cAMP與CRP蛋白質對大腸桿菌厭氧呼吸鏈基因與延胡索酸酵素基因表現之影響

Abstract

cAMP與CRP蛋白質對大腸桿菌厭氧呼吸鏈基因與延胡索酵素基因 表現之影響 學生：邱鈺閔 指導教授：曾慶平 博士 國立交通大學 生物科技研究所 碩士班 摘 要 大腸桿菌(Escherichia coli)在有氧環境下可將葡萄糖經糖解作用和TCA cycle代謝分解，所產生的 NADH 經有氧呼吸鏈酵素傳遞電子給最終接受者氧而產生能量。在厭氧環境下已知的厭氧呼吸酵素基因，包括nitrate reductase(narGHJI)、DMSO/TMAO reductase(dmsABC)及fumarate reductase(frdABCD) 均可被活化，而好氧呼吸基因則被抑制。此外，大腸桿菌在有氧環境下，細胞會利用延胡索酵素進行 TCA cycle 路徑，將 fumarase 轉換為 malate。目前已知在大腸桿菌中有三種延胡索酵素，FumA 酵素在有氧時被大量表現，FumB酵素在厭氧時表現，FumC酵素是在當細胞受高氧應力(high oxygen stress)才被誘發。Fnr與ArcA/B為目前已知大腸桿菌體內兩組重要轉錄調控子，會將外界是否有氧的訊息傳入細胞內而調控上述代謝途徑各基因之表現。就過去的研究了解，在厭氧環境下，厭氧呼吸酵素基因的表現會被 Fnr活化， 但不會受 ArcA/B 所調控， fumA 與fumC基因則受 Fnr 與 ArcA/B 所抑制。但是在 fnr 突變株(Δfnr) 與 arcA 突變株(ΔarcA)或雙突變株(ΔfnrΔarcA) 的情況下，在厭氧或有氧環境下這些基因的表現仍有差距，由此可知，除了 Fnr 與ArcA/B 的調控外，還有其他因子調節這些基因表現。目前已知cAMP是細胞中重要的調控子，可調控細胞中許多的基因表現，本論文中利用構築Δcya、Δcrp及ΔcyaΔcrp雙突變株，來觀察cAMP與CRP蛋白質對上述基因的影響。結果發現 cAMP與CRP蛋白質對narGHJI與dmsABC基因的promoter是扮演repressor的角色，而cAMP與CRP蛋白質對fumA基因與fumC基因具有抑制作用，其中對fumC基因的影響非常顯著。此外，實驗結果也顯示cAMP與CRP蛋白質與上述基因的glucose catabolite repression有相關性。由連續式培養中可知cAMP會影響受生長速率所調控的fumA-lacZ與fumC-lacZ的表現。因此，由研究結果可知cAMP與CRP蛋白質確實是除了Fnr與ArcA/B外另一個影響厭氧呼吸鏈基因及延胡索酵素基因表現的調控因子。

Effect of cAMP and CRP on the Anaerobic Respiratory Genes (narGHJI , dmsABC) and Fumarase Genes (fumA , fumC) Expression in Escherichia coli Student：Yu-Min Chiu Advisor：Dr. Ching-Ping Tseng Institute of Biological Science and Technology National Chiao Tung University Abstract Escherichia coli exhibits diverse respiratory abilities. It synthesizes at least two distinctive cytochrome oxidases during aerobic growth and additional three terminal oxidoreductases for anaerobic respiration. It also contains three biochemically distinct fumarases to catalyze the interconversion of fumarate to L-malate in the TCA cycle. Two broadly acting regulators, Fnr and ArcA, independent control of oxygen-regulated genes have been identified. NarGHJI and dmsABC genes are induced under anaerobic growth condition that are activated by Fnr protein. In addition, fumA and fumC genes are induced under aerobic growth condition that are repressed by Fnr and ArcA. Howerver, in fnr and arcA mutants all of them still show the difference expression during anaerobiosis. Since cAMP is a global control system that regulates many genes in E.coli, it may involve in those genes expression. In order to understand the transcriptional regulation of cAMP and CRP, we constructed cya、crp and double mutants in the wild-type, fnr and arcA mutants to examine the regulation of four reporter fusions. The results showed that mutations in cya and crp increased narGHJI、dmsABC、fumA and fumC expressions, indicating that cAMP and CRP acted as a negative regulatory element. Therefore, cAMP and CRP are important factor related to glucose catabolite. In continuous culture, the results suggested that growth rate regulation of fumA and fumC genes was dependent on cAMP. These findings suggested that in addition to Fnr and ArcA, cAMP and CRP were another factors for regulation of anaerobic respiratory and fumarase genes expression in Escherichia coli.