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標題: Pectobacterium carotovorum subsp. carotovorum 之 c-di-GMP 與 CRP 之協同作用對低分子量細菌素 carocin 基因之調控機制研究
Studies of the regulatory mechanism of the Low-Molecular-Weight Bacteriocin carocin gene through c-di-GMP and CRP cooperation in Pectobacterium carotovorum subsp. carotovorum
作者: 賴瑋婷
Lai, Wei-Ting
關鍵字: 環磷酸腺苷受體蛋白
出版社: 化學系所
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摘要: Pectobacterium carotovorum subsp. carotovorum屬腸道菌科,為植物病原菌。有些Pectobacterium carotovorum subsp. carotovorum菌株在受紫外光刺激誘導下,會產生低分子量細菌素。目前對於低分子量細菌素的訊息傳遞路徑尚未明朗,但本實驗室先前的研究指出,阻斷crp基因的突變株和阻斷dgc基因的突變株,皆失去表現低分子量細菌素carocin基因的能力,因此本研究目的在於探討c-di-GMP與CRP之協同作用對低分子量細菌素carocin基因的調控機制。 我們利用DNASIS MAX軟體分析carocin基因上游序列,從中找出兩個可能為CRP 辨識結合的位置。以此為依據,我們設計兩組30個鹼基對的DNA片段做為探針,探針序列分別包含所預測的CRP結合序列,並於5''端標定上生物素。接著進行生物素化探針沉澱分析實驗,並且製備CRP多株抗體以利西方墨點法的進行。在生物素化探針沉澱分析實驗中,實驗結果顯示CRP確實可以結合於我們所預測的位置,分別命名為CIE-1(carocin induced element-1)和CIE-2(carocin induced element-2)。CIE-1 座落於轉錄起始點上游-112~-117 bp處;CIE-2 則是在-17~-22 bp的位置。我們也發現,紫外光刺激不影響CRP蛋白表現量,卻關乎CRP與DNA的結合。當菌體未受紫外光刺激的情況下,CRP主要結合於CIE-1或CIE-2的其中一處;相反地,當菌體經紫外光刺激誘導的情況下,CRP與CIE-1和CIE-2結合且結合量是一致的。此外,在沒有c-di-GMP的情況下,我們也觀察到CRP與CIE-1和CIE-2的結合量明顯下降。基於這樣的結果,我們推測c-di-GMP是造成CRP異構轉變的因子,即c-di-GMP可能和CRP結合形成複合體,再結合於carocin基因上游序列調控carocin基因的表現。我們也推測紫外光刺激造成菌體內c-di-GMP濃度提升,而c-di-GMP濃度的提升或許是導致CRP由抑制子轉變為活化子的關鍵角色。 總結來說,本研究推測出一個可能的訊息傳遞路徑,來試圖解釋紫外光的刺激如何導致carocin基因表現,以及c-di-GMP和CRP之協同作用於carocin基因調控所扮演的角色。
Pectobacterium carotovorum subsp. carotovorum is a phytopathogenic enterobacterium. Some of Pectobacterium carotovorum subsp. carotovorum strains produced low-molecular-weight-bacteriocin by ultraviolet induction. But litte is known about the signal transduction pathway of low-molecular-weight- bacteriocin carocin gene. The previous studies from our lab has shown that crp-defective mutant and dgc -defective mutant could not express carocin gene respectively. In this study, we want to known that how is the regulatory mechanism of carocin gene throgh cyclic dimeric guanosine monophosphate (c-di-GMP) and cAMP receptor protein(CRP) cooperation? Here, we analyzed the carocin gene upstream sequence by the DNASIS MAX program and this sequence analysis indicated that the carocin gene upstream region contains two putative CRP binding sites. Thus, we designed two 30 bp DNA fragments as biotinylated probes and each probe contains the putative CRP binding site respectively. We thus used the biotinylated probe pull down assay and prepared the CRP polyclonal antibody for western blotting. As a result, in our experiments, CRP binds to two putative CRP sites called CIE-1(carocin induced element-1) and CIE-2(carocin induced element-2). CIE-1 located at positions -112~-117 bp relative to the translation start site, while CIE-2 located at positions -17~-22 bp. Without ultraviolet induction, CRP binds CIE-1 or CIE-2 dominantly in the biotinylated probe pull down assay. With ultraviolet induction, CRP binds to CIE-1 and CIE-2. In addition, in the absence of c-di-GMP, the level of CRP binds to CIE-1 and CIE-2 obviously decreased. Based on the data presented in this study, we think maybe c-di-GMP is a novel effecter for CRP allosteric transition and thus the cooperation of c-di-GMP and CRP regulates the carocin gene. We proposed that ultraviolet induction may cause increasing the level of c-di-GMP concetration and the role of c-di-GMP is to convert CRP from a repressor to an activator. In conclusion, our results attempted to model a signal transduction pathway of the carocin gene expression in response to the ultraviolet induction. In particular, we implicated the roles of the c-di-GMP and CRP cooperation in the carocin gene.
其他識別: U0005-2008201302571400
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