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dc.contributor.authorLu, Chih-Pangen_US
dc.identifier.citationAbendroth J, Murphy P, Sandkvist M, Bagdasarian M, Hol WG (2005) The X-ray structure of the type II secretion system complex formed by the N-terminal domain of EpsE and the cytoplasmic domain of EpsL of Vibrio cholerae. Journal of molecular biology 348: 845-855 Arts J, van Boxtel R, Filloux A, Tommassen J, Koster M (2007) Export of the pseudopilin XcpT of the Pseudomonas aeruginosa type II secretion system via the signal recognition particle-Sec pathway. Journal of bacteriology 189: 2069-2076 Camberg JL, Johnson TL, Patrick M, Abendroth J, Hol WG, Sandkvist M (2007) Synergistic stimulation of EpsE ATP hydrolysis by EpsL and acidic phospholipids. The EMBO journal 26: 19-27 Chen LY, Chen DY, Miaw J, Hu NT (1996) XpsD, an outer membrane protein required for protein secretion by Xanthomonas campestris pv. campestris, forms a multimer. The Journal of biological chemistry 271: 2703-2708 Chen YL, Hu NT (2013) Function-related positioning of the type II secretion ATPase of Xanthomonas campestris pv. campestris. PloS one 8: e59123 Dums F, Dow JM, Daniels MJ (1991) Structural characterization of protein secretion genes of the bacterial phytopathogen Xanthomonas campestris pathovar campestris: relatedness to secretion systems of other gram-negative bacteria. Molecular & general genetics : MGG 229: 357-364 Filloux A (2004) The underlying mechanisms of type II protein secretion. Biochimica et biophysica acta 1694: 163-179 Francetic O, Buddelmeijer N, Lewenza S, Kumamoto CA, Pugsley AP (2007) Signal recognition particle-dependent inner membrane targeting of the PulG Pseudopilin component of a type II secretion system. Journal of bacteriology 189: 1783-1793 Gray MD, Bagdasarian M, Hol WG, Sandkvist M (2011) In vivo cross-linking of EpsG to EpsL suggests a role for EpsL as an ATPase-pseudopilin coupling protein in the Type II secretion system of Vibrio cholerae. Molecular microbiology 79: 786-798 Hu NT, Hung MN, Chiou SJ, Tang F, Chiang DC, Huang HY, Wu CY (1992) Cloning and characterization of a gene required for the secretion of extracellular enzymes across the outer membrane by Xanthomonas campestris pv. campestris. Journal of bacteriology 174: 2679-2687 Hu NT, Lee PF, Chen C (1995) The type IV pre-pilin leader peptidase of Xanthomonas campestris pv. campestris is functional without conserved cysteine residues. Molecular microbiology 18: 769-777 Korotkov KV, Hol WG (2008) Structure of the GspK-GspI-GspJ complex from the enterotoxigenic Escherichia coli type 2 secretion system. Nature structural & molecular biology 15: 462-468 Korotkov KV, Pardon E, Steyaert J, Hol WG (2009) Crystal structure of the N-terminal domain of the secretin GspD from ETEC determined with the assistance of a nanobody. Structure (London, England : 1993) 17: 255-265 Lee HM, Tyan SW, Leu WM, Chen LY, Chen DC, Hu NT (2001) Involvement of the XpsN protein in formation of the XpsL-xpsM complex in Xanthomonas campestris pv. campestris type II secretion apparatus. Journal of bacteriology 183: 528-535 Lee MS, Chen LY, Leu WM, Shiau RJ, Hu NT (2005) Associations of the major pseudopilin XpsG with XpsN (GspC) and secretin XpsD of Xanthomonas campestris pv. campestris type II secretion apparatus revealed by cross-linking analysis. The Journal of biological chemistry 280: 4585-4591 Misic AM, Satyshur KA, Forest KT (2010) P. aeruginosa PilT structures with and without nucleotide reveal a dynamic type IV pilus retraction motor. Journal of molecular biology 400: 1011-1021 