嗜碱盐单胞菌X3中异化型硝酸盐还原酶编码基因簇的功能验证及蛋白结构预测
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摘要
细菌的氮代谢推动环境的氮循环,硝酸盐还原反应是氮代谢途径中重要步骤之一。本文运用酶活测定、qRTPCR和生物信息学软件分析的方法,对嗜碱盐单胞菌(Halomonas alkaliphila)X3中编码细菌异化型硝酸盐还原酶(Dissimilatory nitrate reductase,Nar)不同亚基的基因簇narGYJV进行了功能验证、蛋白结构预测和系统发育分析。研究表明,X3菌株中存在narGYJV基因簇并具有表达活性,测得Nar的酶活为每克蛋白21.415U;预测到的narGYJV编码蛋白功能区域中1～1246氨基酸属于NarG超家族,编码Nar的α亚基;1261～1752氨基酸属于DMSOR-beta-like超家族,编码Nar的β亚基;2061～2279氨基酸编码γ亚基,1802～2018氨基酸编码δ亚基;Nar的二级结构中无规卷曲占37.59%,α螺旋占34.43%,延伸链占18.33%;NarG、NarY和NarV的3D结构与PDB中大肠杆菌(Escherichia coli)的1Q16 3D结构最接近,覆盖率96%～100%。系统发育树显示,菌株X3中NarG与同属菌的遗传距离较近,在不同菌属间虽然功能相似,其同源性较低;NarY与大肠杆菌中的氨基酸序列的同源关系较近,说明异化型硝酸盐还原酶Nar中不同亚基的系统发育地位不同。研究结果表明,Halomonas alkaliphila X3菌株中存在编码Nar的基因簇narGYJV,编码蛋白具有独特的3D结构,不同亚基的系统发育地位不同。研究结果为进一步研究该菌的氮代谢通路选择和调控机制提供了依据。
The nitrogen metabolism of bacteria promotes the environmental nitrogen cycle,and the nitrate reduction reaction is one of the most important steps in nitrogen metabolism pathway.With enzyme activity assay,qRT-PCR and bioinformatic tools,the functional verification,protein structure prediction and phylogenetic analysis were carried out for the gene cluster narGYJVencoding different subunits of dissimilatory nitrate reductase(Nar)in Halomonas alkaliphila X3.The results showed that the enzyme activity of the dissimilatory nitrate reductase was 21.415 Uper gram of protein,and qRT-PCR amplified the target fragments,thus verified the authenticity of the function of narGYJV.It was predicted that 1～1 246 amino acids(aa)of the narGYJVencoding protein belonged to the NarG superfamily and formed theαsubunit of Nar while 1 261～1 752 aa belonged to the DMSOR-beta-like superfamily and formed theβsubunit of Nar,2 061～2 279 aa formed theγsubunit,and 1 802～2 018 aa formed theδsubunit.In the secondary structure of the alfa subunit of Nar,random coil accounted for 37.59%,α-helix accounted for 34.43%and extended chain accounted for 18.33%.The 3 Dstructure of NarG,NarY and NarV were the closest with that of Escherichia coli(1 Q16)in PDB with 96%～100%query coverage.The phylogenetic trees showed that the genetic distance of NarG in X3 was closest to those in the samegenus,but far from those in other genus although they possessed the same function.NarY was closely related to that of E.coli in amino acid sequence,but low in similarity.These findings proved that the genetic evolution of the subunits in the NAR of alienated nitrate reductase was different.Our results provided the basis for the next step in studying the nitrogen metabolism pathway selection and regulation mechanism of the bacterium.
