Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/21724
標題: Characterization of hns Gene of Xanthomonas campestris pv. campestris
十字花科黑腐病菌 hns 基因之特性分析
作者: Wang, Ching-Hua
王清華
關鍵字: 十字花科蔬菜;Xanthomonas campestris;黑腐病;植物病原菌;Clp;H-NS
出版社: 分子生物學研究所
引用: 蕭懿民. (2003). 十字花科黑腐病菌蛋白酶基因 prt1 與纖維酶基因 engA 之轉 錄調控研究. 國立中興大學分子生物研究所博士論文. 陳智華. (2004). 十字花科黑腐病菌 Clp 蛋白之自我調控及對啟動子調控之研 究. 國立中興大學分子生物研究所研究生論文進度期中報告研究計劃書. 張惠雯. (2005). Xanthomonas campestris pv. campestris 主導次要果膠酶 PelB 的基因之特性. 國立中興大學分子生物研究所碩士論文. Atlung, T. & Ingmer, H. (1997). H-NS: a modulator of environmentally regulated gene expression. Mol Microbiol 24, 7-17. Atlung, T. & Hansen, F. G. (2002). Effect of different concentrations of H-NS protein on chromosome replication and the cell cycle in Escherichia coli. J Bacteriol 184, 1843-1850. Badaut, C., Williams, R., Arluison, V., Bouffartigues, E., Robert, B., Buc, H. & Rimsky, S. (2002). The degree of oligomerization of the H-NS nucleoid structuring protein is related to specific binding to DNA. J Biol Chem 277, 41657-41666. Becker, A., Katzen, F., Puhler, A. & Ielpi, L. (1998). Xanthan gum biosynthesis and application: a biochemical/genetic perspective. Appl Microbiol Biotechnol 50, 145-152. Belin, P. & Boquet, P. L. (1994). The Escherichia coli dsbA gene is partly transcribed from the promoter of a weakly expressed upstream gene. Microbiology 140 ( Pt 12), 3337-3348. Bertin, P., Terao, E., Lee, E. H., Lejeune, P., Colson, C., Danchin, A. & Collatz, E. (1994). The H-NS protein is involved in the biogenesis of flagella in Escherichia coli. J Bacteriol 176, 5537-5540. Bertin, P., Benhabiles, N., Krin, E., Laurent-Winter, C., Tendeng, C., Turlin, E., Thomas, A., Danchin, A. & Brasseur, R. (1999). The structural and functional organization of H-NS-like proteins is evolutionarily conserved in gram-negative bacteria. Mol Microbiol 31, 319-329. Birnboim, H. C., and Doly, J. (1979). A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res 7, 1513-1523. Bloch, V., Yang, Y., Margeat, E., Chavanieu, A., Auge, M. T., Robert, B., Arold, S., Rimsky, S. & Kochoyan, M. (2003). The H-NS dimerization domain defines a new fold contributing to DNA recognition. Nat Struct Biol 10, 212-218. Busby, S. & Ebright, R. H. (1997). Transcription activation at class II CAP-dependent promoters. Mol Microbiol 23, 853-859. Ceschini, S., Lupidi, G., Coletta, M., Pon, C. L., Fioretti, E. & Angeletti, M. (2000). Multimeric self-assembly equilibria involving the histone-like protein H-NS. A thermodynamic study. J Biol Chem 275, 729-734. Chan, J. W. & Goodwin, P. H. (1999). The molecular genetics of virulence of Xanthomonas campestris. Biotechnol Adv 17, 489-508. Chen, Y., Shiue, S. J., Huang, C. W., Chang, J. L., Chien, Y. L., Hu, N. T. & Chan, N. L. (2005). Structure and function of the XpsE N-terminal domain, an essential component of the Xanthomonas campestris type II secretion system. J Biol Chem 280, 42356-42363. Chou, F. L., Chou, H. C., Lin, Y. S., Yang, B. Y., Lin, N. T., Weng, S. F. & Tseng, Y. H. (1997). The Xanthomonas campestris gumD gene required for synthesis of xanthan gum is involved in normal pigmentation and virulence in causing black rot. Biochem Biophys Res Commun 233, 265-269. Dame, R. T., Wyman, C. & Goosen, N. (2000). H-NS mediated compaction of DNA visualised by atomic force microscopy. Nucleic Acids Res 28, 3504-3510. Dame, R. T., Wyman, C., Wurm, R., Wagner, R. & Goosen, N. (2002). Structural basis for H-NS-mediated trapping of RNA polymerase in the open initiation