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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 林乃君 | |
dc.contributor.author | Ru-Fen Wu | en |
dc.contributor.author | 吳如芬 | zh_TW |
dc.date.accessioned | 2021-06-15T05:47:24Z | - |
dc.date.available | 2020-12-24 | |
dc.date.copyright | 2010-08-19 | |
dc.date.issued | 2010 | |
dc.date.submitted | 2010-08-18 | |
dc.identifier.citation | Aubert, D. F., Flannagan, R. S., and Valvano, M. A. (2008). A novel sensor kinase-response regulator hybrid controls biofilm formation and type VI secretion system activity in Burkholderia cenocepacia. Infect Immun 76, 1979-1991.
Bönemann, G., Pietrosiuk, A., Diemand, A., Zentgraf, H., and Mogk, A. (2009). Remodelling of VipA/VipB tubules by ClpV-mediated threading is crucial for type VI protein secretion. EMBO J 28, 315-325. Backert, S., and Meyer, T. F. (2006). Type IV secretion systems and their effectors in bacterial pathogenesis. Curr Opin Microbiol 9, 207-217. Ballister, E. R., Lai, A. H., Zuckermann, R. N., Cheng, Y., and Mougous, J. D. (2008). In vitro self-assembly of tailorable nanotubes from a simple protein building block. Proc Natl Acad Sci U S A 105, 3733-3738. Barcelona-Andrés, B., Marina, A., and Rubio, V. (2002). Gene structure, organization, expression, and potential regulatory mechanisms of arginine catabolism in Enterococcus faecalis. J Bacteriol 184, 6289-6300. Beattie, G. A., and Lindow, S. E. (1994). Comparison of the behavior of epiphytic fitness mutants of Pseudomonas syringae under controlled and field Conditions. Appl Environ Microbiol 60, 3799-3808. Bingle, L. E., Bailey, C. M., and Pallen, M. J. (2008). Type VI secretion: a beginner’s guide. Curr Opin Microbiol 11, 3-8. Bladergroen, M. R., Badelt, K., and Spaink, H. P. (2003). Infection-blocking genes of a symbiotic Rhizobium leguminosarum strain that are involved in temperature-dependent protein secretion. Mol Plant Microbe Interact 16, 53-64. Burtnick, M. N., DeShazer, D., Nair, V., Gherardini, F. C., and Brett, P. J. (2010). Burkholderia mallei cluster 1 type VI secretion mutants exhibit growth and actin polymerization defects in RAW 264.7 murine macrophages. Infect Immun 78. Cascales, E. (2008). The type VI secretion toolkit. EMBO Rep 9, 735-741. Christie, P. J. (2004). Type IV secretion: the Agrobacterium VirB/D4 and related conjugation systems. Biochim Biophys Acta 1694, 219-234. Coulthurst, S. J., and Palmer, T. (2008). A new way out: protein localization on the bacterial cell surface via Tat and a novel Type II secretion system. Mol Microbiol 69. Cunnac, S., Lindeberg, M., and Collmer, A. (2009). Pseudomonas syringae type III secretion system effectors: repertoires in search of functions. Curr Opin Microbiol 12, 53-60. da Silva, F. G., Shen, Y., Dardick, C., Burdman, S., Yadav, R. C., de Leon, A. L., and Ronald, P. C. (2004). Bacterial genes involved in type I secretion and sulfation are required to elicit the rice Xa21-mediated innate immune response. Mol Plant Microbe Interact 17, 593-601. Das, S., Chakrabortty, A., Banerjee, R., Roychoudhury, S., and Chaudhuri, K. (2000). Comparison of global transcription responses allows identification of Vibrio cholerae genes differentially expressed following infection. FEMS Microbiol Lett 190, 87-91. Das, S., and Chaudhuri, K. (2003). Identification of a unique IAHP (IcmF associated homologous proteins) cluster in Vibrio cholerae and other proteobacteria through in silico analysis. In Silico Biol 3, 287-300. Deane, J. E., Abrusci, P., Johnson, S., and Lea, S. M. (2010). Timing is everything: the regulation of type III secretion. Cell Mol Life Sci 67, 1065-1075. Enos-Berlage, J. L., Guvener, Z. T., Keenan, C. E., and McCarter, L. L. (2005). Genetic determinants of biofilm development of opaque and translucent Vibrio parahaemolyticus. Mol Microbiol 55, 1160-1182. Filloux, A. (2004). The underlying mechanisms of type II protein secretion. Biochim Biophys Acta 1694, 163-179. Filloux, A., Hachani, A., and Bleves, S. (2008). The bacterial type VI secretion machine: yet another player for protein transport across membranes. Microbiology 154, 1570-1583. Folkesson, A., Löfdahl, S., and Normark, S. (2002). The Salmonella enterica subspecies I specific centisome 7 genomic island encodes novel protein families present in bacteria living in close contact with eukaryotic cells. Res Microbiol 153, 537-545. Gentschev, I., Dietrich, G., and Goebel, W. (2002). The E. coli alpha-hemolysin secretion system and its use in vaccine development. Trends Microbiol 10, 39-45. Henderson, I. R., Navarro-Garcia, F., Desvaux, M., Fernandez, R. C., and Ala’Aldeen, D. (2004). Type V protein secretion pathway: the autotransporter story. Microbiol Mol Biol Rev 68, 692-744. Hsu, F., Schwarz, S., and Mougous, J. D. (2009). TagR promotes PpkA-catalysed type VI secretion activation in Pseudomonas aeruginosa. Mol Microbiol 72, 1111-1125. Hueck, C. J. (1998). Type III protein secretion systems in bacterial pathogens of animals and plants. Microbiol Mol Biol Rev 62, 379-433. Ishikawa, T., Rompikuntal, P. K., Lindmark, B., Milton, D. L., and Wai, S. N. (2009). Quorum sensing regulation of the two hcp alleles in Vibrio cholerae O1 strains. PLoS One 4. Jarchau, T., Chakraborty, T., Garcia, F., and Goebel, W. (1994). Selection for transport competence of C-terminal polypeptides derived from Escherichia coli hemolysin: the shortest peptide capable of autonomous HlyB/HlyD-dependent secretion comprises the C-terminal 62 amino acids of HlyA. Mol Gen Genet 245, 53-60. Katzen, F., Becker, A., Ielmini, M. V., Oddo, C. G., and Ielpi, L. (1999). New mobilizable vectors suitable for gene replacement in gram-negative bacteria and their use in mapping of the 3' end of the Xanthomonas campestris pv. campestris gum operon. Appl Environ Microbiol 65, 278-282. Kolb, A., Busby, S., Buc, H., Garges, S., and Adhya, S. (1993). Transcriptional regulation by cAMP and its receptor protein. Annu Rev Biochem 62, 749-795. L'etoff'e, S., Delepelaire, P., and Wandersman, C. (1996). Protein secretion in gram-negative bacteria: assembly of the three components of ABC protein-mediated exporters is ordered and promoted by substrate binding. EMBO J 15, 5804-5811. Lee, S. W., Han, S. W., Sririyanum, M., Park, C. J., Seo, Y. S., and Ronald, P. C. (2009). A type I-secreted, sulfated peptide triggers XA21-mediated innate immunity. Science 326, 850-853. Leiman, P. G., Basler, M., Ramagopal, U. A., Bonanno, J. B., Sauder, J. M., Pukatzki, S., Burley, S. K., Almo, S. C., and Mekalanos, J. J. (2009). Type VI secretion apparatus and phage tail-associated protein complexes share a common evolutionary origin. Proc Natl Acad Sci U S A 106, 4154-4159. Lindow, S. E. (1993). Novel method for identifying bacterial mutants with reduced epiphytic fitness. Appl Environ Microbiol 59, 1586-1592. Linhartová, I., Bumba, L., Mašín, J., Basler, M., Osička, R., Kamanová, J., Procházková, K., Adkins, I., Hejnová-Holubová, J., Sadílková, L., et al. (2010). RTX proteins: a highly diverse family secreted by a common mechanism. FEMS Microbiol