野油菜黄单胞菌野油菜致病变种一个与胞外多糖产生相关的基因的鉴定
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摘要
野油菜黄单胞菌野菜致病变种(Xcc)是一种能在世界范围内引起十字花科植物致病的革兰氏阴性细菌，其显著特征是能产生胞外多糖(EPS)。胞外多糖一直被认为是Xcc的致病因子之一，同时，由于其特殊的理化性质而被广泛应用于工业生产，因而对胞外多糖生物合成机理的研究在不断深入。本研究中，我们利用转座子Tn5gusA5随机插入诱变Xcc野生型菌株8004，构建了含17，280株突变体的Xcc突变体库。通过热不对称嵌套式PCR(TALL—PCR)和测序确定了转座子Tn5gusA5插入在Xcc基因组中的位置。通过平板检测的方法，在含2％葡萄糖的NYGA平板上从突变体库中筛选到17株胞外多糖缺陷突变体。根据其菌落形态特征，将这些突变体分为3类。致病性检测结果表明，13株突变体丧失了致病力，3株突变体致病力明显降低，而另外2株突变体仍具有与野生型8004相同的致病力。在这17株胞外多糖缺陷突变体中，14株突变体中被突变的ORF的注释功能与胞外多糖的产生相关，而另外3株被突变的ORF与胞外多糖的产生的关系尚不清楚。为了验证这3个ORF与胞外多糖的产生有关，我们成功地构建了这3个ORF的缺失突变体。结果表明Xcc_114缺失突变株的表型与相应ORF的Tn5插入突变株的表型一致，表现为胞外多糖缺陷，而另外两个缺失突变株与野生型8004一致，未表现出胞外多糖缺陷。Xcc_114长756bp，通过NCBI中的blast搜索，结果表明其编码产物与一种跨膜蛋白具有较高的同源性，推测可能与多糖的胞外运输有关。
Xanthomonas campestris pv. campestris (Xcc) is a gram-negative bacterium which can cause black rot on cruciferous plants around the world. One of the products of X.campestris is excellular polysacchride (EPS) named xanthan gum.EPS is always regarded as a pathogenicity factor during interaction between Xcc and host plants, hi addition,EPS was extensively used in many fields of industry, from the food processing to oil drilling, due to its exceptional rheological properties. Extensive researches have been carried out on mechanism of EPS production. In this study, wild type strains 8004 of Xcc were mutagenized by the insertion of transposon Tn5gusA5. An Xcc mutant library was constructed in this way, it holds 17,280 mutants. Insertion sites of mutants were decided by TAIL-PCR and sequencing. 17 mutants deficient in EPS production were screened from the mutant library by the plate method. In terms of their colony morphology, these mutants were classified into three types. We tested their pathogenecities, the results revealed that 11 mutants lost their pathogenecities, 4 mutants decreased their pathogenecities, and 2 mutants shared the same pathogenicities as wild type strain 8004. In the 17 EPS mutants,mutated gene functions of 14 TnSgusAS insertion mutants are known, and 3 gene functions of the other 3 insertion mutants are still unknown. So we carried out deletion to the three corresponding genes of wild type strain 8004, which are Xcc-1^ Xcc-224 and Xcc-3387. The results revealed that the deletion of Xcc-114 leaded to the deficiency in EPS production, and the other two deletion mutants still produce EPS. So we can conclude that Xcc-114 is a gene involved in EPS production. By the searching of NCBI blast, the results revealed that the deduced product encoded by Xcc-114, containing 756bp, showed 36% identities with a transmembrane protein. Its putative function may be relevant to EPS transport.
Xanthomonas campestris pv. campestris (Xcc) is a gram-negative bacterium which can cause black rot on cruciferous plants around the world. One of the products of X.campestris is excellular polysacchride (EPS) named xanthan gum.EPS is always regarded as a pathogenicity factor during interaction between Xcc and host plants, hi addition,EPS was extensively used in many fields of industry, from the food processing to oil drilling, due to its exceptional rheological properties. Extensive researches have been carried out on mechanism of EPS production. In this study, wild type strains 8004 of Xcc were mutagenized by the insertion of transposon Tn5gusA5. An Xcc mutant library was constructed in this way, it holds 17,280 mutants. Insertion sites of mutants were decided by TAIL-PCR and sequencing. 17 mutants deficient in EPS production were screened from the mutant library by the plate method. In terms of their colony morphology, these mutants were classified into three types. We tested their pathogenecities, the results revealed that 11 mutants lost their pathogenecities, 4 mutants decreased their pathogenecities, and 2 mutants shared the same pathogenicities as wild type strain 8004. In the 17 EPS mutants,mutated gene functions of 14 TnSgusAS insertion mutants are known, and 3 gene functions of the other 3 insertion mutants are still unknown. So we carried out deletion to the three corresponding genes of wild type strain 8004, which are Xcc-1^ Xcc-224 and Xcc-3387. The results revealed that the deletion of Xcc-114 leaded to the deficiency in EPS production, and the other two deletion mutants still produce EPS. So we can conclude that Xcc-114 is a gene involved in EPS production. By the searching of NCBI blast, the results revealed that the deduced product encoded by Xcc-114, containing 756bp, showed 36% identities with a transmembrane protein. Its putative function may be relevant to EPS transport.
