茄科作物青枯菌NADH脱氢酶F亚基在细胞运动和致病性中的功能研究（英文）
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摘要
【目的】劳尔氏菌(Ralstonia solanacearum)在茄科作物上引起严重的细菌性青枯病,本研究旨在发掘青枯劳尔氏菌与致病相关的基因。【方法】利用Tn5转座子构建随机插入突变体,分析生物膜形成、细胞运动和致病性;对有表型变化的突变体,运用TAIL-PCR方法鉴定Tn5插入位点,确定所突变的基因。【结果】以模式菌株GMI000为出发菌,总共获得了400个突变体,其中2个突变体不能形成生物膜,在软琼脂平板上的运动能力下降;接种感病番茄植物,这2个突变体都不能引起萎焉症状。TAIL-PCR结果显示,2个突变体的Tn5插入位点都在NADH脱氢酶F亚基(nuoF)中,距离翻译起始位点分别为103-bp和225-bp。ripAY基因启动子推动的nuoF基因互补载体,完全恢复了2个突变体的表型。【结论】NADH脱氢酶复合物是微生物呼吸电子传递链中的第一步催化酶。我们的结果表明,NADH脱氢酶复合物对R. solanacearum生物膜形成、细胞运动和致病性也有重要作用。
[Objective] Ralstonia solanacearum is the causative agent of a devastating bacterial wilt disease in solanaceous plants. The purpose of this work was to identify genes involved in the pathogenesis of R. solanacearum. [Methods] We used a Tn5-based mutagenesis strategy to generate random insertion mutants that were then assayed for biofilm formation, cell motility and pathogenicity. Thermal asymmetric interlaced PCR(TAIL-PCR) was performed to identify the Tn5 insertion site of mutants with altered phenotypes. [Results] A total of 400 mutants were generated in the model strain GMI1000. Two mutants were found to be unable to form biofilms and had reduced swimming and swarming motility on soft agar media. When inoculated into tomato plants, both mutants failed to cause wilting disease symptoms. Both mutants carried a Tn5 insertion within the NADH dehydrogenase subunit F gene(nuo F), and the insertion sites were at 103 bp and 225 bp from the translational start site. A complemented strain expressing nuo F under the control of the rip AY promoter fully restored the wild-type phenotype. [Conclusion] The NADH dehydrogenase complex is the first enzyme in the respiratory electron transport chain in microbes. Our data here indicated that NADH dehydrogenase plays a role in biofilm formation, cell motility and pathogenicity in R. solanacearum.
[Objective] Ralstonia solanacearum is the causative agent of a devastating bacterial wilt disease in solanaceous plants. The purpose of this work was to identify genes involved in the pathogenesis of R. solanacearum. [Methods] We used a Tn5-based mutagenesis strategy to generate random insertion mutants that were then assayed for biofilm formation, cell motility and pathogenicity. Thermal asymmetric interlaced PCR(TAIL-PCR) was performed to identify the Tn5 insertion site of mutants with altered phenotypes. [Results] A total of 400 mutants were generated in the model strain GMI1000. Two mutants were found to be unable to form biofilms and had reduced swimming and swarming motility on soft agar media. When inoculated into tomato plants, both mutants failed to cause wilting disease symptoms. Both mutants carried a Tn5 insertion within the NADH dehydrogenase subunit F gene(nuo F), and the insertion sites were at 103 bp and 225 bp from the translational start site. A complemented strain expressing nuo F under the control of the rip AY promoter fully restored the wild-type phenotype. [Conclusion] The NADH dehydrogenase complex is the first enzyme in the respiratory electron transport chain in microbes. Our data here indicated that NADH dehydrogenase plays a role in biofilm formation, cell motility and pathogenicity in R. solanacearum.
引文
[1] Yagi T, Matsuno-Yagi A. The proton-translocating NADH-quinone oxidoreductase in the respiratory chain:the secret unlocked. Biochemistry, 2003, 42(8):2266–2274.
[2] Hirst J. Mitochondrial complex I. Annual Review of Biochemistry, 2013, 82:551–575.
