野油菜黄单胞菌8004与胞外多糖合成相关基因的鉴定
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摘要
野油菜黄单胞菌野菜致病变种(Xanthomonas campestris pv．campestris，以下简称Xcc)是一种能在全球范围内引起十字花科植物黑腐病的病原性细菌。胞外多糖是Xcc的重要致病因子之一，也是一种重要的工业多糖。本实验室已构建了Xcc 8004的Tn5gusA5插入突变库，并对16462株突变体的EPS产量进行了平板检测，获得了75株胞外多糖产量下降突变株。本工作用2％葡萄糖的NYGA平板上检测，对这75个突变体进行了重新筛选，得到了51株胞外多糖缺陷的突变体，它们分布在45个ORF中。其中11个ORF前人已证实与胞外多糖合成有关：34个ORF未报道与胞外多糖的产生相关，这34个ORF中13个编码的为假设保守蛋白。本工作选择这13个编码的为假设保守蛋白的基因的一个，编号为XC0877，进行进一步研究，以确定该ORF是否与EPS合成有相关。根据Xcc 8004的Tn5gusA5插入突变库的资料，XC0877 ORF中有四个独立的Tn5gusA5插入突变体，其中有两个突变体为胞外多糖缺陷型，另两个突变体为胞外多糖产生正常。我们初步认为这种表型不一致的原因有可能是ORF界定有误。为了确证XC0877 ORF与胞外多糖产生的关系，我们采用同源单交换的方法构建了该ORF的非极性定点整合突变体。并对所得突变体进行了胞外多糖产量检测，出乎意料的是在这些突变体中大约有一半表现为缺陷型而另一半则表现为胞外多糖产生正常。用PCR方法扩增野生型XC0877 ORF(包括启动子)，并克隆到pLAFR3上，进行反式互补，结果未能恢复突变体的胞外多糖的产量。综合以上结果，XC0877 ORF是否与胞外多糖产生有关还需进一步研究。
Xanthomonas campestris pv. campestris (Xcc) is an important pathogen of cruciferous plants, it causes the black rot disease of cruciferous crops worldwide. One of the products of X. campestris is excellular polysacchride(EPS) named xanthan gum. EPS is currently regarded as a pathogenicity factor during interaction between Xcc and host plants. In addition, EPS is also a sort of important polysacchride in industry. The Xcc 8004 mutant library was constructed by the insertion of transposition Tn5gusA5 in the laboratory. The EPS production of 16462 Tn5gwsA5 inserted mutants Xcc 8004 strains were tested and 75 had a fall in EPS production. In this study, the NYGA containing 2% glucose is used to re-select these 75 mutants. The result of re-selecting is 51 strains of EPS production deficiency were obtained. They are distributed in 45 ORFs. Among them, 11 have been verified to be relevant to EPS, and 34 have not been reported to have relationship with EPS. Furthermore, 13 of these 34 ORFs encode conserved hypotheti
cal protein. XC0877 is one of these 13 ORFs and it is used to make a further study in order to verify whether it is relevant to the EPS synthesizing. According to the information of Tn5gusA5 5inserted mutants library, there are 4 independent Tn5gwsA5 inserted mutants in XC0877 in which 2 of them are deficient in EPS production and the other 2 are normal in EPS production. The
reason why there are two phenotypes is thought initiatively as a fault of ORFs defining. In order to prove that XC0877 is related to EPS production, we construct the XC0877 non-polar site-specific insertion mutant. It is found that nearly half of these mutants are deficient in EPS production and the other half is normal in EPS production. Using the way of PCR to amplify wild type XC0877 (including promoter) and then clone it on pLAFR3 to undergo complemtation in trans, the result is that the EPS production of the mutant dose not recover. According to all the results above, whether XC0877 is relevant to EPS production is needed to do further research.