complex at the rrnB P1. J Biol Chem 277, 2146-2150. Dame, R. T., Luijsterburg, M. S., Krin, E., Bertin, P. N., Wagner, R. & Wuite, G. J. (2005). DNA bridging: a property shared among H-NS-like proteins. J Bacteriol 187, 1845-1848. Daniels, M. J., Barber, C. E., Turner, P. C., Cleary, W. G. & Sawczyc, M. K. (1984a). Isolation of mutants of Xanthomonas campestris pv. campestris showing altered pathogenicity. J Gen Microbiol 130, 2447-2455. Daniels, M. J., Barber, C. E., Turner, P. C., Sawczyc, M. K., Byrde, R. J. W. & Fielding, A. H. (1984b). Cloning of genes involved in pathogenicity of Xanthomonas campestris pv. campestris using the broad host range cosmid pLAFR1. Embo J 3, 3323-3328. Daniels, M. J., Collinge, D. B., Dow, J. M., Osbourn, A. E. & Roberts, I. N. (1987). Molecular biology of the interaction of Xanthomonas campestris with plant. Plant Physiol Biochem 25, 353-359. de Crecy-Lagard, V., Glaser, P., Lejeune, P., Sismeiro, O., Barber, C. E., Daniels, M. J. & Danchin, A. (1990). A Xanthomonas campestris pv. campestris protein similar to catabolite activation factor is involved in regulation of phytopathogenicity. J Bacteriol 172, 5877-5883. Dersch, P., Schmidt, K. & Bremer, E. (1993). Synthesis of the Escherichia coli K-12 nucleoid-associated DNA-binding protein H-NS is subjected to growth-phase control and autoregulation. Mol Microbiol 8, 875-889. Dole, S., Nagarajavel, V. & Schnetz, K. (2004). The histone-like nucleoid structuring protein H-NS represses the Escherichia coli bgl operon downstream of the promoter. Mol Microbiol 52, 589-600. Dong, Q. & Ebright, R. H. (1992). DNA binding specificity and sequence of Xanthomonas campestris catabolite gene activator protein-like protein. J Bacteriol 174, 5457-5461. Dorman, C. J., Hinton, J. C. & Free, A. (1999). Domain organization and oligomerization among H-NS-like nucleoid-associated proteins in bacteria. Trends Microbiol 7, 124-128. Dorman, C. J. (2004). H-NS: a universal regulator for a dynamic genome. Nat Rev Microbiol 2, 391-400. Dow, J. M., Feng, J. X., Barber, C. E., Tang, J. L. & Daniels, M. J. (2000). Novel genes involved in the regulation of pathogenicity factor production within the rpf gene cluster of Xanthomonas campestris. Microbiology 146 ( Pt 4), 885-891. Dow, J. M., Milligan, D. E., Jamieson, L., Barber, C. E. & Daniels, M. J. (1989). Molecular cloning of a polygalacturonate lyase gene from Xanthomonas campestris pv. campestris and role of the gene product in pathogenicity. Physiol Mol Plant Pathol 35, 113-120. Dow, J. M., Scofield, G., Trafford, K., Turner, P. C. & Daniels, M. J. (1987). A gene cluster in Xanthomonas campestris pv. campestris required for pathogenicity controls the excretion of polygalacturonate lyase and other enzymes. Physiol Mol Plant Pathol 31, 261-271. Ebright, R. H. (1993). Transcription activation at Class I CAP-dependent promoters. Mol Microbiol 8, 797-802. Esposito, D., Petrovic, A., Harris, R., Ono S., Eccleston J. F., Mbabaali A., Haq I., Higgins C. F., Hinton J. C., Driscoll P. C. & Ladbury J. E. (2002). H-NS oligomerization domain structure reveals the mechanism for high order self-association of the intact protein. J Mol Biol 324, 841-850. Francetic, O., Belin, D., Badaut, C. & Pugsley, A. P. (2000). Expression of the endogenous type II secretion pathway in Escherichia coli leads to chitinase secretion. Embo J 19, 6697-6703. Gallant, C. V., Ponnampalam, T., Spencer, H., Hinton, J. C. & Martin, N. L. (2004). H-NS represses Salmonella enterica