Please use this identifier to cite or link to this item:
標題: Erwinia carotovora subsp. carotovora 低分子量細菌素選殖與表現
Low-Molecular-Weight Bacteriocin Genes Cloning and Expressioin from Erwinia carotovora subsp. carotovora
作者: 翁培化
Weng, Pei-Hua
關鍵字: bacteriocin
genes cloning
出版社: 化學系所
引用: 1. Hoa Anh Nguyen, Toshio Tomita, Morihiko Hirota, Jun Kaneko, Tetsuya Hayashi, and Yoshiyuki Kamio. 2001. DNA Inversion in the Tail Fiber Gene Alters the Host Range Specificity of Carotovoricin Er, a Phage-Tail-Like Bacteriocin of Phytopathogenic Erwinia carotovora subsp. carotovora Er. J. Bacteriol. 183: 6274 - 6281. 2. Horn, N., Fernandez, A., Dodd, HM., Gasson, MJ., Rodriguez, JM. 2004. Nisin-controlled production of pediocin PA-1 and colicin V in nisin- and non-nisin-producing Lactococcus lactis strains. Appl Environ Microbiol. 70: 5030-5032. 3. Phillips, J. A. and A. Kelman. 1982. Direct fluorescent antibody stain procedure applied to insect transmission of Erwinia carotovora. Phytopathol. 72: 898-901. 4. Kloepper, J. W., M. D. Harrison, and J. W. Brewer. 1979. The association of Erwinia carotovora var. atroseptica and Erwinia carotovora var. carotovora with insects in Colorado. American Potato J. 56: 351-361. 5. Campos, E., E. A. Maher, and A. Kelman. 1982. Relationship of Pectolytic clostridia and Erwinia carotovora strains to decay of potato tubers in storage. Plant Dis. 66: 543-546. 6. Malcolmson, J. F. 1959. A study of Erwinia isolates obtained from soft rots and blackleg of potatoes. Trans. Brit. Mycol. Soc. 42: 261-269. 7. Lund, B. M. 1975. Formation of reducing sugars from sucrose by Erwinia species. J. Gen. Microbiol. 88: 367-371. 8. Lund, B. M. and G. M. Wyatt. 1973. The nature of reducing compounds formed from sucrose by Erwinia carotovora var. atroseptica. J. Gen. Microbiol. 78: 331-336. 9. Gavini, F., J. Mergaert, A. Beji, C. Mielcarek, D. Izard, K. Kersters, and J. De Ley. 1989. Transfer of Enterobacter agglomerans (Beijerinck 1888) Ewing and Fife 1972 to Pantoea gen. nov. as Pantoea agglomerans comb. nov. and description of Pantoea dispersa sp. nov. Int. J. Syst. Bacteriol. 39:337-345. 10. Avrova, A. O., Hyman, L. H., Toth, R. L. & Toth, I. K. (2002). Application of amplified fragment length polymorphism fingerprinting for taxonomy and identification of the soft rot bacteria Erwinia carotovora and Erwinia chrysanthemi. Appl Environ Microbiol 68, 1499–1508. 11. Jones, R. D. and W. J. Dowson. 1950. On the bacteria responsible for soft-rot in stored potatoes and the relation of the tuber to invasion by Bacterium carotovorum (Jones) Lehmann and Neumann. Ann. Appl. Biol. 37: 563-569. 12. Brown, M. R. W., H. Anwar, and A. P. Lambert. 1984. Evidence that mucoid Pseudomonas aeruginosa in the cystic fibrosis lung grows under iron-restricted conditions. FEMS Microbiol. Lett. 21:113-117. 13. Chatterjee, A. K., G. E. Buchanan, M. K. Behrens, and M. P.Starr. 1979.Synthesis and excretion of polygalacturonic acid trans-eliminase in Erwinia, Yersinia and Klebsiella species. Can. J. Microbiol. 25: 94-102. 14. Chatterjee, A. K., and M. P. Starr. 