Xanthomonas campestris pv. campestris (Xcc) is an important pathogen of cruciferous plants, it causes the black rot disease of cruciferous crops worldwide. One of the products of X. campestris is excellular polysacchride(EPS) named xanthan gum. EPS is currently regarded as a pathogenicity factor during interaction between Xcc and host plants. In addition, EPS is also a sort of important polysacchride in industry. The Xcc 8004 mutant library was constructed by the insertion of transposition Tn5gusA5 in the laboratory. The EPS production of 16462 Tn5gwsA5 inserted mutants Xcc 8004 strains were tested and 75 had a fall in EPS production. In this study, the NYGA containing 2% glucose is used to re-select these 75 mutants. The result of re-selecting is 51 strains of EPS production deficiency were obtained. They are distributed in 45 ORFs. Among them, 11 have been verified to be relevant to EPS, and 34 have not been reported to have relationship with EPS. Furthermore, 13 of these 34 ORFs encode conserved hypotheti
cal protein. XC0877 is one of these 13 ORFs and it is used to make a further study in order to verify whether it is relevant to the EPS synthesizing. According to the information of Tn5gusA5 5inserted mutants library, there are 4 independent Tn5gwsA5 inserted mutants in XC0877 in which 2 of them are deficient in EPS production and the other 2 are normal in EPS production. The
reason why there are two phenotypes is thought initiatively as a fault of ORFs defining. In order to prove that XC0877 is related to EPS production, we construct the XC0877 non-polar site-specific insertion mutant. It is found that nearly half of these mutants are deficient in EPS production and the other half is normal in EPS production. Using the way of PCR to amplify wild type XC0877 (including promoter) and then clone it on pLAFR3 to undergo complemtation in trans, the result is that the EPS production of the mutant dose not recover. According to all the results above, whether XC0877 is relevant to EPS production is needed to do further research.
引文
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3. Robeson DJ and Cook DR. 1985. Production of low molecular weight carboxylic acids by Xanthomonas capestris pv. carotae in relation to the amino acid composition of the medium and their possible involvement in pathogenesis. Physiological Plant Pathology 26:219-230
4. Williams PH 1980. Black rot: a continuing threat to world crucifers. Plant Disease, 64, 736-742.
5. Alvarez AM and Lou K 1985.Rapid identification of Xanthamonas campestris pv.campestris by ELISA. Plant Dis.69:1082-1086
6. Swings JG, and Civerolo EL. 1993. Xanthornonas 1st edition London: Chapman & Hall, pp.51-55
7. Kennedy JF, and Bradshaw IJ. 1984, Production ,properties and application of xanthan.Prog Ind Microbiol. 19:319-371
8. Smith EF. 1987 A bacterial disease of cruciferous plants. Science N.Y.,5:963
9. Cook AA,Walker JC,and Larson RH. 1952 Studies on the disease cycle of black rot of crucifers.Phytopathology,42:162-167
10. Bretschneider KE,Gonella MG, RobesonDJ.1989.A comparative light-and electron microscopical study of campatible and incompatible interaction between Xanthamonas campestris pv. Campestris and cabbage (Brassica oleracea).Physiol Mol Plant Pathol. 34:285-297
11. Sutton JC,Williams PH. 1970. Relation of xylem plugging to black rot lesion development in cabbage.Can J Bot. 48:391-401