引文
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10． Coplin D L and Cook D. 1990b. Mol. Plant-Microbe Interact.. 41:459-472．
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12． Coplin. D. L , and Majerczak, D. R. 1990． Mol. Plant-Microbe Interact. 3: 286-292．
13． Costerton J W, Cheng K-J, Geesey G G. et al. 1987． J Annu Rev Microbiol. 1987． 41:435-464．
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38． Keen, N.T. 1990． Annu. Rev. Genet. 24:447-463．
39． Kenndey, J. F., and I. J. Bradshaw. 1984． Prog. Ind. Microbiol, 19:319-230
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43． Leong. S.A., Ditta, G. S., Helinski, D. R., 1982, J. Biol. Chem. 257:8724-8730．
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45． Lindgren PB, Peet R C, Panopoulos NJ, 1986, J.Bacteriol. 168:512-522．
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2． Baird, J., P. Sandfrird, and I. Cottrell. 1983． Bio/Technology. 1:778-783．
3． Barrere G C, Barber C E and Daniels M J. 1986． Int. J. Biol. Macromol. 8:372-374
4． Beringer, J. E., Beynon, J. L., Buchanan. Wollaston, A. V. et al. 1998． Nature. 276:633-634
5． Boher B, M. Nicole M P and Geiger J P. 1997． Mol. Plant-Microbe Interact., 10: (7) :803-811．
6． Capage, M. R., D. H. Doherty, M. R. Betlach, and R. W. Vanderslice. March 1987． International patent W087/05938．
7． Chou F L, Chou H C, Lin Y S et al. 1997． Bioph Res Comm. 233:265-269．
8． Cook, A. A, Walker, J. C., and Larson, R. H. 1952． Phytopathol. 42:162-167．
9． Coplin D L and Cook D. 1990a. ilol. Plant-Microbe Interact.. 3(5) :270-279．
10． Coplin D L and Cook D. 1990b. Mol. Plant-Microbe Interact.. 41:459-472．
11． Coplin, D. L , and Cook D. 1990c. Mol. Plant-Microbe Interact. 3:271-279．
12． Coplin. D. L , and Majerczak, D. R. 1990． Mol. Plant-Microbe Interact. 3: 286-292．
13． Costerton J W, Cheng K-J, Geesey G G. et al. 1987． J Annu Rev Microbiol. 1987． 41:435-464．
14． Daniels M J and Leach J E. 1993． In: Swings J. G. and Civerolo E. L., Chapman & Hall. London, pp.301-339．
15． Daniels M J and, Osbourn A E and Tang J L. 1989． NATO ASI Series, Vol. H36． B JJ Lugtenberg ed. Springer-Verlag, Berlin, pp.189-196．
16． de Crecy-Lagard V, Glaser P, Lejeune P. Sismeiro et al. 1990． J Bactetriol. 172:5877-5883．
17． Dianese, J.C., and Schaad. N. W., 1982． Phytopathol., 72:1284-1289．
18． Dos Samtos, R. M. D. B., and Dianses, J. C. 1985 . Phytopathol. 75:581-587．
19． Dow J M. Ocbourn A E, Greer-Wilson T J. et al. 1995． Mol. Plant-Microbe Interact. 8:768-777．
20． Dudman WF. Carbohydr Res, 1978,66;9-23．
21． Flor H H 1955． Phytopathol. 45:680-685．
22． Flor, A. H. 1947． Phytopathol. 45:680-685．
23． Friedman, A.M., Long, S. K., Brown, S. E. et al. 1982． Gene, 18:289-296．
24． Goodman, R. N. and Novacky, A., 1994 , American phytopathological Society, St. Paul, MU
25． Hanahan, D., 1983． .J. Mol. Biol. 166:557．
26． Harding, N. E., J M. Cleary, and L. Ielpi. 1995． P. 495-514． In Y.H. Hui and G. Khachatourians (ed), Food biotechnology microorganisms. VCH Publishers. Inc. New York.
27． Harding, N. E., J. M. Cleary, D. K. Cabanas , I. G. Rosen, and K. S. Kang. 1987． J. bacterial. 169;2854-2861．
28． Hassler, R. A., and D. H. Doherty. 1990． Biotechnol. Prog. 6:182-187．
29． Hickman J and Ashwell G. 1966． J Biol Chem. 241: 1424-1430．
30． Hignett R. C. 1988． Physiol Mol Plant Pathol. 32: 387-394．
31． Hokawat, S. 1988． Dissertation, Universitat Gottingen.
