(3.236.122.9) 您好!臺灣時間:2021/05/09 07:55
字體大小: 字級放大   字級縮小   預設字形  
回查詢結果

詳目顯示:::

我願授權國圖
: 
twitterline
研究生:陳淑娟
研究生(外文):Shu-Chuan Chen
論文名稱:十字花科黑腐病菌中XpsG和XpsH蛋白間之交互作用關係探討
論文名稱(外文):Study of the interaction between XpsG and XpsH of Xanthomonas campestris pv. campestris
指導教授:陳凌雲陳凌雲引用關係
指導教授(外文):Ling-Yun Chen
學位類別:碩士
校院名稱:中山醫學院
系所名稱:生物化學研究所
學門:生命科學學門
學類:生物化學學類
論文種類:學術論文
論文出版年:2001
畢業學年度:89
語文別:中文
論文頁數:54
中文關鍵詞:十字花科黑腐病菌一般分泌路徑類似纖毛結構
外文關鍵詞:Xanthomonas campestrisgeneral secretion pathwaypilus-like structure
相關次數:
  • 被引用被引用:0
  • 點閱點閱:76
  • 評分評分:系統版面圖檔系統版面圖檔系統版面圖檔系統版面圖檔系統版面圖檔
  • 下載下載:0
  • 收藏至我的研究室書目清單書目收藏:0
中 文 摘 要
革蘭氏陰性菌之第二類型胞外蛋白分泌途徑,可分為兩個步驟,先由Sec系統,將胞外蛋白送到胞質週緣區,再由另一群12-14個蛋白所組成的外膜分泌系統,將胞外蛋白從胞質週緣區分泌到胞外。在十字花科黑腐病菌中,此分泌系統是由XpsD-O十二個蛋白所組成,本論文探討的對象是此分泌系統成員之中的XpsGHIJ蛋白。XpsGHIJ蛋白的N端具有和PilA纖毛的N端很高的同源性,因此被稱為pseudopilin。它們被推測可能會形成pilus-like的構造,以連接其他重要的蛋白。當中的XpsG可能的功能是形成一個骨架,以提供分泌系統中的其他蛋白來進行組裝,或是指引被分泌的蛋白通過外膜,然而此類結構至今仍未被證實出來。為了想更進一步了解在十字花科黑腐病菌中,XpsG、H、I、J蛋白,彼此之間是否有交互作用關係?是否四者之間會共同來形成一纖毛類似結構?所以,在本實驗中,構築了四個在其C端皆含His-Tag的重組表現蛋白,分別為XpsG-His6、XpsH-His6、XpsI-His6、XpsJ-His6,再利用Ni-NTA親和性管柱來純化蛋白,以西方墨點法分析。結果發現,當分別將XpsH-His6、XpsI-His6、XpsJ-His6的可溶性蛋白與膜蛋白萃取液,通過Ni-NTA親和性管柱,皆可同時將XpsG蛋白一起純化出來。另外,將XpsG-His6的可溶性蛋白通過Ni-NTA親和性管柱,也可同時將XpsH蛋白一起純化出來。因此證明XpsG蛋白分別與XpsH、I、J蛋白,彼此會有交互作用關係存在。藉由Ni-NTA親和性管柱層析實驗知,在不同基因缺損株,例如在xpsI或xpsJ的菌株中XpsH-His6還會與XpsG有交互作用。同樣的在xpsH或xpsJ的菌株中XpsI-His6還會與XpsG有交互作用;在xpsH或xpsI的菌株中XpsJ-His6會與XpsG有交互作用。由此表示XpsH或I或J蛋白與XpsG蛋白之間的相互作用應與其他pseudopilin無關。若將XpsI-His6、XpsJ-His6的可溶性蛋白通過Ni-NTA親和性管柱,取其流出液再以XpsG抗體進行免疫沉澱,可偵測到XpsG蛋白;另以XpsH抗體進行免疫沉澱,也偵測到少量的XpsG蛋白。由此表示XpsG蛋白與XpsH、I、J四蛋白極可能彼此之間有交互作用。接著分析各個XpsG突變蛋白中與XpsH蛋白之間的交互作用關係變化情形。結果發現XpsG-His6與XpsG-Y142-His6的可溶性與酯溶性蛋白與XpsH有交互作用;而XpsG-D103E-His6、XpsG-Y136-His6則與XpsH無交互作用。由Proteinase K(PK)處理Sphaeroplast的實驗發現XpsG-His6與XpsG-Y142-His6屬於PK-resistence狀態;相對之下,XpsG-D103E-His6與XpsG-Y136-His6則屬於PK-sensitive狀態。綜合以上結果分析,XpsG-D103E-His6與XpsG-Y136-His6無法再與XpsH蛋白形成一複合體型式,可能是因為蛋白構形上已發生改變所導致。由於,GspGHIJ纖毛結構一直無法被純化出來,所以,在本實驗中,構築了在其C端含His-Tag的重組表現蛋白XpsG-His6與XpsH-His6,以嘗試對該蛋白進行純化。結果發現以Guanidine-HCl denature 的方式進行純化,可增加純化的回收率。再利用膠質篩濾層析法分析純化出來的XpsG-His6及XpsH-His6蛋白,結果發現,XpsG蛋白及XpsH蛋白分子量均大於660KDa,表示以denature純化出的XpsG-His6及XpsH-His6重組蛋白在renature後為一大分子high-order。