[3] Yagi T. Purification and characterization of NADH dehydrogenase complex from Paracoccus denitrificans.Archives of Biochemistry and Biophysics, 1986, 250(2):302–311.
[4] Falk-Krzesinski HJ, Wolfe AJ. Genetic analysis of the nuo locus, which encodes the proton-translocating NADH dehydrogenase in Escherichia coli. Journal of Bacteriology,1998, 180(5):1174–1184.
[5] Calhoun MW, Gennis RB. Demonstration of separate genetic loci encoding distinct membrane-bound respiratory NADH dehydrogenases in Escherichia coli. Journal of Bacteriology,1993, 175(10):3013–3019.
[6] Friedrich T, Scheide D. The respiratory complex I of bacteria,archaea and eukarya and its module common with membrane-bound multisubunit hydrogenases. FEBS Letters,2000, 479(1/2):1–5.
[7] Zambrano MM, Kolter R. Escherichia coli mutants lacking NADH dehydrogenase I have a competitive disadvantage in stationary phase. Journal of Bacteriology, 1993, 175(17):5642–5647.
[8] Tran QH, Bongaerts J, Vlad D, Unden G. Requirement for the proton-pumping NADH dehydrogenase I of Escherichia coli in respiration of NADH to fumarate and its bioenergetic implications. European Journal of Biochemistry, 1997,244(1):155–160.
[9] Weerakoon DR, Olson JW. The Campylobacter jejuni NADH:ubiquinone oxidoreductase(complex I)utilizes flavodoxin rather than NADH. Journal of Bacteriology, 2008, 190(3):915–925.
[10] Baradaran R, Berrisford JM, Minhas GS, Sazanov LA. Crystal structure of the entire respiratory complex I. Nature, 2013,494(7438):443–448.
[11] Mansfield J, Genin S, Magori S, Citovsky V, Sriariyanum M,Ronald P, Dow M, Verdier V, Beer SV, Machado MA, Toth I,Salmond G, Foster GD. Top 10 plant pathogenic bacteria in molecular plant pathology. Molecular Plant Pathology, 2012,13(6):614–629.
[12] Mori Y, Inoue K, Ikeda K, Nakayashiki H, Higashimoto C,Ohnishi K, Kiba A, Hikichi Y. The vascular plant-pathogenic bacterium Ralstonia solanacearum produces biofilms required for its virulence on the surfaces of tomato cells adjacent to intercellular spaces. Molecular Plant Pathology, 2016, 17(6):890–902.
[13] Tans-Kersten J, Huang HY, Allen C. Ralstonia solanacearum needs motility for invasive virulence on tomato. Journal of Bacteriology, 2001, 183(12):3597–3605.
[14] Li P, Yin WF, Yan JL, Chen YF, Fu SN, Song SH, Zhou JN,Lyu MF, Deng YY, Zhang LH. Modulation of inter-kingdom communication by Phc BSR quorum sensing system in Ralstonia solanacearum phylotype I strain GMI1000.Frontiers in Microbiology, 2017, 8:1172.
[15] Sun DL, Zhuo T, Hu X, Fan XJ, Zou HS. Identification of a Pseudomonas putida as biocontrol agent for tomato bacterial wilt disease. Biological Control, 2017, 114:45–50.
[16] Zhuo T, Rou W, Song X, Guo J, Fan XJ, Kamau GG, Zou HS.Molecular study on the car AB operon reveals that car B gene is required for swimming and biofilm formation in Xanthomonas citri subsp. citri. BMC Microbiology, 2015, 15:225.
[17] Boucher CA, Barberis PA, Trigalet AP, Demery DA.Transposon mutagenesis of Pseudomonas solanacearum:isolation of Tn5-induced avirulent mutants. Journal of General Microbiology, 1985, 131(9):2449–2457.
[18] Herrero M, de Lorenzo V, Timmis KN. Transposon vectors containing non-antibiotic resistance selection markers for cloning and stable chromosomal insertion of foreign genes in Gram-negative bacteria. Journal of Bacteriology, 1990,172(11):6557–6567.
[19] Better M, Helinski DR. Isolation and characterization of the rec A gene of Rhizobium meliloti. Journal of Bacteriology,1983, 155(1):311–316.