serovar Typhimurium dsbA expression during exponential growth. J Bacteriol 186, 910-918. Gough, C. L., Dow, J. M., Barber, C. E. & Daniels, M. J. (1988). Cloning of two endoglucanase genes of Xanthomonas campestris pv. campestris: Analysis of the role of the major endoglucanase in pathogenesis. Mol Plant Microbe Interact 1, 275-281. Harding, N. E., Cleary, J. M., Cabanas, D. K., Rosen, I. G. & Kang, K. S. (1987). Genetic and physical analyses of a cluster of genes essential for xanthan gum biosynthesis in Xanthomonas campestris. J Bacteriol 169, 2854-2861. Hayat, M. A. & Mancarella, D. A. (1995). Nucleoid proteins. Micron 26, 461-480. Higgins, C. F., Hinton, J. C., Hulton, C. S., Owen-Hughes, T., Pavitt, G. D. & Seirafi, A. (1990). Protein H1: a role for chromatin structure in the regulation of bacterial gene expression and virulence? Mol Microbiol 4, 2007-2012. Hommais, F., Krin, E., Laurent-Winter, C., Soutourina, O., Malpertuy, A., Le Caer, J. P., Danchin, A. & Bertin, P. (2001). Large-scale monitoring of pleiotropic regulation of gene expression by the prokaryotic nucleoid-associated protein, H-NS. Mol Microbiol 40, 20-36. Hsiao, Y. M. & Tseng, Y. H. (2002). Transcription of Xanthomonas campestris prt1 gene encoding protease 1 increases during stationary phase and requires global transcription factor Clp. Biochem Biophys Res Commun 295, 43-49. Hsiao, Y. M., Liao, H. Y., Lee, M. C., Yang, T. C. & Tseng, Y. H. (2005). Clp upregulates transcription of engA gene encoding a virulence factor in Xanthomonas campestris by direct binding to the upstream tandem Clp sites. FEBS Lett 579, 3525-3533. Ish-Horowicz, D., and Burke, J.F. (1981). Rapid and efficient cosmid cloning. Nucleic Acids Res 9, 2989-2998. James, V. & Hugouvieux-Cotte-Pattat, N. (1996). Regulatory systems modulating the transcription of the pectinase genes of Erwinia chrysanthemi are conserved in Escherichia coli. Microbiology 142 ( Pt 9), 2613-2619. Kohler, T., Curty, L. K., Barja, F., van Delden, C. & Pechere, J. C. (2000). Swarming of Pseudomonas aeruginosa is dependent on cell-to-cell signaling and requires flagella and pili. J Bacteriol 182, 5990-5996. Kolb, A., Busby, S., Buc, H., Garges, S. & Adhya, S. (1993). Transcriptional regulation by cAMP and its receptor protein. Annu Rev Biochem 62, 749-795. Lee, T. C., Chen, S. T., Lee, M. C., Chang, C. M., Chen, C. H., Weng, S. F. & Tseng, Y. H. (2001). The early stages of filamentous phage ψLf infection require the host transcription factor, Clp. J Mol Microbiol Biotechnol 3, 471-481. Miller, J. H. (1972). Experiments in molecular genetics. Cold Spring Habor Laboratory, Cold Spring Harbor, N. Y. Munier, H., Gilles, A. M., Glaser, P., Krin, E., Danchin, A., Sarfati, R. & Barzu, O. (1991). Isolation and characterization of catalytic and calmodulin-binding domains of Bordetella pertussis adenylate cyclase. Eur J Biochem 196, 469-474. Nasser, W. & Reverchon, S. (2002). H-NS-dependent activation of pectate lyases synthesis in the phytopathogenic bacterium Erwinia chrysanthemi is mediated by the PecT repressor. Mol Microbiol 43, 733-748. Paula, D. P., Azzoni, A. R., Siqueira, S. F., Catani, C. F., Rosselli, L. K. & de Souza, A. P. (2003). Expression and purification of a putative H-NS nucleoid-associated protein from the phytopathogen Xylella fastidiosa. Protein Expr Purif 32, 61-67. Rescalli, E., Saini, S., Bartocci, C., Rychlewski, L., De Lorenzo, V. & Bertoni, G. (2004). Novel physiological modulation of the Pu promoter of TOL plasmid: negative