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研究生:謝惠如
研究生(外文):Huei Ru Sie
論文名稱:fimR啟動子之鑑定及其對副血鏈球菌FW213fim操縱組之調控分析
論文名稱(外文):Molecular and functional analysis of FimR in Streptococcus parasanguinis FW213
指導教授:陳怡原
指導教授(外文):Y. Y. M. Chen
學位類別:碩士
校院名稱:長庚大學
系所名稱:基礎醫學研究所
學門:醫藥衛生學門
學類:醫學學類
論文種類:學術論文
論文出版年:2008
畢業學年度:96
論文頁數:62
中文關鍵詞:副血鍊球菌FW213FimR蛋白質fimR啟動子
外文關鍵詞:Streptococcus parasanguinis FW213fim operonFimR
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副血鏈球菌 (Streptococcus parasanguinis)是口腔中常見之正常菌叢之一,在特殊情況下,副血鏈球菌可侵入血流而引發亞急性心肌內膜炎。由過去研究結果得知,副血鏈球菌FW213 fimCBA operon可轉錄並轉譯出一負責運送錳離子之ABC-binding cassette transporter,且FimA蛋白質是副血鏈球菌造成心肌內膜炎的主要毒力因子之一。目前已知於fim operon 啟動子 (pfim) 上游包含一段金屬調控蛋白質之保留結合位,且pfim的表現可受錳離子濃度的調控,但此調控是否由一反式調控蛋白質來掌控,至今仍不詳。藉由chromosomal walking 策略,近來本實驗室已在fim operon 下游約2 kbp處發現一與戈登鏈球菌 (Streptococcus gordonii) 中已知之金屬調控蛋白質ScaR有65%高相似性的蛋白質,且由序列比對分析後發現此蛋白質的N端具有一helix-turn-helix(HTH)區塊、金屬結合位(metal binding site)及成雙體(dimerization)的區域,顯示此蛋白質為一金屬轉錄調控蛋白質。本研究藉由副血鏈球菌FW213 pfim-cat融合之重組株觀察pfim活性的變化,發現若將此預測性之金屬調控蛋白質突變後,pfim的活性較野生株約高2倍,證實此蛋白質為一可抑制fim操縱組表現之抑制子(repressor),因此將此蛋白質命名為FimR。若將當菌體培養於不同濃度之錳離子(0 或50 μM Mn2+)及不同pH值(pH 7.5或6.8)的環境下,可發現不論是野生株還是fimR突變株,於低錳環境下pfim活性的表現皆為於高錳環境下的兩倍,而在中性環境下pfim的活性也較在酸性環境下佳,顯示FimR蛋白質並非唯一參與fim操縱組表現之蛋白質,尚具其它轉錄調控蛋白可藉由感應周遭不同濃度的錳離子及pH進而調控pfim的表現。藉由電泳移動率分析之技術證實FimR蛋白質可直接結合上fim操縱組序列,且錳離子及鐵離子可些許影響FimR蛋白質與fim操縱組的結合反應。
The acquisition of transition metal ions is crucial for the viability and in some cases the expression of virulence genes in bacteria. The fimCBA operon of Streptococcus parasanguinis FW213 encodes a high-affinity ABC-type transporter for manganese (Mn2+). In addition to transport manganese, studies have shown that FimA, the lipoprotein of the system, plays a critical role in the development of endocarditis in an animal model. The expression of fimCBA is inhibited by excess amount of Mn2+, and a conserved palindromic regulatory sequence for metalloregulatory proteins is located immediately 5’ to the fimCBA promoter (pfim). To investigate the regulatory mechanism, a chromosome walking approach was employed to search for possible trans-acting regulatory elements. A locus that shared significant homology with Streptococcus gordonii scaR at deduced amino acid level (65 % identity) was identified 2 kbp 3’ to the fimCBA operon. Sequence analysis of this locus revealed that the encoded protein was composed of a helix-turn-helix motif at the N-terminus, followed by a metal binding and dimerization domain, suggesting that this protein was a metalloregulatory DNA-binding protein. To evaluate the function of this locus on pfim expression, a recombinant pfim-cat fusion strain was constructed. The expression of pfim in wild-type strain increased approximately 2-fold in cells cultured in medium with no additional Mn2+ than that in high Mn2+ containing medium (with 50 M Mn2+). Inactivation of this locus resulted in up-regulation of pfim expression, but the expression was still responsive to Mn2+. The derepression of pfim expression in the mutant was restored when a wld-type locus was supplement in trans on a shuttle vector, pDL276. Furthermore, a direct interaction between the protein encoded by this locus and the conserved palindromic regulatory sequence was demonstrated by electrophoresis gel mobility shift assay, and the addition of Mn2+ and Fe2+ was effective in promoting fragment shift. Take together, these data indicated that the presence of this locus was essential for the optimal regulation of fimCBA operon, and thus this locus was designated as fimR.