The nitrogen metabolism of bacteria promotes the environmental nitrogen cycle,and the nitrate reduction reaction is one of the most important steps in nitrogen metabolism pathway.With enzyme activity assay,qRT-PCR and bioinformatic tools,the functional verification,protein structure prediction and phylogenetic analysis were carried out for the gene cluster narGYJVencoding different subunits of dissimilatory nitrate reductase(Nar)in Halomonas alkaliphila X3.The results showed that the enzyme activity of the dissimilatory nitrate reductase was 21.415 Uper gram of protein,and qRT-PCR amplified the target fragments,thus verified the authenticity of the function of narGYJV.It was predicted that 1～1 246 amino acids(aa)of the narGYJVencoding protein belonged to the NarG superfamily and formed theαsubunit of Nar while 1 261～1 752 aa belonged to the DMSOR-beta-like superfamily and formed theβsubunit of Nar,2 061～2 279 aa formed theγsubunit,and 1 802～2 018 aa formed theδsubunit.In the secondary structure of the alfa subunit of Nar,random coil accounted for 37.59%,α-helix accounted for 34.43%and extended chain accounted for 18.33%.The 3 Dstructure of NarG,NarY and NarV were the closest with that of Escherichia coli(1 Q16)in PDB with 96%～100%query coverage.The phylogenetic trees showed that the genetic distance of NarG in X3 was closest to those in the samegenus,but far from those in other genus although they possessed the same function.NarY was closely related to that of E.coli in amino acid sequence,but low in similarity.These findings proved that the genetic evolution of the subunits in the NAR of alienated nitrate reductase was different.Our results provided the basis for the next step in studying the nitrogen metabolism pathway selection and regulation mechanism of the bacterium.
引文
[1]李春阳.海洋细菌来源的功能蛋白对海洋中重要有机物的识别及催化机制[D].济南:山东大学,2016.Li Chun-yang.Mechanistic Insights Into the Recognition and Catalysis of Several Important Oceanic Organic Matters by Bacterial Functional Proteins[D].Jinan:Shandong University,2016.
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[6]姜星宇,姚晓龙,徐会显,等.长江中下游典型湿地沉积物-水界面硝酸盐异养还原过程[J].湖泊科学,2016,28(6):1283-1292.Jiang Xing-yu,Yao Xiao-long,Xu Hui-xian,et al.Dissimilatory nitrate reduction processes between the sediment-water interface in three typical wetlands of middle and lower reaches of Yangtze River[J].Journal of Lake Sciences,2016,28(6):1283-1292.
[7]李小平,方涛,敖鸿毅,等.东湖沉积物中dNaR活性和硝酸盐还原菌的垂向分布[J].中国环境科学,2010,30(2):228-232.LI Xiao-ping,FANG Tao,AO Hong-yi,et al.Vertical variation of dissimilatory nitrate reductase activity and nitrate reducer in the sediments of Lake Donghu[J].China Environmental Science,2010,30(2):228-232.
[8] Tseng C P,Hansen A K,Cotter P,et al.Effect of cell growth rate on expression of the anaerobic respiratory pathway operons frdABCD,dmsABC,and narGHJI of Escherichia coli[J].Journal of Bacteriology,1994,176(21):6599-6605.
[9] Rendon J,Pilet E,Fahs Z,et al.Demethylmenaquinol is a substrate of Escherichia coli,nitrate reductase A(NarGHI)and forms a stable semiquinone intermediate at the NarGHI quinol oxidation site[J].BBA-Bioenergetics,2015,1847(8):739-747.
[10] Paiva J B,Filho R A C P,Pereira E A,et al.The contribution of genes required for anaerobic respiration to the virulence of Salmonella enterica serovar gallinarum for chickens[J].Brazilian Journal of Microbiology,2009,40(4):994-1001.
[11]张小玲.反硝化菌DNF409水体脱氮规律研究及其narG基因的中断[D].武汉:华中农业大学,2006.Zhang Xiao-ling.Denitrification of Denitrifying Bacteria DNF409andnarG Gene Disruption[D].Wuhan:Huazhong Agricultural University,2006.
[12] Blasco F,Pommier J,Augier V,et al.Involvement of the narJ or narW gene product in the formation of active nitrate re-ductase in Escherichia coli.[J].Molecular Microbiology,1992,6(2):221-230.
[13] Kristjansson J K,Hollocher T C.Substrate binding site for nitrate reductase of Escherichia coli is on the inner aspect of the membrane[J].Journal of Bacteriology,1979,137(3):1227-1233.
[14] Pérezrodríguez I,Bohnert K A,Cuebas M,et al.Detection and phylogenetic analysis of the membrane-bound nitrate re-ductase(Nar)in pure cultures and microbial communities from deep-sea hydrothermal vents[J].Fems Microbiology Ecology,2013,86(2):256-267.
[15] Oikawa Y,Sinmura Y,Ishizaka H,et al.Nar is the dominant dissimilatory nitrate reductase under high pressure conditions in the deep-sea denitrifier Pseudomonas sp.MT-1[J].Journal of General&Applied Microbiology,2015,61(1):10-14.