12. Wallis FM,Rijikerg FHJ, Joubert JJ, and Martin MM.1973 .Ultrastructural histopathology of cabbage leaves infected with Xanthamonas campestris.Physiol Plant Pathol.3:371-378
13．唐江涛 2003 《细菌转座Tn5gusA5在野油菜黄单胞菌8004中转座规律分析及生物学验证》 硕士论文，中国南宁
14. Kennedy JG, and Bradshaw IJ. 1984. Production, properties and application of xanthan .Prog Ind Microbiol. 19:319-371
15. Jansson P E,. Kenne L, and Lindberg B. 1975. Structure of extracellular polysaccharide from Xanthomonas campestris. Carbohydr. Res. 45:275-282
16. Coplin DL, andCook D. 1990. Molecular genetics of extracellular polysaccharide biosynthesis
in vascular phytopathogenic bacteria. Mol. Plant-Microbe Interact. 3:271-279
17. Cadmus MC, Rogovin SP, Burton KA,Pittsley JE, Knutson CA 1978. AColonial variation in Xanthomonas campestris NRRL B- 1459 and characterization of the polysaccharide from a variant strain. CanadianJ. Microbiol., 19:942-948,
18. Stankowski JD,Muller BE,Zeller SG. 1993,Location of a second o-acetyl group in xanthan gum by the reductive-cleavage method .Carbohydr Res, 1993,241:321-326
19．姜成林 徐丽华 2001 《微生物资源开发利用》 175～181
20．聂凌鸿 周如金 宁正祥 2003 《黄原胶的特性、发展现状、生产及其应用》 《中国食品添加剂》 2003No 3
21．李兴存，张忠智，王洪君，陈进富，李术元 2002 《黄原胶的性能与应用》日用化学工业 Vol．No．32
22．杨新亭，张良1999 《黄原胶的性能及其应用》 河南农业大学学报 1999，9月 第33卷 增刊
23．赵景林 1994 《黄原胶的特性、生产及应用》 现代化工 1994年第五期
24. Baird J, Sandford P, and Cottrell I. 1983. Industrial applications of some new microbial polysaccharides. Bio/Technology 1:778-783
25. Garcia-Ochoa F ,Santos VE,Casas JA,et al. 2000 Xanthan gum:production,recovery, and properties.Biotechnol Adv, 18:549-579
26. Harding NE, Raffo S, Raimondi A, Cleary JM, and Ielpi L. 1993. Identification, genetic and biochemical analysis of genes involved ih synthesis sugar nucleotide precursors of xanthan gum. J. Gen. Microbiol. 139:447-457
27. Ielpi L, Couso R, and Dankert M. 1981. Lipid-linked intermediates in the biosynthesis of xanthan gum. FEBS Lett. 130:253-256
28. Ielpi L, Couso RO, and Dankert MA 1993. Sequential assembly and polymerization of the polyprenol-linked pentasaccharide repeating unit of the xanthan polysaccharide in Xanthomonas campestris. J. Bacteriol. 175:2490-2500
29. Ielpi L, Couso RO, and Dankert MA. 1983. Xanthan gum biosynthesis: acetylation occurs at the prenyl-phospho-sugar stage. Biochem. Int. 6:323-333.
30. Kplin R, Arnold W, Htte B, Simon R, Wang G, and Ptlhler A. 1992. Genetics of xanthan production in Xanthomonas campestris: the xanA and xanB genes are involved in UDP-glucose and GDP-mannose biosynthesis. J. Bacteriol. 174:191-199
31. Capage MR, Doherty MR, and Vanderslice RW 1987. Recombinant-DNA mediated production of xanthan gum. International patent WO87/05938.
32. Harding NE, Cleary JM, Cabafias DK, Rosen IG and Kang KS 1987. Genetic and physical analyses of a cluster of genes essential for xanthan gum biosynthesis in Xanthomonas campestris. Bacteriol. 169:2854-2861
33. Vanderslice RW, DohertyDH, Capage MA, Betlach MR, Hassler RA, Henderson NM,et al 1988. Genetic engineering of polysaccharide structure in Xanthomonas campestris. Gordon and Breach Science Publishers, p145-157.New York, N.Y
34. Katzen F, Becker A, Zorreguieta A, Pu hler A, Ielpi L. 1996. Promoter analysis of the Xanthomonas campestris pv. campestris gum operon directing the biosynthesis of the xanthan polysaccharide. J Bacteriol 178:4313-4318
35. Chou F L, Chou H C, Lin Y S, Yang B Y, Lin N T, Wenga 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
