碳源、供氧量及細胞生長速率對大腸桿菌延胡索酸脢基因表現之研究：mRNA穩定性及酵素活性之探討

Abstract

中文摘要 延胡索酸瓷（fumarase）為三羧酸循環（tricarboxylic acid cycle）中負責催化延胡索酸（fumarate）與蘋果酸（malate）相互轉換的酵素。已知大腸桿菌延胡索酸瓷含有 fumA，fumB 及 fumC 三種不同的基因。其中 fumA 與 fumB 有很相似的基因序列，所生成的 FumA 及 FumB 是一種含有鐵硫（iron-sulfur）活性中心且為熱不安定性（heat labile）的酵素。fumC 則與真核生物的延胡索酸瓷基因序列有較高的相似性，所生成的 FumC 為熱安定性（heat stable），不需要鐵離子參與其酵素活性反應，並且 superoxide-generating agents 會影響 fumC 基因的表現。 為了研究碳源、供氧量及細胞生長速率對延胡索酸瓷基因表現的影響，我們利用化學恆定（steady-state chemostat）連續式培養法來控制細胞的生長速率，並探討不同碳源、供氧量及細胞生長速率下延胡索酸瓷基因的調控情形。我們以葡萄糖或醋酸為碳源進行培養，分別萃取不同生長速率下的總 RNA 以進行北方墨點轉漬分析，結果顯示 fumA 及 fumC 基因的 mRNA 表現量隨生長速率的上升而下降，而兩種 mRNA 表現量都是以醋酸為碳源時高於以葡萄糖為碳源。此外，這兩種 mRNA 在不同供氧量下的表現亦有所不同。我們利用 rifampin 抑制 mRNA 的轉錄並測定 fumA 及 fumC 的 mRNA 半衰期，結果顯示 fumA 及 fumC 的 mRNA 均隨細胞生長速率變快而愈穩定，而且也是在以醋酸為碳源時高於葡萄糖為碳源時，顯示 fumA 及 fumC 基因的表現在轉錄（後）層次上分別受碳源、供氧量及細胞生長速率影響。 以西方墨點轉漬分析探討改變細胞生長速率對 FumA 蛋白表現量與延胡索酸瓷酵素活性的改變情形，發現生長速率愈快，延胡索酸瓷的酵素活性愈低，但在以醋酸與葡萄糖為碳源時，FumA 蛋白表現量隨生長速率改變在兩者有相反的調控。以上結果顯示延胡索酸瓷在轉譯（後）層次上亦受碳源、供氧量及細胞生長速率影響。

Abstract Escherichia coli contains three biochemically distinct fumarases to catalyze the interconversion of fumarate to L-malate in the tricarboxylic acid (TCA) cycle. While the fumA and fumB genes encode heat-labile, iron-containing fumarases, FumC is a heat-stable fumarase which does not require iron for its activity. It has been reported that synthesis of FumC increased with superoxide radicals, and fumC expression required the SoxR regulatory protein. In order to study how fumA and fumC genes are regulated by different carbon sources, oxygen transfer rates (OTR) and cell growth rates, we examined the expression of fumA and fumC genes in continuous cultures. With Northern blotting analysis, we found that the amount of fumA and fumC mRNA relative to the total RNA decreased with increasing cell growth rate. In addition, we measured the decay of fumA and fumC mRNA after blocking transcription initiation with rifampin and found that the stability of fumA and fumC mRNA increased at high cell growth rates. We also used Western blotting analysis to measure the production of FumA and determined fumarases activity. The results demonstrated that the activity of fumarases was regulated independently by cell growth rates and carbon sources availability. The amount of FumA protien and the activity of FumA and FumC showed the hierarchical control which depends on the OTR that the cell encounters. These results suggested that growth rate-dependent regulation of fumA and fumC genes was mediated at both transcriptional and translational levels.