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研究生:潘亨文
研究生(外文):Heng-Wen Pan
論文名稱:十字花科黑腐病菌XpsE與XpsF蛋白於第二類型胞外蛋白分泌路徑之角色探討
論文名稱(外文):Characterization of the Roles of XpsE and XpsF Proteins in the Type II Secretion Pathway of Xanthomonas campestris pv. campestris
指導教授:呂維茗
指導教授(外文):Wei-Ming Leu
學位類別:碩士
校院名稱:國立中興大學
系所名稱:農業生物科技學研究所
學門:農業科學學門
學類:農業技術學類
論文種類:學術論文
論文出版年:2002
畢業學年度:90
語文別:中文
論文頁數:90
中文關鍵詞:一般分泌途徑
外文關鍵詞:GSPXpsType II secretion pathway
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革蘭氏陰性菌的細胞結構由內而外,依次分為細胞質、內胞膜、胞質週緣區、細胞壁及外胞膜,不同的細菌發展出不同的分泌途徑以將多種蛋白分泌至胞外。第二類型胞外蛋白分泌途徑常見於革蘭氏陰性菌中,又稱為一般分泌途徑 (general secretory pathway; GSP) ,其分泌過程可分成兩個步驟,首先由SEC系統輔助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GSP系統將之分泌到胞外。十字花科黑腐病菌的胞外蛋白分泌系統是由XpsD~N等十一個蛋白組成,其中XpsE是唯一的細胞質蛋白,推測具有ATP-binding motif,可能扮演能量供應者的角色;XpsF蛋白以電腦分析預測其為一個穿過膜三次的內膜蛋白,主體為N端與C端兩個位向細胞質的大片段,但功能未明,本實驗以遺傳與生化實驗策略探討XpsE及XpsF蛋白的角色。首先利用同源性基因置換的方式,製備XpsE蛋白Walker A box單點突變菌株,破壞其與ATP的結合能力,實驗結果證實此菌株無法將澱粉酵素分泌至胞外。此外,在野生株中大量表現XpsF蛋白,發現可導致菌株的分泌能力降低,暗示XpsF蛋白可能與其它相對微量的蛋白分子具有複合體關係;相反的,過量表達XpsE Walker A box單點突變蛋白則不影響分泌。為了進一步解析這些菌株分泌機器的變化,我們以免疫共沉澱實驗策略偵測已知之XpsL/M/N及XpsL/E 等蛋白複合體是否發生明顯的含量變化,並觀察XpsF是否亦參與形成蛋白複合體。結果顯示XpsL/M/N蛋白複合體的形成不會受到上述條件影響,亦未明顯偵測到XpsF蛋白參與複合體的形成。

There are five subcellular compartments including the cytoplasm, inner membrane, periplasm, cell wall and the outer membrane in the gram-negative bacteria. Several distinct routes have evolved for protein export in various bacteria. The type II secretion pathway, also known as the General Secretory Pathway (GSP), is one of such system that widely distributed among bacteria. In this system, the secreted proteins are firstly translocated across the cytoplasmic membrane via the sec machinery. The second step requires 12-14 Gsp proteins for assisting proteins across the outer membrane. These proteins are designated XpsD-N in Xanthomonas campestris pv. campestris. XpsE, the only cytoplasmic protein, is associated with cytoplasmic membrane through interaction with XpsL and may act as a kinase or ATPase. XpsF is predicted as a polytopic inner membrane protein with a small periplasmic loop and two large cytoplasmic domains connected by three transmembrane regions. Roles of XpsF remain unclear thus far. We investigated the roles of XpsE and XpsF involved in the assembly of the secretion machinery by genetic and biochemical strategies. A chromosomal xpsE mutant with point mutation on the Walker A box was constructed by homologous recombination. We concluded that an intact Walker A box is essential for function, although XpsEw did not result in overproduction inhibition. In contrast, overproducing XpsF hampered the secretion process, indicates that XpsF may interact with proteins in a limited amount in the wild type strain. To further resolve the mechanism of secretion, co-immunoprecipitation was performed to detect if there was any qualitative change in the known complexes such as XpsL/M/N and XpsL/E, etc. Simultaneously, roles of XpsF were explored if it involved in complex formation. We concluded that the XpsL/M/N complex formation was not affected in the strain analyzed. Complex formation between XpsF and other Xps proteins was not detected.

中文摘要 I
英文摘要 III
圖目錄 V
縮寫表 VII
前言 1
材料與方法 10
結果 25
討論 35
參考文獻 39
圖表 47
附錄 74

Akrim, M., M. Bally, et al. (1993). "Xcp-mediated protein secretion in Pseudomonas aeruginosa: identification of two additional genes and evidence for regulation of xcp gene expression." Mol Microbiol 10(2): 431-43.
