標題: 生長速率對大腸桿菌延胡索酸酵素活性及fumA與 fumC基因表現之研究
Growth rate – dependent regulation of fumarase activity and fumA as well as fumC genes expression in Escherichia coli
作者: 張琦豔
Chi-Yen Chang
曾慶平
Dr. Ching-Ping Tseng
生物科技學系
關鍵字: 延胡索酸酵素;生長速率;大腸桿菌;mRNA穩定性;酵素活性;fumarase;growth rate;Escherichia coli;mRNA stability;enzyme activity
公開日期: 2000
摘要: 中文摘要 延胡索酸酵素為檸檬酸循環中負責催化延胡索酸與蘋果酸相互轉換之酵素。已知大腸桿菌含有 fumA, fumB 及 fumC 三種不同延胡索酸酵素基因。為探討供氧量及細胞生長速率對各延胡索酸酵素基因表現之影響,本實驗利用化學恆定連續式培養法,藉控制細胞生長速率來觀察大腸桿菌延胡索酸酵素突變株 (△fumA、△fumB 及 △fumC) 中酵素之相互調控關係。結果發現在低供氧量下, FumC 酵素活性在 fumA 突變株中為野生菌株之 6 倍,且在高供氧量下,FumA 酵素活性在 fumC 突變株中亦為野生菌株之 2 倍以上,顯示在不同供氧量下, fumA 與 fumC 基因之表現各有其回饋抑制作用來調控酵素活性;此外,不論在低或高供氧量下,各突變株之延胡索酸酵素活性皆隨生長速率之上升而減少,此現象與野生株活性表現之趨勢相同。 而在調控轉錄層面之研究,當以葡萄糖為碳源培養時,不同生長速率對野生菌株 fumC mRNA 之影響,發現在比生長速率由 0.48/h 上升至 1.2/h 時,其表現量下降 2.3 倍,而穩定性上升 2.5 倍,但相對於所表現之 FumC 酵素活性表現卻又隨生長速率之上升而下降 2.4 倍,顯示在轉錄(後)及轉譯層面,fumC 基因會受生長速率影響而有不同表現。在不同供氧量的條件下,當空氣比例由 0﹪增加至 10﹪時,fumC mRNA 含量低且增加量少,當空氣比例為 20﹪時,mRNA 之含量增加 2 倍,之後隨空氣比例上升 mRNA 之含量變化不大,與 FumC 酵素活性相比較,在空氣比例由 0﹪至 20﹪時,其活性表現量低且變化不大,而在空氣比例介於 20﹪至 100﹪之間時,FumC 酵素活性卻升高約5倍。而在 fumA 基因部分,當空氣比例由 0﹪增加至 5﹪時,fumA mRNA 含量增加 3 倍,之後則隨著氧氣比例的增加而逐漸下降,在空氣比例為 100﹪時含量只剩無氧時的 2 倍,與 FumA 酵素活性相比較,當空氣比例由 0﹪增加至 5﹪時,活性增加 1.5 倍,之後則隨著氧氣比例的增加而下降至無氧時的大小,顯示 fumA 及 fumC 基因在 mRNA 含量與酵素活性層面受通氣量影響之趨勢類似。另一方面,分別以不同碳源 (葡萄糖及醋酸) 培養大腸桿菌野生株,發現 fumC mRNA 穩定性在葡萄糖為碳源時,隨生長速率之增加而上升,若以醋酸為碳源時卻出現相反之結果,顯示碳源在 fumC mRNA 轉錄(後)層面,亦扮演重要之調控角色。
Abstract Escherichia coli contains three biochemically distinct fumarases to catalyze the interconversion of fumarate to L-malate in the tricarboxylic acid (TCA) cycle. In order to study how the fumA and fumC genes are regulated by different air saturation and cell growth rates, we examined the fumarase activity of fumA, fumB and fumC mutants in continuous cultures. The results showed that the fumarase activity of three mutants are regulated by the rate of cell growth as they shown in wild type strain. However, it showed that there is feedback regulation on fumA and fumC genes . At transcriptional level, there are the same variation of fumA and fumC genes between the amount of mRNA and the enzyme activity. In addition, when the specific cell growth rate increase from 0.48/h to 1.2/h, the amount of fumC mRNA decrease 2.3 fold, while the stability of fumC mRNA increase 2.5 fold and the expression of FumC enzyme activity decrease 2.4 fold. Therefore, it showed that expression are dependent on cell growth rate at (post-) transcriptional and translational level. In addition, the regulation of the stability of fumC mRNA are carbon source dependent. The results showed that the growth rate-dependent regulation of fumA and fumC genes was affected by oxygen level and carbon source utilization at both transcriptional and translational levels.
URI: http://140.113.39.130/cdrfb3/record/nctu/#NT890111003
http://hdl.handle.net/11536/66550
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