[20] Kovach ME, Phillips RW, Elzer PH, Roop II RM, Peterson KM. p BBR1MCS:a broad-host-range cloning vector.Biotechniques, 1994, 16(5):800–802.
[21] Liu YG, Whittier RF. Thermal asymmetric interlaced PCR:automatable amplification and sequencing of insert end fragments from P1 and YAC clones for chromosome walking.Genomics, 1995, 25(3):674–681.
[22] Jacobs MA, Alwood A, Thaipisuttikul I, Spencer D, Haugen E, Ernst S, Will O, Kaul R, Raymond C, Levy R, Liu C R,Guenthner D, Bovee D, Olson MV, Manoil C. Comprehensive transposon mutant library of Pseudomonas aeruginosa.Proceedings of the National Academy of Sciences of the United States of America, 2003, 100(24):14339–14344.
[23] Song X, Guo J, Ma WX, Ji ZY, Zou LF, Chen GY, Zou HS.Identification of seven novel virulence genes from Xanthomonas citri subsp. citri by Tn5-based random mutagenesis. Journal of Microbiology, 2015, 53(5):330–336.
[24] Panopoulos NJ, Schroth MN. Role of flagellar motility in the invasion of bean leaves by Pseudomonas phaseolicola.Phytopathology, 1974, 64(11):1389–1397.
[25] Bayot RG, Ries SM. Role of motility in apple blossom infection by Erwinia amylovora and studies of fire blight control with attractant and repellent compounds.Phytopathology, 1986, 76(4):441–445.
[26] Hatterman DR, Ries SM. Motility of Pseudomonas syringae pv. glycinea and its role in infection. Phytopathology, 1989,79(3):284–289.
[27] Hawes MC, Smith LY. Requirement for chemotaxis in pathogenicity of Agrobacterium tumefaciens on roots of soil-grown pea plants. Journal of Bacteriology, 1989,171(10):5668–5671.
[2] Hirst J. Mitochondrial complex I. Annual Review of Biochemistry, 2013, 82:551–575.
[3] Yagi T. Purification and characterization of NADH dehydrogenase complex from Paracoccus denitrificans.Archives of Biochemistry and Biophysics, 1986, 250(2):302–311.
[4] Falk-Krzesinski HJ, Wolfe AJ. Genetic analysis of the nuo locus, which encodes the proton-translocating NADH dehydrogenase in Escherichia coli. Journal of Bacteriology,1998, 180(5):1174–1184.
[5] Calhoun MW, Gennis RB. Demonstration of separate genetic loci encoding distinct membrane-bound respiratory NADH dehydrogenases in Escherichia coli. Journal of Bacteriology,1993, 175(10):3013–3019.
[6] Friedrich T, Scheide D. The respiratory complex I of bacteria,archaea and eukarya and its module common with membrane-bound multisubunit hydrogenases. FEBS Letters,2000, 479(1/2):1–5.
[7] Zambrano MM, Kolter R. Escherichia coli mutants lacking NADH dehydrogenase I have a competitive disadvantage in stationary phase. Journal of Bacteriology, 1993, 175(17):5642–5647.
[8] Tran QH, Bongaerts J, Vlad D, Unden G. Requirement for the proton-pumping NADH dehydrogenase I of Escherichia coli in respiration of NADH to fumarate and its bioenergetic implications. European Journal of Biochemistry, 1997,244(1):155–160.
[9] Weerakoon DR, Olson JW. The Campylobacter jejuni NADH:ubiquinone oxidoreductase(complex I)utilizes flavodoxin rather than NADH. Journal of Bacteriology, 2008, 190(3):915–925.
[10] Baradaran R, Berrisford JM, Minhas GS, Sazanov LA. Crystal structure of the entire respiratory complex I. Nature, 2013,494(7438):443–448.
[11] Mansfield J, Genin S, Magori S, Citovsky V, Sriariyanum M,Ronald P, Dow M, Verdier V, Beer SV, Machado MA, Toth I,Salmond G, Foster GD. Top 10 plant pathogenic bacteria in molecular plant pathology. Molecular Plant Pathology, 2012,13(6):614–629.