[16] Blasco F,Pommier J,Augier V,et al.Involvement of the narJ or narW gene product in the formation of active nitrate reductase in Escherichia coli[J].Molecular Microbiology,2010,6(2):221-230.
[17]李洪鹏,李秋芬,张艳,等.浅海养殖环境复合生态净化菌群的筛选及其净化功能研究[J].渔业科学进展,2009,30(2):46-53.Li Hong-peng,Li Qiu-fen,Zhang Yan,et al.Screening of ecocompound bacteria from coastal aquaculture environment and study on their purification ability[J].Progress in Fishery Sciences,2009,30(2):46-53.
[18]孙雪梅,李秋芬,张艳,等.一株海水异养硝化-好氧反硝化菌系统发育及脱氮特性[J].微生物学报,2012,52(6):687-695.Sun Xue-mei,Li Qiu-fen,Zhang Yan,et al.Phylogenetic analysis and nitrogen removal characteristics of a heterotrophic nitrifying-aerobic denitrifying bacteria strain from marine environment[J].Acta Microbiologica Sinica,2012,52(6):687-695.
[19]张艳,李秋芬,成钰,等.水体中盐单胞菌属(Halomonas)细菌实时荧光定量PCR检测方法的建立及应用[J].应用与环境生物学报,2016,22(1):140-145.Zhang Yan,Li Qiu-fen,Cheng Yu,et al.Establishment and application of a real-time PCR method for Halomonas in water[J].Chinese Journal of Applied and Environmental Biology,2016,22(1):140-145.
[20]王修芳,刘庆慧,吴垠,等.中国明对虾(Fenneropenaeus chinensis)coat-ε基因全长cDNA克隆及组织分布[J].渔业科学进展,2016,37(4):147-152.Wang Xiu-fang,Liu Qing-hui,Wu Yin,et al.cDNA cloning of coat-epsilon gene and its tissue distribution in Fenneropenaeus chinensis[J].Progress in Fishery Sciences,2016,37(4):147-152.
[21] Pal R R,Khardenavis A A,Purohit H J.Identification and monitoring of nitrification and denitrification genes in Klebsiella pneumoniae,EGD-HP19-C for its ability to perform heterotrophic nitrification and aerobic denitrification[J].Functional&Integrative Genomics,2015,15(1):63-76.
[22] And L P S,Barber M J.Assimilatory nitrate reductase:Functional properties and regulation[J].Annual Review of Plant Biology,1990,41(1):225-253.
[23] Richardson D J,Berks B C,Russell D A,et al.Functional,biochemical and genetic diversity of prokaryotic nitrate reduc-tases[J].Cellular&Molecular Life Sciences Cmls,2001,58(2):165-178.
[24] González P J,Correia C,Moura I,et al.Bacterial nitrate reductases:Molecular and biological aspects of nitrate reduction[J].Journal of Inorganic Biochemistry,2006,100(5-6):1015-1023.
[25]李丹,李忠磊,邵雷,等.细菌硝酸盐还原中的关键酶影响细菌耐药性的研究进展[J].中国抗生素杂志,2015,40(7):481-485.Li Dan,Li Zhong-Lei,ShaoLei,et al.The research progress of bacterial resistance affected by the key enzymes in bacterial nitrate reduction[J].Chinese Journal of Antibiotics,2015,40(7):481-485.
[26]杨航,黄钧,刘博.异养硝化-好氧反硝化菌Paracoccus pantotrophus ATCC 35512的研究进展[J].应用与环境生物学报,2008,14(4):585-592.Yang Hang,Huang Jun,Liu Bo.Advances in research of heterotrophic nitrification-aerobic denitrification strain paracoccus pantotrophus ATCC 35512[J].Chin J Appl Environ Biol,2008,14(4):585-592.
[27] Vetriani C,Voordeckers J W,Crespomedina M,et al.Deep-sea hydrothermal vent Epsilonproteobacteriaencode a con-served and widespread nitrate reduction pathway(Nap)[J].Isme Journal,2014,8(7):1510-1521.
[28] Blasco F,Dos Santos J P,Magalon A,et al.NarJ is a specific chaperone required for molybdenum cofactor assembly in nitrate reductase A of Escherichia coli[J].Molecular Microbiology,1998,28(3):435-447.
