,並於其上產生孔洞,菌因此死亡。我們98年度已證明本地的痢疾桿菌的一個 colicin 質體與大腸菌的pColIb-P9質體有很高的同質性,質體上具有colIb、imm基因、及可使較多colicin Ib釋出菌體外的 ibfA 基因。如果使大腸桿菌大量產生immunity 蛋白或IbfA蛋白,則菌生長曲線皆無法上升,顯示二蛋白可能皆有增加膜通透性的能力。我們假設 immunity蛋白與IbfA蛋白皆可將colicin Ib 釋出,但機制不同。前者為透過colicin 的不同domain 與內外膜上包括 immunity 蛋白、translocator蛋白及receptor蛋白等膜蛋白的交互作用,將colicin Ib由內膜移至外膜上,而後者為藉由活化 type I ABCtransporter 分泌系統,將colicin Ib 直接由細胞質中釋出至培養基中。本計畫主要目的為證明此假設。方法為購買各種與colicin Ib 有關的膜蛋白的缺失株與野生株,將質體上的colIb、imm、與ibfA三基因加上不同的tag及產生三基因的變異;我們檢查三基因蛋白及其變異蛋白在各種膜蛋白的缺失株與野生株中,是否會與膜蛋白產生交互作用,是否會釋出colicin Ib於外膜上或培養基中,三基因蛋白在膜蛋白皆為野生株中的構形。亦將檢查colicin Ib在具有imm或ibfA基因質體的野生株中的構形並比較,則可證明或修正我們的假設。
URI: http://hdl.handle.net/11455/52220
其他識別: NSC99-2311-B005-001-MY3
文章連結: http://grbsearch.stpi.narl.org.tw/GRB/result.jsp?id=2130912&plan_no=NSC99-2311-B005-001-MY3&plan_year=99&projkey=PA9907-2818&target=plan&highStr=*&check=0&pnchDesc=%E5%A4%A7%E8%85%B8%E8%8F%8C%E7%B4%A0Colicin+Ib+%E9%87%8B%E5%87%BA%E6%A9%9F%E5%88%B6%E4%B9%8B%E7%A0%94%E7%A9%B6
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