Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/92219
標題: Xanthomonas 非致病性菌株中 hrp 基因之表現
Expression of hrp genes in a nonpathogenic Xanthomonas strain
作者: 陳正哲
Cheng-Che Chen
關鍵字: 黃原菌;hrpX;穿孔蛋白;溶菌酶;Xanthomonas;hrpX;holin;lysozyme
引用: 李佳霓 (2006) Xanthomonas 溶裂型噬菌體 phiL7 與 phiXo411 之探討。國立中興大學分子生物學研究所博士論文。 張昕淳 (2002) 十字花科蔬菜黑腐病菌的β-lactam類抗生素敏感性突變株 XKBLA 之分析。國立中興大學分子生物學研究所碩士論文。 莊明芬 (2012) 以植物誘導行啟動子表現外源基因的重組線狀噬菌體之構築。國立中興大學分子生物學研究所碩士論文。 蔡任濠 (2010) 噬菌體 phiL7 溶裂基因串之功能探討。國立中興大學分子生物學研究所碩士論文。 鄭宜寧 (2012) 以植物誘導行啟動子表現溶裂基因的模式系統之建立。國立中興大學分子生物學研究所碩士論文。 Arlat, M., Gough, C.L., Barber, C.E., Boucher, C., & Daniels, M.J. (1991) Xanthomonas campestris contains a cluster of hrp genes related to the larger hrp cluster of Pseudomonas solanacearum. Mol Plant Microbe Interact 4:593-601. Arnold, D. L., Pitman, A., & Jackson, R. W. (2003) Pathogenicity and other genomic islands in plant pathogenic bacteria. Mol Plant Pathol 4: 407-420. Borysowski, J., Weber-Dabrowska, B., & Go´rski, A. (2005). Bacteriophage endolysins as a novel class of antibacterial agents. Exp Biol Med 231: 366-377. Guo, Y., Figueiredo, F., Jones, J., & Wang, N. (2011) HrpG and HrpX Play Global Roles in Coordinating Different Virulence Traits of Xanthomonas axonopodis pv. citri Mol Plant Microbe Interact 24:649-61 He, S.Y., Nomura, K., & Whittam, T.S. (2004) Type III protein secretion mechanism in mammalian and plant pathogens. Biochim Biophys Acta 1694: 181–206. Hilbi, H., Moss, J. E., Hersh, D., Chen, Y., Arondel, J., Banerjee, S., Flavell, R. A., Yuan, J., Sansonetti, P. J., & Zychlinsky, A. (1998) Shigella-induced Apoptosis Is Dependent on Caspase-1 Which Binds to IpaB. J Biol Chem 273 : 32895–32900. Huang, D. L., Tang, D. J., Liao, Q., Li, X. Q., He, Y. Q., Feng, J. X., Jiang, B. L., Lu, G. T., & Tang, J. L. (2009) The Zur of Xanthomonas campestris is involved in hypersensitive response and positively regulates the expression of the hrp cluster via hrpX but not hrpG. Mol Plant Microbe Interact 22: 321–329. Huguet, E., Hahn, K., Wengelnik, K., & Bonas, U. (1998) hpaA mutants of Xanthomonas campestris pv. vesicatoria are affected in pathogenicity but retain the ability to induce host-specific hypersensitive reaction. Mol. Microbiol. 29:1379-90. Koebnik, R., Kruger, A., Thieme, F., Urban, A., & Bonas, U. (2006) Specific binding of the Xanthomonas campestris pv. vesicatoria AraC-type transcriptional activator HrpX to plant-inducible promoter boxes. J Bacteriol 188: 7652-7660. Krupovic, M., Daugelavicius, R., & Bamford, D. H. (2007). A novel lysis system in PM2, a lipid-containing marine double-stranded DNA bacteriophage. Mol. Microbiol. 