土壤中可编码乌头酸异构酶的芽胞杆菌菌株筛选及鉴定
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摘要
【目的】以芽胞杆菌(Bacillus)为筛选对象,分离土壤中可编码乌头酸异构酶(aconitate isomerase,AI)的革兰氏阳性(Gram positive,G+)菌株,以丰富对AI分布的科学认识,为其生物学功能研究奠定理论与材料基础。【方法】采用土样高温预处理法、含反式乌头酸(trans-aconitic acid,TAA)唯一碳源的ACO固体平板培养法,结合16S rDNA基因序列同源性分析,筛选能够编码AI的芽胞杆菌目的菌株。【结果】共分离得到22株能够利用TAA碳源的细菌菌株,成功鉴定了其中的16株,分别为巨大芽胞杆菌(Bacillus megaterium) 2株,阿氏芽胞杆菌(Bacillus aryabhattai) 7株,短小芽胞杆菌(Bacillus pumilus) 1株,未鉴定到种的芽胞杆菌(Bacillus sp.) 6株;且它们所含AI编码基因与已知AI基因在序列上存在差异。【结论】首次证明可编码AI的芽胞杆菌细菌种类具有多样性,暗示G+细菌广泛编码AI的可能性,更新了AI几乎只在G–细菌中分布的观点,为后续深入挖掘AI基因及其生物学功能研究提供更多可用微生物资源。
[Objective] Purpose of this work was to specifically screen the Gram positive(G+) bacteria of Bacillus that could encode the aconitate isomerase(AI) enzyme, to enrich our understanding of the distribution of AI and to provide theoretical and material basis for further research. [Methods] Through heat pretreatment of soil sample,plate cultivation by using ACO solid medium containing trans-aconitic acid(TAA) as the sole carbon source and the 16 S rDNA sequences homologous analysis, the Bacillus target strains that encode AI can be isolated. [Results]We totally isolated 22 bacterial strains that could utilize TAA carbon from the ACO plate, and 16 of which were successfully classified as: 2 strains of Bacillus megaterium, 7 strains of Bacillus aryabhattai, one Bacillus pumilus strain, and 6 undetermined Bacillus sp. strains; besides, we found that the AI-coding genes of these 16 Bacillus isolates were different from the already cloned ones in DNA sequence. [Conclusion] The species diversity of Bacillus bacteria encoding AI enzyme is rich, indicating the encoding ability in more G+ bacteria and updating the old opinion that considered AI distribution mainly in G– hosts. Our research provided available microbial resource for further studies on the identification of AI gene as well as its biological significance.
[Objective] Purpose of this work was to specifically screen the Gram positive(G+) bacteria of Bacillus that could encode the aconitate isomerase(AI) enzyme, to enrich our understanding of the distribution of AI and to provide theoretical and material basis for further research. [Methods] Through heat pretreatment of soil sample,plate cultivation by using ACO solid medium containing trans-aconitic acid(TAA) as the sole carbon source and the 16 S rDNA sequences homologous analysis, the Bacillus target strains that encode AI can be isolated. [Results]We totally isolated 22 bacterial strains that could utilize TAA carbon from the ACO plate, and 16 of which were successfully classified as: 2 strains of Bacillus megaterium, 7 strains of Bacillus aryabhattai, one Bacillus pumilus strain, and 6 undetermined Bacillus sp. strains; besides, we found that the AI-coding genes of these 16 Bacillus isolates were different from the already cloned ones in DNA sequence. [Conclusion] The species diversity of Bacillus bacteria encoding AI enzyme is rich, indicating the encoding ability in more G+ bacteria and updating the old opinion that considered AI distribution mainly in G– hosts. Our research provided available microbial resource for further studies on the identification of AI gene as well as its biological significance.
引文
[1]Du CY,Cao SY,Shi XY,Nie XT,Zheng JS,Deng Y,Ruan LF,Peng DH,Sun M.Genetic and biochemical characterization of a gene operon for trans-aconitic acid,a novel nematicide from Bacillus thuringiensis.Journal of Biological Chemistry,2017,292(8):3517-3530.
