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研究生:黃慧軒
研究生(外文):Hui-Hsuan Huang
論文名稱:檸檬酸對轉糖鏈球菌的生長及酸耐受性的影響
論文名稱(外文):The effect of citrate on the growth and acid tolerance of Streptococcus mutans
指導教授:賈景山
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:微生物學研究所
學門:生命科學學門
學類:微生物學類
論文種類:學術論文
論文出版年:2004
畢業學年度:92
語文別:中文
論文頁數:48
中文關鍵詞:酸耐受性檸檬酸轉糖鏈球菌
外文關鍵詞:acid tolerancecitrateStreptococcus mutans
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轉糖鏈球菌的產酸性與酸耐受性是造成齲齒的重要致病因子之一。先前實驗已證明,先利用弱酸刺激對數生長期的細菌會誘發酸耐受性反應(Acid tolerance response,ATR)可以增加在強酸環境下的存活。此種對數生長期的ATR需要上游調控作用與下游調控作用以調控蛋白質合成。本實驗室先前已發現citBZC operon為會受到酸鹼值5.5與外生性檸檬酸下游調控的基因,顯示此operon在轉糖鏈球菌的ATR上扮演某種角色。
在本篇實驗中分析野生株GS-5R與isogenic mutants之間耐酸性的差異,並驗證了生長期與細胞濃度皆可調控轉糖鏈球菌的酸適應力。我們發現citB mutant可以在致死性酸鹼值下稍微增加存活率,且比野生株與其他突變株能維持較高的胞內酸鹼值。培養基中僅含檸檬酸(citrate)無法支持轉糖鏈球菌生長,但外加檸檬酸到培養基中會產生dose-dependent抑制生長的現象。利用defined medium我們發現此種抑制生長的現象是由於檸檬酸螯合掉金屬離子所致。檸檬酸可以幫助轉糖鏈球菌產生酸耐受性,特別對未經弱酸適應的citB mutant效果最為顯著;而單獨剔除掉citM(citrate transporter)的突變株卻仍然保有外加檸檬酸可增加耐酸性的現象,推測檸檬酸可能在細胞外就具有幫助產生酸耐受性的作用;由基因資料庫比對找到在轉糖鏈球菌中含有檸檬酸發酵途徑中必需酵素的gene cluster,其中包括一組two component system genes,實驗室中已建構了其sensor 與regulator的突變株並預備分析並比較其酸耐受性。由實驗結果初步瞭解,檸檬酸除了在細胞內可以發揮部分緩衝溶液系統的功用外,或許在細胞外就可當作一個訊息分子誘發轉糖鏈球菌的酸耐受性。
Streptococcus mutans is considered to be an important etiological agent of dental caries. Its ability to tolerate and grow in low-pH environments is critical to its survival and pathogenicity. Previous studies have demonstrated that acid shock of exponential cells from pH 7.5 to 5.5 resulted in the induction of an acid tolerance response (ATR) increasing survival at low pH (3.5 – 3.0). This exponential “ATR” requires both up-regulation and down-regulation of protein synthesis. We found previously that the citBZC operon is down-regulated after exposure to a low pH 5.5 and in the presence of exogenous citrate,suggesting this operon might play a role in ATR of S. mutans.
In this study, we analyzed the ATR of wild type strain GS-5R and several isogenic mutant strains,and confirmed that both growth phase and cell density can modulate acid adaptation in S. mutans. We found that the citB mutant exhibited slightly better survival in the killing pH and maintain a higher level of intracellular pH than parental or citZ and citBZC mutant strains. Citrate alone could not sustain the growth of S. mutans, and exogenous addition of citrate could inhibit the growth of S. mutans in a dose-dependent manner. Using defined medium, we found the inhibition of growth was resulted form the chelating effect of metal ions by citrate. Citrate also enhances the ATR of S. mutans,particularly in unadapted cells and CitB mutant. The enhancement was still observable in a CitM mutant (defective in citrate transporter), suggesting that exogenous citrate in extracellular environment might play a role in triggering the ATR. From the genomic bank we found a putative gene cluster related to citrate fermentation pathway, including a two-component system. Isogenic mutants of the two-component sensor and regulator have been constructed in our laboratory and ready to analyze and compare their acid survival capability. Preliminary results suggested that citrate enhance ATR of S. mutans both through its buffering effect inside and through signaling events outside of the bacterial cell
目錄

中文摘要
Abstract
目錄……………………………………………………………………………………i
表次……………………………………………………………………………………ii
圖次…………………………………………………………………………………...iii
壹、 緒論………………………………………………………………………………1
貳、 實驗材料與方法…………………………………………………………...…...10
參、 結果……………………………………………………………………………..15
肆、 討論……………………………………………………………………………..25
伍、 表………………………………………………………………………………..28
陸、 圖………………………………………………………………………………..35
柒、 參考文獻………………………………………………………………………..43
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