1977. Donor strains of the soft-rot bacterium Erwinia chrysanthemi and conjugational transfer of the pectolytic capacity. J. Bacteriol. 132: 862-869. 15. Chatterjee, A. K., and M. P. Starr. 1980. Genetics of Erwinia species. Annu. Rev. Microbiol. 34: 645-676. 16. Peter Reeves. 1965. The bacteriocins. Bacteriol. Rev. 29: 24-45. 17. Spelaung, S. R. and S. K. Harlander. 1989. Inhibition of foodborne bacterial pathogens by bacteriocins from Lactococcus lactis and Pediococcus pentosaceus. J. Food Prot. 52: 856-862. 18. Lewus, C. B., A. Kaiser and T. J. Montville. 1991. Inhibition of foodborne bacterial pathogens by bacteriocins from lactic acid bacteria isolated from meat. Appl. Environ. Microbiol. 57: 1683-1688. 19. Tagg, J. R., A. S. Dajani and L. W. Wannamaker. 1976. Bacteriocin of gram-positive bacteria. Bacteriol. Rev. 40: 722-756. 20. Tagg, J. R. 1991. Bacterial BLIS. ASM News. 57:611. 21. Klaenhammer, T. R. 1993. Genetics of bacteriocins produced by lactic acidbacteria. FEMS Microbiol. Rev. 12: 39-85. 22. Kordel, M. and H.-G. Sahl. 1986. Susceptibility of bacterial eukaryotic, and artificial membranes to the disruptive action of the cationic peptides pep 5 and nisin. FEMS Microbiol. Lett. 34: 139-144. 23. Abee, T., T. R. Klaenhammer and L. Letellier. 1994. Kinetic studies of the action of lactacin F, a bacteriocin produced by Lactobacillus johnsonii that forms poration complexes in the cytoplasmic membrane. Appl. Environ. Microbiol. 60 (3):1006-1013. 24. Hurst, A. 1981. Nisin. Advances Appl. Microbiol. 27: 85-123. 25. Mørtvendt, C. J., J. Nissen-Meyer, K. Sletten and I. F. Nes. 1991. Purification and amino acid squence of lactocin S, a bacteriocin produced by Lactobacillus sake L45. Appl. Environ. Microbiol. 57: 1829-1834. 26. Piard, J. C., P. M. Muriana, M. J. Desmazeaud and T. R. Klaenhammer. 1992. Purification and partial characterization of lacticin 481, a lanthionine-containing bacteriocin produced by Lactobacillus lactis subsp. lactis CNRZ481. Appl. Environ. Microbiol. 58: 279-284. 27. Stoffels, G., H.-G. Sahl and A. Gudmundsdottir. 1993. Carnocin UI49, a potential biopreservative produced by Carnobacterium piscicola: large scale purification and activity against various Gram-positive bacteria including Listeria spp. Int. J. Food Microbiol. 20: 199-210. 28. Stoffels, G., H.-G. Sahl and A. Gudmundsdottir. 1993. Carnocin UI49, a potential biopreservative produced by Carnobacterium piscicola: large scale purification and activity against various Gram-positive bacteria including Listeria spp. Int. J. Food Microbiol. 20: 199-210. 29. Bhunia, A. K., M. C. Johnson, and B. Ray. 1988. Purification, characterization and antimicrobial spectrum of a bacteriocin produced by Pediococcus acidilactici. J. Appl. Bacteriol. 65: 261-268. 30. Van Belkum, M. J., Kok, J., Venema, G., Holo, H., Nes, I. F., Konings, W. N., and T. Abee. 1991. The bacteriocin lactococcin A specification increases the permeability of lactococcal cytoplasmic membranes in a voltage-indenpent, protein-mediated manner. J. Bacteriol. 173: 7934-7941. 31. Kaiser, A. L. and T. J. Montville. 