[2]Rao MRR,Altekar WW.Aconitate isomerase.Biochemical and Biophysical Research Communications,1961,4(2):101-105.
[3]Watanabe K,Katsuhara M,Nakao H,Sato M.Detection and molecular analysis of plant-and insect-associated bacteria harboring aconitate isomerase involved in biosynthesis of trans-aconitic acid as antifeedant in brown planthoppers.Current Microbiology,1997,35(2):97-102.
[4]都萃颖.小分子杀线虫毒素反式乌头酸生物合成途径及该毒素应用于植物根结线虫防治的研究.华中农业大学博士学位论文,2017.
[5]Yuhara K,Yonehara H,Hattori T,Kobayashi K,Kirimura K.Enzymatic characterization and gene identification of aconitate isomerase,an enzyme involved in assimilation of trans-aconitic acid,from Pseudomonas sp.WU-0701.The FEBS Journal,2015,282(22):4257-4267.
[6]Ruan LF,Crickmore N,Peng DH,Sun M.Are nematodes a missing link in the confounded ecology of the entomopathogen Bacillus thuringiensis?Trends in Microbiology,2015,23(6):341-346.
[7]Peng DH,Lin J,Huang Q,Zheng W,Liu GQ,Zheng JS,Zhu L,Sun M.A novel metalloproteinase virulence factor is involved in Bacillus thuringiensis pathogenesis in nematodes and insects.Environmental Microbiology,2016,18(3):846-862.
[8]Ruan LF,Wang HH,Cai G,Peng DH,Zhou H,Zheng JS,Zhu L,Wang XX,Yu HQ,Li S,Geng C,Sun M.A two-domain protein triggers heat shock pathway and necrosis pathway both in model plant and nematode.Environmental Microbiology,2015,17(11):4547-4565.
[9]曹诗云.苏云金芽胞杆菌中一种新毒素CT-A的结构鉴定及合成关键基因的定位.华中农业大学硕士学位论文,2012.
[10]刘要.苏云金杆菌芽胞中华亚种中一种具有杀线虫活性新毒素的初步鉴定及其与苏云金素的代谢关系.华中农业大学硕士学位论文,2009.
[11]Klinman JP,Rose IA.Purification and kinetic properties of aconitate isomerase from Pseudomonas putida.Biochemistry,1971,10(12):2253-2259.
[12]Bravo A,Likitvivatanavong S,Gill SS,Soberón M.Bacillus thuringiensis:a story of a successful bioinsecticide.Insect Biochemistry and Molecular Biology,2011,41(7):423-431.
[13]Liu GH,Liu B,Wang JP,Che JM,Zhu YJ,Ge CB,Chen Z.Advances in taxonomy and application of Bacillus.Microbiology,2017,44(4):949-958.(in Chinese)刘国红,刘波,王阶平,车建美,朱育菁,葛慈斌,陈铮.芽胞杆菌分类与应用研究进展.微生物学通报,2017,44(4):949-958.
[14]Long W,Chen CS.Application of 16S rRNA sequence in bacteria indication.Journal of Beijing Technology and Business University(Natural Science Edition),2006,24(5):10-12.(in Chinese)龙雯,陈存社.16S rRNA测序在细菌鉴定中的应用.北京工商大学学报(自然科学版),2006,24(5):10-12.
[15]Gürtler V,Stanisich VA.New approaches to typing and identification of bacteria using the 16S-23S rDNA spacer region.Microbiology,1996,142(1):3-16.
[16]Li XY,Huang HD,Lu XZ,Yang HP,Wang YS,Cao XJ.Screening of dominant bacteria for PHA accumulation in activated sludge and structure analysis of its product.Environmental Science&Technology,2009,32(9):124-128.(in Chinese)李晓雁,黄海东,卢显芝,杨红澎,王燕森,曹晓娟.活性污泥中PHA合成优势菌的筛选及产物结构分析.环境科学与技术,2009,32(9):124-128.
