臺灣博碩士論文加值系統

毒素-抗毒素(Toxin-Antitoxin)基因組包含了毒素及能抑制其作用的抗毒素分子。除了一些質體或嗜菌體的基因序列之外，這樣的基因組亦廣泛的存在於細菌的染色體上。許多研究指出這些位在染色體上的毒素-抗毒素系統(Toxin-Antitoxin system)參與調控細菌的多細胞行為，例如：生物膜(biofilm)和子實體(fruiting body)的形成，然而這樣子的系統是否影響其他類型的多細胞行為，如細菌的表面移行(swarming)，目前仍不清楚。Swarming為複雜的細菌表面多細胞行為，藉由不同的化學及物理訊號調控，細菌能夠從非移行的細胞(vegetative cells)分化出能夠在表面群聚移行的細胞(swarmer cells)。於此論文研究成果中，我們發現Serratia marcescens在swarming發展過程中大約有90%的非移行細胞有死亡的現象。透過實驗結果，我們更進一步指出這樣的現象與ccdAB(Sm)，一個位於染色體上的Toxin-Antitoxin system有所關聯。此外，在將ccdAB(Sm)自染色體剔除後，此變異株有提早啟動表面移行的現象。我們更進一步發現實驗室之前所研究的一套二元系統，RssAB亦參與調控細菌於swarming發展中的細胞死亡，並且RssAB可以直接調控ccdAB(Sm)的基因表現。總和所有結果，此研究證實了在Serratia marcescens swarming發展的過程中，RssAB與CcdAB(Sm) 共同參與調控細胞死亡的現象。

Toxin-antitoxin (TA) modules are gene pairs specifying for a toxin and its cognate antitoxin and are found on the chromosomes of many bacteria including pathogens. It has been demonstrated that chromosomal TA systems influence bacterial multicellular behaviors such as biofilm formation and fruiting body formation. However, the role of TA systems in swarming behavior, another type of bacterial multicellular behavior, remains elusive. Swarming is a complex multicellular behavior requires the integration of chemical and physical signals, which leads to the physiological and morphological differentiation of the bacteria from nonmotile vegetative cells into highly-motile swarmer cells. Here we demonstrated that approximately 90% of cells undergo cell death in vegetative cell population during swarming development in Serratia marcescens. A pair of chromosomal TA module named ccdAB(Sm) was identified. Deletion of ccdAB(Sm) caused dramatic reduction of dead cells during swarming development and showed precocious swarming motility. Interestingly, deletion of rssBA, a two-component system regulating swarming initiation, also reduced cell death during swarming. Furthermore, the transcription of ccdAB(Sm) and their upstream genes are negatively regulated by RssAB signaling which led to cell death in early swarming development. Taken together, these findings provide evidences that RssAB regulates cell death through a chromosomal TA module, CcdAB(Sm), in swarming development.

Contents
指導教授推薦書
口試委員會審定書
長庚大學授權書 ⅲ
致謝 ⅳ
中文摘要 ⅴ
Abstract ⅵ
Contents ⅶ
Figure contents ⅸ
Table contents ⅹ
Chapter 1 Introduction
1.1 Serratia marcescens 1
1.2 Bacterial multicellular behavior 1
1.3 Bacterial programed cell death 2
1.4 Toxin-antitoxin systems 3
1.5 Two-component system RssA-RssB regulates swarming behavior in S. marcescen 4
1.6 Aim of this study 5
Chapter 2 Materials and methods
2.1 Bacteria strains, plasmids, and primers 6
2.2 Bacteria medium and growth conditions 8
2.3 Swarming motility assay 8
2.4 Bacterial LIVE/DEAD Staining 9
2.5 Determining the effect of CcdB toxin overproduction 9
2.6 Postsegregational killing assay 9
2.7 Reverse transcription-PCR (RT-PCR) assay 10
2.8 Electrophoretic mobility shift assay (EMSA) 11
Chapter 3 Results
3.1 Cell death during swarming development in S. marcescens 12
3.2 Identification of chromosomal TA system, ccdABSm, in S. marcescens 12
3.3 ccdABSm is involved in cell death during swarming development 14
3.4 RssAB affects cell death in swarming development 15
3.5 RssAB down-regulates expression of ccdABSm 16
3.6 RssAB signaling induces cell death in early swarming development 18
Chapter 4 Discussion 20
Chapter 5 Reference 24

Figure contents
Fig.3-1 Population-specific cell death during swarming in S. marcescens CH-1. 29
Fig.3-2 Sequence alignment of CcdBSm homolog and location of the CcdBSm gene on the chromosome. 30
Fig.3-3 The effect of the overproduction of CcdBSm toxins on S. marcescens survival in solid and liquid media. 31
Fig.3-4 ccdABSm mediates postsegregational killing (PSK) in S. marcescens. 32
Fig.3-5 Deletion of chromosomal ccdABSm reduces cell death during swarming. 33
Fig.3-6 The effect of ccdAB TA system during swarming development. 34
Fig.3-7 RssAB regulates cell death in swarming development. 35
Fig.3-8 Expression of ccdABSm was indirectly regulated by RssAB. 36
Fig.3-9 ccdABSm co-transcribe with upstream genes. 37
Fig.3-10 Signaling of RssAB results in cell death during swarming development. 38
Fig.3-11 Proposed models of how does RssAB regulate swarming initiation in S. marcescens 39

Table contents
Table 2-1. Bacteria strains used in this study 6
Table 2-2. The plasmid used in this study 7
Table 2-3. The primers used in this study 8

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