1996. Purification of the bacteriocin Bavaricin MN and characterization of its mode of action against Listeria monocytogenes Scott A cells and lipid vesicles. Appl. Environ. Microbiol. 62: 4529-4535. 32. Joerger, M. C. and T. R. Klaenhammer. 1986. Characterization and purification of helveticin J and evidence for a chromosomally determined bacteriocin produced by Lactobacillus helveticus 481. J. Bacteriol. 167 (2): 439-446. 33. Rammelsberg, M. and F. Radler. 1990. Antibacterial polypeptides of Lactobacillus species. J. Appl. Bacteriol. 69: 177-184. 34. Hamon, Y., and Y. Peron. 1961. Les propriete antagonistes reciproques parmi les Erwinia. Discussion de la position taxonomique de ce genre. C. R. Acad. Sci. 253:913-915. 35. Itoh, Y., K. Izaki, and H. Takahashi. 1978. Purification and characterization of a bacteriocin from Erwinia carotovora. J. Gen. Appl. Microbiol. 24: 27–39. 36. Kamimiya, S., K. Izaki, and H. Takahashi. 1977. Bacteriocins in Erwinia aroideae with tail like structure of bacteriophages. Agric. Biol. Chem. 41: 911–912. 37. Lewus, C. B., S. Sun and T. J. Montville. 1992. Production of an amylase-sensitive bacteriocin by an typical Leuconostoc paramesenteroides strain. Appl. Environ. Microbiol. 58: 143-149. 38. Be´ne´detti, H., M. Frenette, D. Baty, M. Knibiehler, F. Pattus, and C. Lazdunski. 1991. Individual domains of colicins confer specificity in colicin uptake, in pore-properties and in immunity requirements. J. Mol. Biol. 217: 429–439. 39. J. Konisky. 1982. Colicins and other bacteriocins with established modes of action, Annu. Rev. Microbiol. 36: 125–144. 40. A.P. Pugsley. 1984. The ins and outs of colicins. Part I: Productions, and translocation across membranes, Microbiol. Sci. 1: 168–175. 41. A.P. Pugsley. 1984 The ins and outs of colicins. Part II: Lethal action, immunity and ecological implications, Microbiol. Sci. 1: 203–205. 42. R. James, C. Kleanthous and G.R. Moore. 1996. The biology of E colicins: paradigms and paradoxes, Mircobiology 142: 1569–1580. 43. W.A. Cramer, M. Lindeberg and R. Taylor. 1999. The best offense is a good defense, Nat. Struct. Biol. 6: 295–297. 44. Toba M, Masaki H, Ohta T. 1988. Colicin E8, a DNase which indicates an evolutionary relationship between colicins E2 and E3. J Bacteriol 170: 3237–3242 45. Lazdunski, C. J., E. Bouveret, A. Rigal, L. Journet, R. Lloubes, and H. Benedetti. 1998. Colicin import into Escherichia coli cells. Journal of Bacteriology 180:4993-5002. 46. Soelaiman S, Jakes K, Wu N, Li C, Shoham M. 2001. Crystal structure of colicin E3: implications for cell entry and ribosome inactivation. Mol Cell 8: 1053–1062 47. K. Tomita, T. Ogawa, T. Uozumi, K. Watanabe and H. Masaki. 2000. A cytotoxic ribonuclease which specifically cleaves four isoaccepting arginine tRNAs at their anticodon loops, Proc. Natl. Acad. Sci. USA 97:8278–8283. 48. Duen-Yau Chuang, Ampaabeng G. Kyeremeh, Yuichi Gunji, Yoshiyuki Takahara, Yoshio Ehara, and Toshio Kikumoto. 1999. Identification and Cloning of an Erwinia carotovora subsp. carotovora Bacteriocin Regulator Gene by Insertional Mutagenesis. J. Bacteriol. 181: 1953-1957. 