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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 賴信志(Hsin-Chih Lai) | |
dc.contributor.author | Jun-Rong Wei | en |
dc.contributor.author | 魏君容 | zh_TW |
dc.date.accessioned | 2021-06-13T06:23:30Z | - |
dc.date.available | 2008-02-09 | |
dc.date.copyright | 2006-02-09 | |
dc.date.issued | 2006 | |
dc.date.submitted | 2006-01-23 | |
dc.identifier.citation | Reference
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/34701 | - |
dc.description.abstract | 本篇論文主要是以分生及生化的方法,分別探討Serratia marcescens在洋菜基表面的多細胞移行行為之分子機制,包括在菌株CH-1的flagella-dependent swarming 現象與菌株SS-1的flagella-independent sliding 現象。Swarming為細菌重要多細胞行為,本實驗室為進一步了解其中機制,以transposon mutagenesis篩選負向調控因子,本論文的一部分即在證實其中RssA-RssB這套二元系統(two-component system)的生化功能。純化出的RssA蛋白質,證實可以自體磷酸化並磷酸化其對應之RssB蛋白質,並透過RssB與DNA結合的能力,調控下游基因表現。
Serratia marcescens SS-1擁有一套quorum sensing訊息調控機制---SpnIR系統。透過SpnI所產生的訊息分子---AHL,和其所對應的調控蛋白SpnR, S. marcescens的sliding,毒素分子及許多次級代謝物都被微妙地調控。此基因組位於一個Tn3 family的transposon中,並仍具有在不同的質體與染色體間傳遞的活性,此結果為quorum-sensing基因組位於transposon中的首例。而SpnR也證實會負向影響此transposon的跳躍頻率。其上的SpnT被大量表現時,S. marcescens SS-1的sliding與色素分泌都會被抑制,但實驗結果推測SpnT並不透過與SpnIR系統之互動調控上述表現型。 | zh_TW |
dc.description.abstract | The main focus of this thesis is to explore underlying mechanism of multicellular surface translocation behavior in Serratia marcescens, including flagella-dependent swarming behavior in strain CH-1 and flagella-independent sliding behavior in strain SS-1. In our lab, we had screened several potential negatively regulators involved in the swarming behaviour through transposon mutagenesis. In part of my thesis, I had proved the biochemical function of a two-component pair—RssA and RssB. In vitro assay confirmed the auto-phosphorylation activity of RssA and its phospho-relay activity to RssB. After phosphorylated, RssB was proved to bind target DNA.
The SpnIR quorum sensing system in Serratia marcescens strain SS-1 regulates flagellum-independent multi-cellular surface migration (sliding motility) and the production of nuclease, biosurfactant, and the red pigment, prodigiosin. spnTIR was found located on a Tn3 family transposon, TnTIR. TnTIR has been proved to transpose between plasmids and chromosomes. SpnIR functions in the new host and negatively regulates the TnTIR transposition frequency. It’s the first report of a quorum sensing system located on a transposon and do great contribution reveal the horizontal transfer and evolutionary mechanism of quorum-sensing genes and alter the way we perceive regulation of bacterial multicellular behaviour. SpnT may function as a negative regulator of surface-dependent migration and secondary metabolite production independent of SpnIR, and possibly through interfering with DNA replication and subsequently cell division. | en |
dc.description.provenance | Made available in DSpace on 2021-06-13T06:23:30Z (GMT). No. of bitstreams: 1 ntu-95-F89424001-1.pdf: 7316629 bytes, checksum: 0917e62547cda993dc1e3fd88b4a311e (MD5) Previous issue date: 2006 | en |
dc.description.tableofcontents | Contents
