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研究生:黃冠曄
研究生(外文):Kuan Yeh Huang
論文名稱:唾液鏈球菌57.Ⅰvic操縱子之功能及其表現之分析
論文名稱(外文):Molecular and functional analysis of the Streptococcus salivarius 57.Ⅰ vic operon
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:2009
畢業學年度:96
論文頁數:52
中文關鍵詞:唾液鏈球菌訊息傳遞自我調控
外文關鍵詞:streptococcussalivariusvictwo component signal transduction system
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摘要

Two component signal transduction system( TCS )為細菌體內主要的訊息傳遞以及調節系統,此系統參與細菌生理代謝、壓力適應、抗藥性、勝任性,以及毒性因子表現等之調控。VicRK TCS( 在某些菌種稱為YycFG )是一廣泛存於革蘭氏陽性菌,且對於細菌的毒性,生長,壓力適應均扮演重要調控角色的系統。在唾液鏈球菌也發現到此一系統的存在,透過引子延伸實驗發現在-16發現一extended啟動子序列,且在-132位置發現一VicR-P結合的保留性序列,透過RT-PCR及EMSA實驗結果發現VicR-P可以一抑制子的角色逆向調控vic 操縱子的表現。vic啟動子的活性會在細菌生長至飽和期時降至最低,並且在pH中性環境下的活性也比酸性環境下好。在比較野生株、VicK及VicX突變株的功能分析實驗中發現,VicK去活性株在氧化壓力適應,生長及生物膜生合成的能力上都比野生株與VicX去活性株來的差。由此得知VicRK TCS對於細菌生長初期扮演著重要的角色,同時VicRK TCS也參與唾液鏈球菌的氧化壓力適應反應。由VicR-P所引導的回饋逆向調控可精確地控制vic操縱子的表現量,及由其所調控下游基因的表現。
ASTRACT

Two-component signal transduction system (TCS) is the major signal transduction and regulation system in bacteria. Cellular functions such as metabolic activity, stress responses, drug resistance, competence, and virulence all are regulated by TCS. The VicRK (also known as YycFG) TCS, encoded by vic operon, is widely identified in Gram-positive bacteria, and its impact in cell wall biogenesis, and virulence gene expression has been demonstrated in various streptococcal species, Staphylococcus aureus, and Bacillus subtilis. Recent study in our laboratory revealed that a vicRKX operon is present in Streptococcus salivarius and the expression of this operon has been confirmed by RT-PCR. To analyze the expression of vic operon in S. salivarius, a promoter-chloramphenicol resistance gene fusion (pvic-cat) was constructed and integrated into the lacZ locus in this study. The promoter activities in cells grown at different conditions were examined by measuring CAT activity. The expression of pvic was regulated by both growth stages and culture pH, with lower expression in cells grown in the stationary stages and under acidic pH. Transcription start site was located 62 bp 5’ to the ATG. An extended -10 promoter sequence was found in the corresponding region and a regulator binding sequence proposed for S. pneumoniae VicR was located at -132 bp. Furthermore, the amount of vicR-specific message, measured by RT-PCR, was up-regulated in the vicK-deficient strain comparing to the wild-type strain grown under the same condition. These results suggested that the expression of pvic was negatively autoregulated by phosphorylated VicR. Functional analysis indicated that an intact vicK, but not the vicX, was essential for optimal oxidative stress responses and biofilm formation. Thus, it is likely that Vic TCS is involved in a global regulatory circuit in S. salivarius.
目錄
指導教授推薦書
口試委員審定書
誌謝 IV
摘要 V
ASTRACT VI
目錄 VII
圖表目錄 IX
一、 草綠色鏈球菌屬(VIRIDANS STREPTOCOCCI) - 1 -
二、 唾液鏈球菌(STREPTOCOCCUS SALIVARIUS) - 1 -
三、 口腔鏈球菌的酸性適應( ACID ADAPTATION ) - 2 -
四、 尿素酶 (UREA AMIDOHYDROLASES 【EC 3.5.1.5】) - 3 -
五、 TWO-COMPONENT SIGNAL TRANSDUCTION SYSTEM(TCS) - 4 -
1. Sensor kinase (SK) - 5 -
2. Response regulator (RR) - 6 -
六、 PHOSPHORELAY SIGNAL TRANSDUCTION SYSTEM - 7 -
七、 TCS的重要性 - 7 -
八、 VICRK(又名YYCFG)TCS對於鏈球菌之重要性 - 8 -
九、 實驗目標 - 9 -
材料與方法 - 11 -
一、 菌株、質體、引子與生長環境 - 11 -
二、 VICX突變株之建構 - 11 -
三、 啟動子重組株之建構 - 12 -
四、 LYSATE的製備與CAT報導基因活性分析 - 12 -
五、 RNA萃取與反轉錄酶聚合酶連鎖生合成反應(RT-PCR) - 13 -
六、 MBP-VICR融合蛋白質的純化與ELECTROPHORESIS MOBILITY SHIFT ASSAY(EMSA) - 13 -
七、 引子延伸實驗(PRIMER EXTENSION) - 14 -
八、 環境壓力對於唾液鏈球菌生長抑制實驗 - 14 -
九、 生物膜生合成分析(BIOFILM FORMATION ASSAY) - 15 -
結果 - 16 -
一、 VIC操縱子與其啟動子的結構與比對 - 16 -
二、 PVIC活性分析 - 16 -
三、 野生株與突變株RT-PCR結果分析 - 17 -
四、 VICR與PVIC間的交互作用 - 17 -
五、 VIC TCS的功能分析 - 17 -
討論 - 19 -
圖 - 23 -
表 - 33 -

圖表目錄

圖一:引子延伸結果 - 23 -
圖二:pvic-cat重組株(KY3)之建構與確認 - 24 -
圖三:唾液鏈球菌pvic-cat重組株在不同生長時期的CAT活性。 - 25 -
圖四:唾液鏈球菌pvic-cat重組株培養在TH-KPO4 (pH=7.5) 與TH-HCl (pH=5.5)下之CAT 活性。 - 26 -
圖五:藉由RT-PCR探討vicR在野生株與SF31的mRNA表現量 - 27 -
圖六:MBP-VicR與Pvic結合 - 28 -
圖七:VicX突變株之建構與確認 - 29 -
圖八:唾液鏈球菌57.I野生株及其突變株生長曲線分析 - 30 -
圖九:唾液鏈球菌57.I野生株及其突變株的生物膜生合成能力。 - 31 -
圖十:唾液鏈球菌 pvic調控路徑之假說 - 32 -
表一、本實驗所用之菌株與質體 - 33 -
表二、實驗所用之引子 - 34 -
表三:VicRKX同源性蛋白質比對結果 - 35 -
表四:野生株與突變株在各種壓力下所能生長的最低濃度 - 36 -
表五:唾液鏈球菌VicR目標基因上游序列以及S. pneumoniae 與B. subtilis 之VicR(YycF)目標基因已發表之上游序列 - 37 -
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