Rev. Liu, H., Coulthurst, S. J., Pritchard, L., Hedley, P. E., Ravensdale, M., Humphris, S., Burr, T., Takle, G., Brurberg, M. B., Salmond, G. P., et al. (2008). Quorum sensing coordinates brute force and stealth modes of infection in the plant pathogen Pectobacterium atrosepticum. PLoS Pathog 4. Ma, L. S., Lin, J. S., and Lai, E. M. (2009). An IcmF family protein, ImpLM, is an integral inner membrane protein interacting with ImpKL, and its walker a motif is required for type VI secretion system-mediated Hcp secretion in Agrobacterium tumefaciens. J Bacteriol 191, 4316-4329. Mattinen, L., Nissinen, R., Riipi, T., Kalkkinen, N., and Pirhonen, M. (2007). Host-extract induced changes in the secretome of the plant pathogenic bacterium Pectobacterium atrosepticum. Proteomics 7, 3527-3537. Mougous, J. D., Cuff, M. E., Raunser, S., Shen, A., Zhou, M., Gifford, C. A., Goodman, A. L., Joachimiak, G., Ordoñez, C. L., Lory, S., et al. (2006). A virulence locus of Pseudomonas aeruginosa encodes a protein secretion apparatus. Science 312, 1526-1530. Mougous, J. D., Gifford, C. A., Ramsdell, T. L., and Mekalanos, J. J. (2007). Threonine phosphorylation post-translationally regulates protein secretion in Pseudomonas aeruginosa. Nat Cell Biol 9, 797-803. Nicoloff, H., Arsène-Ploetze, F., Malandain, C., Kleerebezem, M., and Bringel, F. (2004). Two arginine repressors regulate arginine biosynthesis in Lactobacillus plantarum. J Bacteriol 186, 6059-6069. Pallen, M., Chaudhuri, R., and Khan, A. (2002). Bacterial FHA domains: neglected players in the phospho-threonine signalling game? Trends Microbiol 10, 556-563. Pallen, M. J. (2002). The ESAT-6/WXG100 superfamily -- and a new Gram-positive secretion system? Trends Microbiol 10, 209-212. Parsons, D. A., and Heffron, F. (2005). sciS, an icmF homolog in Salmonella enterica serovar Typhimurium, limits intracellular replication and decreases virulence. Infect Immun 73, 4338-4345. Pell, L. G., Kanelis, V., Donaldson, L. W., Howell, P. L., and Davidson, A. R. (2009). The phage lambda major tail protein structure reveals a common evolution for long-tailed phages and the type VI bacterial secretion system. Proc Natl Acad Sci U S A 106, 4160-4165. Phadnis, S. H., Ilver, D., Janzon, L., Normark, S., and Westblom, T. U. (1994). Pathological significance and molecular characterization of the vacuolating toxin gene of Helicobacter pylori. Infect Immun 62, 1557-1565. Pieper, R., Huang, S. T., Robinson, J. M., Clark, D. J., Alami, H., Parmar, P. P., Perry, R. D., Fleischmann, R. D., and Peterson, S. N. (2009). Temperature and growth phase influence the outer-membrane proteome and the expression of a type VI secretion system in Yersinia pestis. Microbiology 155, 498-512. Plumbridge, J. (2001). DNA binding sites for the Mlc and NagC proteins: regulation of nagE, encoding the N-acetylglucosamine-specific transporter in Escherichia coli. Nucleic Acids Res 29, 506-514. Pukatzki, S., Ma, A. T., Revel, A. T., Sturtevant, D., and Mekalanos, J. J. (2007). Type VI secretion system translocates a phage tail spike-like protein into target cells where it cross-links actin. Proc Natl Acad Sci U S A 104, 15508-15513. Pukatzki, S., Ma, A. T., Sturtevant, D., Krastins, B., Sarracino, D., Nelson, W. C., Heidelberg, J. F., and Mekalanos, J. J. (2006). Identification of a conserved bacterial protein secretion system in Vibrio cholerae using the Dictyostelium host model system. Proc Natl Acad Sci U S A 103, 1528-1533. Purcell, M., and Shuman, H. A. (1998). The Legionella pneumophila icmGCDJBF genes are required for killing of human macrophages. Infect Immun 66, 