DNA 超螺旋結構對大腸桿菌厭氧呼吸酵素基因表現與調控之研究

Abstract

大腸桿菌 (Escherichia coli) 為兼性厭氧細菌，在有氧環境下細 胞可將葡萄糖經 glycolysis pathway 及 TCA cycle 代謝分解，並將 產生的 NADH 經有氧呼吸鏈酵素 cytochrome o oxidase (cyoABCDE) 與 cytochrome d oxidase (cydABC) 傳遞電子給最終接受者氧而產生能量． 無氧環境下，厭氧呼吸酵素基因包括 nitrate reductase (narGHJI)， DMSO/TMAO reductase (dmsABC) 及 fumarate reductase(frdABCD) 會被 活化，而上述好氧呼吸基因則被抑制．ArcA-ArcB 與 Fnr 為目前已知大 腸桿菌體內兩組重要轉錄調控子，它們會將外界是否有氧的訊息傳入細胞 內而調控上述基因的表現．以前的研究瞭解在無氧環境下，這些厭氧呼吸 酵素基因的表現會被 Fnr 活化，但不受 ArcA-ArcB 所調控．雖然 Fnr 在無氧環境下可調控這些厭氧呼吸酵素的基因表現，但在無氧及有氧環境 下這些厭氧呼吸酵素基因之表現於 fnr 突變株中仍有 2~5 倍的差距，由 此可知除 Fnr外尚有其它因子也參與厭氧呼吸酵素基因表現的調控．因為 不同含氧環境已證實會改變 DNA 超螺旋結構，所以我們認為 DNA 超螺旋 結構可能會參與調控厭氧呼吸基因的表現． 大腸桿菌內 DNA 超螺旋結構 主要是由會增加和減少 negative supercoils 的 DNA gyrase和 topoisomerase I，經彼此互相協調以達到一個 topology 的平衡．因此 本論文中利用不同的 gyrase 抑制劑，及構築 topoisomeraseI 及 gyrase B subunit 的突變株，分別使菌體內 DNA 超螺旋結構變得鬆散及 捲緊，以觀察厭氧呼吸基因在 in vivo 中受其調控及表現．結果發現在 無氧環境下當 DNA 超螺旋結構較鬆散時 frdA-lacZ 與 dmsA-lacZ的表 現都被活化，而 narG-lacZ 表現則是被抑制．反之當 DNA 超螺旋結構捲 緊時，frdA-lacZ 與 dmsA-lacZ 的表現被抑制而 narG-lacZ 表現則被活 化．這三種厭氧呼吸基因受 DNA 超螺旋結構改變之影響在有氧環境下都 較無氧環境下明顯，而對 fnr 突變株之厭氧呼吸基因的影響又較野生株 為顯著．因此我們認為 Fnr 在細胞中可能扮演避免 DNA 超螺旋結構變化 過大而影響細胞正常生理功能的角色．DNA 超螺旋結構對厭氧呼吸基因表 現之影響除 frdA-lacZ 外，對 dmsA-lacZ 與 narG-lacZ 有可能是經由 影響 Fnr 後再去調控該基因之表現．從本研究中發現 DNA 超螺旋結構在 in vivo 中確實是除 Fnr 外，影響厭氧呼吸基因表現的另一調控因子． Escherichia coli exhibits diverse respiratory abilities. It synthesizes atleast two distinctive cytochrome oxidase (cytochrome o oxidase and cytochrome d oxidase) during aerobic growth and can produce an additional terminal oxidoreductase, nitrate reductase(narGHIJ), DMSO/TMAO reductase( dmsABC), and fumarate reductase(frdABCD), for anaerobic respiration with the alternative electron acceptors. Although the anaerobic genes are activated by the Fnr, they still have two to five folds induction during anaerobic growth in fnr mutants. Hence Fnr might not be the sole requirement for the anaerobic induction of anaerobic genes. A topological state of the bacterial chromosome is important for transcription, replication and recombination. To determine how the anaerobic genes are regulated in response to a variety of DNA supercoiling, including inhibitors of gyrase and topoisomerase mutants, we examined their expression by using lacZ repoter fusions in wild-type and fnr mutant strains. The effect of DNA supercoling on the expression of anaerobic respiratory genes was more conspicuous when cells were growth under aerobic condition and fnr mutants. These results infer that Fnr may be a factor for the balance of DNA superhelicity. When the DNA supercoling was relaxed, expression of frdA-lacZ and dmsA-lacZ fusion were activated under anaerobic growth, but narG-lacZ expression was repressed. In contrast, the expression of frdA-lacZ and dmsA-lacZ fusion were repressed and narG-lacZ expression increased when DNA supercoiling became more negative. These findings suggested that in addition to Fnr, DNA︿ upercoiling was another factor for the regulation of anaerobic respiratory gene expression.