標題: 選殖 cAMP 進入大腸桿菌過程之相關基因
Cloning of the genetic components involved in cAMP uptake in Escherichia coli
作者: 羅愛雁
Ai-Yen Lo
曾慶平
分子醫學與生物工程研究所
關鍵字: 運送子;跳躍子;突變;transporter;cAMP;transposon;mutagenesis
公開日期: 2005
摘要: Cyclic adenosine monophosphate(cAMP)已知是許多真核與原核生物重要調控因子。文獻指出,大腸桿菌可以利用外加的 cAMP 來進行體內的生理調節,且吸收 cAMP 的過程並非滲透作用(permeation),而是藉載體蛋白質(carrier protein)擴散作用達成,但是對於 cAMP 如何穿透大腸桿菌的雙層膜進入菌體的機制尚不清楚。為了研究 cAMP 進入大腸桿菌過程是利用何種蛋白質當作載體,以及是否有其他分子參與,本研究利用Tn5 隨機突變技術(Tn5 transposon mutagenesis),以大腸桿菌 K12 的 cya 突變株為母株建立一個突變株庫(mutant library),希望能以此作為基礎來了解 cAMP 通過雙層膜時參與之蛋白質,並藉此探討 cAMP 進入大腸桿菌的機制。 篩選方法設計上,由於大腸桿菌需要有 cAMP 存在才能利用MacConkey agar 中乳糖而使菌落呈現紅色,反之不能利用則呈現白色菌落,在利用 Tn5 隨機突變技術建立突變株庫後,選出白色菌落的突變株,之後再將菌株培養在以琥珀酸當作碳源的培養液中,觀察生長狀況以排除因乳糖操縱組(lac operon)突變而造成無法利用乳糖的現象;剩下的突變株再利用對 crp基因作聚合酶連鎖反應,觀察放大片段大小來排除因 crp(cAMP receptor protein)基因突變造成無法利用乳糖的現象。剩餘突變株即是本研究之目標菌株。 目前從突變株庫中經過三次篩選得到 23 個無法利用外加 cAMP而呈現白色菌落的菌株,且其中 20 株已實際測定菌體內 cAMP 含量,經過突變基因定序分析發現,膜構造改變、莢膜組成改變、運送蛋白質至外膜機制、一些轉錄調控因子以及代謝基因都可能影響到 cAMP 進入大腸桿菌體內,或許 c-di-GMP 以及 AI-2 也都參與其中。
Cyclic adenosine monophosphate(cAMP)is known to be the important regulator in both prokaryotes and eukaryotes. Previous studies showed that exogenous cAMP could recover the phenomenon caused by cAMP mutation, and proposed that the uptake of cAMP into E.coli was performed by facilitated diffusion process. However, the mechanism is still unclear. To investigate whether any other molecules take part in the process of cAMP import and the functions of the molecules, mutant library was established by sending transposon Tn5 into E.coli K12 cya mutant strain by electroporation. Selecting candidate mutant strains by indirect(MacConkey agar、succinate and PCR of crp gene)and direct methods(measurements of intracellular cAMP after adding exogenous cAMP). 15 mutant clones were analyzed. The results indicated that the changes of membrane structures、 capsules、secretion of membrane protein、some regulator genes and some genes involved in metabolism may interfere with the process of cAMP import. Besides, c-di-GMP and AI-2 may also play the roles in the process of cAMP uptake. These findings need further studies to be confirmed.
URI: http://140.113.39.130/cdrfb3/record/nctu/#GT009229503
http://hdl.handle.net/11536/76960
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