中文摘要 i Abstract ii Contents iii Figure contents vii Table contents ix Chapter 1 General Introduction 1.1 Serratia marcescens, a unique bacterium in Enterobacteriaceae 1 1.2 Bacterial multicellularity 3 1.3 Differentiation, multicellular behaviour, and surface translocation of S. marcescens 5 1.4 Bacterial signaling system – the two-component system 6 1.5 Bacterial cell-cell communication – the quorum sensing system 8 1.6 Quorum sensing in Serratia and the spnIR quorum sensing system 10 1.7 Horizontal gene transfer, and mobile quorum-sensing unit 12 Chapter 2 Materials and methods 2.1 Bacterial strains 15 2.2 Plasmids 16 2.3 Primers 17 2.4 Enzymes and chemicals 18 2.5 Isolation of plasmid DNA 18 2.6 Preparation of bacterial chromosomal DNA 18 2.7 Extraction of DNA from agarose gels 19 2.8 Restriction enzyme digestion 19 2.9 Calcium chloride Transformation 20 2.10 Eletroporation 20 2.11 Blue-white screening for recombinant plasmids 21 2.12 Colony-PCR screening for recombinant plasmids 22 2.13 DNA sequencing 22 2.14 Plasmid transfer from E. coli S17-1 to S. marcescens by conjugation 23 2.15 Southern blot analysis 24 2.16 DIG-detection assay 24 2.17 Strategies used to knock out spnT 25 2.18 Construction of recombinant plasmids for different purpose 26 2.19 Transposition assay. 29 2.20 RNA extraction 30 2.21 RNA Gel Electrophoresis (Agarose/Formaldehyde) 30 2.22 RT-PCR (reverse transcription-PCR) 31 2.23 Primer extension 31 2.24 Northern blot 31 2.25 Expression of His-tagged recombinant proteins 32 2.26 Protein electrophoresis 32 2.27 Purification of His-tagged recombinant proteins 33 2.28 Antibody preparation 34 2.29 Western blot analysis of SpnT 34 2.30 In vitro phosphorylation and phosphotransfer assay 35 2.31 Gel mobility shift assay 36 2.32 Mobility assay 36 2.33 N-Acyl homoserine lactone assay 37 2.34 Biosurfactant assay 38 2.35 Electron microscopy 39 2.36 Flow cytometry 40 Chapter 3 Biochemical characterization of RssA-RssB, a two-component signal transduction system regulating swarming behavior in Serratia marcescens 3.1 Introduction 42 3.2 A potential two-component system involved in regulation of S. marcescens CH-1 swarming 43 3.3 Autophosphorylation of cRssA and phosphotransfer to RssB. 44 3.4 His(248) or Asp(51) is crucial for RssA or RssB function. 45 3.5 RssB binds directly to its own promoter. 47 3.6 Phosphorylated RssB shows higher DNA binding affinity. 49 3.7 rssB promoter activity is activated in S. marcescens CH-1DA. 51 3.8 Discussion 52 Chapter 4 A Mobile Quorum Sensing System in Serratia marcescens 4.1 Introduction 55 4.2 Duplication of spnTIR region. 57 4.3 The region of mobile spnTIR unit. 59 4.4 Identification and phylogenetic analysis of the TnTIR transposon. 61 4.5 TnTIR transposition. 63 4.6 Elimination of tnpA inactivates TnTIR transposition. 67 4.7 SpnR negatively regulates TnTIR transposition. 67 4.8 SpnIR functions in a LuxIR-negative S. marcescens strain CH-1. 68 4.9 Discussion 69 Chapter 5 spnT, a novel gene involved in regulation of sliding motility, nucleoid aggregation, cell division, and secondary metabolite production 5.1 Introduction 73 5.2 Identification and analysis of spnT. 75 5.3 Synthesis of SpnT peaks as cells enter stationary phase. 78 5.4 SpnT does not regulate SpnI-dependent AHL synthesis and SpnR does not regulate spnT expression. 78 5.5 SpnT inhibits biosurfactant production independent of SpnIR. 80 5.6 spnT over-expression leads to aberrant cell morphology, inhibition of nucleoid segregation and cell division in S. marcescens SS-1. 81 5.7 SpnT regulates cellular DNA content and cell division in E. coli. 84 5.8 SpnT may act through PriA pathway. 85 5.9 SpnT inhibits swarming but not swimming in S. marcescens strain CH-1. 