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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 賴信志 | |
dc.contributor.author | Yung-Lin Chang | en |
dc.contributor.author | 張永麟 | zh_TW |
dc.date.accessioned | 2021-06-13T02:01:23Z | - |
dc.date.available | 2007-08-08 | |
dc.date.copyright | 2007-08-08 | |
dc.date.issued | 2007 | |
dc.date.submitted | 2007-07-06 | |
dc.identifier.citation | Adler, J. & Templeton, B. (1967). The effect of environmental conditions on the motility of Escherichia coli. Journal of general microbiology 46, 175-184.
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/30334 | - |
dc.description.abstract | Serratia marcescens是一種會產生紅色色素,隸屬腸道桿菌科 (Enterobacteriaceae)中靈菌屬 (Serratia)的革蘭氏陰性菌。其為一伺機性致病菌,易造成院內感染,而導致免疫低下的病人致病。Swarming為其細菌群體在固體培養基表面移行的一種特殊型態。S. marcescens 野生株CH-1在30°C時,具有在0.8% 培養基上swarming的現象,但在37°C的環境下,卻無swarming的表現型態。為進一步瞭解其中的機制,利用轉位子突變(mini-Tn5-km1) 的方式,在37°C找到數株precocious-swarming mutant。對其中一株進行研究發現轉位子嵌入在sspA ORF(open reading frame)上,sspA的產物是lipoprotein,實驗室學長已經確定sspA缺乏是造成precocious swarming的原因之一。因此我選擇了sspA附近的ORF—rfaD作為研究主題,根據序列比對,推測此基因可能與細菌的LPS合成有關。rfaD的蛋白產物為nucleoside-diphosphate-sugar epimerases,主要是在LPS core合成的步驟將ADP-D-Glycero-D-manno-heptose轉為ADP-L-Glycero-D-manno-heptose。因此我針對野生株CH-1進行了rfaD基因的剔除得到了rfaD knock strain-YL121,可惜的是發現rfaD對於S. marcescens的swarming並無顯著影響;而觀察其LPS也發現到YL121與CH-1的LPS pattern並無差異,故推測在CH-1裡或許有其他的基因可以補償rfaD的功能。另有一株WW100,轉位子嵌入的位置,在manA ORF上游約211 nucleotides處,此位置位於預測promoter region的範圍內。推測可能是由於此基因的表現量改變,造成37°C時的表現型與野生株有所差異。而目前已知manA製造出的蛋白質mannose-6-phosphate isomerase,其功能為轉換fructose-6-P (Fru-6-P) 及 mannose-6-P (Man-6-P),是連接能量代謝及mannose代謝的橋樑。為了進一步研究37°C時precocious-swarming的原因是否導因於manA基因的過量表現或者降低表現,我們針對野生株CH-1進行了manA的基因剔除(WW101)以及overexpression(WW102)的實驗。取得突變株後,觀察其與CH-1在37°C時swarming的表現,推測manA的過量表現應為造成precocious-swarming的原因。我則著手確認manA的基因與蛋白表現是否是過量,並且針對mannose代謝與glucose代謝進行實驗。根據實驗發現葡萄糖能量代謝途徑對於S. marcescens的swarming比LPS合成途徑來的重要。另外也測量CH-1、WW100、WW101的ATP增加量與NADH/NAD ratio,最後推測WW100的manA過量表現,導致能量代謝途徑的活性增加,造成precocious swarming的行為。 | zh_TW |
dc.description.abstract | Serratia marcescens is a red-pigmented gram-negative enteric organism. In addition, it is an opportunistic human pathogen and causes many nosocomial infections. Swarming is a specialized form of bacterial surface translocation when bacteria were cultured on surface of solid media. S. marcescens CH-1 swarms on 0.8% LB agar at 30°C, but not at 37°C. In order to understand the underlying mechanism of temperature-dependent regulation of swarming behavior, transposon mutagenesis had been performed to screen for the mutants that swarm at 37°C. One precocious-swarming mutant was isolated and further experiments showed that insertion site of transposon lay in the predicted open reading frame ‘sspA’. The sspA encoded a lipoprotein which was uncharacterized. This gene was characterized by others in our lab. I undertake another open reading frame, rfaD, which convers ADP-D-Glycero-D-manno-heptose to ADP-L-Glycero-D-manno-heptose in LPS synthesis pathway. In order to investigate whether the rfaD was involved in swarming, I use homologues recombination to knock out rfaD in CH-1, and performed series assay to identify the gene function. Furthermore I found that this potential rfaD mutant was not involved in swarming and LPS synthesis. The underlying mechanism is unknown. Another part of this thesis is manA, due to a previously screened precocious swarming strain WW100 which was inserted in manA promoter by transposon. The manA was predicted to encode a protein named mannose-6-phosphate