regulatory role of the TurA protein of Pseudomonas putida in the response to suboptimal growth temperatures. J Biol Chem 279, 7777-7784. Ryu, S., Ramseier, T. M., Michotey, V., Saier, M. H., Jr. & Garges, S. (1995). Effect of the FruR regulator on transcription of the pts operon in Escherichia coli. J Biol Chem 270, 2489-2496. Sandford, P. A. & Baird, J. (1983). Industrial utilization of polysaccharide. In The polysaccharide, pp. 411-490. Edited by G. O. Aspinall: New York Academic Press. Schroder, O. & Wagner, R. (2002). The bacterial regulatory protein H-NS--a versatile modulator of nucleic acid structures. Biol Chem 383, 945-960. Smyth, C. P., Lundback, T., Renzoni, D., Siligardi G., Beavil R., Layton M., Sidebotham J. M., Hinton J. C., Driscoll P. C., Higgins C. F. & Ladbury J. E. (2000). Oligomerization of the chromatin-structuring protein H-NS. Mol Microbiol 36, 962-972. Sonden, B. & Uhlin, B. E. (1996). Coordinated and differential expression of histone-like proteins in Escherichia coli: regulation and function of the H-NS analog StpA. Embo J 15, 4970-4980. Soutourina, O., Kolb, A., Krin, E., Laurent-Winter, C., Rimsky, S., Danchin, A. & Bertin, P. (1999). Multiple control of flagellum biosynthesis in Escherichia coli: role of H-NS protein and the cyclic AMP-catabolite activator protein complex in transcription of the flhDC master operon. J Bacteriol 181, 7500-7508. Tang, J. L., Gough, C. L., Barber, C. E., Dow, J. M. & Daniels, M. J. (1987). Molecular cloning of protease gene(s) from Xanthomonas campestris pv. campestris: Expression in Escherichia coli and role in pathogenicity. Mol Gen Genet 1987, 443-448. Tendeng, C. & Bertin, P. N. (2003). H-NS in Gram-negative bacteria: a family of multifaceted proteins. Trends Microbiol 11, 511-518. Tendeng, C., Soutourina, O. A., Danchin, A. & Bertin, P. N. (2003). MvaT proteins in Pseudomonas spp.: a novel class of H-NS-like proteins. Microbiology 149, 3047-3050. Thorne, L., Tansey, L. & Pollock, T. J. (1987). Clustering of mutations blocking synthesis of xanthan gum by Xanthomonas campestris. J Bacteriol 169, 3593-3600. Tseng, Y. H., Choy, K. T., Hung, C. H., Lin N. T., Liu J. Y., Lou C. H., Yang B. Y., Wen F. S., Weng S. F. & Wu J. R. (1999). Chromosome map of Xanthomonas campestris pv. campestris 17 with locations of genes involved in xanthan gum synthesis and yellow pigmentation. J Bacteriol 181, 117-125. Ueguchi, C., Suzuki, T., Yoshida, T., Tanaka, K. & Mizuno, T. (1996). Systematic mutational analysis revealing the functional domain organization of Escherichia coli nucleoid protein H-NS. J Mol Biol 263, 149-162. Ueguchi, C., Seto, C., Suzuki, T. & Mizuno, T. (1997). Clarification of the dimerization domain and its functional significance for the Escherichia coli nucleoid protein H-NS. J Mol Biol 274, 145-151. Ussery, D. W., Hinton, J. C., Jordi, B. J., Granum P. E., Seirafi A., Stephen R. J., Tupper A. E., Berridge G., Sidebotham J. M. & Higgins C. F. (1994). The chromatin-associated protein H-NS. Biochimie 76, 968-980. Wang, T. W. T., Y. H. (1992). Electrotransformation of Xanthomonas campestris by RF DNA of filamentous phage ψLf. Lett Appl Microbiol 14, 65-68. Williams, P. H. (1980). Black rot: A continuing threat to world crucifers. Plant Dis 64, 736-742. Woldringh, C. L. & Odijk, T. (1999). Structure of DNA within the bacterial cell: physics and physiology, p. 171–187. In R. L. Charlebois (ed.), Organization of the prokaryotic genome. American Society for Microbiology, Washington, D.C.
