两类产NO的亚硝酸盐还原酶的分布,结构与序列比较及宏基因组分析
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摘要
【目的】为了体现亚硝酸盐还原酶在环境中氮生物循环的重要性,研究了它们的分布情况。【方法】利用现有亚硝酸盐还原酶序列在已经测序的基因组数据库中进行查找,研究该酶的分布情况,通过多序列比对比较了它们的序列相似性,通过构建系统发育树比较其进化关系,并利用宏基因组学的方法研究了它们在海洋宏基因组中的分布。【结果】分析结果显示,两类亚硝酸盐还原酶在已测序的细菌和古生菌基因组中分别有397和812个,分别占总量的8%和15.7%,几乎所有的古生菌都含有Ⅱ类酶;它们自身的序列相似性很高,在Ⅰ类酶和Ⅱ类酶中底物结合位点以及Ⅱ类酶的铜离子结合位点保守性都很高,显示该酶序列保守性与其环境功能相适应的特点;进化分析显示它们可能具有共同的进化来源;在海洋宏基因组中,平均每100000读数中分别有6个Ⅰ类和35个Ⅱ类,且2类酶都在热带南太平洋区域有最大分布。【结论】NIR在氮的生物循环及环境修复中可能起到重要作用。
[Objective] To reflect the importance of nitrite reductase(NIR) in the environment, we studied its distribution. [Methods] The sequences of NIR were searched in the sequenced genome database at NCBI based on previous reported NIR sequences. The sequence similarity was done by multiple sequence alignment, and phylogenetic relationship was evaluated via constructing the phylogenetic tree. Furthermore, their distribution in the marine metagenome was studied by metagenomics. [Results] The homologues of these two enzymes were 397 and 812 strains in sequenced genome, and the proportion was 8 and 15.7 percent, respectively. Almost all of archaea containing type II NIR. They have high identity by multiple sequence alignment analysis. The cofactor, the substrate and the cooper binding sites in type II were high conserved, suggesting that these enzymes had the specific function in denitrification. Phylogenetic analysis showed the two enzymes may have the common ancestor. In marine metagenome analysis, type I and II have 6 and 35 reads per 100000 reads, respectively, the two types of NIRs have the biggest proportion at the tropical south pacific area. [Conclusion] Collectively, we suggested NIR, especially type II, play a key role in bioremediation of nitrogen contamination.
[Objective] To reflect the importance of nitrite reductase(NIR) in the environment, we studied its distribution. [Methods] The sequences of NIR were searched in the sequenced genome database at NCBI based on previous reported NIR sequences. The sequence similarity was done by multiple sequence alignment, and phylogenetic relationship was evaluated via constructing the phylogenetic tree. Furthermore, their distribution in the marine metagenome was studied by metagenomics. [Results] The homologues of these two enzymes were 397 and 812 strains in sequenced genome, and the proportion was 8 and 15.7 percent, respectively. Almost all of archaea containing type II NIR. They have high identity by multiple sequence alignment analysis. The cofactor, the substrate and the cooper binding sites in type II were high conserved, suggesting that these enzymes had the specific function in denitrification. Phylogenetic analysis showed the two enzymes may have the common ancestor. In marine metagenome analysis, type I and II have 6 and 35 reads per 100000 reads, respectively, the two types of NIRs have the biggest proportion at the tropical south pacific area. [Conclusion] Collectively, we suggested NIR, especially type II, play a key role in bioremediation of nitrogen contamination.
引文
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[9]Larkin MA,Blackshields G,Brown NP,Chenna R,Mc Gettigan PA,Mc William H,Valentin F,Wallace IM,Wilm A,Lopez R,Thompson JD,Gibson TJ,Higgins DG.Clustal Wand clustal X version 2.0.Bioinformatics,2007,23(21):2947-2948.
[10]Rusch DB,Halpern AL,Sutton G,Heidelberg KB,Williamson S,Yooseph S,Wu DY,Eisen JA,Hoffman JM,Remington K,Beeson K,Tran B,Smith H,Baden-Tillson H,Stewart C,Thorpe J,Freeman J,Andrews-Pfannkoch C,Venter JE,Li K,Kravitz S,Heidelberg JF,Utterback T,Rogers YH,Falcón LI,Souza V,Bonilla-Rosso G,Eguiarte LE,Karl DM,Sathyendranath S,Platt T,Bermingham E,Gallardo V,Tamayo-Castillo G,Ferrari MR,Strausberg RL,Nealson K,Friedman R,Frazier M,Venter JC.The sorcerer II global ocean sampling expedition:northwest Atlantic through eastern tropical Pacific.PLo S Biology,2007,5(3):e77.