36. Bcckcr A,Katzen F, Puhler A, et al.1998.Xanthan gum biosynthesis and application :a biochemical/genetic perspective .Appl Microbiol Biotechnol,50; 145-152
37. Katzen F, Ferreiro DU, Oddo CG, Ielmini MV, BeckerA, Pu hlerA, Ielpi L 1998 Xanthomonas campestris pv. campestris gum mutants: effects on xanthan biosynthesis and plant virulence. JBacteriol 180:1607-1617
38. Hassler RA,Doherty DH. 1990 Genetic engineering of polysaccharide structure:production of variants of xanthan gum in Xanthomonas campestris. Biotechnol prog 6(3): 182-7
39. Adrian A. Vojnov ,Angeles Zorreguieta et al. 1998 .Evidence for a role for the gumB and gumC gene products in the formation of xanthan from its pentasaccharide repeating unit by Xanthomonas campestris. Microbiology. 144:1487-1493
40. de Crecy-Lagard V de, Glaser P, Lejeune P, Sismeiro O, Barber CE,Daniels M J, Danchin A. 1990 A Xanthomonas campestris pv.campestris protein similar to catabolite activation factor is in-volved in regulation of phytopathogenicity. J Bacteriol 172:5877-5883
41. Tang JL, Liu YN, Barber CE, Dow J M, Wootton JC and DanielsM.J. 1991. Genetic and molecular analysis of a cluster of rpf genes involved in positive regulation of synthesis of extracellular enzymes and polysaccharide in Xanthomonas campestris pathovar campestris. Mol. Gen. Genet. 226:409-417
42. Tang JL, Gough CL, Daniels MJ 1990 Cloning of genes involved in negative regulation of production of extracellular enzymes and polysaccharide ofXanthomonas campestris pathovar carnpestris. Mol Gen Genet 222:157-160
43. Daniels MJ, Barber CE, Dow JM, Han B,Liddle SA ,Newman MA,et al 1993. Bacterial genes required for pathogenesis: interaction between Xanthomonas and crucifers. In: Mechanisms of Plant Defence Responses 53-63.
44. Poplawsky A R,Chun W. 1998 Xanthomonas campestris pv. Carapestris requires a functional pigB for epiphytic survival and host infection. Mol Plant Microbe Interact, 11:466-475
45. Daniels MJ, Barber CE, Dow JM, Han B, Liddle SA et al 1993. Plant and bacterial genes involved in interactions between Xanthomonas and crucifers. In: Advances in Molecular Genetics of Plant-Microbe Interactions 2:423-433
46. Barrére GC, Barber CE,andDaniels MJ 1986. Molecular cloning of genes involved in the production of the extracellular polysaccharide xanthan by Xanthomonas campestris pv. campestris. Int. J. Biol. Macromol. 8:372-374
47．陆光涛 1997 《水稻白叶枯病诱导抗性初探》 硕士论文 中国南宁
48．李有志 1998 《野油菜黄单胞菌野油菜致病型胞外多糖产生有关的—基因族的鉴定和分析》，博士论文，中国武汉
49. Shaw J.J, Settles LG ,Kado CI. 1988 Transposon Tn4431 mutagenesis of Xanthomonas campestris pv. campestris : characterization of a nonpathogenic mutant and cloning of a locus for pathogenicity. Molecular Plant Microbe Interactions, 1: 39-45, 1988
50. Barber CE, Tang J L, Feng JX, Pan M Q, et al 1997. A novel regulatory system required for pathogenicity of Xanthomonas campestris is mediated by a small diffusible signal, molecule Mol. Microbiol. 24:555-566
51．查东兴 1996 《甘蓝黑腐病菌胞外多糖产生的分子遗传学研究》博士论文，中国武汉
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2. Coplin DL , and Cook D. 1990. Molecular genetics of extracellular polysaccharide biosynthesis in vascular phytopathogenic bacteria. Mol. Plant-Microbe Interact. 3:271-279
3. Robeson DJ and Cook DR. 1985. Production of low molecular weight carboxylic acids by Xanthomonas capestris pv. carotae in relation to the amino acid composition of the medium and their possible involvement in pathogenesis. Physiological Plant Pathology 26:219-230
