生長速率對大腸桿菌細胞能量狀態、DNA 超螺旋結構與 cAMP 濃度調控之研究

Abstract

中文摘要 微生物可利用不同的受質來獲得能量，大腸桿菌生長在不同養分環境下會有不同的能量狀態，我們利用連續式培養法以改變細胞的生長速率，來研究不同碳源在不同的生長速率下對細菌體內能量變化的影響。結果我們發現，生長速率會改變菌體中 energy charge ，當生長速率加快時細胞內的ATP、ADP 等高能物質含量也隨之提高。而相較之下， AMP 在細胞內的含量不高，而且不受細胞生長速率所調控。在低生長速率時三種 adenylate 化合物之間的含量差異較小；隨著生長速率愈快 ATP、ADP、AMP 的含量差異也愈大。藉由 ATP/ADP 比例的變化也發現，生長較快速的細胞其 ADP 對 ATP 有較高的轉換率，而此現象不受碳源的種類所影響 (carbon source independent)。 DNA 超螺旋結構與 cAMP 是細胞中重要的調控子， 此二系統可調控許多原核細胞中的基因表現。在此論文中，我們以連續式培養方式也證明了生長速率可調控 DNA 超螺旋結構的鬆緊程度與細胞內的 cAMP 濃度。當我們以質體 pBR322 檢測不同碳源與生長速率對 DNA 超螺旋結構改變的影響時，發現質體超螺旋結構與 ATP/ADP 比例二者間為正相關，ATP/ADP 比例愈高 DNA 超螺旋結構愈緊密，雖然能量狀態為影響 DNA 超螺旋結構的主要因子，但在高生長速率時 homeostatic control 的影響會大於 thermodynamic control，而使 DNA 超螺旋結構不至於旋轉過緊，以維持正常的生理功能。在 cAMP 方面，經由我們所構築的 pcya-lacZ protein fusion 在不同生長速率下表現，證明adenylate cyclase 會隨著生長速率而增加表現量，但細胞內 cAMP 的含量卻會隨細胞生長速率加快而下降。由於生長速率增加時會降低 adenylate cyclase 被phosphoenopyruvate phosphotransferase system (PTS) 活化的機率，因此細胞中 cAMP 的濃度主要取決於 adenylate cyclase 的活性而非其表現量。 藉由以上實驗我們證明了，不同碳源與生長速率均會影響細胞內的能量狀態，生長速率改變可經由 ATP/ADP 比例的變化進而影響 DNA 超螺旋結構與控制細胞內 cAMP 濃度來影響各基因表現。

Abstract Bacteria can generate energy by using different carbon sources. The energy state of Escherichia coli varied with nutrient and growth conditions. To study how cell growth rate affects the level of energy in E. coli, the chemostate culture was used in this study. The results showed that energy charge changed with cell growth rate which high energy materials ATP and ADP increased at high cell growth rate, but AMP content was remained at low level. In addition, high difference of adenylate compounds and ATP/ADP ratio were found at high growth rate and these result are carbon source independent. DNA supercoiling and cAMP are global control systems that regulate many genes in E. coli. Our result demonstrated that DNA supercoiling level and cAMP concentration in E. coli were also regulated by cell growth rate. DNA supercoiling strongly depends on the cellular energy state at low ATP/ADP ratio. Homeostatic control mechanism attenuated thermodynamic control to prevent the over negative DNA supercoiled when ATP/ADP ratio was high. In order to understand the transcriptional regulation of adenylate cyclase, we also constructed cya-lacZ protein fusion to exame how adenylate cyclase expressed at different growth rates. In contrast to cAMP concentration decreased at high cell growth rate, the cya gene expression was increased. These results suggest that intracellular cAMP concentrations depended on the activity of adenylate cyclase instead of its gene expression. In this dissertation, we have showed that different carbon source and growth rate resulted in varied intracellular energy states. Growth rate can regulate gene expression by changing DNA supercoiling level and cellular cAMP concentration. These findings may be important for the regulation of some growth-rate-dependent genes in E.coli.