32． Ielpi, L. , R. Couso. and M. Dandert. 1981． FEBS Lett. 130:253-256．
33． Ielpi. L, R.O. Couso, and M. A. Dankert. 1993 J Bacteriol. 175:2490-2500．
34． Ielpi. L., R. O. Couse, M. A. Dankert. 1983． Biochen. Int. 6:323-333．
35． Jansson, P. E. L. Keene. and B. Lindberg. 1975． Carbohydr. Res. 45:275-282．
36． Kamdar HV, Kamoun S and Kado CI. 1993． J Bactereriol. 175:2017-2025．
37． Katzen F, Becker. A., Zorrenguieta. A., Ielpi. L. 1996． J.Bacteriol. 178(14) : 4313-4318．
38． Keen, N.T. 1990． Annu. Rev. Genet. 24:447-463．
39． Kenndey, J. F., and I. J. Bradshaw. 1984． Prog. Ind. Microbiol, 19:319-230
40． Kingsley M and Bohlool B B. 1992． Appl Environ Microbiol. 58:1095-1101．
41． Klement, Z. 1963． Nature. 199:299-300．
42． Leoeman M, Van Pelt J A, Den Ouden F M, et al. 1995． Phytopathol. 85:1021-1027．
43． Leong. S.A., Ditta, G. S., Helinski, D. R., 1982, J. Biol. Chem. 257:8724-8730．
44． Liideritz, O., Freudenberg, M. A., Galanos, C., Lehmann, V., Rietsch, E. T. and Shaw, D.H. 1982． Vol. 17, Academic Press, New York, pp79-151．
45． Lindgren PB, Peet R C, Panopoulos NJ, 1986, J.Bacteriol. 168:512-522．
46． Liu Yao-Guang and Robert F. Whittier. Genomics, 1995, 25(3) :674-681．
47． Liu Yao-Guang, Norihiro Mitsukawa, Rober F. Whittier. The Plant Journal. 1995,8(3) :457-463．
48． Manurelli A T and Sansonetti P J. 1998． Annu Rev Microbiol. 42:127-150．
49． Mark T Kingsley, Dean W Gabriel, Gary C Marlow et al. 1993． J Bacteriol 9:5839-5850．
50． Marzocca, M. P., N. E. Harding., E. A. Petroni, J. M. Cleary., and L. Ielpi. 1991． J. Bacteriol. 113:7519-7524．
51． Mazzucchi V, Bazzi C and Pupill P. 1979． Physiol. 14:19-30．
52． Mazzuchi U and Pupillo P. 1976． Physiol Plant Pathol. 9:101-112．
53． McNeil m et al. Carbohydr Res, 1986,146:307-326．
54． Melton, L. D., Mindt, L., Ress, D. A., and Sanderson, G. R. 1976． Carbo Hydr. Res. 46: 245-257．
55． Newman M-A, Conrads-Strauch J, Scofield G, et al. 1994． MPMI. 7:556-565．
56． Nidaido H and Vaara M. 1985． Microbiol Rev. 49:1-32．
57． Osbourn, A. E. Clarke, B. R., Stevens, B. J. H., and Daniels, M. J. 1990． Mol. Gen Gent. 222:145-151．
58． Parder C T, Kloder A W, Schnaitman C A et al. 1992． J Bacteriol. 1992． 174:2525-2583．
59． Pettitt. D. J., 1979． P. 263-280． In. J. M. V. Blanshard and J. R. Mitchell (ed). Polysaccharides in food . Butterworths. London.
60． Puvanesarajah V et al. J bacteriol, 1987, 169:137-141．
61． Ramirez M E, Fucikovsky L, Garcia-Jimenez F et al. 1988． Appl Microbiol Bicroiol, 29:5-10．
62． Reinhard Koplin, G E Wang, Barbara Hotte. Et al. 1993． Bacteriol. P. 7786-7792．
63． Robeson, F. J., and Cook, D. R. 1985． Physiologyical Plant Pathology, 26:219-230．
64． Rudolph, K. W. E., Gross, M., et al. 1989． NATOASI Series, Vol, H27, Springer Verlag, Berlin. pp127-218．
65． Rudolph, K., Ebrahim-Nesbat, F., Mendgen, K. andthiele, C. 1987． Proceedings of the Sixth International Conference on Plant Pathogenic Bacteria, Maryland, 2-7 June 1985． Martinus Nijhoff Publishers, Dordrecht, pp604-612．
66． Schaad, N. W. 1989． Page 6875 in: Petection of Bacteria in Seed and Other Planting Material. A. W. Saettler, N. W. Schaad, and D. A. Roth, eds. American Phytopathologyical Society, St, Panl, Minn
67． Sdchoonejans E, Expert D and Toussaint A. 1987． Bacteriol. 169: 4011-4017．
68． Sharon S B and Signer E. 1990． Genes Development. 4:344-356．
69． Staska wics, B., Dahlbeck D, Keen, N. et al. 1987． J. Bacteriol. 169:5789-5794．
70． Staskawicz, B. J., Dahlbeck, D., and Keen. N. T. 1984． Max(L) Merr.Proc. Natl. Acad. Sci, USA. 981:6024-6028．
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