臺灣博碩士論文加值系統

唾液鏈球菌(Streptococcus salivarius)是人類口腔裡少數已知可以產生大量尿素酶的正常菌叢，其存在對於維持口腔的酸鹼值、蛀牙的防預、及保持口腔中正常菌叢的生態平衡均有很大的貢獻。唾液鏈球菌利用Two-component signal-transduction systems (TCSTSs)去偵測外界環境的刺激並且調控內部基因的表現，使其能夠迅速地適應環境的各種變化。根據一與唾液鏈球菌在演化上具親源性的嗜熱性鏈球菌(Streptococcus thermophilus LMG 18311)的基因體序列，成功地在唾液鏈球菌的染色體上找出兩組與covRS具同源性之TCSTSs，並且利用RT-PCR證實此兩組基因組在唾液鏈球菌中的表現。為分析此二組TCSTSs的功能，以insertion inactivation的方式將此二TCSTSs的HK基因破壞掉，建立突變株JC01及JC07。分析顯示野生株及突變株JC01及JC07在酸性環境(pH 3.0)及高鹽(6M)環境的存活率並沒有顯著的差異，然而在H2O2 (0.08%)的環境中，突變株JC01及JC07的存活率較野生株的存活率來得低。為了能系統性地分析此二組TCSTSs的功能，進一步以二維蛋白質電泳比較野生株與突變株差異，且發現在JC01中數個與糖解作用有關的蛋白質表現來得比野生株低，而與細胞膜組成及脂質合成有關的酵素在JC07中的表現較野生株高。由實驗結果顯示，推測rr01/hk01及rr07/hk07在唾液鏈球菌中參與不同的訊息傳遞路徑，然而均與抗氧化壓力的偵測有關。

Streptococcus salivarius is one of the most abundant and highly ureolytic bacteria in the oral cavity. The expression of S. salivarius urease is subject to environmental signals, with optimal expression in cells grown at acidic environment. The differential expression of urease in response to external pH is crucial for maintaining oral pH homeostasis and preventing carie formation, however the molecular mechanism used by S. salivarius to detect pH signals is not yet defined. To initiate the analysis on how S. salivarius senses and responds to external stimuli, this study focuses on two independent two-component signal transduction systems (TCSTS), RR/HK01 and RR/HK07 of S. salivarius. To determine the functions of each system in signal transduction, genes encoding histidine kinases (HK) of the two TCSTSs were insertionally inactivated to generate strains JC01 (with mutation in hk01) and JC07 (with mutation in hk07), respectively. No significant difference in survival rates was observed between the wild-type and mutant strains when exposed to acidic (pH 3.0) or high salt (6M) conditions, indicating that neither systems was involved in sensing pH or osmolarity stressors. When exposing to an oxidative stressor, H2O2 (0.08%), a lower survival rate was consistently observed in both JC01 and JC07, comparing to wild-type strain. Systemic proteomic analysis revealed that three enzymes involved in carbohydrate metabolism were down-regulated in JC01, whereas enzymes involved in membrane biogenesis and lipid metabolism were up-regulated in JC07. Thus, it is likely that these two systems are involved in different signaling pathways in S. salivarius, with overlapping in oxidative stress responses.

指導教授推薦書............................................
口試委員會審定書..........................................
授權書...................................................iii
誌謝.....................................................iv
中文摘要..................................................v
英文摘要..................................................vi
目錄.....................................................vii
圖表目錄.................................................viii
緒論.....................................................1
材料與方法................................................15
實驗結果..................................................25
討論.....................................................31
圖.......................................................37
表.......................................................54
參考文獻..................................................58

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