Anderson, D. M. and O. Schneewind (1999). "Type III machines of Gram-negative pathogens: injecting virulence factors into host cells and more." Curr Opin Microbiol 2(1): 18-24.
Ball, G., V. Chapon-Herve, et al. (1999). "Assembly of XcpR in the cytoplasmic membrane is required for extracellular protein secretion in Pseudomonas aeruginosa." J Bacteriol 181(2): 382-8.
Bally, M., A. Filloux, et al. (1992). "Protein secretion in Pseudomonas aeruginosa: characterization of seven xcp genes and processing of secretory apparatus components by prepilin peptidase." Mol Microbiol 6(9): 1121-31.
Binet, R., S. Letoffe, et al. (1997). "Protein secretion by Gram-negative bacterial ABC exporters--a review." Gene 192(1): 7-11.
Bleves, S., A. Lazdunski, et al. (1996). "Membrane topology of three Xcp proteins involved in exoprotein transport by Pseudomonas aeruginosa." J Bacteriol 178(14): 4297-300.
Bleves, S., R. Voulhoux, et al. (1998). "The secretion apparatus of Pseudomonas aeruginosa: identification of a fifth pseudopilin, XcpX (GspK family)." Mol Microbiol 27(1): 31-40.
Charkowski, A. O., H. C. Huang, et al. (1997). "Altered localization of HrpZ in Pseudomonas syringae pv. syringae hrp mutants suggests that different components of the type III secretion pathway control protein translocation across the inner and outer membranes of gram-negative bacteria." J Bacteriol 179(12): 3866-74.
Chen, L. Y., D. Y. Chen, et al. (1996). "XpsD, an outer membrane protein required for protein secretion by Xanthomonas campestris pv. campestris, forms a multimer." J Biol Chem 271(5): 2703-8.
Driessen, A. J., P. Fekkes, et al. (1998). "The Sec system." Curr Opin Microbiol 1(2): 216-22.
Dums, F., J. M. Dow, et al. (1991). "Structural characterization of protein secretion genes of the bacterial phytopathogen Xanthomonas campestris pathovar campestris: relatedness to secretion systems of other gram-negative bacteria." Mol Gen Genet 229(3): 357-64.
Fath, M. J. and R. Kolter (1993). "ABC transporters: bacterial exporters." Microbiol Rev 57(4): 995-1017.
Filloux, A., G. Michel, et al. (1998). "GSP-dependent protein secretion in gram-negative bacteria: the Xcp system of Pseudomonas aeruginosa." FEMS Microbiol Rev 22(3): 177-98.
Hardie, K. R., S. Lory, et al. (1996). "Insertion of an outer membrane protein in Escherichia coli requires a chaperone-like protein." Embo J 15(5): 978-88.
Holland, I. B., M. A. Blight, et al. (1990). "The mechanism of secretion of hemolysin and other polypeptides from gram-negative bacteria." J Bioenerg Biomembr 22(3): 473-91.
Hu, N. T., M. N. Hung, et al. (1992a). "Molecular cloning, characterization and nucleotide sequence of the gene for secreted alpha-amylase from Xanthomonas campestris pv. campestris." J Gen Microbiol 138(Pt 8): 1647-55.
Hu, N. T., M. N. Hung, et al. (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(8): 2679-87.
Hu, N. T., M. N. Hung, et al. (1995a). "Subcellular location of XpsD, a protein required for extracellular protein secretion by Xanthomonas campestris pv. campestris." Microbiology 141(Pt 6): 1395-406.
Hu, N. T., P. F. Lee, et al. (1995). "The type IV pre-pilin leader peptidase of Xanthomonas campestris pv. campestris is functional without conserved cysteine residues." Mol Microbiol 18(4): 769-77.
Hu, N. T., M. N. Hung, et al. (1998). "Insertion mutagenesis of XpsD, an outer-membrane protein involved in extracellular protein secretion in Xanthomonas campestris pv. campestris." Microbiology 144(Pt 6): 1479-86.
Hu, N. T., W. M. Leu, et al. (2002). "XpsG, the major pseudopilin in Xanthomonas campestris pv. campestris, forms a pilus-like structure between cytoplasmic and outer membranes." Biochem J 365(Pt 1): 205-11.
Jacob-Dubuisson, F., C. Locht, et al. (2001). "Two-partner secretion in Gram-negative bacteria: a thrifty, specific pathway for large virulence proteins." Mol Microbiol 40(2): 306-13.