Abstract
In Gram negative bacteria, the export of proteins through the type II secretory pathway requires two steps. Proteins are firstly assisted by the well-characterized Sec system to cross the inner membrane, then require secretory machinery constituted by 12-14 components to overcome the outer membrane barrier. In Xanthomonas campestris, the secretory machinery is assembled by twelve members, XpsD-O. We aim to investigate the structure and function of XpsG, -H, -I, and -J proteins in this machinery. XpsG-J proteins were assumed to form pilus-like structure as they all possess an N-terminal sequence highly homologous with that of PilA, a pilin protein. Among them the most abundant component, XpsG, may play a structural role in formation of the pilus-like structure, thereby the assembled machinery can well span the two membranes and/or the secreted proteins can be guided through. However, no such structure had been demonstrated yet. To this end, four plasmids that express XpsG-His6, XpsH-His6, XpsI-His6, or XpsJ-His6, were constructed and transformed into their respective chromosomal knock-out mutants. Cell extracts containing either soluble or membrane proteins were assayed by Ni-NTA affinity chromatography separately. Western blot analysis of the eluent showed that XpsG was co-eluted either with XpsH-His6, XpsI-His6, or XpsJ-His6. Conversely, XpsH was co-eluted with XpsG-His6. Furthermore, immunoprecipitation assays performed on the Ni-NTA eluent of XpsI-His6-, or XpsJ-His6-containing soluble cell extract revealed that XpsG could be co-precipitated by the anti-XpsH antiserum. These results strongly argue for the protein-protein interactions among XspG, -H, -I and -J.
To further elucidate the roles of XpsG, several mutations were introduced into XpsG-His6 protein. Among the generated mutants, the retention of XpsH by XpsG-D103E-His6 or XpsG-Y136-His6, but not XpsG-Y142-His6, was affected. Coincidentally, both XpsG-D103E-His6 and XpsG-Y136-His6, but not WT or XpsG-Y142-His6, were proteinase K-sensitive in semi-permeabilized spheroplast. This result indicates that the conformation of D103E or Y136 proteins may be drastically changed, therefore affected their protein-protein interactions with XpsH. Several approaches aimed to purify the pili-like complex from wild type Xanthomonas were failed previously, therefore we took advantage of the His6-tag on proteins and peiform the purification by Ni-NTA resin. However, both XpsG-His6 and XpsH-His6 could only been efficiently purified under denatured conditions. After gel-filtration chromatography analysis on the refolded purified proteins separately, both XpsG or XpsH proteins were found to be eluted at the fraction with molecular weight larger than 600 kDa, suggesting the formation of a high-order or aggegated structure.