49. Bennik, MH., Verheul, A., Abee, T., Naaktgeboren-Stoffels, G., Gorris, LG.,Smid, EJ. 1997. Interactions of nisin and pediocin PA-1 with closely related lactic acid bacteria that manifest over 100-fold differences in bacteriocin sensitivity. Appl Environ Microbiol. 63: 3628-36. 50. Jabrane, A., Sabri, A., Compere, P., Jacques, P., Vandenberghe, I., Van Beeumen, J., Thonart, P. 2002. Characterization of serracin P, a phage-tail-like bacteriocin, and its activity against Erwinia amylovora, the fire blight pathogen. Appl Environ Microbiol. 68: 5704-10. 51. Fredericq, P. 1957. Colicins. Annu. Rev. Microbiol.11:7-22. 52. Jack, R. W., Tagg, J. R., and B. Ray. 1995. Bateriocins of gram-positive bateria. Microb. Rev. 59:171-200 53. Gantotti, B. V., K. L. Kindle, and S. V. Beer. 1981. Transfer of the drug-resistance transposon Tn5 to Erwinia herbicola and the iduction of insertion mutation. Microbiol. 6:417-425 54. Beringer, J. E., J. L. Beynon, A. V. Buchanan-Wollaston, and A. W. B.Johnston. 1978. Transfer of the drug resistance transposon Tn5 to Rhizobium. Nature 276:633–634 55. Sambrook, J., Fritsch, E. F., and Maniatis, T. 1989. Molecular Cloning: a laboratory manual, 2nd ed. Cold Spring Laboratory press, Cold Spring Harbor,N.Y. 56. 李國鏞,張邦彥 1990. 分子選殖(遺傳工程理論及實驗手冊)藝軒圖書出版社326-329 57. Liu, Y.G. and Whittier, R.F. 1995. Thermal asymmetric interlaced PCR: automatable amplification and sequencing of insert end fragments from P1 and YAC clones for chromosome walking.Genomics 25: 674–681. 58. Hanahan,D. 1983. Studies on transformation of Escherichia coli with plasmids. J.Mol. Biol. 166:577-580 59. Sambrook, J., Russell, D.W., Molecular Cloning: A Laboratory Manual, the third edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 2001. 60. Raquel Tobes and Juan L. Ramos. AraC-XylS database: a family of positive transcriptional regulators in bacteria. Nucleic Acids Res. 2002 January 1; 30(1): 318–321 61. Gregory V. Plano. Modulation of AraC family member activity by protein ligands. Mol Microbiol. 2004 Oct; 54(2):287-290. 62. Hauben, L., Moore, E. R. B., Vauterin, L., Steenackers, M., Mergaert, J., Verdonck, L. & Swings, J. (1998). Phylogenetic position of phytopathogens within the Enterobacteriaceae. Syst Appl Microbiol 21, 384–397. 63. Kageyama, B., M. Nakae, S. Yagi, and T. Sonoyama. 1992. Pantoea punctata sp. nov., Pantoea citrea sp. nov., and Pantoea terrea sp. nov. isolated from fruit and soil samples Intl. J. Syst. Bacteriol. 42: 203–210. 64. Mergaert, J., L. Verdonck, and K. Kersters. 1993. Transfer of Erwinia ananas (synonym, Erwinia uredovora) and Erwinia stewartii to the genus Pantoea emend. As Pantoea ananas (Serrano 1928) comb. Nov. and Pantoea stewartii (Smithe 1898) comb. nov., respectively, and description of Pantoea stewartii subsp. indologenes subsp. nov. Intl. J. Syst. Bacteriol. 43: 162–173. 65. Brenner, D. J., G. R. Fanning, and A. G. Steigerwalt. 1973. Deoxyribonucleic acid relatedness among Erwinia and other Enterobacteriaceae: the gall, wilt and dry-necrosis organism (Genus Erwinia Winslow et al. sensu stricto). Intl. J. Syst. Bacteriol. 24: 197–204.
