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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 賴信志 | |
| dc.contributor.author | Po-Chi Soo | en |
| dc.contributor.author | 蘇伯琦 | zh_TW |
| dc.date.accessioned | 2021-06-13T05:58:39Z | - |
| dc.date.available | 2008-08-02 | |
| dc.date.copyright | 2006-08-02 | |
| dc.date.issued | 2006 | |
| dc.date.submitted | 2006-06-27 | |
| dc.identifier.citation | Adams M , Jia Z (2005) Structural and biochemical analysis reveal pirins to possess quercetinase activity. J Biol Chem 280: 28675-28682.
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/34219 | - |
| dc.description.abstract | 靈菌之表面移行是屬於鞭毛運動，細胞分化及細胞族群密度的多細胞行為。目前已知flhDC 及 Quorum-Sensing 系統為調控靈菌表面移行的重要因子，但仍然還有許多未知的調控機制尚須要進一步探索。靈菌是一種受溫度調控而影響表面移行的細菌，當溫度在低溫30度時會表現出表面移行行為，但當溫度升高到37度時，則表面移行的行為就會被抑制。為了進一步探索這受溫度調控之表面移行行為的機制，我們利用跳躍子來篩選出在37度會表面移行的突變株。這些會提早表面移行的突變株中有兩株分別突變在dapASm-nlpBSm 基因位及pirinSm基因，這兩隻突變株分別跟細胞壁中的peptidoglycan 合成及Pyruvate dehydrogenase (PDH) 酵素活性有關。 在dapASm-nlpBSm 基因突變株中，經由進一步實驗證實，nlpBSm 基因所表現出的lipoprotein NlpBSm 和靈菌的表面移行有關。DapASm 則跟維持細胞型態及細胞膜相關之表現型有關。另一零菌PirinSm 蛋白，在進一步實驗中發現到會和PDH 酵素中的E1次單元形成結合體，且因此結合會由於抑制E1次單元活性，導至整個PDH酵素活性被抑制。且在pirinSm 突變株中E1 次單元活性、PDH酵素活性及細胞ATP的產量分別比野生株多約150%、40%及120%。因此，我們認為靈菌的PirinSm 會經由抑制PDH 酵素活性導至TCA cycle能量調控受到壓抑。 | zh_TW |
| dc.description.abstract | Swarming migration of Serratia marcescens requires both flagellar motility and cellular differentiation and is a population-density-dependent behavior. While the flhDC and quorum-sensing systems have been characterized as important factors regulating S. marcescens swarming, the underlying molecular mechanisms are currently far from being understood. Serratia swarming is thermoregulated and is characterized by continuous surface migration on rich swarming agar surfaces at 30 degrees C but not at 37 degrees C. To further elucidate the mechanisms, identification of specific and conserved regulators that govern the initiation of swarming is essential. We performed transposon mutagenesis to screen for S. marcescens strain CH-1 mutants that swarmed at 37 degrees C. Analysis of a 'precocious-swarming' mutant revealed that the defect in a conserved dapASm-nlpBSm genetic locus and pirinSm gene which are closely related to the synthesis of bacterial cell wall peptidoglycan and pyruvate dehydrogenase (PDH) enzyme activity, respectively. In dapASm-nlpBSm mutant, further complementation and gene knockout studies showed that nlpBSm, which encodes a membrane lipoprotein, NlpBSm, but not dapASm, is specifically involved in swarming regulation. We also present evidence that DapASm is involved in the determination of cell-envelope-associated phenotypes and that NlpBSm is involved in the regulation of swarming motility. In S. marcescens PirinSm, protein pull-down and bacterial two-hybrid assays followed by SDS-PAGE and electrospray ionization (ESI) MASS-MASS analyses showed pyruvate dehydrogenase (PDH) E1 subunit as a component interacting with PirinSm. Functional analyses showed that both PDH E1 subunit activity and PDH enzyme complex activity were inhibited by PirinSm in S. marcescens CH-1. Accordingly, the PDH E1, PDH enzyme complex activities and cellular ATP concentration were increased for up to 250 %, 140 % and 220 % respectively in S. marcescens CH-1 pirinSm mutant. Our results showed that PirinSm plays a regulatory role in the process of pyruvate catabolism to acetyl-CoA through interaction with PDH E1 subunit and inhibiting PDH complex activity in S. marcescens CH-1, suggesting PirinSm be an important protein involved in determining direction of pyruvate metabolism on whether to go towards the tricarboxylic acid (TCA) cycle or the fermentation pathways. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-13T05:58:39Z (GMT). No. of bitstreams: 1 ntu-95-D91424001-1.pdf: 18839449 bytes, checksum: a5ecbdd592d8a994a135397e444adb73 (MD5) Previous issue date: 2006 | en |
| dc.description.tableofcontents | 中文摘要 ……………………………………………………… I
Abstract ……………………………………………………… II Chapter 1 General introduction 1.1 Bacterial differentiatian and multicellular behaviour ……………………1 1.1.1 Specific examples of differentiation and development among bacteria ……… 2 1.2 Bacteria1 swarming migration …………………………………………………… 3 1.2.1 Frequently identified but poor1y understood ………………………… 3 1.2.2 The diversity of bacterial swarming …………………………………………3 1.2.3 The swarming of Vibrio parahaemolyticus ………………………………………4 1.2.4 The multicellular swarming behaviour of Proteus mirabilis ………………4 1.3 The swarming of Serratia marcescens …………………………………………6 1.3.1 S. marcescens is an important opportunistic pathogen ……………………… 1.3.2 The multicellular swarming behaviour of S. marcescens ………………………6 1.4 The factors involved in bacteria motility ………………………………………7 1.4.1 Bacterial swarming ………………………………………………………7 1.4.2 Flagella-dependent swarming motility ………………………………8 1.4.3 Biosurfactant production is essential for the swarming motility …………8 1.4.4 Bacteria biofilm ……………………………………………………………9 1.5 Bacterial lipoprotein …………………………………………………………11 1.5.1 Lipoproteins in Brucella spp. …………………………………………11 1.5.2 Lipoproteins in Campylobacter jejuni …………………………………..