Kagami, Y., M. Ratliff, et al. (1998). "Type II protein secretion by Pseudomonas aeruginosa: genetic suppression of a conditional mutation in the pilin-like component XcpT by the cytoplasmic component XcpR." Mol Microbiol 27(1): 221-33.
Kamoun, S. and C. I. Kado (1990). "A plant-inducible gene of Xanthomonas campestris pv. campestris encodes an exocellular component required for growth in the host and hypersensitivity on nonhosts." J Bacteriol 172(9): 5165-72.
Kamoun, S., E. Tola, et al. (1992). "Rapid generation of directed and unmarked deletions in Xanthomonas." Mol Microbiol 6(6): 809-16.
Kazmierczak, B. I., D. L. Mielke, et al. (1994). "pIV, a filamentous phage protein that mediates phage export across the bacterial cell envelope, forms a multimer." J Mol Biol 238(2): 187-98.
Kumamoto, C. A. (1990). "SecB protein: a cytosolic export factor that associates with nascent exported proteins." J Bioenerg Biomembr 22(3): 337-51.
Lauer, P., N. H. Albertson, et al. (1993). "Conservation of genes encoding components of a type IV pilus assembly/two-step protein export pathway in Neisseria gonorrhoeae." Mol Microbiol 8(2): 357-68.
Lee, C. A. (1997). "Type III secretion systems: machines to deliver bacterial proteins into eukaryotic cells?" Trends Microbiol 5(4): 148-56.
Lee, H. M., K. C. Wang, et al. (2000). "Association of the cytoplasmic membrane protein XpsN with the outer membrane protein XpsD in the type II protein secretion apparatus of Xanthomonas campestris pv. campestris." J Bacteriol 182(6): 1549-57.
Lee, H. M., S. W. Tyan, et al. (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(2): 528-35.
Lu, H. M., S. T. Motley, et al. (1997). "Interactions of the components of the general secretion pathway: role of Pseudomonas aeruginosa type IV pilin subunits in complex formation and extracellular protein secretion." Mol Microbiol 25(2): 247-59.
Michel, G., S. Bleves, et al. (1998). "Mutual stabilization of the XcpZ and XcpY components of the secretory apparatus in Pseudomonas aeruginosa." Microbiology 144(Pt 12): 3379-86.
Michel, L. O., M. Sandkvist, et al. (1995). "Specificity of the protein secretory apparatus: secretion of the heat-labile enterotoxin B subunit pentamers by different species of gram- bacteria." Gene 152(1): 41-5.
Nouwen, N., N. Ranson, et al. (1999). "Secretin PulD: association with pilot PulS, structure, and ion-conducting channel formation." Proc Natl Acad Sci U S A 96(14): 8173-7.
Nouwen, N., H. Stahlberg, et al. (2000). "Domain structure of secretin PulD revealed by limited proteolysis and electron microscopy." Embo J 19(10): 2229-36.
Possot, O., C. d'Enfert, et al. (1992). "Pullulanase secretion in Escherichia coli K-12 requires a cytoplasmic protein and a putative polytopic cytoplasmic membrane protein." Mol Microbiol 6(1): 95-105.
Possot, O. and A. P. Pugsley (1994). "Molecular characterization of PulE, a protein required for pullulanase secretion." Mol Microbiol 12(2): 287-99.
Possot, O. M., L. Letellier, et al. (1997). "Energy requirement for pullulanase secretion by the main terminal branch of the general secretory pathway." Mol Microbiol 24(3): 457-64.
Possot, O. M. and A. P. Pugsley (1997). "The conserved tetracysteine motif in the general secretory pathway component PulE is required for efficient pullulanase secretion." Gene 192(1): 45-50.
Possot, O. M., M. Gerard-Vincent, et al. (1999). "Membrane association and multimerization of secreton component pulC." J Bacteriol 181(13): 4004-11.
Possot, O. M., G. Vignon, et al. (2000). "Multiple interactions between pullulanase secreton components involved in stabilization and cytoplasmic membrane association of PulE." J Bacteriol 182(8): 2142-52.
Pugsley, A. P., C. d'Enfert, et al. (1990). "Genetics of extracellular protein secretion by gram-negative bacteria." Annu Rev Genet 24: 67-90.
Pugsley, A. P. (1993a). "The complete general secretory pathway in gram-negative bacteria." Microbiol Rev 57(1): 50-108.
Pugsley, A. P. (1993b). "Processing and methylation of PuIG, a pilin-like component of the general secretory pathway of Klebsiella oxytoca." Mol Microbiol 9(2): 295-308.
Pugsley, A. P. and O. Possot (1993). "The general secretory pathway of Klebsiella oxytoca: no evidence for relocalization or assembly of pilin-like PulG protein into a multiprotein complex." Mol Microbiol 10(3): 665-74.