4. Williams PH 1980. Black rot: a continuing threat to world crucifers. Plant Disease, 64, 736-742.
5. Alvarez AM and Lou K 1985.Rapid identification of Xanthamonas campestris pv.campestris by ELISA. Plant Dis.69:1082-1086
6. Swings JG, and Civerolo EL. 1993. Xanthornonas 1st edition London: Chapman & Hall, pp.51-55
7. Kennedy JF, and Bradshaw IJ. 1984, Production ,properties and application of xanthan.Prog Ind Microbiol. 19:319-371
8. Smith EF. 1987 A bacterial disease of cruciferous plants. Science N.Y.,5:963
9. Cook AA,Walker JC,and Larson RH. 1952 Studies on the disease cycle of black rot of crucifers.Phytopathology,42:162-167
10. Bretschneider KE,Gonella MG, RobesonDJ.1989.A comparative light-and electron microscopical study of campatible and incompatible interaction between Xanthamonas campestris pv. Campestris and cabbage (Brassica oleracea).Physiol Mol Plant Pathol. 34:285-297
11. Sutton JC,Williams PH. 1970. Relation of xylem plugging to black rot lesion development in cabbage.Can J Bot. 48:391-401
12. Wallis FM,Rijikerg FHJ, Joubert JJ, and Martin MM.1973 .Ultrastructural histopathology of cabbage leaves infected with Xanthamonas campestris.Physiol Plant Pathol.3:371-378
13．唐江涛 2003 《细菌转座Tn5gusA5在野油菜黄单胞菌8004中转座规律分析及生物学验证》 硕士论文，中国南宁
14. Kennedy JG, and Bradshaw IJ. 1984. Production, properties and application of xanthan .Prog Ind Microbiol. 19:319-371
15. Jansson P E,. Kenne L, and Lindberg B. 1975. Structure of extracellular polysaccharide from Xanthomonas campestris. Carbohydr. Res. 45:275-282
16. Coplin DL, andCook D. 1990. Molecular genetics of extracellular polysaccharide biosynthesis
in vascular phytopathogenic bacteria. Mol. Plant-Microbe Interact. 3:271-279
17. Cadmus MC, Rogovin SP, Burton KA,Pittsley JE, Knutson CA 1978. AColonial variation in Xanthomonas campestris NRRL B- 1459 and characterization of the polysaccharide from a variant strain. CanadianJ. Microbiol., 19:942-948,
18. Stankowski JD,Muller BE,Zeller SG. 1993,Location of a second o-acetyl group in xanthan gum by the reductive-cleavage method .Carbohydr Res, 1993,241:321-326
19．姜成林 徐丽华 2001 《微生物资源开发利用》 175～181
20．聂凌鸿 周如金 宁正祥 2003 《黄原胶的特性、发展现状、生产及其应用》 《中国食品添加剂》 2003No 3
21．李兴存，张忠智，王洪君，陈进富，李术元 2002 《黄原胶的性能与应用》日用化学工业 Vol．No．32
22．杨新亭，张良1999 《黄原胶的性能及其应用》 河南农业大学学报 1999，9月 第33卷 增刊
23．赵景林 1994 《黄原胶的特性、生产及应用》 现代化工 1994年第五期
24. Baird J, Sandford P, and Cottrell I. 1983. Industrial applications of some new microbial polysaccharides. Bio/Technology 1:778-783
25. Garcia-Ochoa F ,Santos VE,Casas JA,et al. 2000 Xanthan gum:production,recovery, and properties.Biotechnol Adv, 18:549-579
26. Harding NE, Raffo S, Raimondi A, Cleary JM, and Ielpi L. 1993. Identification, genetic and biochemical analysis of genes involved ih synthesis sugar nucleotide precursors of xanthan gum. J. Gen. Microbiol. 139:447-457
27. Ielpi L, Couso R, and Dankert M. 1981. Lipid-linked intermediates in the biosynthesis of xanthan gum. FEBS Lett. 130:253-256
28. Ielpi L, Couso RO, and Dankert MA 1993. Sequential assembly and polymerization of the polyprenol-linked pentasaccharide repeating unit of the xanthan polysaccharide in Xanthomonas campestris. J. Bacteriol. 175:2490-2500
29. Ielpi L, Couso RO, and Dankert MA. 1983. Xanthan gum biosynthesis: acetylation occurs at the prenyl-phospho-sugar stage. Biochem. Int. 6:323-333.
30. Kplin R, Arnold W, Htte B, Simon R, Wang G, and Ptlhler A. 1992. Genetics of xanthan production in Xanthomonas campestris: the xanA and xanB genes are involved in UDP-glucose and GDP-mannose biosynthesis. J. Bacteriol. 174:191-199
31. Capage MR, Doherty MR, and Vanderslice RW 1987. Recombinant-DNA mediated production of xanthan gum. International patent WO87/05938.
