野油菜黄单胞菌中编码磷酸葡萄糖异构酶的基因与致病性相关
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摘要
病原菌从寄主中获得营养是病原菌致病所必需的，碳素营养是所需营养的基本成分。在缺乏葡萄糖等六碳糖的环境中，微生物会通过糖异生途径利用非糖物质来合成葡萄糖以维持生长。我们实验室的实验结果已证明野油菜黄单胞菌中编码糖异生途径的两个关键酶的基因(ppsA基因和maeB基因)的突变体的致病力显著低于野生型菌株，这表明糖异生途径是野油菜黄单胞菌致病所必需的。六磷酸葡萄糖异构酶(PGI)是糖异生途径中另一个关键酶，在野油菜黄单胞菌中编码该酶的基因是pgi基因(Xcc 8004基因组中基因编号为XC2379)。为了明确该基因是否在糖异生途径中起重要作用及其与Xcc致病性的关系，我们用质粒定点整合突变方法构建了pgi基因的突变体。碳源利用检测结果表明，pgi突变体对多种碳源，例如柠檬酸、琥珀酸、延胡索酸乙酸、乳糖等的利用能力很差，这说明该基因在糖异生途径中起重要作用。我们用剪叶接种法在Xcc的寄主植物满身红萝卜上检测了pgi突变体的致病力，接种十天后，pgi突变体的病斑长度是3.6mm，而野生型的病斑程度是9.6 mm。这一结果证明pgi基因是黄单胞菌致病所必需的。以上结果结合本实验室前期已证实的ppsA和maeB基因是黄单胞菌致病所必需的结论，进一步说明了糖异生途径在黄单胞菌致病的重要性。
The ability to acquire nutrients from the host is essential for a pathogen to cause disease. Among those nutrients, carbon nutrient is one of the most important elements. Like other organisms, pathogens may use gluconeogenesis to synthesize glucose from non-sugar compounds when there is not sufficient hexose in their niche. The importance of the gluconeogenesis in Xcc virulence have been demonstrated recently in our laboratory. Glucose-6-phosphate isomerase (PGI) is another key enzyme in gluconueogenesis. According to Xcc 8004 genome annotation, Glucose-6-phosphate isomerase was encoded by pgi gene (ORF XC2379 ). To determined the role of this gene in gluconeogenesis and in pathogenesis of Xcc, mutants of pgi gene were constructed by homology plasmid integration method. The carbon utilization ability of the resulting mutant was tested, results showed that the pgi mutant grow poorly in non-carbon MMX medium with acetate, citrate, lactose, pyruvate, succinate, fumarate, or malate as sole carbon source. This in
dicated that pgi gene is involved in gluconueogenesis. The virulence of pgi
mutant was tested on Chinses radish (Raphanus sativus L. var. radical us Pers.) by leaf clipping, ten days post-inoculation, lesion length with a mean of 9.9 mm was observed on leaves inoculated with the wild type strain, while mean lesion lengths of 3.6 mm were observed with pgi mutant. This result demonstrated that pgi gene is required for full virulence of Xcc. These results combined with the previously findings in our laboratory that ppsA and maeB, genes encoding key enzymes in gluconeogenesis, are required for Xcc virulence, strongly indicated that the gluconeogenic pathway is important for Xcc virulence.
The ability to acquire nutrients from the host is essential for a pathogen to cause disease. Among those nutrients, carbon nutrient is one of the most important elements. Like other organisms, pathogens may use gluconeogenesis to synthesize glucose from non-sugar compounds when there is not sufficient hexose in their niche. The importance of the gluconeogenesis in Xcc virulence have been demonstrated recently in our laboratory. Glucose-6-phosphate isomerase (PGI) is another key enzyme in gluconueogenesis. According to Xcc 8004 genome annotation, Glucose-6-phosphate isomerase was encoded by pgi gene (ORF XC2379 ). To determined the role of this gene in gluconeogenesis and in pathogenesis of Xcc, mutants of pgi gene were constructed by homology plasmid integration method. The carbon utilization ability of the resulting mutant was tested, results showed that the pgi mutant grow poorly in non-carbon MMX medium with acetate, citrate, lactose, pyruvate, succinate, fumarate, or malate as sole carbon source. This in
dicated that pgi gene is involved in gluconueogenesis. The virulence of pgi
mutant was tested on Chinses radish (Raphanus sativus L. var. radical us Pers.) by leaf clipping, ten days post-inoculation, lesion length with a mean of 9.9 mm was observed on leaves inoculated with the wild type strain, while mean lesion lengths of 3.6 mm were observed with pgi mutant. This result demonstrated that pgi gene is required for full virulence of Xcc. These results combined with the previously findings in our laboratory that ppsA and maeB, genes encoding key enzymes in gluconeogenesis, are required for Xcc virulence, strongly indicated that the gluconeogenic pathway is important for Xcc virulence.
