大腸桿菌環單磷酸腺苷受體蛋白參與激酶與磷酸酶轉錄層次調控之研究

Abstract

磷酸化作用在生理功能中扮演關鍵角色，會調控許多細胞新陳代謝過程，這些過程依賴於激酶和磷酸酶的作用。環磷酸腺苷受體蛋白（CRP）是一個廣泛的轉錄因子，在大腸桿菌中已知調控超過400個基因，其中有26個基因是編碼激酶和磷酸酶的基因，然而CRP對於激酶和磷酸酶基因的轉錄調控之研究目前還沒有完整的研究資訊。 因此本論文結合生物資訊CRP結合位序列預測工具，初步篩選在大腸桿菌中202激酶和磷酸酶基因，發現44個目標基因，在其啟動子上可能具有CRP結合位，進一步利用electrophoretic mobility shift assay (EMSA)和即時定量real-time PCR的技術，來驗證CRP對這些基因的調控關係，以達到系統性並廣泛地找出新的CRP調節之激酶和磷酸酶基因。結果顯示在44個目標基因中，有39 個基因確實受到CRP調控。 在上述的研究中我們發現，編碼trehalose-6-phosphate phosphatase的基因otsB，在crp突變的菌株中表現量增加68倍。進一步利用DNase I footprinting和lacZ reporter fusion的實驗，來驗證otsB啟動子區的CRP結合位點。這些實驗結果證實CRP在轉錄層級中抑制otsBA基因的表現。過去已知當大腸桿菌在高滲透壓下時，CRP的基因和蛋白表現都會下降。所以我們推測低CRP表現導致otsBA基因的表現增加，而產生更多海藻糖作為抗滲透壓物質。此推論進一步藉由染色體上的CRP結合位點突變和TLC檢測海藻糖含量得到驗證。總結我們的實驗發現證實cAMP-CRP可作為反應滲透壓的調節子，使大腸桿菌在不斷變化的環境條件下利用此機制來適應滲透壓的改變。

Phosphorylation plays a crucial role in biological functions and controls cellular process. These processes depend on the highly regulated and opposing actions of kinases and phosphatases. Cyclic AMP receptor protein (CRP) is a global regulator, which is known to regulate more than 400 genes in Escherichia coli. Among these genes, 26 genes are kinase and phosphatase-related genes. However, the comprehensive mechanism of transcriptional regulation of CRP on kinase and phosphatase genes is still unclear. To clarify novel CRP-regulated kinase and phosphatase genes, CRP binding sites were predicted by computational model. After screening 202 kinase and phosphatase genes, 44 targets were selected. The roles of CRP on these genes were further examined by using electrophoretic mobility shift assay (EMSA) and real-time PCR. The results demonstrated that 39 of the 44 genes were regulated by CRP directly. Interestingly, we found that otsB, encoding trehalose-6-phosphate phosphatase increased 68 fold in crp mutant. The CRP binding site on otsB promoter region was presented by DNase I footprinting and lacZ reporter fusion assay. All of these results indicated that CRP was a repressor of otsBA in E. coli. It suggested that when E. coli under high osmolarity in exponentially phase, the low CRP level caused the expression level of otsBA operon to increase to produce more trehalose as osmoprotectant through derepression of CRP. The hypothesis was further verified by site-directed mutation of CRP binding site and TLC assay. To sum up, cAMP-CRP as an osmoregulator gave E. coli an efficiency way to acquire long survival time to adapt high osmolarity in changing environment conditions.