摘要: Erwinia carotovora subsp. carotovor (E.c.c.)常造成植物的軟腐病,目前為止對於此菌種所造成的細菌性軟腐病的防治仍找不到有效的方法。此研究的目標為找到一種廣泛且有效力抑制E.c.c.生長的細菌素,希望能以非農藥方式,達成軟腐菌對植物病害防治。針對本實驗室以及本校植病所曾國欽老師實驗室所保存的E.c.c.,進行細菌素活性調查,發現本實驗室所保存的3F-3菌株所分泌的細菌素具有高度的抑菌活性。 首先利用跳躍因子Tn5在染色體上進行插入突變,阻斷3F-3菌株低分子量細菌素產生後,我們利用TAIL-PCR的方法增幅並定序各個突變菌株被Tn5阻斷的周圍序列,經由美國國家衛生研究院醫學圖書館的資料庫(NCBI blast)以DNA或胺基酸序列比對,結果發現突變株TF1-2所阻斷位置為細菌素生產基因,此段基因是未曾被發現過的新規細菌素基因,因此我們把它命名為carocin S2。 由3F-3 菌株的genomic DNA分離出長度為5705bp的片段,它包含6個open reading frames (ORFs), 其中ORF2轉譯後胺基酸序列與colicin D activity蛋白有38%的同質性,colicin D屬於核醣核酸水解酶; ORF3轉譯後胺基酸序列與colicin D immunity 蛋白有32%的同質性,colicin D immunity蛋白的作用是抑制colicin D activity蛋白活性保護宿主細胞。因此我們將ORF2命名為caroS2K而ORF3命名為caroS2I,這兩段基因合稱為carocin S2。將此DNA片段連接於pBR3222 和pMCL210質體建構出pBR322-119 和pMCL210-119以轉形作用分別送入DH5α、Ea1068以及TF1-2三個沒有生產細菌素能力的菌株,實驗發現帶有pMCL210-119質體的菌株均能表現出低分子量細菌素活性,因此我們成功的將E.c.c.低分子量細菌素基因選殖並表現出它的抗生效果。
The Erwinia carotovora subsp. carotovovra (E.c.c.) causes soft-rot disease of many plant species. Despite its economic importance, no efficient method has been found to control this disease. The bacterium produces the antibacterial substances called bacteriocins owning the ability to identify the similar bacteria and kills them. We want to find out the bacteriocin of E.c.c. being able to kill most of subspecies, and use the biological control this disease. By bacterial mating experiment a transposon Tn5 insertion mutant TF1-2 was obtained, which lost the low-molecular-weight bacteriocin(LMWB) producing ability. After the first TAIL-PCR experiment, the PCR products were isolated and the DNA sequences were determined from the Tn5 insertion junction site. The DNA probe was prepared from TAIL-PCR result and it was used in Southern hybridization experiments. A 5705bp fragment was isolated from 3F-3 DNA library, and it contains six open reading frames (ORFs). The ORF2 encodes a protein 38% identical to colicin D activity protein, which is ribonuclease activity. The ORF3 encodes a protein 32% identical to immunity protein of colicin D, which inhibition the bacteriocin activity to host cell. It was therefore proposed that ORF2 be designated as caroS2K, and ORF3 as caroS2I. The two genes were named carocin S2 gene. We cloned the carocin S2 gene into pBR322 or pMCL210 vector, and the expression plasmids pBR322-119 and pMCL210-119 were obtained. After pBR322-119 or pMCL210-119 into E. coli DH5α, Ea1068, or TF1-2 cells, the results showed that DH5α/pMCL210-119, Ea1068/pMCL210-119 and TF1-2/pMCL210-119 can produce the low-molecular-weight bacteriocin by bacteriocin assay experiments. This result suggests that the carocin S2 gene is a LMWB genes contain a killing protein and an immunity protein.
其他識別: U0005-2806200617261900
Appears in Collections:化學系所



Items in DSpace are protected by copyright, with all rights reserved, unless otherwise indicated.