碳源與生長速率對大腸桿菌DNA 超螺旋結構、ATP生合成基因與細胞分裂基因調控之研究

Abstract

微生物可利用不同受質獲取能量，大腸桿菌代謝碳源產生能量的途徑與生長速率亦隨受質不同而改變。本論文以醋酸、葡萄糖、甘油與琥珀酸四種不同碳源作為大腸桿菌之生長基質，結果發現不同碳源會造成細胞生長速率改變，相對地代謝途徑上與 ATP 生合成相關基因的表現量也發生變化（pgk、 pyk、ackA、sucCD、atpI）。由於 DNA 超螺旋結構受 ATP/ADP 比例所調控，研究發現細胞內ATP 與 ADP 含量以及 ATP/ADP比例會隨碳源不同而上升或下降，當生長速率加快時，因 ATP/ADP 比例與 gyrase 表現量受到調控而上升，因此促使 DNA 超螺旋結構較生長速率緩慢時來得緊密。 此外 ppGpp 為大腸桿菌在貧瘠環境下的重要調控因子，實驗結果顯示 ppGpp 並不影響大腸桿菌體內 ATP/ADP 比例與 DNA 超螺旋結構的改變，但卻觀察到 ppGpp 會使細胞分裂基因 ftsZ 表現上升，且對 minC 與 minD 基因進行負向調控。本研究也發現細胞大小會隨生長速率加快而變大，以不同碳源為生長基質時，造成細胞分裂基因（ftsZ, minC, minD）的表現有明顯差異。當大腸桿菌生長在碳源貧乏的環境時，菌體型態會趨向長絲狀並呈現鍊狀結構，因此推測 ppGpp 有可能透過影響細胞分裂基因的表現而改變細胞大小以及菌體型態。本實驗藉由研究碳源如何改變大腸桿菌生長速率， ATP 與 ADP 含量及細胞內 ATP/ADP 比例、 DNA 超螺旋結構、細胞分裂等結果，可作為基礎分子生物學研究提供一個重要的模式發現。

To understand the expression of ATP generation and cell division genes in different carbon sources, we individually used acetate, glucose, glycerol or succinate as a sole carbon for energy source. The results of this study showed that the expression of ATP generating genes in metabolic pathway varied with carbon sources and ATP concentration increased with cell growth rate. Comparision with the wild-type strain and relA spoT double mutant, the growth rate and ATP yields were changed, but ATP/ADP ratio remained at the same level. DNA supercoiling was dependent on ATP/ADP ratio. Whereas ppGpp did not change DNA supercoiling. Under various growth conditions, fast-growing E. coli cells were larger than slowly growing ones. Starvation of the cells resulted in filamentous morphology. In addition, the results clearly showed that relA spoT double mutant had more filamentous than wild-type cells regardless acetate or glucose as carbon substrates. It was also notable that the filamentous features provided the phenotypic clues for ppGpp function. However, the morphology raised the possibility of indirect, rather than direct, effects of ppGpp. It indicated that a link between the levels of ppGpp and cell division, which ppGpp could act as a positive regulator of the expression of ftsZ gene. Deficiency of ppGpp (relA spoT double mutant) drastically reduced the expression of minC and minD. These results also suggested that ppGpp was important factor involved in the regulation of cell cycle of E. coli under starvation condition.