[11]Howard EC,Sun SL,Biers EJ,Moran MA.Abundant and diverse bacteria involved in DMSP degradation in marine surface waters.Environmental Microbiology,2008,10(9):2397-2410.
[12]Cai XL,Luo JF,Lin WT,Tian GL.Microbial community in nitrogen cycle of aquaculture water of the Pearl River Delta.Acta Microbiologica Sinica,2012,52(5):645-653.(in Chinese)蔡小龙,罗剑飞,林炜铁,田国梁.珠三角养殖水体中参与氮循环的微生物群落结构.微生物学报,2012,52(5):645-653.
[13]Nurizzo D,CutruzzolàF,Arese M,Bourgeois D,Brunori M,Cambillau C,Tegoni M.Conformational changes occurring upon reduction and NO binding in nitrite reductase from Pseudomonas aeruginosa.Biochemistry,1998,37(40):13987-13996.
[14]Nurizzo D,Silvestrini MC,Mathieu M,CutruzzolàF,Bourgeois D,Fül?p V,Hajdu J,Brunori M,Tegoni M,Cambillau C.N-terminal arm exchange is observed in the 2.15?crystal structure of oxidized nitrite reductase from Pseudomonas aeruginosa.Structure,1997,5(9):1157-1171.
[15]Antonyuk SV,Strange RW,Sawers G,Eady RR,Hasnain SS.Atomic resolution structures of resting-state,substrate-and product-complexed Cu-nitrite reductase provide insight into catalytic mechanism.Proceedings of the National Academy of Sciences of the United States of America,2005,102(34):12041-12046.
[16]Adman ET,Godden JW,Turley S.The structure of copper-nitrite reductase from Achromobacter cycloclastes at five p H values,with NO2-bound and with type II copper depleted.The Journal of Biological Chemistry,1995,270(46):27458-27474.
[17]Li Y,Hodak M,Bernholc J.Enzymatic mechanism of copper-containing nitrite reductase.Biochemistry,2015,54(5):1233-1242.
[18]Martiny AC,Kathuria S,Berube PM.Widespread metabolic potential for nitrite and nitrate assimilation among Prochlorococcus ecotypes.Proceedings of the National Academy of Sciences of the United States of America,2009,106(26):10787-10792.
[19]Zeng W,Li L,Yang YY,Zhang Y,Wang SY.Short-cut nitrification and denitrification in A2O process treating domestic wastewater.China Environmental Science,2010,30(5):625-632.(in Chinese)曾薇,李磊,杨莹莹,张悦,王淑莹.A2O工艺处理生活污水短程硝化反硝化的研究.中国环境科学,2010,30(5):625-632.
[20]Ma Y,Wang SY,Zeng W,Peng YZ,Zhou L.A/O pilot-scale nitrogen removal process treating domestic wastewaterⅠ.The study of short-cut nitrification and denitrification.Acta Scientiae Circumstantiae,2006,26(5):703-709.(in Chinese)马勇,王淑莹,曾薇,彭永臻,周利.A/O生物脱氮工艺处理生活污水中试(一)短程硝化反硝化的研究.环境科学学报,2006,26(5):703-709.
[21]Zhang L,Cui LH.The denitrification status and its research progress of construction wetland.Chinese Agricultural Science Bulletin,2012,28(5):268-272.(in Chinese)张玲,崔理华.人工湿地脱氮现状与研究进展.中国农学通报,2012,28(5):268-272.
[22]Wu YF,LüXW,Zhong ZP,Shi J,Xu W.Influencing factors of nitrogen removal from stream water using special riverine ecosystem.Journal of Central South University(Science and Technology),2011,42(2):539-545.(in Chinese)吴义锋,吕锡武,仲兆平,史静,徐微.河渠岸坡特定生态系统的脱氮效率及影响因素.中南大学学报(自然科学版),2011,42(2):539-545.