64, 1635-1648. Leyns, F., De Cleene, M., Swings, J. G., & De Ley, J. (1984) The host range of the genus Xanthomonas. Bot. Rev. 50: 308-356. Li, R. F., Lu, G. T., Li, L., Su, H. Z., Feng, G. F., Chen, Y., He, Y. Q., Jiang, B. L., Tang, D. J., & Tang, J. L. (2014) Identification of a putative cognate sensor kinase for the two-component response regulator HrpG, a key regulator controlling the expression of the hrp genes in Xanthomonas campestris pv. campestris. Environ Microbiol. 16:2053-71 Lorenz, C., Kirchner, O., Egler, M., Stuttmann, J., Bonas, U., & Büttner, D. (2008) HpaA from Xanthomonas is a regulator of type III secretion. Mol. Microbiol. 69:344-60. Lukacik, P., Barnard, T. J. et al. (2012) Structural engineering of a phage lysin that targets gram-negative pathogens. Proc. Natl. Acad. Sci. USA. 109:9857-9862. Miller, F. (1972) Glycopeptides of human immunoglobulins. 3. The use and preparation of specific glycosidases. Immunochemistry 9:217-28 Mole, B. M., Baltrus, D. A., Dangl, J. L., and Grant, S. R. (2007) Global virulence regulation networks in phytopathogenic bacteria. Trends Microbiol 15: 363-371. Park, T., Struck, D. K., Dankenbring, C. A., Young, R. (2007) The pinholin of lambdoid phage 21: control of lysis by membrane depolarization. J Bacteriol. 189:9135-9 Rossier, O., G, Van den Ackerveken & U. Bonas (2000) HrpB2 and HrpF from Xanthomonas are type III-secreted proteins and essential for pathogenicity and recognition by the host plant. Mol. Microbiol. 38:828-38. Schweizer, H. D. (1993) Small broad-host-range gentamycin resistance gene cassettes for site-specific insertion and deletion mutagenesis. Biotechniques. 15:831-4. Sukchawalit, R., Vattanaviboon, P., Sallabhan, R., & Mongkolsuk, S. (1999 ) Construction and characterization of regulated L-arabinose-inducible broad host range expression vectors in Xanthomonas. FEMS microbiol.Lett. 181:217-223 Van den Mooter, M., & Swings, J. (1990) Numerical analysis of 295 phenotypic features of 266 Xanthomonas strains and related strains and an improved taxonomy of the genus. Int. J. Syst. Bacteriol. 40:348-369. Vauterin, L., Swings, J., & Kersters, K. Gillis, M., Mew, T. W., Schroth, M. N., Palleroni, N. J., Hilderbrand, D. C., Stead, D. E., Civerolo, E. L., Hayward, A. C., Maraite, H., Stall, R. E., Vidaver, A. K. & Bradbury, J. F. (1990). Towards an improved taxonomy of Xanthomonas'. Int J Syst Bacteriol 40: 312–316. Young, R. (1992) Bacteriophage lysis: mechanism and regulation. Microbiol Rev. 56:430-81 Young, R. (2014) Phage lysis: three steps, three choices, one outcome. J Microbiol. 52:243-258 Zychlinsky, A., Kenny, B., Menard, R., Prevost, M. C., Holland, I.B., & Sansonetti, P.J. (1994) IpaB mediates macrophage apoptosis induced by Shigella flexneri. Mol. Microbiol. 11: 619–627.