[17]Zhang SS,Li ZJ,Yan YC,Zhang CL,Li J,Zhao BS.Bacillus urumqiensis sp.nov.,a moderately haloalkaliphilic bacterium isolated from a salt lake.International Journal of Systematic and Evolutionary Microbiology,2016,66(6):2305-2312.
[18]Orioli GA,Thompson JF.Aconitate accumulation in wheat seedlings.Botanical Gazette,1990,151(1):30-37.
[19]Stout PR,Brownell J,Burau RG.Occurrences of trans-aconitate in range forage species.Agronomy Journal,1966,59(1):21-24.
[20]Thompson JF,Schaefer SC,Madison JT.Determination of aconitate isomerase in plants.Analytical Biochemistry,1990,184(1):39-47.
[21]Thompson JF,Schaefer SC,Madison JT.Role of aconitate isomerase in trans-aconitate accumulation in plants.Journal of Agricultural and Food Chemistry,1997,45(9):3684-3688.
[22]Burau R,Stout PR.Trans-aconitic acid in range grasses in early spring.Science,1965,150(3697):766-767.
[23]Jaitz L,Mueller B,Koellensperger G,Huber D,Oburger E,Puschenreiter M,Hann S.LC-MS analysis of low molecular weight organic acids derived from root exudation.Analytical and Bioanalytical Chemistry,2011,400(8):2587-2596.
[24]Savka MA,Farrand SK.Modification of rhizobacterial populations by engineering bacterium utilization of a novel plant-produced resource.Nature Biotechnology,1997,15(4):363-368.
[25]Salomone JY,Crouzet P,de Ruffray P,Otten L.Characterization and distribution of tartrate utilization genes in the grapevine pathogen Agrobacterium vitis.Molecular Plant-Microbe Interactions,1996,9(5):401-408.
[26]Salomone JY,Szegedi E,Cobanov P,Otten L.Tartrate utilization genes promote growth of Agrobacterium spp.on grapevine.Molecular Plant-Microbe Interactions,1998,11(8):836-838.
[27]Koch M,Delmotte N,Ahrens CH,Omasits U,Schneider K,Danza F,Padhi B,Murset V,Braissant O,Vorholt JA,Hennecke H,Pessi G.A link between arabinose utilization and oxalotrophy in Bradyrhizobium japonicum.Applied and Environmental Microbiology,2014,80(7):2092-2101.
[28]Oresnik IJ,Pacarynuk LA,O’Brien SAP,Yost CK,Hynes MF.Plasmid-encoded catabolic genes in Rhizobium leguminosarum bv.trifolii:evidence for a plant-inducible rhamnose locus involved in competition for nodulation.Molecular Plant-Microbe Interactions,1998,11(12):1175-1185.
[29]Jones JT,Haegeman A,Danchin EGJ,Gaur HS,Helder J,Jones MGK,Kikuchi T,Manzanilla-López R,Palomares-Rius JE,Wesemael WML,Perry RN.Top 10 plant-parasitic nematodes in molecular plant pathology.Molecular Plant Pathology,2013,14(9):946-961.
[30]Zasada IA,Halbrendt JM,Kokalis-Burelle N,LaMondia J,McKenry MV,Noling JW.Managing nematodes without methyl bromide.Annual Review of Phytopathology,2010,48:311-328.
[31]de Waele D,Elsen A.Challenges in tropical plant nematology.Annual Review of Phytopathology,2007,45:457-485.
[32]孙明,曹诗云,都萃颖,聂相涛,施祥雨,彭东海,阮丽芳.反式乌头酸产生菌及应用.中国:CN103421703B.2015-05-20.
[33]孙明,曹诗云,聂相涛,都萃颖,彭东海,阮丽芳.反式乌头酸在防治农业有害生物中的应用.中国:CN103416400A.2013-12-04.