86 5.10 Discussion 87 Chapter 6 Concluding discussion 92 Reference 102 Appendix IStandard buffers, solutions 117 Appendix IIPublications arising from this thesis 123 Figure contents FIG. 1-1 Modular Design of Histidine Kinases and Response Regulators 7 FIG. 1-2 The role of acyl-HSL mediated quorum sensing genes (spnI/R) in regulation of secondary metabolism, exoenzyme production and surface translocation in S. marcescens. 12 FIG. 3-1 Comparison of RssA blocks and RssB domains with two-component family proteins and purification of proteins used in this study. 43 FIG. 3-2 Autophosphorylation of cRssA, cRssA(H248A) and transphosphorylation of RssB or RssB(D51E) by cRssA. 46 FIG. 3-3 In vitro RssB--DNA gel mobility shift assay. 48 FIG. 3-4 Effect of in vitro phosphorylation to RssB on DNA binding capability. 50 FIG. 3-5 rssB transcription level is elevated in S. marcescens CH-1DA. 51 FIG. 4-1 Restriction and physical map of the 13 kb spnTIR locus in S. marcescens SS-1. 57 FIG 4-2 Strategies used for disruption of spnT. 58 FIG. 4-3 spnTIR region is mobile in S. marcescens SS-1. 60 FIG. 4-4 Sequences and designs of TnP1 and TnP2 and the PCR results. 62 FIG. 4-5. Analyses of TnTIR. 62 FIG. 4-6 Transposition of TnTIR::Sm and production of AHL signals. 65 FIG. 4-7 Swarming and pigment synthesis of the LuxIR-negative strain S. marcescens CH-1 68 FIG. 5-1 spnT and spnI form an operon. 76 FIG. 5-2 Physical maps of spnT and its homologues identified in S. marcescens SS-1 and three other bacterial strains. 77 FIG. 5-3 Purification, cellular location and production of SpnT as a function of growth. 78 FIG. 5-4 SpnT does not affect synthesis of AHLs. 80 FIG. 5-5 spnT over-expression reduces biosurfactant production and sliding independent of spnR. 81 FIG. 5-6 spnT over-expression results in cell elongation and chromosomal DNA aggregation in S. marcescens SS-1. 82 FIG. 5-7 spnT over-expression leads to cell elongation and chromosomal DNA aggregation in Escherichia coli. 84 FIG. 5-8 spnT represses swarming but not swimming in S. marcescens CH-1. 87 FIG. 6-1 Summary of the two major sensory, regulatory systems (ovals) involved in swarm cell differentiation and surface motility. 93 Table contents Table 1-1 Quorum sensing genes, signals and regulated phenotypes in Serratia 10 Table 2-1 The bacteria strains used in this thesis 15 Table 2-2 The plasmids used in this thesis 16 Table 2-3 The primers used in this thesis. 17 Table 4-1 TnTIR transposition sites and sequences in target plasmids pBAD18-Kan and pDM4. 66 Table 6-1 G+C content in the chromosome or plasmid, which contain spnT homologue. 95 | |
dc.language.iso | en | |
dc.title | Serratia marcescens多細胞表面移行行為之調控:
二元系統RssA-RssB與跳躍子TnTIR之角色 | zh_TW |
dc.title | Regulation of multicellular surface translocation behaviour in Serratia marcescens:
Roles of a two-component system RssA-RssB and a transposon TnTIR | en |
dc.type | Thesis | |
dc.date.schoolyear | 94-1 | |
dc.description.degree | 博士 | |
dc.contributor.oralexamcommittee | 陳培哲,王錦堂,黃介辰,鄧麗珍 | |
dc.subject.keyword | 細菌多細胞行為,二元系統,跳躍子,群體感應系統, | zh_TW |
dc.subject.keyword | bacterial multicellular behavior,two-component system,transposon,quorum-sensing system, | en |
dc.relation.page | 123 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2006-01-24 | |
dc.contributor.author-college | 醫學院 | zh_TW |
dc.contributor.author-dept | 醫學檢驗暨生物技術學研究所 | zh_TW |
顯示於系所單位: | 醫學檢驗暨生物技術學系 |
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