生長速率對大腸桿菌細胞內能量狀態、DNA 超螺旋結構與呼吸鏈基因 cyo, cyd 調控 之研究

Abstract

微生物可利用不同的碳源來得到能量，大腸桿菌在不同養分及環境下會有不同的能量狀態。本實驗室利用連續式發酵法改變細胞生長速率，來研究在不同碳源及不同生長速率下對菌體體內能量狀態的影響。從實驗結果得知，以葡萄糖、醋酸、琥珀酸及甘油四種不同碳源培養時，在生長速率快時，菌體中內 ATP/ADP ratio 以及 energy charge 皆會隨著增高。 另外，在 ATP/ADP ratio 與 DNA 超螺旋結構的關係方面，從之前實驗室所做出的結果我們知道當生長速率增加時 ATP/ADP ratio 愈高，且 DNA 超螺旋結構愈緊。而在本論文實驗中我們將生長速率控制在一定值 (k = 1.2) 且將碳源的濃度加倍，我們發現 ATP/ADP ratio 在高濃度的碳源時其值會較高，且 DNA 超螺旋結構也相對呈現較緊密的狀態，因此我們認為 DNA 超螺旋結構的鬆緊主要是取決於 ATP/ADP ratio 而與生長速率無關。 在研究檸檬酸循環 (TCA cycle) 時發現，succinate dehydrogenase, malate dehydrogenase 及 fumarate dehydrogenase 等基因之表現量會隨著生長速率的上升而下降，而 TCA cycle 是產生 NADH + H+ 進入呼吸鏈去產生能量的主要來源，而我們實驗也發現細胞內 ATP 的含量會隨著生長速率的上升而上升。藉由偵測有氧呼吸鏈中 cytochrome o oxidase 及 cytochrome d oxidase 酵素之表現量，我們發現以葡萄糖、醋酸、琥珀酸及甘油四種不同碳源培養時此兩種酵素之表現量皆隨著生長速率的上升而下降，所以我們推測當生長速率增加時，能量的來源可能是經由 substrate level phosphorylation 而來的。此外我們也發現到 cyo, cyd 基因有 catabolite repression 的現象，當我們將 cya 基因突變掉之後菌體因無法產生 cAMP 則 catabolite repression 的現象便消失，因此我們認為 cAMP 是調控 cyo, cyd 基因表現的重要因子。

Bacterial can generate energy by using different carbon sources. The energy state of Escherichia coli varied with nutrient and growth conditions. In this study we used continuous culture to study how the different cell growth rates and different carbon sources affect the energy state of E. coli. The result showed that the ATP/ADP ration and energy charge were all raised with cell growth rate when glucose, acetate, succinate, glycerol were used as carbon sources. In the study of relationship between ATP/ADP ratio and DNA supercoiling, had observed that ATP/ADP ratio raise with increasing cell growth rate and more negative DNA supercoiling. In this study, we also used the continuous culture and control the cell at constant growth rate (k = 1.2), to determine the effect of carbon source concentration on the ATP/ADP ratio and the DNA supercoiling. The result suggested that DNA supercoiling is dependent on ATP/ADP ratio but not on cell growth rate. It has been reported that the TCA cycle enzymes, such as succinate dehydrogenase, malat dehydrogenase, fumarate dehydrogenase, expression decreased with increasing cell growth rate. Since the NADH+H+ is the major product of TCA cycle which will into the respiratory chain to generate ATP. In this study, we observed that the ATP concentration increased with increasing cell growth rate. Also, we measured expression of the two respiratory genes (cyoABCDE, cydAB), and we found these two genes expression decreased with increasing cell growth rate. This results suggested that at the high growth rate, the energy may be generate from substrate level phosphorylation. In addition, cyo and cyd genes showed the phenomenon of catabolite repression, and the phenomenon was disappeared in the cya mutant strain (the strain can not generate cAMP). Therefore, it suggested that cAMP is an important factor related to cyo and cyd gene expression at different growth rate.