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研究生:黃維君
研究生(外文):Wei-chun Huang
論文名稱:TraT和EF-Tu蛋白質在大腸桿菌對大腸桿菌素敏感性機制中所扮演的角色
論文名稱(外文):The role of TraT and EF-Tu in the mechanism of E. coli sensitive to colicin E7
指導教授:胡小婷
指導教授(外文):Shiau-Ting Hu
學位類別:碩士
校院名稱:國立陽明大學
系所名稱:微生物及免疫學研究所
學門:生命科學學門
學類:微生物學類
論文種類:學術論文
論文出版年:2004
畢業學年度:92
語文別:中文
中文關鍵詞:大腸桿菌素E7大腸桿菌TraT蛋白質EF-Tu蛋白質
外文關鍵詞:colicin E7E. coliTraT proteinEF-Tu protein
相關次數:
  • 被引用被引用:1
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摘要
大腸桿菌素E7(ColE7)是一個去氧核醣核酸酶,具有三個不同的結構區域,N端是穿透功能區(Translocation domain,T domain);中間是受器結合功能區(receptor binding domain,R domain);C端是毒性功能區(Toxic domain,T2A-DNase domain)。目前推測ColE7進入細胞膜間區域時,其T domain會與Tol系統蛋白質的TolB作用,並經蛋白酶切割後,與T2A-DNase domain分離,T2A-DNase domain可進入細菌細胞質中,分解去氧核醣核酸。本論文的主要目的在研究Tol系統蛋白質以外,是否仍有其他的蛋白質參與ColE7之修飾切割的過程。
首先利用轉位子建立JM110菌株的突變基因庫,篩選出對ColE7敏感的突變株,結果發現三株突變株其外膜蛋白質的TraT皆有增加,為調查TraT是否與細菌對ColE7之敏感有關,於是利用只有單一複本數及大量複本數的質體,選殖帶有完整啟動子的traT基因,將此二質體轉殖進入大腸桿菌株DH5α,看看是否因其TraT蛋白質表現量增加,使細菌對ColE7較敏感。結果顯示,TraT蛋白質之大量或少量增加,並不影響細菌對ColE7之敏感,故推測應有其他蛋白質參與此機制中,而非單一TraT蛋白質就可造成細菌對ColE7敏感。
本實驗室之前的研究顯示,對ColE7敏感之DH5α突變株5D1,其外膜蛋白質中含有大量的EF-Tu蛋白質,為了解EF-Tu之增加是否為造成5D1菌株對ColE7敏感之因子,我們建構可大量表現EF-Tu蛋白質之質體pQE70TufB並轉殖進入DH5α菌株,結果顯示EF-Tu大量表現會使細菌對ColE7敏感性上升4倍以上。此外之前的in vitro實驗發現EF-Tu蛋白質會與TR domain有交互作用。本論文利用細菌雙雜交系統證明EF-Tu蛋白質確實會與 TR domain進行交互作用,且得知其不會與T2A-DNase domain、Tol系統蛋白質有交互作用。已知TolB蛋白質會與TR domain進行交互作用,欲了解EF-Tu與TR domain 之作用是否會影響到TolB蛋白質與TR domain結合?於是利用細菌雙雜交系統測試,結果顯示TolB與EF-Tu之間會互相影響對方與TR domian的鍵結。已知EF-Tu除了做為轉譯作用的延長因子外,亦具有GTPase及chaperone-like 修飾蛋白質功能,本論文欲了解GTPase功用之喪失是否會影響到細菌對ColE7之敏感性,因此將EF-Tu之第84個胺基酸突變,使其喪失GTP水解功能後,再予以測試,結果發現突變的EF-Tu蛋白質表現量不如野生型的EF-Tu蛋白質。但是此少量增加的突變EF-Tu仍會造成細菌對ColE7之敏感性些許上升,故我們推測,EF-Tu在細菌膜間區域時,或許可扮演修飾TR domain的一個角色,此修飾功能可能並不會與EF-Tu的GTP 水解功能有關。
Abstract
Colicin E7 (ColE7) is a DNase bacteria toxin, which comprises of three genes: cea, cei, cel. There are three domains within cei gene; from N terminal to C terminal is translocation domain (T domain), receptor domain (R domain) and toxic domain (T2A-DNase domain). It has been hypothesized that when ColE7 entering the periplasm, colicin TR domain can be processed by protease and separated from T2A-DNase domain in the periplasm. Then T2A-DNase domain can enter the cytoplasm to carry out its toxic function on DNA. In this study, we aim to find out any other protein participated in processing colE7 beside the Tol system protein.
First, we established a JM110 strain mutant library by transposon Tn5, and selected some mutants that were sensitive to Colicin E7. All of the sensitive mutants showed increased expression of TraT protein in their outer membrane. We cloned the single or multiple copies of complete traT gene with its own promoter, and transformed it into E. coli DH5α strain. We examined whether there is any relation between over-expressing TraT protein and colicin sensitivity. According to the data, neither large nor small amount increasing of TraT protein can affect the colicin sensitivity. Therefore, we conclude that the TraT protein alone could not make E. coli sensitive to ColE7. Instead, other proteins together with TraT may confer this sensitivity. There are other proteins involved in this sensitive mechanism, instead of TraT protein alone in making E. coli sensitive to ColE7.
We observed that the DH5α sensitive mutant, 5D1, has increased expression of EF-Tu protein more than wild DH5α strain in their outer membrane protein. To clarify whether EF-Tu protein renders 5D1 sensitive to colE7, we constructed a pQE70TufB clone which can over-express the EF-Tu protein. We transformed the plasmid into E. coli DH5α strain. E. coli DH5α strain with over-expressed EF-Tu protein is four-fold more sensitive than DH5α strain without EF-Tu protein. Besides, previously EF-Tu protein has been observed to interact with colicin TR domain in vitro. We found that only the TR domain, but not the T2A-DNase domain or Tol system protein, can interact with EF-Tu protein in bacterial two-hybrid system. We would like to find out if there is any effect between EF-Tu and TolB when they both interact with colicin TR. The data showed that TolB will influence EF-Tu interaction with TR domain in bacterial two-hybrid system and vice versa. EF-Tu protein is not only a translation elongation factor, but also a GTPase and chaperone-like protein. To investigate whether the effect of EF-Tu on the bacterial sensitivity to ColE7 is related to the GTPase function, the 84th residue of EF-Tu was mutated from His to Ala. It was found this mutant protein would not be produced as much as nonmutated protein. However, the small amount of mutant EF-Tu protein resulted in a little increase of the bacterial sensitivity to colE7. According to the data, we hypothesize that EF-Tu may play a part role in colicin TR domain processing in the periplasm. The mechanism may not be related to the EF-Tu energy change or the conformation changed by GTP hydrolysis function.
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潘逸萱.(2002). 陽明大學生化所碩士論文:亞精胺與大腸桿菌素位移相關機制
之研究.
鄭文軒.(2003).陽明大學微免所碩士論文:大腸桿菌DH5α 菌株之danA-突變株
對大腸桿菌素E7 之敏感機制.
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