[2]Rao MRR,Altekar WW.Aconitate isomerase.Biochemical and Biophysical Research Communications,1961,4(2):101-105.
[3]Watanabe K,Katsuhara M,Nakao H,Sato M.Detection and molecular analysis of plant-and insect-associated bacteria harboring aconitate isomerase involved in biosynthesis of trans-aconitic acid as antifeedant in brown planthoppers.Current Microbiology,1997,35(2):97-102.
[4]都萃颖.小分子杀线虫毒素反式乌头酸生物合成途径及该毒素应用于植物根结线虫防治的研究.华中农业大学博士学位论文,2017.
[5]Yuhara K,Yonehara H,Hattori T,Kobayashi K,Kirimura K.Enzymatic characterization and gene identification of aconitate isomerase,an enzyme involved in assimilation of trans-aconitic acid,from Pseudomonas sp.WU-0701.The FEBS Journal,2015,282(22):4257-4267.
[6]Ruan LF,Crickmore N,Peng DH,Sun M.Are nematodes a missing link in the confounded ecology of the entomopathogen Bacillus thuringiensis?Trends in Microbiology,2015,23(6):341-346.
[7]Peng DH,Lin J,Huang Q,Zheng W,Liu GQ,Zheng JS,Zhu L,Sun M.A novel metalloproteinase virulence factor is involved in Bacillus thuringiensis pathogenesis in nematodes and insects.Environmental Microbiology,2016,18(3):846-862.
[8]Ruan LF,Wang HH,Cai G,Peng DH,Zhou H,Zheng JS,Zhu L,Wang XX,Yu HQ,Li S,Geng C,Sun M.A two-domain protein triggers heat shock pathway and necrosis pathway both in model plant and nematode.Environmental Microbiology,2015,17(11):4547-4565.
[9]曹诗云.苏云金芽胞杆菌中一种新毒素CT-A的结构鉴定及合成关键基因的定位.华中农业大学硕士学位论文,2012.
[10]刘要.苏云金杆菌芽胞中华亚种中一种具有杀线虫活性新毒素的初步鉴定及其与苏云金素的代谢关系.华中农业大学硕士学位论文,2009.
[11]Klinman JP,Rose IA.Purification and kinetic properties of aconitate isomerase from Pseudomonas putida.Biochemistry,1971,10(12):2253-2259.
[12]Bravo A,Likitvivatanavong S,Gill SS,Soberón M.Bacillus thuringiensis:a story of a successful bioinsecticide.Insect Biochemistry and Molecular Biology,2011,41(7):423-431.
[13]Liu GH,Liu B,Wang JP,Che JM,Zhu YJ,Ge CB,Chen Z.Advances in taxonomy and application of Bacillus.Microbiology,2017,44(4):949-958.(in Chinese)刘国红,刘波,王阶平,车建美,朱育菁,葛慈斌,陈铮.芽胞杆菌分类与应用研究进展.微生物学通报,2017,44(4):949-958.
[14]Long W,Chen CS.Application of 16S rRNA sequence in bacteria indication.Journal of Beijing Technology and Business University(Natural Science Edition),2006,24(5):10-12.(in Chinese)龙雯,陈存社.16S rRNA测序在细菌鉴定中的应用.北京工商大学学报(自然科学版),2006,24(5):10-12.
[15]Gürtler V,Stanisich VA.New approaches to typing and identification of bacteria using the 16S-23S rDNA spacer region.Microbiology,1996,142(1):3-16.
[16]Li XY,Huang HD,Lu XZ,Yang HP,Wang YS,Cao XJ.Screening of dominant bacteria for PHA accumulation in activated sludge and structure analysis of its product.Environmental Science&Technology,2009,32(9):124-128.(in Chinese)李晓雁,黄海东,卢显芝,杨红澎,王燕森,曹晓娟.活性污泥中PHA合成优势菌的筛选及产物结构分析.环境科学与技术,2009,32(9):124-128.