摘要: 
Xanthomonas 為革蘭氏陰性的植物病原菌,其 hypersensitive response and pathogenicity (hrp) 基因為宿主植物的致病因子。在植物體內或以貧瘠培養基 (XVM2) 培養會啟動此菌 hrp 基因之表現。HrpX 與 HrpG 為主要調控 hrp 基因的轉錄因子,而 HrpX 又會受到 HrpG 的調控,若將兩基因突變後,菌株會失去病原性。Xc11A 為自然情況下產生之無病原性突變株,無法感染寄主植物產生病徵,其 hrpX mRNA 表現量極低。為了探討 hrp 基因表現與菌株 Xc11A 病原性之間的關係,以微陣列分析法 (microarray) 分析比較培養於 LB 及 XVM2 的 Xc17 及 Xc11A 之 hrp 基因表現。發現相較於 LB,在 XVM2 中,Xc17 大部分的 hrp 基因表現是上升的;而在XVM2 中,Xc11A 大部分的hrp 基因表現低於 Xc17,但 hrpG 表現量卻比 Xc17 高1.8倍。研究指出 HrpG 需被磷酸化才有活性調控下游的基因。因此,推測 Xc11A 失去病原性可能與 HrpG 無活性有關。本實驗中構築了可被誘導表現 HrpXhis 之質體,並證明此質體可表現有活性的 HrpXhis,啟動下游基因。構築帶有以 lac promoter持續表現 HrpXhis 質體之轉殖株,感染宿主後,此轉殖株無病原性。推測質體可能於感染初期即失去。因此以同源重組方式將可持續表現 HrpXhis 之片段插入染色體中,將獲得的突變株感染宿主植物,植物並未出現病徵。推測 hrpX 表現量低不是造成 Xc11 與 Xc11A 失去病原性的主要因素。
噬菌體 phiL7 為可專一感染 Xc17 的溶裂型噬菌體,帶有四個溶裂基因p27 (holin)、p28 (lysozyme)、p29 (Rz) 與 p29.1 (Rz1)。前人研究發現共同表現 p27 與 p28 對 Xc17 的抑菌效果最好。本研究進一步了解 p27 與 p28對 DH5α 及 Xag 生長的影響,發現單獨表現 p28 對 DH5α 的生長沒有影響,若同時表現 p27 與 p28 對兩測試菌體的抑菌效果最為顯著。這些溶裂蛋白質的表現會造成細菌死亡,但菌體並未裂解。此外,構築完成重組質體,可成功表現與 Glutathione S-transferase (GST) 融合的蛋白 GST-p27 與 GST-p28。兩蛋白質表現後會造成菌體死亡,證明融合蛋白具有活性。GST-p27 表現後雖為可溶性蛋白質,但表現量很低,不利於純化。GST-p28 在低溫環境下可形成可溶性蛋白質,經純化並濃縮後的蛋白產量很低,以至於無法進行後續相關實驗。

The hrp (hypersensitive response and pathogenicity) genes of genus Xanthomonas encode factors that are required for pathogenicity in host plants and hypersensitive response in non-host plants. These genes are expressed in planta or when cultured in minimal medium (XVM2). HrpX and HrpG are the main transcriptional regulators for hrp genes, while HrpX is regulated by HrpG; mutation in which causes the loss of pathogenicity. X. campestris pv. campestris strain 11A (Xc11A), a spontaneous avirulent mutant derived from Xc11, has an extremely low level of hrpX mRNA. Microarray analysis performed here showed that expression of most Xc17 hrp genes was enhanced in XVM2, which was generally higher than those of Xc11A, except that the level of hrpG was 1.8 times higher than that in Xc17. Since it has previously been shown that phosphorylation of HrpG is required for regulation of the downstream genes, it is possible that the loss of pathogenicity in Xc11A is correlated to defect in HrpG. With this in mind, a HrpXhis expression plasmid, that could express active HrpXhis, was constructed. Then subcloning was performed by placing the hrpXhis gene downstream of the lac promoter for constructive expression of HrpXhis, which was then introduced into Xc11 and Xc11A, which were used to infect the host plant. Transformants with this construct integrated into the chromosome were also made. Data showed that none of these constructs confers pathogenicity. The above results suggest that low levels of hrpX expression may not be the main reason for Xc11 and Xc11A to lose virulence.
The lytic phage phiL7, specifically infecting Xc17, has four lytic genes: p27 (holin), p28 (lysozyme), p29 (Rz) and p29.1 (Rz1). It has previously been shown that co-expression of p27 and p28 has the strongest antibacterial effect on Xc17. This study showed that expressing p28 alone did not affect the growth of DH5α. However, co-expression of p27 and p28 readily killed DH5α and Xag, although no cell lysis was caused. Furthermore, recombinant plasmids which could express GST-p27 and GST-p28 proteins, both with antibacterial effects, were also constructed; however, the expression levels were too low for further studies.
URI: http://hdl.handle.net/11455/92219
Rights: 同意授權瀏覽/列印電子全文服務,2018-08-28起公開。
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