摘要: 
The gram-negative plant pathogenic Xanthomonas campestris pv. campestris causes black rot in crucifers. The virulence of this bacterium is mainly depending on its ability to produce large mounts of an exopolysaccharide (EPS, also called xanthan gum) and several extracellular enzymes, the virulence factors. It is known that the global transcription factor Clp (cAMP receptor protein-like protein) is involved in the production and secretion of these virulence factors. Using the consensus Clp-binding sequences (CBS) predicted in our laboratory for genome-wide search, 46 genes with at least one CBS located in the upstream region, suggesting possible regulation by Clp, were found in X. campestris pv. campestris strain 17. One of them was XCC0637, which was predicted to be a DNA-binding protein genes belonging to functional category Ⅲ possibly involved in macromolecule metabolism. It was designated as an X. campestris pv. campestris H-NS, because it possesses an H-NS conserved domain and shares 72, 62, 46 and 35% identities with the global regulator H-NS from Stenotrophomonas maltophilia, Xylella fastidiosa, Rhodopseudomonas palustris and Escherichia coli, respectively. In enterobacteria, H-NS regulates gene expression primarily as a transcriptional repressor, with only a few examples required for a direct positive control. In addition, H-NS and CRP in E. coli regulate gene expression independently. In this study, gel retardation and Phns-lacZ transcriptional fusion assays show that Clp positively regulates expression of hns by direct binding to the promoter. An hns mutan was constructed by insertional mutation. Testing with this mutant indicated that, relative to the wild-type, i) production of EPS and extracellular enzymes was slightly reduced, ii) appearance of the black rot symptom was slightly delayed, by about one day slower, iii) swarming motility was drastically reduced. Finally, transcriptional fusion assays also demonstrated that the X. campestris pv. campestris H-NS exerts negative control on the expression of hns, pelB and xpsE and positive control on the expression of engA.

革蘭氏陰性的 Xanthomonas campestris pv. campestris 為十字花科蔬菜黑腐病 (black rot) 的植物病原菌,此菌所產生之大量胞外多醣 (Exopolysaccharide,亦稱為xanthan gum) 及胞外酵素為本菌之重要致病因子。 目前只知 Clp (cAMP receptor protein-like protein) 為一 global transcription factor ,參與調控胞外酵素以及胞外多醣之產生與分泌。 本實驗室前人將 X. campestris pv. cmpestris 已知的 Clp-binding sequences (CBSs) 加以彙整找出其 genome 中 46 個可能受 Clp 調控的基因。 其中屬於第 III 類基因 (macromolecule metabolism)的 XCC0637 被預測為一 DNA binding protein。 因為其具有 H-NS conserved domain,且與 Stenotrophomonas maltophilia、Xylella fastidiosa、Rhodopseudomonas palustris 以及 Escherichia coli 的 H-NS 之相同度 (identity) 分別為 72%、62%、46% 以及 35%,因此將之命名為 hns。 已知 H-NS 為一 global transcription regulator,負向調控一些基因的表現;不過,也有少數基因可受其正向調控。 在 E. coli,H-NS 與 CRP各自獨立調控一些基因的表現,但並沒有 CRP 可調控 hns 基因表現的相關報導。 由於 Xanthomonas 的 H-NS 還沒有被報導過,本研究利用 Clp 與 hns 上游片段之 gel retardation 試驗,以及透過 Phns-lacZ transcriptional fusion construct 分析野生株 Xc17 以及 clp 突變株 TC817 的 hns 上游片段之啟動子活性,確認 Clp 可經由與 hns 上游區域結合而正向調控 hns 基因表現。 另外構築 hns 突變株 HNSM,進行其特性分析,結果顯示 hns 的缺失造成 Xc17 之胞外酵素分泌能力、胞外多醣產量略為下降,swarming motility 下降 0.5 ㎝,感染十字花科植物時,病徵的出現較野生株晚約一天。 最後,透過 promoter-lacZ transcriptional fusion construct,分析野生株 Xc17 以及 hns 突變株 HNSM 的 hns、prt1、engA、pelB 以及 xpsE 上游片段之啟動子活性,確定 H-NS 可負向調控 hns、pelB 以及 xpsE,正向調控 engA,但並不會對 prt1 基因施以調控。
URI: http://hdl.handle.net/11455/21724
其他識別: U0005-0902200701585700
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