指導教授推薦書
口試委員會審定書
授權書 iii
誌謝 iv
摘要 v
ABSTRACT vi
目錄 viii
圖表目錄 x
序論 - 1 -
一、 草綠色鏈球菌 (Viridans streptococci)及其主要致病 - 1 -
二、 副血鏈球菌 (Streptococcus parasanguinis) - 1 -
三、 副血鏈球菌之毒力因子及fim operon - 2 -
四、 fim operon 之同源性系統及其功能 - 5 -
五、 fim operon 之同源性系統的分子調控 - 7 -
六、實驗動機 - 9 -
實驗材料與方法 - 11 -
一、 所使用之菌株、質體、培養基及生長條件 - 11 -
二、 DNA之分離及操作方法 - 11 -
三、 重組菌株之建構 - 12 -
四、 以引子延伸法(primer extension)分析fimR轉錄啟始點 - 14 -
五、 氯胺醇乙醯移轉酶(Chloroamphenical acetyltransferase,CAT)活性分析試驗 - 15 -
六、 FimR蛋白質誘導試驗及純化 - 16 -
七、 電泳移動率分析試驗(Electrophoretic Mobility Shift Assay,EMSA) - 16 -
實驗結果 - 18 -
一、 副血鏈球菌中ScaR蛋白質同源體 (homolog) 的發現、比對分析結果及啟動子鑑定 - 18 -
二、 副血鏈球菌pfim-cat融合重組株及含fimR突變之重組株的建立 - 19 -
三、 副血鏈球菌pfim-cat融合重組株及及其fimR突變衍生株之pfim活性分析 - 19 -
四、 錳離子對pfim活性的影響 - 19 -
五、 pfim活性的表現受到環境中不同pH值的調控 - 20 -
六、 FimR蛋白質表現的誘導及純化 - 20 -
七、 電泳移動率分析試驗 - 20 -
討論 - 22 -
結果圖 - 27 -
表 - 39 -
參考文獻 - 42 -

圖表目錄
圖 一、副血鏈球菌fim操縱子及其週邊ORF之示意圖。 - 27 -
圖 二、fimR轉錄起始點及fimR啟動子之鑑定。 - 28 -
圖 三、(A)副血鏈球菌fimR之衍生胺基酸序列比對分析。(B)FimR與其相關蛋白質比對的結果。 - 30 -
圖 四、副血鏈球菌 pfim-cat融合重組株的建構。 - 31 -
圖 五、副血鏈球菌pfim-cat融合重組株fimR突變的建構(FHR2)。 - 32 -
圖 六、pfim-cat 在野生株及fimR突變株之CAT活性分析及fimR基因之反式回補試驗(in trans complementation)。 - 33 -
圖 七、野生株及fimR突變株在不同錳離子濃度下之pfim活性之分析。 - 34 -
圖 八、野生株及fimR突變株在不同酸鹼環近中之pfim活性之分析。 - 35 -
圖 九、FimR蛋白質誘導表現及純化。 - 36 -
圖 十、FimR蛋白質與pfim之電泳移動率分析試驗。 - 37 -
圖 十一、環境金屬離子之FimR蛋白質與pfim之電泳移動率分析試驗 - 38 -

表 一、所用之選殖珠、質體 - 39 -
表 二、所用之引子 - 40 -
表 三、Semi-defined medium(SDM)成分表 - 41 -
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