Reichow SL, Korotkov KV, Hol WG, Gonen T (2010) Structure of the cholera toxin secretion channel in its closed state. Nature structural & molecular biology 17: 1226-1232 Sandkvist M (2001) Biology of type II secretion. Molecular microbiology 40: 271-283 Sandkvist M, Bagdasarian M, Howard SP, DiRita VJ (1995) Interaction between the autokinase EpsE and EpsL in the cytoplasmic membrane is required for extracellular secretion in Vibrio cholerae. The EMBO journal 14: 1664-1673 Sandkvist M, Hough LP, Bagdasarian MM, Bagdasarian M (1999) Direct interaction of the EpsL and EpsM proteins of the general secretion apparatus in Vibrio cholerae. Journal of bacteriology 181: 3129-3135 Satyshur KA, Worzalla GA, Meyer LS, Heiniger EK, Aukema KG, Misic AM, Forest KT (2007) Crystal structures of the pilus retraction motor PilT suggest large domain movements and subunit cooperation drive motility. Structure (London, England : 1993) 15: 363-376 Savvides SN, Yeo HJ, Beck MR, Blaesing F, Lurz R, Lanka E, Buhrdorf R, Fischer W, Haas R, Waksman G (2003) VirB11 ATPases are dynamic hexameric assemblies: new insights into bacterial type IV secretion. The EMBO journal 22: 1969-1980 Shiue SJ, Chien IL, Chan NL, Leu WM, Hu NT (2007) Mutation of a key residue in the type II secretion system ATPase uncouples ATP hydrolysis from protein translocation. Molecular microbiology 65: 401-412 Shiue SJ, Kao KM, Leu WM, Chen LY, Chan NL, Hu NT (2006) XpsE oligomerization triggered by ATP binding, not hydrolysis, leads to its association with XpsL. The EMBO journal 25: 1426-1435 Strom MS, Nunn DN, Lory S (1993) A single bifunctional enzyme, PilD, catalyzes cleavage and N-methylation of proteins belonging to the type IV pilin family. Proceedings of the National Academy of Sciences of the United States of America 90: 2404-2408 Tsai RT, Leu WM, Chen LY, Hu NT (2002) A reversibly dissociable ternary complex formed by XpsL, XpsM and XpsN of the Xanthomonas campestris pv. campestris type II secretion apparatus. The Biochemical journal 367: 865-871 Yamagata A, Tainer JA (2007) Hexameric structures of the archaeal secretion ATPase GspE and implications for a universal secretion mechanism. The EMBO journal 26: 878-890en_US
dc.description.abstract十字花科黑腐病菌中的第二型分泌系統 (T2SS) 除了含有內、外胞膜蛋白,並含有一個胞內蛋白XpsE,可以區分成N端及C端兩個獨立區域。胺基酸序列比對顯示其C端區域含有四個高度保留性之nucleotide binding motifs,與ATPase活性相關。根據本實驗室的研究顯示: XpsE與ATP結合後聚集形成六聚體,進而與內膜蛋白XpsL結合,參與第二型分泌系統之運轉。先前研究發現K331M突變會導致XpsE ATPase活性降低,但仍維持部分與ATP結合的能力,KMRA (K331M, R504A) 雙突變則會使XpsE失去與ATP結合的能力,不再形成六聚體。若將XpsE與其同源性蛋白比對,發現其R385胺基酸可能扮演著Arginine finger的角色,推測可穩定ATP水解過程中的中間產物,E357此胺基酸不但同源性極高,且其同源性蛋白PaPilT結構顯示此胺基酸參與和鎂離子的結合,可能也與ATPase活性相關。D387可能扮演著XpsE兩分子界面相互結合之重要胺基酸。先前研究顯示上述胺基酸突變均會使XpsE失去分泌功能。本研究使用綠色螢光蛋白 (superfolder GFP; sfGFP),融合於XpsE C端並利用廣寄主性載體送入十字花科黑腐病菌中表現,以觀察XpsE或其突變蛋白在活體細胞之分佈情形。先前研究顯示孔道蛋白XpsD不存在時,螢光蛋白在細胞邊緣聚集成點,反之則分散於細胞質中,進ㄧ步分析顯示XpsE蛋白僅在參與分泌時呈現與分泌系統結合的現象,分泌完成則解散至細胞質中。為探討XpsE的ATPase活性在其聚散中扮演的角色,本研究利用廣寄主性載體攜帶 xpsER385A-sfgfp、xpsED387A-sfgfp及xpsEE357Q-sfgfp,並觀察綠色螢光在胞內的分佈情形,發現當孔道蛋白XpsD不在時XpsER385A-sfGFP及XpsEE357Q-sfGFP呈現明顯的點狀分佈,說明ATPase活性對於XpsE的聚集並非必要,恢復孔道蛋白XpsD使得XpsER385A-sfGFP點狀分佈的數量減少,強度減弱但並非完全解散,而XpsEE357Q-sfGFP的點狀分佈則維持不變,暗示XpsE的解散可能需要ATPase的ATP水解活性。XpsED387A-sfGFP的觀察結果顯示無論孔道蛋白XpsD存在與否,皆無明顯的螢光點存在,暗示D387A突變可能破壞了XpsE與XpsE之間的結合。zh_TW