目錄
頁次
圖次………………………………………………………………..1
附錄說明…………………………………………………………..3
縮寫表……………………………………………………………..4
一、中文摘要……………………………………………………..5
二、英文摘要……………………………………………………..7
三、前言…………………………………………………………..9
四、實驗材料與方法…………………………………………….14
五、結果………………………………………………………….20
六、討論………………………………………………………….25
七、參考文獻…………………………………………………….29
八、圖表………………………………………………………….33
九、附錄………………………………………………………….47

參 考 文 獻
Anderson, D. M., and O. Schneewind. 1997. A mRNA signal for the type III secretion of Yop proteins by Yersinia enterocolitica. Science. 278 : 1140-1143.
Ball, G., Chapon-Herve, V., Bleves, S., Michel, G., and Bally, M., 1999 Assembly for XcpR in the cytoplasmic membrane is required for extracellular protein secretion in Pseudomonas areuginosa. J. Bacteriol. 181 : 382-388.
Bleves, S., R. Voulhoux, G. Michel, A. Lazdunski, J.Tommassen, and A. Filloux. 1998. The secretion apparatus of Pseudomonas aeruginosa : idenfication of fifth pseudopilin, XcpX ( GspK family ). Mol. Microbiol. 27 : 31-40.
Binet, R., S. Letoffe, J. M. Ghigo, P. Delepelaire, and C. Wandersman. 1997. Protein secretion by Gram-negative bacterial ABC exports-a review. Gene 192 : 7-11.
Bitter, W., M. Koster, M. Latijnhouwers, H. de Cock, and J. Tommassen. 1998. Formation of oligomeric rings by XcpQ and PilQ, which are involved in protein transport across the outer membrane of Pseudomonas aeruginosa. Mol. Microbiol. 27 : 209-219.
Chen, L.-Y., Chen, D.-Y., Miaw, J., and Hu. N.-T. 1996. XpsD, an outer membrane protein require for protein secretion by Xanthomonas campestris pv. campestris, forms a multimer. J. Biol. Chem. 271 : 2701-2708.
Cornelis, G. R. and Wolf-Watz, H. 1997. The Yersinia Yop virulon : a bacterial system for subverting eukaryotic cells. Mol. Microbiol. 23 : 861-867.
Economou, A. 1998. Bacterial preprotein transloase : mechanism and comformational dynamics of a processive enzyme. Mol. Microbial. 27 : 511-518.
Fath, M. J., and Kolter, R., 1993. ABC transporters: bacteria exporters. Microbiol. Rev. 57. 995-1017.
Fernandez, L. A., and Berenguer, J., 2000. Secretion and assembly of regular surface in gram-negative bacteria, FEMS Microbiology Review, 24. 21-44.
Filloux, A., G. Michel, and M. Bally. 1998. Gsp-dependent protein secretionin Gram-negative bacteria : the Xcp system of Pseudomonas aeruginosa. FEMS Microbiol. Rev. 22 : 177-198.
Forest, K. T., and Tainer, J. A., 1997. Type-4-pilus-structure outside to inside and top to bottom —a minireview. Gene. 192. 165-169.
Guilvout, I., K. R. Hardie, N. Sauvonnet, and A. P. Pugsley. 1999. Genetic dissection of the outer membrane secretin PulD: Are there distinct domains for multimerization and secretion specificity ? J. Bacteriol. 181 : 7212-7220.
Hardie, K. R., Lory, S., and Pugsley, A. P., 1996. Insertion of an outer membrane protein in Escherichia coli requires a chaperon-like protein. EMBO J., 15 : 978-988.
Lee H.-M., Tyan S.-W., Leu W.-M., Chen L.-Y., Chen D. C., and Hu N.-T. 2001. Involvement of the XpsN Protein in Formation of the XpsL-XpsM complex in Xanthomonas campestris pv. campestris Type II Secretion Apparatus. J. Bacteriol. 183 : 528-535.
Hu, N.-T., Hing, M.-N., Huang, A.-M., Tsai, H.-F., Yang, B.-Y,. Chow, T.-Y., and Tseng, Y.-H., 1992a. Molecular cloning, characterization and nucleotide sequence of the gene for secreted a-amylase from Xanthomonas campestris pv. campestris. J.Gen. Microbiol. 138 : 1647-1655.
Hu, N.-T., Hung, M.-N., Chiou, S.-J., Tang, F., Chiang, D.-C., Huang, H.-Y., and Wu, C.-Y., 1992b. Cloning and characterization of a gene required for the secretion of extracellular enzymes across the outer membrane by Xanthomonas campestris pv. campestris. J. Bacteriol. 174 : 2679-2687.
Kazmierczak, B. I., Mielke, D.L., Russel, M., and Model, P., 1994. pIV, a filamentous phage protein that mediates phage exprot across the bacterial cell envelope, forms a multimer. J. Mol. Biol. 238 : 187-198.