[2]赵娜.短程硝化—反硝化聚磷菌脱氮除磷技术的研究与应用进展[J].辽宁化工,2017(1):99-102.Zhao Na.Research progress and application of denitrification and phosphorus removal technology with shortcut nitrification and denitrifying phosphorus accumulating bacteria[J].Liaoning Chemical Industry,2017(1):99-102.
[3] Morozkina E V,Zvyagilskaya R A.Nitrate reductases:Structure,functions,and effect of stress factors[J].Biochemistry Biokhimiia,2007,72(10):1151--1160.
[4]许苗苗.净菜、泡菜贮藏过程中亚硝酸盐的变化及控制[D].泰安:山东农业大学,2010.Xu Miao-miao.Changes and Control of Nitrite in Fresh-Cut Vegetables and Pickle[D].Tai'an:Shandong Agricultural University,2010.
[5]杨立志.海杆菌NY-4的反硝化代谢特征及nirS基因的异源表达[D].南京:南京工业大学,2016.Yang Li-Zhi.Characteristics of Denitrifying Metabolism and Heterologous Expression of nirSGene in Marinobacter sp.NY-4[D].Nanjing:Nanjing University of Technology,2016.
[6]姜星宇,姚晓龙,徐会显,等.长江中下游典型湿地沉积物-水界面硝酸盐异养还原过程[J].湖泊科学,2016,28(6):1283-1292.Jiang Xing-yu,Yao Xiao-long,Xu Hui-xian,et al.Dissimilatory nitrate reduction processes between the sediment-water interface in three typical wetlands of middle and lower reaches of Yangtze River[J].Journal of Lake Sciences,2016,28(6):1283-1292.
[7]李小平,方涛,敖鸿毅,等.东湖沉积物中dNaR活性和硝酸盐还原菌的垂向分布[J].中国环境科学,2010,30(2):228-232.LI Xiao-ping,FANG Tao,AO Hong-yi,et al.Vertical variation of dissimilatory nitrate reductase activity and nitrate reducer in the sediments of Lake Donghu[J].China Environmental Science,2010,30(2):228-232.
[8] Tseng C P,Hansen A K,Cotter P,et al.Effect of cell growth rate on expression of the anaerobic respiratory pathway operons frdABCD,dmsABC,and narGHJI of Escherichia coli[J].Journal of Bacteriology,1994,176(21):6599-6605.
[9] Rendon J,Pilet E,Fahs Z,et al.Demethylmenaquinol is a substrate of Escherichia coli,nitrate reductase A(NarGHI)and forms a stable semiquinone intermediate at the NarGHI quinol oxidation site[J].BBA-Bioenergetics,2015,1847(8):739-747.
[10] Paiva J B,Filho R A C P,Pereira E A,et al.The contribution of genes required for anaerobic respiration to the virulence of Salmonella enterica serovar gallinarum for chickens[J].Brazilian Journal of Microbiology,2009,40(4):994-1001.
[11]张小玲.反硝化菌DNF409水体脱氮规律研究及其narG基因的中断[D].武汉:华中农业大学,2006.Zhang Xiao-ling.Denitrification of Denitrifying Bacteria DNF409andnarG Gene Disruption[D].Wuhan:Huazhong Agricultural University,2006.
[12] Blasco F,Pommier J,Augier V,et al.Involvement of the narJ or narW gene product in the formation of active nitrate re-ductase in Escherichia coli.[J].Molecular Microbiology,1992,6(2):221-230.
[13] Kristjansson J K,Hollocher T C.Substrate binding site for nitrate reductase of Escherichia coli is on the inner aspect of the membrane[J].Journal of Bacteriology,1979,137(3):1227-1233.
[14] Pérezrodríguez I,Bohnert K A,Cuebas M,et al.Detection and phylogenetic analysis of the membrane-bound nitrate re-ductase(Nar)in pure cultures and microbial communities from deep-sea hydrothermal vents[J].Fems Microbiology Ecology,2013,86(2):256-267.
[15] Oikawa Y,Sinmura Y,Ishizaka H,et al.Nar is the dominant dissimilatory nitrate reductase under high pressure conditions in the deep-sea denitrifier Pseudomonas sp.MT-1[J].Journal of General&Applied Microbiology,2015,61(1):10-14.