2245-2255. Records, A. R., and Gross, D. C. (2010). Sensor kinases RetS and LadS regulate Pseudomonas syringae type VI secretion and virulence factors. J Bacteriol. Robinson, J. B., Telepnev, M. V., Zudina, I. V., Bouyer, D., Montenieri, J. A., Bearden, S. W., Gage, K. L., Agar, S. L., Foltz, S. M., Chauhan, S., et al. (2009). Evaluation of a Yersinia pestis mutant impaired in a thermoregulated type VI-like secretion system in flea, macrophage and murine models. Microb Pathog 47, 243-251. Roest, H. P., Mulders, I. H. M., Spaink, H. P., Wijffelman, C. A., and Lugtenberg, B.J.J. (1997). A Rhizobium leguminosarum biovar trifolii locus not localized on the sym plasmid hinders effective nodulation on plants of the pea cross-inoculation group. Mol Plant Microbe Interact 10, 938-941. Sandkvist, M. (2001). Biology of type II secretion. Mol Microbiol 40, 271-283. Schell, M. A., Ulrich, R. L., Ribot, W. J., Brueggemann, E. E., Hines, H. B., Chen, D., Lipscomb, L., Kim, H. S., Mrázek, J., Nierman, W. C., et al. (2007). Type VI secretion is a major virulence determinant in Burkholderia mallei. Mol Microbiol 64, 1466-1485. Schlieker, C., Zentgraf, H., Dersch, P., and Mogk, A. (2005). ClpV, a unique Hsp100/Clp member of pathogenic proteobacteria. Biol Chem 386, 1115-1127. Southey-Pillig, C. J., Davies, D. G., and Sauer, K. (2005). Characterization of temporal protein production in Pseudomonas aeruginosa biofilms. J Bacteriol 187, 8114-8126. Suarez, G., Sierra, J. C., Erova, T. E., Sha, J., Horneman, A. J., and Chopra, A. K. (2010). A type VI secretion system effector protein, VgrG1, from Aeromonas hydrophila that induces host cell toxicity by ADP ribosylation of actin. J Bacteriol 192, 155-168. Suarez, G., Sierra, J. C., Sha, J., Wang, S., Erova, T. E., Fadl, A. A., Foltz, S. M., Horneman, A. J., and Chopra, A. K. (2008). Molecular characterization of a functional type VI secretion system from a clinical isolate of Aeromonas hydrophila. Microb Pathog 44, 344-361. Sun, G. W., Chen, Y., Liu, Y., Tan, G. Y., Ong, C., Tan, P., and Gan, Y. H. (2010). Identification of a regulatory cascade controlling Type III Secretion System 3 gene expression in Burkholderia pseudomallei. Mol Microbiol 76, 677-689. Szabò, I., Brutsche, S., Tombola, F., Moschioni, M., Satin, B., Telford, J. L., Rappuoli, R., Montecucco, C., Papini, E., and Zoratti, M. (1999). Formation of anion-selective channels in the cell plasma membrane by the toxin VacA of Helicobacter pylori is required for its biological activity. EMBO J 18, 5517-5527. Tekaia, F., Gordon, S. V., Garnier, T., Brosch, R., Barrell, B. G., and Cole, S. T. (1999). Analysis of the proteome of Mycobacterium tuberculosis in silico. Tuber Lung Dis 79. Ulrich, R. L., DeShazer, D., Hines, H. B., and Jeddeloh, J. A. (2004). Quorum sensing: a transcriptional regulatory system involved in the pathogenicity of Burkholderia mallei. infect Immun 72, 6589-6596. Wang, J., Li, C., Yang, H., Mushegian, A., and Jin, S. (1998). A novel serine/threonine protein kinase homologue of Pseudomonas aeruginosa is specifically inducible within the host infection site and is required for full virulence in neutropenic mice. J Bacteriol 180, 6764-6768. Wang, W., Wang, Q., Xiao, J., Liu, Q., Wu, H., Xu, L., and Zhang, Y. (2009). Edwardsiella tarda T6SS component evpP is regulated by esrB and iron, and plays essential roles in the invasion of fish. Fish and shellfish immunology 27, 469-477. Wang, Y. Y. (2009). Characterization of type VI secretion systems in Pseudomonas syringae pv. tomato DC3000. In Agricultural Chemistry (Taipei, National