Koster, M., Bitter, W., de Cock, H., Allaoui, A.,Cornelis, G. R., and Tommassen, J., 1997. The outer membrane component, YscC, of the Yop secretion machinery of Yersinia enterocolitica forms a ring-shaped multimeric complex. Mol. Microbiol., 26 : 789-797.
Larsen, R. A., M. G. Thmas. And K. Postle. 1999. Protonomotive force, ExbB and lignd-bond FepA drive comformational changes in TonB. Mol. Microbiol. 31 : 1809-1824.
Lee, C. A. 1997. Type III secretion systems : machines to deliver bacterial proteins into eukaryotic cells. Trends Microbiol. 5 : 148-156.
Linderoth, N. A., M. N. Simon, and M. Russel. 1997. The filamentous phage pIV multimer visualized by scanning transmission electron microscopy. Science. 278 : 1635-1638.
Michel, G., Bleves, S., Ball, G., Lazdunski, A. and Filloux, A., 1998.
Mutual stabilization of the XcpZ and XcpY components of the secretory apparatus in Pseudomonas aeruginosa. Microbiology, 144 : 3379-3386.
Nouwen, N., Stahlberg, H., Pugsley, A. P., and Engel, A., 2000. Domain structure of secretin PulD revealed by limited proteolysis and electro microscopy. EMBO J. 19 : 2229-2236.
Nouwen, N., Ranson, N., Saibil, H., Wolpensinger, B., Engel, A., and Pugsley, A. P., 1999. Secretin PulD: association with pilot PulS, structure, and ion-conducting channel formation. Proc. Natl. Acad. Sci. USA. 96 :
8173-8177.
Nunn, D. N., and Lory, S., 1993. Cleavage. Methylation. and localization of the Pseudomonas aeruginosa export proteins XcpT. —U.
—V. and —W. J. Bacteriol., 175 : 4375-4382.
Nouwen, N., H. Stahlberg, A. P. Pugsley, and A. Engel. 2000. Domain structure of secretin PulD revealed by limited proteolysis and electron microscopy. EMBO J. 19 : 2229-2236.
Possot, O. M., Vignon, G., Bomchil, N., Ebel, F., and Pugsley, A. P., 2000. Multiple interactions between pullulanase secretion components involved in stabilization and cytoplasmic membrane association of PulE. J. Bacteriol. 182 : 2142-2152.
Russel, M. 1998. Macromolecular assembly and secretion across the bacterial cell envelope: type II protein systems. J. Mol. Biol., 279 : 485-499.
Sandkvist, M., Hough, L. P., Bagdasarian, M. M., and Bagdasarian, M., 1999. Direct interaction of the EpsL and EpsM protein of the general secretion apparatus in Vibrio cholerae. J. Bacteriol. 181 : 3129-3135.
Sandkvist, M. 2001a. Type II secretion and pathogenesis. Infect. Immun. 69 : 3523-3535.
Sandkvist, M. 2001b. Biology of type II secretion. Mol. Microbiol., 40 : 271-283.
Sauvonnet, N., Vignon, G., Pugsley, A. P., and Gounon, P., 2000. Pilus formation and protein secretion by the same machinery in Escherichia coli. EMBO J., 19 : 2221-2228.
Sauvonnet, N., Vignon, G., Pugsley, A. P., and Gounon, P., 2000. PpdD type IV pilin of Escherichia coli K-12 can be assembled into pili in Pseudomonas aeruginosa. J. Bacteriol. 182 : 2142-2152.
Turner, L. H., Cano. Lara, J., Nunn, D. N. and Lory, S., 1993. Mutations in the consensus ATP-binding sites of XcpR and PilB eliminate extracellular protein secretion and pilus biogenesis in Pseudomonas aeruginosa. J. Bacteriol. 175 4962-4969.
Thomas, J. D, P. J. Reeves, and G. P. Salmond. 1997. The general secretion pathway of Erwinia carotovora subsp. Carotovora : analysis of the membrane topology of OutC and OutF. Microbiology. 143 : 713-720.

QRCODE
 
 
 
 
 
                                                                                                                                                                                                                                                                                                                                                                                                               
第一頁 上一頁 下一頁 最後一頁 top
無相關論文
 
系統版面圖檔 系統版面圖檔