[16] Blasco F,Pommier J,Augier V,et al.Involvement of the narJ or narW gene product in the formation of active nitrate reductase in Escherichia coli[J].Molecular Microbiology,2010,6(2):221-230.
[17]李洪鹏,李秋芬,张艳,等.浅海养殖环境复合生态净化菌群的筛选及其净化功能研究[J].渔业科学进展,2009,30(2):46-53.Li Hong-peng,Li Qiu-fen,Zhang Yan,et al.Screening of ecocompound bacteria from coastal aquaculture environment and study on their purification ability[J].Progress in Fishery Sciences,2009,30(2):46-53.
[18]孙雪梅,李秋芬,张艳,等.一株海水异养硝化-好氧反硝化菌系统发育及脱氮特性[J].微生物学报,2012,52(6):687-695.Sun Xue-mei,Li Qiu-fen,Zhang Yan,et al.Phylogenetic analysis and nitrogen removal characteristics of a heterotrophic nitrifying-aerobic denitrifying bacteria strain from marine environment[J].Acta Microbiologica Sinica,2012,52(6):687-695.
[19]张艳,李秋芬,成钰,等.水体中盐单胞菌属(Halomonas)细菌实时荧光定量PCR检测方法的建立及应用[J].应用与环境生物学报,2016,22(1):140-145.Zhang Yan,Li Qiu-fen,Cheng Yu,et al.Establishment and application of a real-time PCR method for Halomonas in water[J].Chinese Journal of Applied and Environmental Biology,2016,22(1):140-145.
[20]王修芳,刘庆慧,吴垠,等.中国明对虾(Fenneropenaeus chinensis)coat-ε基因全长cDNA克隆及组织分布[J].渔业科学进展,2016,37(4):147-152.Wang Xiu-fang,Liu Qing-hui,Wu Yin,et al.cDNA cloning of coat-epsilon gene and its tissue distribution in Fenneropenaeus chinensis[J].Progress in Fishery Sciences,2016,37(4):147-152.
[21] Pal R R,Khardenavis A A,Purohit H J.Identification and monitoring of nitrification and denitrification genes in Klebsiella pneumoniae,EGD-HP19-C for its ability to perform heterotrophic nitrification and aerobic denitrification[J].Functional&Integrative Genomics,2015,15(1):63-76.
[22] And L P S,Barber M J.Assimilatory nitrate reductase:Functional properties and regulation[J].Annual Review of Plant Biology,1990,41(1):225-253.
[23] Richardson D J,Berks B C,Russell D A,et al.Functional,biochemical and genetic diversity of prokaryotic nitrate reduc-tases[J].Cellular&Molecular Life Sciences Cmls,2001,58(2):165-178.
[24] González P J,Correia C,Moura I,et al.Bacterial nitrate reductases:Molecular and biological aspects of nitrate reduction[J].Journal of Inorganic Biochemistry,2006,100(5-6):1015-1023.
[25]李丹,李忠磊,邵雷,等.细菌硝酸盐还原中的关键酶影响细菌耐药性的研究进展[J].中国抗生素杂志,2015,40(7):481-485.Li Dan,Li Zhong-Lei,ShaoLei,et al.The research progress of bacterial resistance affected by the key enzymes in bacterial nitrate reduction[J].Chinese Journal of Antibiotics,2015,40(7):481-485.
[26]杨航,黄钧,刘博.异养硝化-好氧反硝化菌Paracoccus pantotrophus ATCC 35512的研究进展[J].应用与环境生物学报,2008,14(4):585-592.Yang Hang,Huang Jun,Liu Bo.Advances in research of heterotrophic nitrification-aerobic denitrification strain paracoccus pantotrophus ATCC 35512[J].Chin J Appl Environ Biol,2008,14(4):585-592.
[27] Vetriani C,Voordeckers J W,Crespomedina M,et al.Deep-sea hydrothermal vent Epsilonproteobacteriaencode a con-served and widespread nitrate reduction pathway(Nap)[J].Isme Journal,2014,8(7):1510-1521.
[28] Blasco F,Dos Santos J P,Magalon A,et al.NarJ is a specific chaperone required for molybdenum cofactor assembly in nitrate reductase A of Escherichia coli[J].Molecular Microbiology,1998,28(3):435-447.