isomerase whose function is interconverting fructose-6-phosphate and mannose-6-phosphate, and link the glucose and mannose metabolic pathway. A manA knock-out strain was constructed. Also, manA overexperssion in S. marcescens CH-1 was performed to test the WW100 precocious swarming phenotype. I concluded that this phenotype was due to manA overexpression. Subsequently, ATP assay, NADH/NAD ratio and LPS pattern were determined. The precocious swarming phenotype was due to increase in TCA cycle activity. Whether the LPS plays a role in this manA mutant phenotype remained to be determined. | en |
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dc.description.tableofcontents | Contents
中文摘要 01 Abstract 02 Chapter 1: Introduction 03 1.1Serratia marcescens is an important opportunistic pathogen 03 1.2Bacterial multicellular behavior 03 1.3Swarming behavior 04 1.3.1 Frequently identified but poorly understood 04 1.3.2 The diversity of bacterial swarming 04 1.4The factor that involved in bacteria swarming 05 1.4.1 Bacterial signaling system – Quorum sensing system 05 1.4.2 Quorum sensing in Serratia and the spnIR quorum-sensing system 06 1.4.3 Bacterial signaling system – the two-component system 07 1.4.4 Flagellum-dependent swarming motility 08 1.5 Biosurfactant production is essential for swarming motility 10 1.6 LPS are involved in swarming 10 1.7 The role of ATP in swarming behavior 11 Chapter 2: Materials and methods 13 2.1 Bacterial strains, plasmids and primers 13 2.1.1 Bacteria strains 13 2.1.2 plasmids 14 2.1.3 primers 14 2.2 Materials and reagents 14 2.2.1 Kit 14 2.2.2 Medium 14 2.2.3 Antibiotics 15 2.2.4 Enzymes and chemicals 15 2.2.5 Electrophoresis reagents 16 2.2.6 Southern blot hybridization reagents 16 2.2.7 Plasmid DNA isolation reagents 17 2.2.8 Chromosomal DNA purification reagents 17 2.2.9 General chemicals 17 2.2.10 Miscellaneous 18 2.2.11 Equipment and Supplies 18 2.3 Experimental procedures 20 2.3.1 Isolation of plasmid DNA 20 2.3.2 Purification of chromosomal DNA 20 2.3.3 Extraction of DNA from agarose gel 21 2.3.4 Restriction enzyme digestion 21 2.3.5 Ligation reaction 21 2.3.6 Calcium chloride transformation 21 2.3.6.1 Preparation of transformation-competent cells 21 2.3.6.2 Transformation with DNA 21 2.3.7 Electroporation. 22 2.3.7.1 Preparation of electroporation-comptement cells 22 2.3.7.2 Electroporation of DNA 22 2.3.8 Blue-white screening for recombinant plasmids 22 2.3.9 Sequence analysis and databank comparison 23 2.3.10 Plasmid transfer from E. coli S17-1 to S. marcescens by conjugation 23 2.3.11 Southern Blot Hybridization 24 2.3.12 DIG-detection assay 24 2.3.13 Strategies used to knockout rfaD 24 2.3.14 Strategies used to knock out manA 25 2.3.15 Construction of recombinant plasmid for gene overexpression 26 2.3.16 RNA technique 26 2.3.16.1 RNA extraction 26 2.3.16.2 Agarose/Formaldehyde Gel Electrophoresis 26 2.3.16.3 RT-PCR (reverse transcription-PCR) 26 2.3.17 Mobility assay 27 2.3.17.1 Swarming assay of S. marcescens CH-1 27 2.3.17.2 Swimming assay of S. marcescens CH-1 27 2.3.18 Measurement of bacterial growth 27 2.3.19 Quantitation of cell attachment ability 27 2.3.20 Cellular ATP concentration assay. 28 2.3.21 Measurement of NAD and NADH concentrations. 28 Chapter 3 : RfaD might be involved in swarming regulation in Serratia marcescens 30 3.1 Introduction 30 3.2 RfaD is require for LPS synthesis 32 3.3 Construction of rfad insertion-deletion mutant S. marcescens YL121 derived from S. marcescens CH-1. 34 3.4 Characterization of YL121 phenotypes. 