引文
1. Amey, R.C., Mills, ER., Bailey, A., Foster, G.D. 2003. Investigating the role of a Verticilliura fungicola beta-1,6-glucanase during infection of Agaricus bisporus using targeted gene disruption. Fungal Genetics and Biology, 39:264-275.
2. Bhriain, N.N., Brown, N.L., Lund, P.A., 1989. Mercury resistance in bacteria. In D.A. Hopwood and K.F. Chater (eds.) Genetics of Bacterial Diversity. Academic Press, New York, pp. 176-195.
3. Bogs, J., and Geider, K., 2000. Molecular Analysis of Sucrose Metabolism of Erwinia arnylovora and influence on Bacterial Virulence. J. Bactedol. 182: 5351-5358.
4. Bonas, U., Fenselau, S., Horns, T., Mric, C., Moussian, B., Pierre, M., Wengelnik, K.,and den Ackerveken, V, 1994. hrpx and avirulence gene of Xanthomonas campestris pv. esicatoria controlling the interaction with peper and tomato. Adcances in molecular genetics of plant-microbe in teractions.3: 58-64.
5. Chico-Calero, I., Suarez, M., Gonzalez-Zorn, B., Scortti, M., Slaghuis, J., Goebel, W., European Listeria Genome Consortium, T., Vazquez-Boland, J. A. 2002. Hpt, a bacterial homolog of the microsomal glucose-6-phosphate translocase, mediates rapid intracellular proliferation in Listeria. Proc. Natl. Acad. Sci. U. S. A. 99: 431-436.
6. De Crécy-Lagard, V., Glaser P., Lejeune P., Sismeiro, Barber, C.E., Daniels, M.J and 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.
7. da Silva Correia, J., Soldau, K., Christen, U., et al. 2001. Lipopoly-saaccharide is in close proximity to each of the proteins in its membrane receptor complex. J.Biol, 276:21129-21135.
8. Dean, RA., Timberlake, W.E., 1989. Production of cell wall-degrading enzymes by Aspergillus nidulans: a model system for fungal pathogenesis of plants. Plant
Cell. 3:265-73.
9. Foster, A.J., Jenkinson, J.M., Talbot, N.J. 2003. Trehalose synthesis and metabolism are required at different stages of plant infection by Magnaporthe grisea. EMBO J. 22, 225-235.
10. Foulongne, V., Bourg, G., Cazevieille, C., Michaux-Charachon, S. & O'Callaghan, D. 2000. Identification of Brucella suis Genes Affecting Intracellular Survival in an In Vitro Human Macrophage Infection Model by Signature-Tagged Transposon Mutagenesis Infect. Immun. 68:1297-1303.
11. Gaurivaud, P., Danet, J.L., Laigret, F, et al, 2000. Fructose utilization and phytopathogenicity of Spiroplasma citri. Mol Plant Microbe Interact. 13(10):1145-55
12. Gaynor, E.C., Cawthraw, S., Manning, G., MacKichan, J.K., Falkow, S., Newell, D. G. 2004. The Genome-Sequenced Variant of Campylobacter jejuni NCTC 11168 and the Original Clonal Clinical Isolate Differ Markedly in Colonization, Gene Expression, and Virulence-Associated Phenotypes. J. Bacteriol. 186:503-517.
13. Gold, S., Nishio, S., Tsuyumu, S., and Keen, N.T. 1992. Analysis of the pelE promoter in Erwinia chrysanthemi EC16. Mol. Plant-Microbe Interact. 5: 170-178.
14. Ji, P., Wilson, M. 2003. Enhancement of Population Size of a Biological Control Agent and Efficacy in Control of Bacterial Speck of Tomato through Salicylate and Ammonium Sulfate Amendments. Appl. Environ. Microbiol. 69: 1290-1294.
15. Langen, H., Gray, C., Roeder, D., Juranville, J.-F., Taka' cs, B., and Fountoulakis, M. 1997. From genome to proteome: Protein map of Haemophilus influenzae. Electrophoresis 18, 1184-1192.
16. Leite, R.P., Minsavage, G.V., Bonas, U., and Stall, R.E. 1994. Detection and identification of plant pathogenic strains of Xanthomonas based on amplification of DNA sequences related to the hrp genes of Xanthomonas campestris pv. vesicatoria. Appl Environ Microbiol 60:1068-1077.