………12 1.5.3 The function of lipoproteins ………………………………………………… 12 1.6 meso-diaminopimelate (m-DAP) and bacterial cell wall …………………………13 1.7 Main questions and works in this thesis ……………………………………14 Chapter 2 Materials and Methods 2.1 Bacterial strains, plasmids and primers …………………………………………15 2.2 Materials and reagents ……………………………………………………………16 2.3 Experimental procedures …………………………………………………………22 2.4 Detection of luciferase activity …………………………………………29 2.5 Construction of S. marcescens CH-1 nlpBSm and dapASm insertion deletion mutants ………………………………………………………………………29 2.6 Complementation of precocious-swarming mutants with dapASm and nlpBSm ………30 2.7 Cell attachment assay …………………………………………………………30 2.8 Measurement of hemolysin activity and pattern of cell-surface-associated proteins …………………………………………………………………………30 2.9 Transmission electron microscopy (TEM).……………………………………31 2.10 Scanning electron microscopy (SEM) …………………………………………31 2.11 Cytotoxicity assay . ………………………………………………………….31 2.12 Globomycin assay ……………………………………………………………31 2.13 Hypotonic tolerance assay ………………………………………………………32 2.14 Construction of a S. marcescens CH-1 pirinSm insertion deletion mutant S. marcescens PC103 …………………………………………32 2.15 GST Pull-Down Assay ………………………………………………………33 2.16 Protein Identification ………………………………………………33 2.17 Enzyme Assays ……………………………………………………… 2.18 Acetyl-CoA Concentration Assay ……………………………………………35 2.19 Bacterial Two-hybrid Screening ………………………………………………35 2.20 ATP assay ……………………………………………………………35 Chapter 3 Characterization of the dapA-nlpB Genetic Locus Involved in Regulation of Swarming Motility, Cell Envelope Architecture, Hemolysin Production, and Cell Attachment Ability in Serratia marcescens 3.1 Introduction ………………………………………………………………37 3.2 Results ………………………………………………………………38 3.2.1 A Serratia marcescens mutant with aberrant precocious swarming behavior …………………………………………………………38 3.2.2 Characterization of the locus interrupted by transposon …………………39 3.2.3 nlpBSm is specifically involved in swarming regulation ……………………40 3.2.4 NlpBSm is a constitutively expressed membrane lipoprotein ………………41 3.2.5 dapASm is involved in the determination of cell wall integrity, cell attachment ability, and hemolysin production in S. marcescens …………42 3.2.6 PC100 and PC102 show an increase in S. marcescens cytotoxicity ………44 3.3 Discussion ………………………………………………44 Chapter 4 Characterization of the pirin Genetic Locus Involved in Regulation of Swarming Motility, Hemolysin Production, and Cell Attachment Ability in Serratia marcescens 4.1 Introduction …………………………………………………48 4.2 Results …………………………………………………………49 4.2.1 A Serratia marcescens mutant with aberrant precocious swarming behavior ………………………………………………………………………..49 4.2.2 Characterization of the locus interrupted by transposon ………………..…50 4.2.3 flhDC and flagellum expression were increased in PirinSm mutant strain …51 4.2.4 PirinSm is involved in the cell attachment ability and hemolysin production in S. marcescens ……………………………………………………51 4.2.5 PirinSm is a heat and stationary phase induced Protein …………………52 4.3 Discussion and Conclusion ………………………………52 Chapter 5 Pirin regulates pyruvate catabolism through interaction with pyruvate dehydrogenase E1 subunit and modulating pyruvate dehydrogenase activity 5.1 Introduction ……………………………………………………………………54 5.2 Results ……………………………………………………………………56 5.2.1 A Pirin ortholog is identified in Serrati marcescens …………………………56 5.2.2 S. marcescens PirinSm interacts with E1 subunit of pyruvatedehy drogenase ………………………………………………………………………57 5.2.3 Inhibition of PDH E1 activity by PirinSm ……………………………………58 5.2.4 PDH enzyme complex activity is inhibited by PirinSm in S. marcescens ……60 5.2.5 Cellular ATP concentration is increased in S. marcescens PC103 …………61 5.3 Discussion ……………………………………………………63 Chapter 6 General discussions …………………………………68 Reference list ……………………………………………………72 | |
| dc.language.iso | en | |
| dc.subject | 多細胞行為 | zh_TW |
| dc.subject | 靈菌 | zh_TW |
| dc.subject | multucellular brhavior | en |
| dc.subject | Serratia marcescens | en |
| dc.title | 靈菌多細胞表面移行行為之調控― DapASm-NlpBSm及 PirinSm之調控角色 | zh_TW |
| dc.title | Regulation of multicellular surface translocation behaviour in Serratia marcescens ― Role of DapASm-NlpBSm and PirinSm regulation | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 94-2 | |
| dc.description.degree | 博士 | |
| dc.contributor.oralexamcommittee | 王錦堂,胡小婷,鄧麗珍,鄧述諄 | |
| dc.subject.keyword | 靈菌,多細胞行為, | zh_TW |
| dc.subject.keyword | Serratia marcescens,multucellular brhavior, | en |
| dc.relation.page | 82 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2006-06-28 | |
| 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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