臺灣博碩士論文加值系統

戈登鏈球菌是牙菌斑的早期成員之一。口腔細菌可因宿主的進食啟動糖解，造成環境酸化，進而破壞健康菌叢生態平衡並引發蛀牙。因應環境酸化，口腔細菌也需適時啟動適應機制，以利生存。為了解戈登鏈球菌在口腔生態的生理調控及抗壓反應，本計畫針對戈登鏈球菌 pH 訊息調控系統及其對抗酸反應的影響進行分析。因雙分子調控系統 (two component systems) 是細菌主要的訊息調控系統，本計畫利用對 pH敏感的尿素酶啟動子結合報導基因 (pureI-cat) 來觀察雙分子系統突變對戈登鏈球菌的影響。由分析13對功能未知雙分子系統得知，第8及第13對雙分子訊息調控系統在酸性的環境下抑制尿素酶啟動子的表現，暗示此二對系統參與pH訊息調控路徑。第8對突變株在耐酸性測試中 (acid killing assay) 的存活率較野生株生差，但第13對突變並不影響戈登鏈球菌的存活率。此外，第8對突變株在含巴拉刈環境下的生長速率較野生株快，而第13對系統則較慢，故此二系統也參與氧化壓力反應。為全面研究此二系統可能參與的調控途徑，此計畫也分析此二突變株與野生株蛋白質體間的差異。分別在第8及第13對系統突變株發現8及4個表現量不同於野生株的蛋白質點，而這些蛋白質多參與糖類以及氨基酸代謝途徑，顯示此二系統可藉調控代謝參與戈登鏈球菌的抗壓反應。

Streptococcus gordonii CH1 is one of the early colonizers of the dental plaque. The two-component system (TCS) is the major signal transduction system in bacteria. Since pH alteration is a frequent event occurring in the oral cavity, this study aims to identify TCSs that are potentially involved in the pH signaling in S. gordonii. To monitor the effects of mutations in TCS in pH signaling, the promoterless chloramphenicol acetyltransferase gene (cat) was fused with the pH-sensitive urease promoter (pureI) of Streptococcus salivarius and integrated into the gtfG of S. gordonii (SL17). Mutants of each TCS in SL17 background were generated by allelic exchange, and the expression of the pureI in cells grown at acidic pH was examined. The result revealed that TCS08- and TCS13-mutant strains exhibited aberrant cat expression comparing to the wild-type strain at pH 5.5. A lower survival rate at pH 3 was also detected in TCS08, but not in TCS13 mutant strain, comparing to the wild-type strain. The growth rate of TCS08 mutant was faster than the wild-type strain in the presence of paraquat, whereas TCS13 mutant grew more slowly than the wild-type strain. To identify the potential targets regulated by the two systems, the proteomes of these two strains were analyzed. 8 and 4 protein spots were indentified from TCS08 and TCS13 mutants, respectively, that are differentially expressed from the wild-type strain. Most of the identified proteins are involved in the metabolism, suggesting that these two systems participate in stress responses through modulating metabolic activities.

指導教授推薦書.....................................................................................i
口試委員會審定書....................................................................................ii
長庚大學授權書.....................................................................................iii
致謝.....................................................................................................iv
中文摘要....................................................................................................v
Abstract..........................................................................................vi
Table of Contents............................................................................vii
List of Figures.....................................................................................viii
List of Tables........................................................................................ix
Introduction.....................................................................................1
Materials and Methods.............................................................11
Results........................................................................................18
Discussion......................................................................................26
References.......................................................................................31
Figures...............................................................................................40
Tables..........................................................................................54

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