17. Li, H.Q., Matsuda, I. , Fujise, Y. & Ichiyama, A. 1999. Short-chain acyl-Co-A-dependent production of oxalate from oxaloacetate by Burkholderia glumae, a
plant pathogen which causes grain rot and seedling rot of rice via the oxalate production. J Biochem. 126: 243±253.
18. Lorenz, M.C., Fink, GR., 2001. The glyoxylate cycle is required for fungal virulence. Nature. 412:83-86.
19. Lorenz, M.C., Fink GR., 2002 Life and Death in a Macrophage: Role of the Glyoxylate Cycle in Virulence. Eukaryotic Cell: 4: 657-662.
20. McKinney. J.D., Honer zu Bentrup, K., Munoz-Elias. E.J., Miczak, A., Chen, B., Chan, W.T, Swenson, D., Sacchettini, J.C., Jacobs, W.R., Jr, Russell DG. 2000. Persistence of Mycobacterium tuberculosis in macrophages and mice requires the glyoxylate shunt enzyme isocitrate lyase. Nature. 406:735-738.
21. Milenbachs, A.A, Brown, D.P, Moors, M., and Youngman, P. 1997. Carbon source regulation of virulence gene expression in Listeria monocytogenes. Mol Microbiol 23: 1075-1085.
22. Morris, V.L., Jackson, M.G., Ainsworth, T. and Cuppels, D.A. 1995. Isolation and sequence analysis of the Pseudomonas ayringae pv. tomato gene encoding a 2,3-diphosphglycerate-independent phosphglyceromutase. J.bacterial. 177:1723-1733.
23. Rahm, L., Mindrinos, M.N., and Panopoulos, N.J., 1992. Plant and environmental signals control the expression of hrpX genes in Pseudomonas syringae pv. phasecolicola. J.Bacteriol. 174:3499-3507.
24. Reverchon, S., Expert, D. and J. Robert-Bandouy. 1998. The cyclic AMP receptor protein is the main activator of pectinoluysis genes in Erwinia chrysanthemi. J.Bacteriol. 179:3500-3508.
25. Rietsch, A., Wolfgang, M.C., Mekalanos, J.J. et al. 2004. Effect of Metabolic Imbalance on Expression of Type Ⅲ Secretion Genes in Pseudomonas aeruginosa Infect. Immun. 72: 1383-1390.
26. Ripio, M.T., Brehm, K., Lara, M., Suarez, M., et al. 1997. Glucose-1-phosphate utilization by Listeria monocytogenes is PrfA dependent and coordinately expressed with virulence factors. J.Bacteriol. 179:7174-71800
27. Romeo, T., 1998. Global regulation by the small RNA-binding protein CsrA and
the non-coding RNA molecule CsrB. Mol Microbiol. 29:1321-30.
28. Schulte, R., and Bonas, U., 1992. Expression of the Xanthomonas campestris pv. vesicatioria ahrp gene cluster, which determines pahtogenicity and hypersensivity on pepper and tomato, is plant inducible. J.Bacteriol. 174:815-823.
29. Solomon, P.S; Tan, K.C., Olive, R.P. 2003. The nutrient supply of pathogenic fungi; a fertile field for study. Molecular Plant Pathology. 4: pp. 203-210.
30. Somerville, G.A., Said-Salim, B., Wickman, J.M, et al. 2003. Correlation of acetate catabolism and growth yield in Staphylococcus aureus: implications for host-pathogen interactions. Infect Immun. 71:4724-32.
31. Sonnleitner, E., Hagens, S., Rosenau, F., et al. 2003. Reduced virulence of a hfq mutant of Pseudomonas aeruginosa O1. Microb Pathog. 35:217-28.
32. Timm, J., Post, F.A., Linda-Gail Bekker, L.-G., et al. 2003. Differtial expression of iron, carbon-, and oxygen-responsibe mycobacterial genes in the lungs of chronically infected mice and tuberculosis patients. 10014321-16326.
33. Tuckman, D., Donnell, R.J., et al. 1997. Interruption of the phosphoglucose isomerase gene results in glucose auxotrophy in Mycobacterium smegmatis. J.Bacteriol. 179:2724-2730.
34. Tung, S.Y., and Kuo, T.H., 1999. Requirement for phosphoglucose isomerase of Xanthomonas campestris in pathogenesis of citrus canker. Applied and Environmental Microbiology. 65: 5564-5570.
35. Vim, E.R, Kalivoda, KoA, Deszo, E.L, et al. 2004. Diversity of microbial sialic acid metabolism. Microbiol Mol Biol. 68:132-53.
