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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 賴信志 | |
| dc.contributor.author | Yu-Tze Horng | en |
| dc.contributor.author | 洪育芝 | zh_TW |
| dc.date.accessioned | 2021-06-13T01:36:58Z | - |
| dc.date.available | 2010-08-08 | |
| dc.date.copyright | 2007-08-08 | |
| dc.date.issued | 2007 | |
| dc.date.submitted | 2007-07-14 | |
| dc.identifier.citation | Adler,J., and Templeton,B. (1967) The effect of environmental conditions on the motility of Escherichia coli. J Gen Microbiol 46: 175-184.
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/30110 | - |
| dc.description.abstract | 靈菌 (Serratia marcescens) 能夠在30 oC時在固體表面上能以swarming的方式移動，但在37 oC時這種現象被抑制，已知細菌swarming需利用鞭毛並分泌biosurfactant，但調控swarming的機制並不清楚。我們利用Tn5跳躍子選擇出在37 oC也能進行Swarming的突變株，其中兩株突變株是分別被跳躍子破壞rssA基因及rssC基因。RssA和RssB為一套two-component system，而RssA為sensor kinase，RssC則預測為醯基轉移脢(acyl-transferase)。而我進一步利用Electrophoretic mobility shift assays及Modified chromatin immunoprecipitation (ChIP) assay證明不論是in vivo或in vitro，磷酸化的RssB可結合(bind)到flhDCSm的promoter， 並以DNase I footprinting assay確定RssB在flhDCSm promoter的位置。此外在Primer extension的實驗也確定flhDCSm的兩個transcriptional start sites， P1和P2，經由promoter activity assay發現RssA-RssB主要調控遠端的promoter，P2。在我第二部份的研究顯示rssC突變株表現出泳動能力增強，鞭毛增加，capsule多醣體增加，細胞的附著能力降低，以及Lipopolysaccharide pattern (脂多醣體)的改變。在電子顯微鏡下，突變株呈現不規則的表面，顯示rssC可能影響outermembrane合成。此外，分析rssC promoter region發現一段類似FlhDC 結合的區域(binding site)，將rssC promoter接luxCDABE分析promoter activity在野生株及flhDCSm突變株是否不同，發現rssC promoter活性在flhDCSm突變株比在野生株較低。綜合實驗結果，flhDCSm可直接受到RssA-RssB的負向調控，由於flhDCSm是啟動鞭毛系統的第一層基因，因此證明RssB-RssA是透過抑制flhDCSm而抑制鞭毛，另一方面也能如鞭毛系統的弟二層基因一樣受到FlhDC的活化。 | zh_TW |
| dc.description.abstract | Swarming in Serratia marcescens is a specialized form of bacterial surface translocation. S. marcescens cells swarm at 30℃ but not at 37℃. The mechanism of swarming, however, is not clear. Our previous studies showed both mutated rssA, encoded a sensor kinase, and mutated rssC, which was predicted to encode a 364 amino-acid polypeptide showing high identity to members of acyl-transferase protein family, revealed precocious swarming phenotypes at 37℃. I showed that direct interaction of phosphorylated RssB (RssB~P) with the flhDCSm promoter region in vivo by electrophoretic mobility shift assays and in vitro by modified chromatin immunoprecipitation (ChIP) assay. The DNase I footprinting assay determine the RssB~P binding site on the flhDCSm promoter region. Primer extension analysis revealed two transcriptional start sites, P1 and P2, of flhDCSm. Analysis of promoter activity was shown that RssA-RssB mainly regulated P2 promoter than P1. In the second part, rssC mutant showed pleiotropic phenotypes including increased swimming motility, flagellum production and capsule polysaccharide production and decreased cell attachment ability. Lipopolysaccharide (LPS) pattern analysis indicated rssC mutant shows a significantly more intense O antigen sugar unit band ladder. Transmission electron microscopy and atomic force microscopy observation showed altered surface morphology in rssC mutant. Further DNA sequence analysis showed a potential FlhDC binding region identified in the rssC promoter region, and the rssC transcription activity was confirmed to be up-regulated by FlhDC. In conclusion, my data showed that activated RssA-RssB signaling directly interacts with the flhDCSm promoter, and inhibits its transcriptional activity, leading to reduced flagellum expression and swarming in S. marcescens. Furthermore FlhDC also activates transcriptional level of rssC. Deficiency of RssC leads to precocious swarming in S. marcescens. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-13T01:36:58Z (GMT). No. of bitstreams: 1 ntu-96-D92424001-1.pdf: 4122742 bytes, checksum: 98f481ec08ed1d9d59d23727eafd1de7 (MD5) Previous issue date: 2007 | en |
| dc.description.tableofcontents | Chapter 1 General Introduction
