轉糖鏈球菌（Streptococcus mutans）不但是人類口腔中造成齲齒的主要致病菌，也可進入血液循環附著於心臟瓣膜上進而引起感染性心內膜炎。先前研究證實轉糖鏈球菌可利用GlnR調控數個參與氮源代謝的基因組，且此經GlnR啟動的調控可增加轉糖鏈球菌抗酸的能力。近期經序列分析及比對，我們在轉糖鏈球菌發現一TnrA的同源體，並發現 TnrA也參與GlnR regulon調控。由基因表現結果發現，GlnR在氮源多的環境下會抑制 GlnR regulon (glnRA, citBZC, glnP及gdhA) 的表現。TnrA在氮源多時會抑制citBZC並活化glnRA的表現；而在氮源缺乏時則活化Smu.807的表現。此外，無論在哪個氮源環境下，gdhA的表現皆受到GlnR及TnrA的抑制。藉由電詠移動率分析證實GlnR及TnrA蛋白質皆可直接與GlnR box序列結合。此外，GlnR及TnrA突變株的抗酸能力比野生株差，但其生物膜的形成則皆較野生株好。由此可知，GlnR及TnrA皆參與GlnR regulon代謝活化的調控，且此調控與抗酸能力及生物膜形成相關，至於 TnrA 是否在轉糖鏈球菌中還有參與其他的調控機制，仍有待進一步的分析。

Streptococcus mutans is the etiologic agent for dental caries, and an opportunistic cause of bacteremia and infective endocarditis. A GlnR regulon that is essential for the acid tolerance response (ATR) was recently identified in S. mutans GS5. The same study also identified a tnrA homolog in S. mutans. This study aims to determine the function of the TnrA homolog in the regulation of GlnR regulon and possibly the ATR. Expression analysis revealed that GlnR negatively regulated the expression of the GlnR regulon (glnRA, citBZC, glnP, and gdhA) under nitrogen excess condition, whereas the TnrA homolog repressed the expression of citBZC and activated glnRA under nitrogen excess condition, and activated Smu.807 expression under nitrogen limiting condition. The expression of gdhA was repressed by both TnrA and GlnR regardless the nitrogen conditions. The results of gel mobility shift assays confirmed the regulation by GlnR and TnrA was via a direct binding to the GlnR box located 5’ to the GlnR regulon genes. Deletion of glnR resulted in a reduced survival rate at lethal acidic pH (pH 3) comparing to the wild-type strain throughout 60 minutes, whereas the tnrA deletion only influenced the survival rate in the initial stage (30 minutes), suggesting that the GlnR and TnrA modulates the ATR differently. Furthermore, both the GlnR and TnrA mutant strains form thicker biofilms than the wild-type strain under the same growth condition. Taken together, the GlnR/TnrA regulon regulates the metabolic activity, the stress responses and the biofilm formation of S. mutans.

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授授權書 iii
誌謝 iv
中文摘要 vi
Abstract vii
Table of Contents viii
List of Figures xi
List of Tables xii
Introduction 1
1. Viridans streptococci 1
2. Streptococcus mutans 2
3. The pathogenic capacity of S. mutans 2
4. The nitrogen metabolic circuit in bacteria 4
5. GlnR/TnrA regulon in Gram positive bacteria 4
6. The GlnR regulon in S. mutans 7
7. Motive 8
Materials and Methods 9
Bacterial strains and growth media 9
General DNA manipulations 10
Construction of the glnR, tnrA and tnrA-glnR
double knockout mutants 10
Growth kinetics 11
RNA isolation and Reverse Transcription (RT)-PCR 11
Construction of promoter-cat integration construct for
S. mutans GS5 13
CAT assay 13
Construction of pMAL-GlnR and pMAL-TnrA fusions and
protein purification 14
Electrophoretic Mobility Shift Assay (EMSA) 15
Acid killing assay 16
Biofilm assay 17
Results 18
1. Construction of the glnR-deficient, the tnrA-deficient
and the tnrA-glnR double knockout mutants 18
2. Growth kinetic of S. mutans GS5 and the mutants 18
3. To determine the impact of above mutations and the
nutrient conditions in the GlnR regulon expression 19
i. glnA expression 19
ii. citZ expression 20
iii. glnP expression 21
iv. Smu.807 expression 22
v. gdhA expression 22
4. The protein purification of pMAL-GlnR and pMAL-TnrA
fusion 23
5. In vitro EMSA 24
6. Both GlnR and TnrA are essential for S. mutans GS5 to
survive at pH 3 25
7. Biofilm formation of S. mutans GS5 and the mutants 26
Discussion 27
Figures 32
Tables 53
References 61

List of Figures
Fig. 1. Construction of the glnR and tnrA deletion derivatives………32
Fig. 2. Growth kinetic…………………………………………………34
Fig. 3. Expression of glnA in wild-type S. mutans GS5 and its deletion derivatives under various nutirent conditions..………………..35
Fig. 4. Expression of citZ in S. mutans GS5 and its deletion derivatives in various media……………………………………..…………38
Fig. 5. Expression of glnP in S. mutans GS5 and its deletion derivatives in various media……………………………………..…………40
Fig.6. Expression of Smu807 in S. mutans GS5 and its deletion derivatives in various media………………………………..…42
Fig. 7. Expression of gdhA in S. mutans GS5 and its deletion derivatives in various media………………………………..………………44
Fig. 8. SDS-PAGE analysis of the Mal-GlnR fusion……………….....46
Fig. 9. SDS-PAGE analysis of the Mal-TnrA fusion………………...47
Fig. 10. In vitro EMSA demonstrating the interaction between GlnR and the promoters of GlnR regulon genes……………….………....48
Fig. 11. In vitro EMSA demonstrating the interaction between TnrA and the promoters of GlnR regulon genes..………………………...49
Fig. 12. Acid killing assay………………………………………………50
Fig. 13. The biofilm formation of S. mutans GS5 and the mutants……51

List of Tables
Table 1. Bacteria strains and plasmids used in this study.……………....53
Table 2. Chemical defined medium (FMC) used in this study…….……55
Table 3. Biofilm medium (BM) used in this study…………………57
Table 4. Primer used in this study………………………………………58

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