36. Wengelnik, K.& bonas, U., 1996. HrpXv, an AraC-type regulator, activates exp;ression of five of the six loci in the hrp cluster of Xanthomonas campestris pv. esicatoria. J.Bacteriol. 178:3462-3469.
2. Bhriain, N.N., Brown, N.L., Lund, P.A., 1989. Mercury resistance in bacteria. In D.A. Hopwood and K.F. Chater (eds.) Genetics of Bacterial Diversity. Academic Press, New York, pp. 176-195.
3. Bogs, J., and Geider, K., 2000. Molecular Analysis of Sucrose Metabolism of Erwinia arnylovora and influence on Bacterial Virulence. J. Bactedol. 182: 5351-5358.
4. Bonas, U., Fenselau, S., Horns, T., Mric, C., Moussian, B., Pierre, M., Wengelnik, K.,and den Ackerveken, V, 1994. hrpx and avirulence gene of Xanthomonas campestris pv. esicatoria controlling the interaction with peper and tomato. Adcances in molecular genetics of plant-microbe in teractions.3: 58-64.
5. Chico-Calero, I., Suarez, M., Gonzalez-Zorn, B., Scortti, M., Slaghuis, J., Goebel, W., European Listeria Genome Consortium, T., Vazquez-Boland, J. A. 2002. Hpt, a bacterial homolog of the microsomal glucose-6-phosphate translocase, mediates rapid intracellular proliferation in Listeria. Proc. Natl. Acad. Sci. U. S. A. 99: 431-436.
6. De Crécy-Lagard, V., Glaser P., Lejeune P., Sismeiro, Barber, C.E., Daniels, M.J and 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.
7. da Silva Correia, J., Soldau, K., Christen, U., et al. 2001. Lipopoly-saaccharide is in close proximity to each of the proteins in its membrane receptor complex. J.Biol, 276:21129-21135.
8. Dean, RA., Timberlake, W.E., 1989. Production of cell wall-degrading enzymes by Aspergillus nidulans: a model system for fungal pathogenesis of plants. Plant
Cell. 3:265-73.
9. Foster, A.J., Jenkinson, J.M., Talbot, N.J. 2003. Trehalose synthesis and metabolism are required at different stages of plant infection by Magnaporthe grisea. EMBO J. 22, 225-235.
10. Foulongne, V., Bourg, G., Cazevieille, C., Michaux-Charachon, S. & O'Callaghan, D. 2000. Identification of Brucella suis Genes Affecting Intracellular Survival in an In Vitro Human Macrophage Infection Model by Signature-Tagged Transposon Mutagenesis Infect. Immun. 68:1297-1303.
11. Gaurivaud, P., Danet, J.L., Laigret, F, et al, 2000. Fructose utilization and phytopathogenicity of Spiroplasma citri. Mol Plant Microbe Interact. 13(10):1145-55
12. Gaynor, E.C., Cawthraw, S., Manning, G., MacKichan, J.K., Falkow, S., Newell, D. G. 2004. The Genome-Sequenced Variant of Campylobacter jejuni NCTC 11168 and the Original Clonal Clinical Isolate Differ Markedly in Colonization, Gene Expression, and Virulence-Associated Phenotypes. J. Bacteriol. 186:503-517.
13. Gold, S., Nishio, S., Tsuyumu, S., and Keen, N.T. 1992. Analysis of the pelE promoter in Erwinia chrysanthemi EC16. Mol. Plant-Microbe Interact. 5: 170-178.
14. Ji, P., Wilson, M. 2003. Enhancement of Population Size of a Biological Control Agent and Efficacy in Control of Bacterial Speck of Tomato through Salicylate and Ammonium Sulfate Amendments. Appl. Environ. Microbiol. 69: 1290-1294.
15. Langen, H., Gray, C., Roeder, D., Juranville, J.-F., Taka' cs, B., and Fountoulakis, M. 1997. From genome to proteome: Protein map of Haemophilus influenzae. Electrophoresis 18, 1184-1192.
16. Leite, R.P., Minsavage, G.V., Bonas, U., and Stall, R.E. 1994. Detection and identification of plant pathogenic strains of Xanthomonas based on amplification of DNA sequences related to the hrp genes of Xanthomonas campestris pv. vesicatoria. Appl Environ Microbiol 60:1068-1077.
17. Li, H.Q., Matsuda, I. , Fujise, Y. & Ichiyama, A. 1999. Short-chain acyl-Co-A-dependent production of oxalate from oxaloacetate by Burkholderia glumae, a
plant pathogen which causes grain rot and seedling rot of rice via the oxalate production. J Biochem. 126: 243±253.