Taiwan University), pp. 68. Weber, B., Hasic, M., Chen, C., Wai, S. N., and Milton, D. L. (2009). Type VI secretion modulates quorum sensing and stress response in Vibrio anguillarum. Environmental Microbiology 11, 3018-3028. Whalen, M. C., Innes, R. W., Bent, A. F., and Staskawicz, B. J. (1991). Identification of Pseudomonas syringae pathogens of Arabidopsis and a bacterial locus determining avirulence on both Arabidopsis and soybean. Plant Cell 3, 49-59. Williams, S. G., Varcoe, L. T., Attridge, S. R., and Manning, P. A. (1996). Vibrio cholerae Hcp, a secreted protein coregulated with HlyA. Infect Immun 64, 283-289. Wu, H. Y., Chung, P. C., Shih, H. W., Wen, S. R., and Lai, E. M. (2008). Secretome analysis uncovers an Hcp-family protein secreted via a type VI secretion system in Agrobacterium tumefaciens. J Bacteriol 190, 2841-2850. Xiao, Y., Lu, Y., Heu, S., and Hutcheson, S. W. (1992). Organization and environmental regulation of the Pseudomonas syringae pv. syringae 61 hrp cluster. J Bacteriol 174, 1734-1741. Zhang, Y. M., and Rock, C. O. (2009). Transcriptional regulation in bacterial membrane lipid synthesis. J Lipid Res 50, S115-119. Zheng, J., Tung, S. L., and Leung, K. L. (2005). Regulation of a type III and a putative secretion system in Edwardsiella tarda by EsrC is under the control of a two-component system, EsrA-EsrB. Infect Immun 73, 4127-4137 | |
dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/47094 | - |
dc.description.abstract | 革蘭氏陰性菌常利用各種分泌系統外泌酵素及作用蛋白,以達到調控生理、生存或感染寄主之功能。目前已知且廣泛研究的分泌系統有第一型至第五型,除了組成與分泌機制不同之外,其外泌基質也有所差異。第六型分泌系統為2006年才被確認之新分泌系統,生物資訊分析結果顯示此分泌系統的基因叢集廣泛存在於多種細菌基因體中,並具有至少13種高度保守性基因。今已證實第六型分泌系統在多種動物與植物病原菌中受到調控並影響病原性,然而詳細作用機制及功能仍未瞭解透徹。 Pseudomonas syringae pv. tomato DC3000為造成細菌性斑點病之病原菌。分析此菌基因體資料庫得知其具兩套第六型分泌系統之基因叢集,其基因表現或功能研究才剛起步。因此,本研究的目的為瞭解此菌兩套第六型分泌系統之調控情形,以及可能扮演之角色。首先,由半定量reverse transcriptase PCR及報導系統可得知此菌的兩套分泌系統基因皆有表現,而第二套分泌系統基因表現較第一套強;其基質hcp基因則只偵測到第二套的表現。藉研究相關基因突變株之感染能力與葉表存活能力變化發現,第二套重要基因突變對番茄、阿拉伯芥等寄主的感染能力與葉表存活能力具些微影響。利用報導基因偵測第六型分泌系統啟動子在進入寄主體內後的表現,發現在感染六小時後有下降趨勢。由這些結果推論,P. s. pv. tomato DC3000的第六型分泌系統和其他動物與植物病原菌不同,可能在病原性上是不需要的或是有負面效應。 | zh_TW |
dc.description.abstract | For the proposes of survival, regulation of physiology and infection, Gram negative bacteria may utilize specialized apparatus to secrete enzymes, toxin or effector proteins. To date, five types of secretion systems (T1SS to T5SS) have been well studied. Type VI secretion system (T6SS) was formally named in 2006. In silico analysis suggested that the highly conserved gene clusters encoding T6SS are widespread in a broad range of microbes, and at least 13 conserved genes have been identified so far. Accumulating evidences connected T6SS to virulence in many animal and plant pathogens; however, the secretory mechanisms and functions remain largely unclear so far. Pseudomonas syringae pv. tomato DC3000 is the causing agent of tomato speck disease. Previous in silico analysis identified two copies of type VI gene clusters in PstDC3000 genome from Pseudomonas genome database, and studies on the gene expression, regulatory mechanisms and functions of T6SSs in PstDC3000 were just initiated two years ago. Therefore, the objectives of this study were to investigate gene expression and functions of T6SSs in PstDC3000. Based on the results of semi-quantitative RT-PCR, expression levels of the t6ss2 cluster and its putative substrate hcp-2 were higher than that of the t6ss1 cluster in all growth conditions we tested, while expression of hcp-1 was barely detectable. Furthermore, in the pathogenicity assay and epiphytic fitness analysis on tomato and Arabidopsis, the single or double mutants of t6ss2-related genes showed slightly redunction in their ability to colonize inside or on host plants. The in planta promoter activity assay showed decreased levels of the promoters of the t6ss2 cluster and the hcp-2 suggested that, unlike animal pathogens or certain plant pathogens, T6SS may play a minor or a negative role in virulence. | en |