Pugsley, A. P. (1996). "Multimers of the precursor of a type IV pilin-like component of the general secretory pathway are unrelated to pili." Mol Microbiol 20(6): 1235-45.
Pugsley, A. P., O. Francetic, et al. (1997). "Recent progress and future directions in studies of the main terminal branch of the general secretory pathway in Gram-negative bacteria--a review." Gene 192(1): 13-9.
Py, B., L. Loiseau, et al. (1999). "Assembly of the type II secretion machinery of Erwinia chrysanthemi: direct interaction and associated conformational change between OutE, the putative ATP-binding component and the membrane protein OutL." J Mol Biol 289(3): 659-70.
Py, B., L. Loiseau, et al. (2001). "An inner membrane platform in the type II secretion machinery of Gram-negative bacteria." EMBO Rep 2(3): 244-8.
Reeves, P. J., D. Whitcombe, et al. (1993). "Molecular cloning and characterization of 13 out genes from Erwinia carotovora subspecies carotovora: genes encoding members of a general secretion pathway (GSP) widespread in gram-negative bacteria." Mol Microbiol 8(3): 443-56.
Russel, M. (1994). "Mutants at conserved positions in gene IV, a gene required for assembly and secretion of filamentous phages." Mol Microbiol 14(2): 357-69.
Russel, M. (1998). "Macromolecular assembly and secretion across the bacterial cell envelope: type II protein secretion systems." J Mol Biol 279(3): 485-99.
Salmond, G. P. and P. J. Reeves (1993). "Membrane traffic wardens and protein secretion in gram-negative bacteria." Trends Biochem Sci 18(1): 7-12.
Sandkvist, M., V. Morales, et al. (1993). "A protein required for secretion of cholera toxin through the outer membrane of Vibrio cholerae." Gene 123(1): 81-6.
Sandkvist, M., M. Bagdasarian, et al. (1995). "Interaction between the autokinase EpsE and EpsL in the cytoplasmic membrane is required for extracellular secretion in Vibrio cholerae." Embo J 14(8): 1664-73.
Sandkvist, M. and M. Bagdasarian (1996). "Secretion of recombinant proteins by Gram-negative bacteria." Curr Opin Biotechnol 7(5): 505-11.
Sandkvist, M., L. O. Michel, et al. (1997). "General secretion pathway (eps) genes required for toxin secretion and outer membrane biogenesis in Vibrio cholerae." J Bacteriol 179(22): 6994-7003.
Sandkvist, M., L. P. Hough, et al. (1999). "Direct interaction of the EpsL and EpsM proteins of the general secretion apparatus in Vibrio cholerae." J Bacteriol 181(10): 3129-35.
Sandkvist, M., M. Bagdasarian, et al. (2000). "Characterization of the multimeric Eps complex required for cholera toxin secretion." Int J Med Microbiol 290(4-5): 345-50.
Sandkvist, M., J. M. Keith, et al. (2000). "Two regions of EpsL involved in species-specific protein-protein interactions with EpsE and EpsM of the general secretion pathway in Vibrio cholerae." J Bacteriol 182(3): 742-8.
Sandkvist, M. (2001a). "Type II secretion and pathogenesis." Infect Immun 69(6): 3523-35.
Sandkvist, M. (2001b). "Biology of type II secretion." Mol Microbiol 40(2): 271-83.
Swings, J. G., and Civerolo, E. C. (1993). Xanthomonas. Chapters 1-2.
Thomas, J. D., P. J. Reeves, et al. (1997). "The general secretion pathway of Erwinia carotovora subsp. carotovora: analysis of the membrane topology of OutC and OutF." Microbiology 143(Pt 3): 713-20.
Tommassen, J., A. Filloux, et al. (1992). "Protein secretion in Pseudomonas aeruginosa." FEMS Microbiol Rev 9(1): 73-90.
Turner, L. R., J. C. Lara, et al. (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(16): 4962-9.
Turner, L. R., J. W. Olson, et al. (1997). "The XcpR protein of Pseudomonas aeruginosa dimerizes via its N-terminus." Mol Microbiol 26(5): 877-87.
Wandersman, C. and P. Delepelaire (1990). "TolC, an Escherichia coli outer membrane protein required for hemolysin secretion." Proc Natl Acad Sci U S A 87(12): 4776-80.
Wandersman, C. (1992). "Secretion across the bacterial outer membrane." Trends Genet 8(9): 317-22.
Wandersman, C. and S. Letoffe (1993). "Involvement of lipopolysaccharide in the secretion of Escherichia coli alpha-haemolysin and Erwinia chrysanthemi proteases." Mol Microbiol 7(1): 141-50.

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