36 3.5 Brief discussion 38 Chapter 4: ManA, mannose-6-phosphate, regulate swarming in Serratia marcescens 39 4.1 manA transposon mutant, a precocious-swarming strain 39 4.2 Brief introduction of mannose-6-phosphate isomerase 39 4.3 Construction of manA insertion-deletion mutant S. marcescens WW101 derived from S. marcescens CH-1. 42 4.4 Characterization of WW101 phenotypes. 42 4.5 manA is unique gene for interconversion of M-6-P and F-6-P in S. marcescens 43 4.6 ATP is essential in swarming behavior 44 4.7 Aim of this thesis 46 4.8 manA overexpressed in WW100 46 4.9 Mannose-6-phosphate isomerase overproduction stimulates swarming at 37°C. 46 4.10 manA overexpression leading to increasing of TCA activity 47 4.11 Disscussion 49 Appendix I-rfaD sequence 53 Appendix II-manA sequence 56 Reference 62 Figure contents Fig. 1.1 The model of two component system. 08 Fig. 1.2 Composition of the flhDC regulon. 09 Fig. 1.3 Model of the inner and outer membranes of E. coli K-12. 11 Fig. 3.1 32 Fig. 3.2 Lipopolysaccharide synthesis pathway (from KEGG). 34 Fig. 3.3 Vector design & primer design.. 35 Fig. 3.4 Results of PCR using primer pairs designed to target the two different regions. 35 Fig. 3.5 Southern blot hybridization analyses for confirmation of YL121 genotype. 36 Fig. 3.6 Growth dynamics of CH-1 and YL121 at 30°C and 37°C. 36 Fig. 3.7 Swarming of CH-1 and YL121. 37 Fig. 3.8 The swimming motility of CH-1 and YL121. 37 Fig. 3.9 The LPS pattern assay of S.marcescens. strains. 38 Fig. 4.1 The physical map of the 2.9 kb DNA fragment cloned from S. marcescens CH-1 chromosomal DNA. 39 Fig. 4.2 The PMI functions in carbohydrate metabolism. 40 Fig. 4.3 Hypothesized mannose metabolic pathway in Cryptococcus neoformans. 41 Fig. 4.4 Construction of the plasmid pWW101 carrying interrupted and deleted recombinant manA regions. 42 Fig. 4.5 The growth dynamics of WW100, WW101 and CH-1 at 30°C and 37°C. 43 Fig. 4.6 Swarming of CH-1, WW100 and WW101. 43 Fig. 4.7 Survival assay of CH-1, WW100, and WW101 on various carbohydrate source plates. 44 Fig. 4.8 Pathwat of O antigen biosynthesis. 45 Fig. 4.9 Quantification of manA RNA and protein in S. marcescens strains. 46 Fig. 4.10 manA gene in-trans over-expression in CH-1 and WW102 and swarming phenotype at 37°C. 47 Fig. 4.11 Swarming assay at different glucose and mannose concentration. 48 Fig. 4.12 ATP assay of S.marcescens strains. 48 Fig. 4.13 NADH/NAD ratio in S. marcescens strains. 49 Fig. 4.14 Hypothesis of role of manA in Serratia marcescens swarming behavior. 51 Fig. 4.15 Simplified scheme of the cytosolic and ER part of the N-glycosylation pathway. 52 Table contents Table 1.1 AHL-dependent quorum sensing systems identified in Serratia spp. 6 Table 2.1 The bacteria strains used in this thesis were listed followed. 13 Table 2.2 The plasmids used in this thesis. 13 Table 2.3 The primers used in this thesis. 14 Table 3.1 the potential ORF which were near by transposon inserted. 30 | |
dc.language.iso | en | |
dc.title | 靈菌多細胞表面移行行為之調控:rfaD與manA之角色 | zh_TW |
dc.title | Regulation of swarming behavior in Serratia marcescens-Roles of rfaD and manA | en |
dc.type | Thesis | |
dc.date.schoolyear | 95-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 胡小婷,鄧麗貞,廖淑貞,賈景山 | |
dc.subject.keyword | 靈菌,表面移行, | zh_TW |
dc.subject.keyword | serratia,swarming, | en |
dc.relation.page | 77 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2007-07-09 | |
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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