[12] Mori Y, Inoue K, Ikeda K, Nakayashiki H, Higashimoto C,Ohnishi K, Kiba A, Hikichi Y. The vascular plant-pathogenic bacterium Ralstonia solanacearum produces biofilms required for its virulence on the surfaces of tomato cells adjacent to intercellular spaces. Molecular Plant Pathology, 2016, 17(6):890–902.
[13] Tans-Kersten J, Huang HY, Allen C. Ralstonia solanacearum needs motility for invasive virulence on tomato. Journal of Bacteriology, 2001, 183(12):3597–3605.
[14] Li P, Yin WF, Yan JL, Chen YF, Fu SN, Song SH, Zhou JN,Lyu MF, Deng YY, Zhang LH. Modulation of inter-kingdom communication by Phc BSR quorum sensing system in Ralstonia solanacearum phylotype I strain GMI1000.Frontiers in Microbiology, 2017, 8:1172.
[15] Sun DL, Zhuo T, Hu X, Fan XJ, Zou HS. Identification of a Pseudomonas putida as biocontrol agent for tomato bacterial wilt disease. Biological Control, 2017, 114:45–50.
[16] Zhuo T, Rou W, Song X, Guo J, Fan XJ, Kamau GG, Zou HS.Molecular study on the car AB operon reveals that car B gene is required for swimming and biofilm formation in Xanthomonas citri subsp. citri. BMC Microbiology, 2015, 15:225.
[17] Boucher CA, Barberis PA, Trigalet AP, Demery DA.Transposon mutagenesis of Pseudomonas solanacearum:isolation of Tn5-induced avirulent mutants. Journal of General Microbiology, 1985, 131(9):2449–2457.
[18] Herrero M, de Lorenzo V, Timmis KN. Transposon vectors containing non-antibiotic resistance selection markers for cloning and stable chromosomal insertion of foreign genes in Gram-negative bacteria. Journal of Bacteriology, 1990,172(11):6557–6567.
[19] Better M, Helinski DR. Isolation and characterization of the rec A gene of Rhizobium meliloti. Journal of Bacteriology,1983, 155(1):311–316.
[20] Kovach ME, Phillips RW, Elzer PH, Roop II RM, Peterson KM. p BBR1MCS:a broad-host-range cloning vector.Biotechniques, 1994, 16(5):800–802.
[21] Liu YG, Whittier RF. Thermal asymmetric interlaced PCR:automatable amplification and sequencing of insert end fragments from P1 and YAC clones for chromosome walking.Genomics, 1995, 25(3):674–681.
[22] Jacobs MA, Alwood A, Thaipisuttikul I, Spencer D, Haugen E, Ernst S, Will O, Kaul R, Raymond C, Levy R, Liu C R,Guenthner D, Bovee D, Olson MV, Manoil C. Comprehensive transposon mutant library of Pseudomonas aeruginosa.Proceedings of the National Academy of Sciences of the United States of America, 2003, 100(24):14339–14344.
[23] Song X, Guo J, Ma WX, Ji ZY, Zou LF, Chen GY, Zou HS.Identification of seven novel virulence genes from Xanthomonas citri subsp. citri by Tn5-based random mutagenesis. Journal of Microbiology, 2015, 53(5):330–336.
[24] Panopoulos NJ, Schroth MN. Role of flagellar motility in the invasion of bean leaves by Pseudomonas phaseolicola.Phytopathology, 1974, 64(11):1389–1397.
[25] Bayot RG, Ries SM. Role of motility in apple blossom infection by Erwinia amylovora and studies of fire blight control with attractant and repellent compounds.Phytopathology, 1986, 76(4):441–445.
[26] Hatterman DR, Ries SM. Motility of Pseudomonas syringae pv. glycinea and its role in infection. Phytopathology, 1989,79(3):284–289.
[27] Hawes MC, Smith LY. Requirement for chemotaxis in pathogenicity of Agrobacterium tumefaciens on roots of soil-grown pea plants. Journal of Bacteriology, 1989,171(10):5668–5671.
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