18. Lorenz, M.C., Fink, GR., 2001. The glyoxylate cycle is required for fungal virulence. Nature. 412:83-86.
19. Lorenz, M.C., Fink GR., 2002 Life and Death in a Macrophage: Role of the Glyoxylate Cycle in Virulence. Eukaryotic Cell: 4: 657-662.
20. McKinney. J.D., Honer zu Bentrup, K., Munoz-Elias. E.J., Miczak, A., Chen, B., Chan, W.T, Swenson, D., Sacchettini, J.C., Jacobs, W.R., Jr, Russell DG. 2000. Persistence of Mycobacterium tuberculosis in macrophages and mice requires the glyoxylate shunt enzyme isocitrate lyase. Nature. 406:735-738.
21. Milenbachs, A.A, Brown, D.P, Moors, M., and Youngman, P. 1997. Carbon source regulation of virulence gene expression in Listeria monocytogenes. Mol Microbiol 23: 1075-1085.
22. Morris, V.L., Jackson, M.G., Ainsworth, T. and Cuppels, D.A. 1995. Isolation and sequence analysis of the Pseudomonas ayringae pv. tomato gene encoding a 2,3-diphosphglycerate-independent phosphglyceromutase. J.bacterial. 177:1723-1733.
23. Rahm, L., Mindrinos, M.N., and Panopoulos, N.J., 1992. Plant and environmental signals control the expression of hrpX genes in Pseudomonas syringae pv. phasecolicola. J.Bacteriol. 174:3499-3507.
24. Reverchon, S., Expert, D. and J. Robert-Bandouy. 1998. The cyclic AMP receptor protein is the main activator of pectinoluysis genes in Erwinia chrysanthemi. J.Bacteriol. 179:3500-3508.
25. Rietsch, A., Wolfgang, M.C., Mekalanos, J.J. et al. 2004. Effect of Metabolic Imbalance on Expression of Type Ⅲ Secretion Genes in Pseudomonas aeruginosa Infect. Immun. 72: 1383-1390.
26. Ripio, M.T., Brehm, K., Lara, M., Suarez, M., et al. 1997. Glucose-1-phosphate utilization by Listeria monocytogenes is PrfA dependent and coordinately expressed with virulence factors. J.Bacteriol. 179:7174-71800
27. Romeo, T., 1998. Global regulation by the small RNA-binding protein CsrA and
the non-coding RNA molecule CsrB. Mol Microbiol. 29:1321-30.
28. Schulte, R., and Bonas, U., 1992. Expression of the Xanthomonas campestris pv. vesicatioria ahrp gene cluster, which determines pahtogenicity and hypersensivity on pepper and tomato, is plant inducible. J.Bacteriol. 174:815-823.
29. Solomon, P.S; Tan, K.C., Olive, R.P. 2003. The nutrient supply of pathogenic fungi; a fertile field for study. Molecular Plant Pathology. 4: pp. 203-210.
30. Somerville, G.A., Said-Salim, B., Wickman, J.M, et al. 2003. Correlation of acetate catabolism and growth yield in Staphylococcus aureus: implications for host-pathogen interactions. Infect Immun. 71:4724-32.
31. Sonnleitner, E., Hagens, S., Rosenau, F., et al. 2003. Reduced virulence of a hfq mutant of Pseudomonas aeruginosa O1. Microb Pathog. 35:217-28.
32. Timm, J., Post, F.A., Linda-Gail Bekker, L.-G., et al. 2003. Differtial expression of iron, carbon-, and oxygen-responsibe mycobacterial genes in the lungs of chronically infected mice and tuberculosis patients. 10014321-16326.
33. Tuckman, D., Donnell, R.J., et al. 1997. Interruption of the phosphoglucose isomerase gene results in glucose auxotrophy in Mycobacterium smegmatis. J.Bacteriol. 179:2724-2730.
34. Tung, S.Y., and Kuo, T.H., 1999. Requirement for phosphoglucose isomerase of Xanthomonas campestris in pathogenesis of citrus canker. Applied and Environmental Microbiology. 65: 5564-5570.
35. Vim, E.R, Kalivoda, KoA, Deszo, E.L, et al. 2004. Diversity of microbial sialic acid metabolism. Microbiol Mol Biol. 68:132-53.
36. Wengelnik, K.& bonas, U., 1996. HrpXv, an AraC-type regulator, activates exp;ression of five of the six loci in the hrp cluster of Xanthomonas campestris pv. esicatoria. J.Bacteriol. 178:3462-3469.