[17]Zhang SS,Li ZJ,Yan YC,Zhang CL,Li J,Zhao BS.Bacillus urumqiensis sp.nov.,a moderately haloalkaliphilic bacterium isolated from a salt lake.International Journal of Systematic and Evolutionary Microbiology,2016,66(6):2305-2312.
[18]Orioli GA,Thompson JF.Aconitate accumulation in wheat seedlings.Botanical Gazette,1990,151(1):30-37.
[19]Stout PR,Brownell J,Burau RG.Occurrences of trans-aconitate in range forage species.Agronomy Journal,1966,59(1):21-24.
[20]Thompson JF,Schaefer SC,Madison JT.Determination of aconitate isomerase in plants.Analytical Biochemistry,1990,184(1):39-47.
[21]Thompson JF,Schaefer SC,Madison JT.Role of aconitate isomerase in trans-aconitate accumulation in plants.Journal of Agricultural and Food Chemistry,1997,45(9):3684-3688.
[22]Burau R,Stout PR.Trans-aconitic acid in range grasses in early spring.Science,1965,150(3697):766-767.
[23]Jaitz L,Mueller B,Koellensperger G,Huber D,Oburger E,Puschenreiter M,Hann S.LC-MS analysis of low molecular weight organic acids derived from root exudation.Analytical and Bioanalytical Chemistry,2011,400(8):2587-2596.
[24]Savka MA,Farrand SK.Modification of rhizobacterial populations by engineering bacterium utilization of a novel plant-produced resource.Nature Biotechnology,1997,15(4):363-368.
[25]Salomone JY,Crouzet P,de Ruffray P,Otten L.Characterization and distribution of tartrate utilization genes in the grapevine pathogen Agrobacterium vitis.Molecular Plant-Microbe Interactions,1996,9(5):401-408.
[26]Salomone JY,Szegedi E,Cobanov P,Otten L.Tartrate utilization genes promote growth of Agrobacterium spp.on grapevine.Molecular Plant-Microbe Interactions,1998,11(8):836-838.
[27]Koch M,Delmotte N,Ahrens CH,Omasits U,Schneider K,Danza F,Padhi B,Murset V,Braissant O,Vorholt JA,Hennecke H,Pessi G.A link between arabinose utilization and oxalotrophy in Bradyrhizobium japonicum.Applied and Environmental Microbiology,2014,80(7):2092-2101.
[28]Oresnik IJ,Pacarynuk LA,O’Brien SAP,Yost CK,Hynes MF.Plasmid-encoded catabolic genes in Rhizobium leguminosarum bv.trifolii:evidence for a plant-inducible rhamnose locus involved in competition for nodulation.Molecular Plant-Microbe Interactions,1998,11(12):1175-1185.
[29]Jones JT,Haegeman A,Danchin EGJ,Gaur HS,Helder J,Jones MGK,Kikuchi T,Manzanilla-López R,Palomares-Rius JE,Wesemael WML,Perry RN.Top 10 plant-parasitic nematodes in molecular plant pathology.Molecular Plant Pathology,2013,14(9):946-961.
[30]Zasada IA,Halbrendt JM,Kokalis-Burelle N,LaMondia J,McKenry MV,Noling JW.Managing nematodes without methyl bromide.Annual Review of Phytopathology,2010,48:311-328.
[31]de Waele D,Elsen A.Challenges in tropical plant nematology.Annual Review of Phytopathology,2007,45:457-485.
[32]孙明,曹诗云,都萃颖,聂相涛,施祥雨,彭东海,阮丽芳.反式乌头酸产生菌及应用.中国:CN103421703B.2015-05-20.
[33]孙明,曹诗云,聂相涛,都萃颖,彭东海,阮丽芳.反式乌头酸在防治农业有害生物中的应用.中国:CN103416400A.2013-12-04.
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