1.1 Coordinated multicellular behaviours in bacteria 1 1.2 Swarming behaviour in Serratia marcescens 2 1.2.1 S. marcescens is a human pathogen 2 1.2.2 S. marcescens shows multicellular swarming behaviour 2 1.3 Many bacterial species exhibit swarming behaviours 3 1.4 The flagellum motility system is essential for swarming 4 1.5 Hierarchical organization of flagellar systems in enterobacteria 5 1.6 Upstream regulators of the flhDC operon 6 1.7 The RssA-RssB two-component system is involved in swarming regulation in S. maecescens 7 1.8 Acyltransferase plays an important role in lipopolysaccharide synthesis 8 1.9 Main questions and works in this study 9 Chapter 2 Materials and Methods 2.1 Bacterial strains and plasmids and primers 11 2.2 Materials and reagents 13 2.2.1 Chemical reagents 13 2.2.2 Kits 14 2.2.3 Enzymes 14 2.2.4 Medium 15 2.2.5 Antibiotics 15 2.2.6 DNA electrophoresis reagents 15 2.2.7 Southern blot hybridization reagents 16 2.2.8 Plasmid DNA isolation reagents 17 2.2.9 Biofilm formation assay reagents 17 2.2.10 Equipment and Supplies 17 2.2.11 Miscellaneous 19 2.3 Experimental procedures 19 2.3.1 Mini-Tn5km1 mutagenesis 19 2.3.2 Southern blot hybridization 20 2.3.2.1 Preparation of DNA probe 20 2.3.2.2 Transfer of DNA to membrane 21 2.3.2.3 Prehybridization and hybridization 21 2.3.2.4 Detection of homologous DNA on Southern blots 21 2.3.3 Cloning the mutant flanking DNA 22 2.3.4 Preparation of bacterial chromosomal DNA 22 2.3.5 Purification of DNA from agarose gel 23 2.3.5.1 Gel electrophoresis of DNA 23 2.3.5.2 Purification of DNA from low melting agarose gel 23 2.3.6 Construction of recombinant plasmids 23 2.3.6.1 Preparation of vector and insert DNA 23 2.3.6.2 DNA ligation 23 2.3.7 Transformation of DNA into bacterial cells 24 2.3.7.1 Preparation of transformation-competent cells 24 2.3.7.2 Transformation with DNA 24 2.3.8 Electrotransformation 24 2.3.8.1 Preparation of transformation-competent cells 25 2.3.8.2 Transformation with DNA by electroporator 25 2.3.9 Selection of recombinant plasmids 25 2.3.10 Isolation of plasmid DNA 25 2.3.11 Nucleotide sequencing and analysis 26 2.3.12 Cloning of PCR products 27 2.3.12.1 Components of PCR 27 2.3.12.2 Amplification of DNA by PCR 27 2.3.13 Surface translocation assays 27 2.3.13.1 Agar plates for swarming assay 28 2.3.13.2 Agar plates for swimming assay: 28 2.3.14 Detection of luciferase activity. 28 2.3.15 Construction of S. marcescens CH-1 rssCSm insertion and deletion mutants. 28 2.3.16 Cell attachment assay. 29 2.3.17 Transmission electron microscopy (TEM). 29 2.3.18 Atomic force microscopy (AFM) 29 2.3.19 Extraction of lipopolysaccharide (LPS) and polysaccharide (PS). 30 2.3.20 PS characterization. 31 2.3.21 LPS characterization. 31 2.3.22 Gel mobility shift assay 31 2.3.23 Reverse transcription-PCR (RT-PCR) assay. 32 2.3.24 Identification of transcriptional start site(s) in flhDCSm. 33 2.3.25 DNase I footprinting. 34 2.3.26 Modified chromatin immunoprecipitation assay. 35 Chapter 3 The two component system RssA-RssB regulates swarming through modulating flhDCSm promoter activity 37 3.1 Introduction 37 3.2 Results 38 3.2.1 RssA negatively regulates the flagellar motility system 38 3.2.2 Phosphorylated RssB interacts with the flhDCSm upstream promoter i 40 3.2.3 Determination of specific RssB~P binding site within the Fa region 42 3.2.4 RssA reduces transcriptional level of flhDCSm expression 42 3.2.5 In vivo RssAB signaling and binding of RssB~P with the flhDCSm promoter 44 3.3 Discussion 45 Chapter 4 A potential acyltransferase, RssC, regulates S. marcescens swarming 58 4.1 Introduction 58 4.2 Results 59 4.2.1 A Serratia marcescens precocious swarming mutant 59 4.2.2 Characterization of the genetic locus interrupted by transposon 60 4.2.3 RssC determines cell surface morphology, flagellar motiltiy, and cell attachment ability 61 4.2.4 S. marcescens PC105 shows increase in polysaccharide production 63 4.2.5 S. marcescens PC105 shows denser O-antigen pattern than CH-1 63 4.2.6 The rssCsm promoter activity is up-regulated by FlhDC 64 4.3 Discussion 64 Chapter 5 General discussion 73 Reference 77 | |
| dc.language.iso | en | |
| dc.subject | 表面移行 | zh_TW |
| dc.subject | 靈菌 | zh_TW |
| dc.subject | 二元系統RssA-RssB | zh_TW |
| dc.subject | swarming | en |
| dc.subject | Serratia marcescens | en |
| dc.subject | two-component system | en |
| dc.title | 靈菌表面移行行為之調控:-二元系統RssA-RssB調控flhDC進而影響表面移行-RssC, 醯基轉移脢調控靈菌表面移行 | zh_TW |
| dc.title | Regulation of swarming behaviour in Serratia marcescens:
-The two component system RssA-RssB regulates swarming through modulating flhDC promoter activity-A potential acyltransferase, RssC, regulates S. marcescens swarming | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 95-2 | |
| dc.description.degree | 博士 | |
| dc.contributor.oralexamcommittee | 王錦堂,胡小婷,鄧麗珍,史有伶 | |
| dc.subject.keyword | 靈菌,二元系統RssA-RssB,表面移行, | zh_TW |
| dc.subject.keyword | Serratia marcescens,two-component system,swarming, | en |
| dc.relation.page | 87 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2007-07-16 | |
| 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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