dc.description.provenance | Made available in DSpace on 2021-06-15T05:47:24Z (GMT). No. of bitstreams: 1 ntu-99-R97623004-1.pdf: 2161352 bytes, checksum: 32c225eb28e44448706ff450b8744b75 (MD5) Previous issue date: 2010 | en |
dc.description.tableofcontents | Index………………………………………………………………………I
List of Tables and Figures………………………………………III List of Appendix……………………………………………………VI Chinese Abstract……………………………………………………VII English Abstract…………………………………………………VIII Introduction……………………………………………………………1 Discovery of Type VI Secretion System (T6SS)…………………5 Functions of Type VI Secretion Systems …………………………9 Regulation of Type VI Secretion Systems………………………12 Pseudomonas syringae pv. tomato DC3000....................13 Objectives……………………………………………………………15 Materials and Methods………………………………………………16 Bacterial strains and growth conditions………………………16 Plant materials and growth conditions…………………………16 Preparation of electrocompetent cells…………………………16 DNA manipulation……………………………………………………16 Generation of Hcp-2 antibody………………………………………17 Secretion Assay………………………………………………………18 Semi-quantitative reverse transcriptase-PCR…………………19 Pathogenicity test …………………………………………………20 Epiphytic assay………………………………………………………21 Construction of GUS reporter system in PstDC3000……………21 Determination of β-glucuronidase (GUS) activity……………22 Determination of β-glucuronidase (GUS) activity in N. benthamiana leaves.......................................23 Results…………………………………………………………………24 Expression of t6ss cluster in all growth conditions………………………………….....................24 Analysis of upstream regions of the t6ss clusters and hcp genes…….…………..…...................................24 The promoters of t6ss2 clusters and hcp-2 gene exhibited stronger activities………................................25 Hcp-2 proteins was secreted into culture medium in a T6SS2-dependent manner.......................................…27 T6SSs might play a minor role in pathogenicity of PstDC3000…………………................................…27 Expression of t6ss gene clusters and hcp genes was down-regulated after entry of N. benthamiana……………………………………………………………28 T6SSs do not contribute to epiphytic fitness in PstDC3000.................................................29 Discussion………………………………………………………………30 References………………………………………………………………35 | |
dc.language.iso | en | |
dc.title | Pseudomonas syringae pv. tomato DC3000 第六型分泌系統表現與功能之分析 | zh_TW |
dc.title | Characterization of the Expression and Function of Type VI Secretion Systems in Pseudomonas syringae pv. tomato DC3000 | en |
dc.type | Thesis | |
dc.date.schoolyear | 98-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 鄧文玲,賴爾?,曾顯雄,劉瑞芬 | |
dc.subject.keyword | 革蘭式陰性菌,Pseudomonas syringae pv. tomato DC3000,病原菌,第六型分泌系統,hcp基因, | zh_TW |
dc.subject.keyword | Gram negative bacteria,Pseudomonas syringae pv. tomato DC3000,pathogens,Type VI secretion system,hcp, | en |
dc.relation.page | 71 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2010-08-19 | |
dc.contributor.author-college | 生物資源暨農學院 | zh_TW |
dc.contributor.author-dept | 農業化學研究所 | zh_TW |
顯示於系所單位: | 農業化學系 |
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