32. Harding NE, Cleary JM, Cabafias DK, Rosen IG and Kang KS 1987. Genetic and physical analyses of a cluster of genes essential for xanthan gum biosynthesis in Xanthomonas campestris. Bacteriol. 169:2854-2861
33. Vanderslice RW, DohertyDH, Capage MA, Betlach MR, Hassler RA, Henderson NM,et al 1988. Genetic engineering of polysaccharide structure in Xanthomonas campestris. Gordon and Breach Science Publishers, p145-157.New York, N.Y
34. Katzen F, Becker A, Zorreguieta A, Pu hler A, Ielpi L. 1996. Promoter analysis of the Xanthomonas campestris pv. campestris gum operon directing the biosynthesis of the xanthan polysaccharide. J Bacteriol 178:4313-4318
35. Chou F L, Chou H C, Lin Y S, Yang B Y, Lin N T, Wenga 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
36. Bcckcr A,Katzen F, Puhler A, et al.1998.Xanthan gum biosynthesis and application :a biochemical/genetic perspective .Appl Microbiol Biotechnol,50; 145-152
37. Katzen F, Ferreiro DU, Oddo CG, Ielmini MV, BeckerA, Pu hlerA, Ielpi L 1998 Xanthomonas campestris pv. campestris gum mutants: effects on xanthan biosynthesis and plant virulence. JBacteriol 180:1607-1617
38. Hassler RA,Doherty DH. 1990 Genetic engineering of polysaccharide structure:production of variants of xanthan gum in Xanthomonas campestris. Biotechnol prog 6(3): 182-7
39. Adrian A. Vojnov ,Angeles Zorreguieta et al. 1998 .Evidence for a role for the gumB and gumC gene products in the formation of xanthan from its pentasaccharide repeating unit by Xanthomonas campestris. Microbiology. 144:1487-1493
40. de Crecy-Lagard V de, Glaser P, Lejeune P, Sismeiro O, Barber CE,Daniels M J, Danchin A. 1990 A Xanthomonas campestris pv.campestris protein similar to catabolite activation factor is in-volved in regulation of phytopathogenicity. J Bacteriol 172:5877-5883
41. Tang JL, Liu YN, Barber CE, Dow J M, Wootton JC and DanielsM.J. 1991. Genetic and molecular analysis of a cluster of rpf genes involved in positive regulation of synthesis of extracellular enzymes and polysaccharide in Xanthomonas campestris pathovar campestris. Mol. Gen. Genet. 226:409-417
42. Tang JL, Gough CL, Daniels MJ 1990 Cloning of genes involved in negative regulation of production of extracellular enzymes and polysaccharide ofXanthomonas campestris pathovar carnpestris. Mol Gen Genet 222:157-160
43. Daniels MJ, Barber CE, Dow JM, Han B,Liddle SA ,Newman MA,et al 1993. Bacterial genes required for pathogenesis: interaction between Xanthomonas and crucifers. In: Mechanisms of Plant Defence Responses 53-63.
44. Poplawsky A R,Chun W. 1998 Xanthomonas campestris pv. Carapestris requires a functional pigB for epiphytic survival and host infection. Mol Plant Microbe Interact, 11:466-475
45. Daniels MJ, Barber CE, Dow JM, Han B, Liddle SA et al 1993. Plant and bacterial genes involved in interactions between Xanthomonas and crucifers. In: Advances in Molecular Genetics of Plant-Microbe Interactions 2:423-433
46. Barrére GC, Barber CE,andDaniels MJ 1986. Molecular cloning of genes involved in the production of the extracellular polysaccharide xanthan by Xanthomonas campestris pv. campestris. Int. J. Biol. Macromol. 8:372-374
47．陆光涛 1997 《水稻白叶枯病诱导抗性初探》 硕士论文 中国南宁
48．李有志 1998 《野油菜黄单胞菌野油菜致病型胞外多糖产生有关的—基因族的鉴定和分析》，博士论文，中国武汉
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