dc.description.abstractThe type II secretion system (T2SS) of Xanthomonas campestris pv. campestris contains, in addition to several inner and outer membrane proteins, a cytoplasmic protein XpsE, which can be divided into an N and a C domain. Four conserved nucleotide binding motifs that are related to its ATPase activity are located in the C domain. Based on previous observations made in this laboratory, the XpsE forms hexamer after it binds to ATP and further interacts with the inner membrane protein XpsL, participating the secretion process. Previous studies showed that the K331M mutation caused reduction in the XpsE ATPase activity, but the mutated protein kept part of the ATP-binding activity. The double mutation K331M, R504A (KMRA), however caused the XpsE deficient in ATP-binding, and the mutated protein was unable to form hexamer. Sequence alignment of XpsE with its homologous proteins reveals that the R385 residue may act as the “ariginine finger” stabilizing the intermediate of ATP hydrolysis. The E357 residue is equivalent to a conserved glutamate in a homologous protein PaPilT that was suggested to bind Mg2+ in the crystal structure, presumably being essential in ATP hydrolysis. An equivalent of the D387 residue was predicted to be significant in the XpsE-XpsE interaction. Previous studies indicated mutation of the residues described above caused the XpsE nonfunctional in T2S. In this study, for monitoring cellular distribution of XpsE, I fused the superfolder GFP (sfGFP) to its C-terminus. Using a broad host-range plasmid, I introduced the fused gene into X. campestris pv. campestris in order to observe the distribution of XpsE or its variants in the cell. Previous studies have shown that in the xpsD- strain, the fluorescent protein-labeled XpsE appeared as foci at the cell boundary. In contrast, the XpsE was diffused in the cytoplasm. Further analysis suggested the XpsE is associated with the T2SS only when taking part in the secretion process. When secretion is completed, the XpsE returns to diffused state in the cytoplasm. To examine the significance of ATP hydrolysis by XpsE in its foci formation or disassembly, I used a broad host-range plasmid to carry the xpsER385A-sfgfp、xpsED387A-sfgfp or xpsEE357Q-sfgfp gene and observed distribution of green fluorescence in the cell. In absence of the secretin XpsD, both XpsER385A-sfGFP and XpsEE357Q-sfGFP exhibited distinct foci, implicating ATPase activity is not essential for the assembly of XpsE. Introduction of a plasmid encoding the secretin XpsD made XpsER385A-sfGFP foci reduced in number and fluoresecence intensity. However, the XpsEE357Q-sfGFP foci number or intensity remained unchanged, agreeing with the hypothesis that ATP hydrolysis is required for XpsE dissolution. As contrast, regardless of the presence or absence secretin XpsD, the XpsED387A-sfGFP appeared diffused implicating D387A mutation may have destroyed the interaction between XpsE and XpsE.en_US
dc.description.tableofcontents摘要 I Abstract II 目次 IV 前言 1 材料與方法 7 一、 菌種 7 二、 質體 7 三、 培養基、試劑及緩衝溶液 7 四、 質體DNA萃取 (Preparation of plasmid DNA) 7 五、 限制酵素剪切DNA (Restriction enzyme digestion) 7 六、 接合作用 (Ligation) 8 七、 勝任細胞製備 (Preparation of competent cell) 8 八、 轉型作用 (transformation) 8 九、 電穿孔作用 (electroporation) 8 十、 蛋白質電泳 (SDS-PAGE) 9 十一、 西方點墨法 (western blot) 9 十二、 螢光顯微鏡 (fluorescence microscopy) 10 十三、 細菌螢光點之量化 11 結果 12 討論 17 參考文獻 21 圖一、十字花科黑腐病菌第二型分泌機器組成蛋白之分佈。 24 附錄一、菌種 42 附錄二、質體 43 附錄三、培養基、試劑及緩衝溶液 45zh_TW
dc.subjectXanthomonas campestris pv. campestrisen_US
dc.subjectType II secretion systemen_US
dc.subjectGeneral secretion pathwayen_US
dc.titleXpsE ATPase活性在其與第二型分泌機器結合或解離中的重要性zh_TW
dc.titleThe importance of XpsE ATPase activity in its association with or dissociation from T2SSen_US
dc.typeThesis and Dissertationzh_TW
item.fulltextwith fulltext-
item.openairetypeThesis and Dissertation-
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