[23]Fu RB,Yang HZ,Gu GW,Zhang Z.Nitrogen removal from rural sewage by subsurface horizontal-flow in artificial wetlands.Technology of Water Treatment,2006,32(1):18-22.(in Chinese)付融冰,杨海真,顾国维,张政.潜流人工湿地对农村生活污水氮去除的研究.水处理技术,2006,32(1):18-22.
[24]Peng YZ,Sun HW,Yang Q.The biochemical reaction mechanism and kinetics of partial nitrification.Acta Scientiae Circumstantiae,2008,28(5):817-824.(in Chinese)彭永臻,孙洪伟,杨庆.短程硝化的生化机理及其动力学.环境科学学报,2008,28(5):817-824.
[25]Qu Y,Zhang PY,Yu DS,Guo SS,Yang RX.Bioaugmentation for shortcut nitrification in SBR treating for sewage containing sea water by nitrification-aerobic denitrification bacteria.Environmental Science,2010,31(10):2376-2384.(in Chinese)曲洋,张培玉,于德爽,郭沙沙,杨瑞霞.异养硝化/好氧反硝化菌生物强化含海水污水的SBR短程硝化系统初探.环境科学,2010,31(10):2376-2384.
[26]Mu LP,Huang J,Gou S.Denitrification by heterotrophic nitrification microbial consortium and aerobic granular sludge.Chinese Journal of Applied&Environmental Biology,2009,15(3):356-360.(in Chinese)牟丽娉,黄钧,苟莎.异养硝化微生物菌剂及其好氧颗粒污泥的脱氮试验.应用与环境生物学报,2009,15(3):356-360.
[27]Zhou SL,Huang TL,Bai SY,He XX.Nitrogen removal characteristics of mixed aerobic denitrification bacteria under in-situ biological inoculation.Acta Microbiologica Sinica,2016,56(4):590-602.(in Chinese)周石磊,黄廷林,白士远,何秀秀.贫营养好氧反硝化菌群的水库原位投菌脱氮特性.微生物学报,2016,56(4):590-602.
[2]Kuznetsova S,Knaff DB,Hirasawa M,Lagoutte B,Sétif P.Mechanism of spinach chloroplast ferredoxin-dependent nitrite reductase:spectroscopic evidence for intermediate states.Biochemistry,2004,43(2):510-517.
[3]Gamble TN,Betlach MR,Tiedje JM.Numerically dominant denitrifying bacteria from world soils.Applied and Environmental Microbiology,1977,33(4):926-939.
[4]CutruzzolàF.Bacterial nitric oxide synthesis.Biochimica et Biophysica Acta(BBA)-Bioenergetics,1999,1411(2/3):231-249.
[5]Vijgenboom E,Busch JE,Canters GW.In vivo studies disprove an obligatory role of azurin in denitrification in Pseudomonas aeruginosa and show that azu expression is under control of rpo S and ANR.Microbiology,1997,143(9):2853-2863.
[6]Paraskevopoulos K,Hough MA,Sawers RG,Eady RR,Hasnain SS.The structure of the Met144Leu mutant of copper nitrite reductase from Alcaligenes xylosoxidans provides the first glimpse of a protein-protein complex with azurin II.Journal of Biological Inorganic Chemistry,2007,12(6):789-796.
[7]Ichiki H,Tanaka Y,Mochizuki K,Yoshimatsu K,Sakurai T,Fujiwara T.Purification,characterization,and genetic analysis of Cu-containing dissimilatory nitrite reductase from a denitrifying halophilic archaeon,Haloarcula marismortui.Journal of Bacteriology,2001,183(14):4149-4156.
[8]Zumft WG.Cell biology and molecular basis of denitrification.Microbiology and Molecular Biology Reviews,1997,61(4):533-616.
[9]Larkin MA,Blackshields G,Brown NP,Chenna R,Mc Gettigan PA,Mc William H,Valentin F,Wallace IM,Wilm A,Lopez R,Thompson JD,Gibson TJ,Higgins DG.Clustal Wand clustal X version 2.0.Bioinformatics,2007,23(21):2947-2948.
[10]Rusch DB,Halpern AL,Sutton G,Heidelberg KB,Williamson S,Yooseph S,Wu DY,Eisen JA,Hoffman JM,Remington K,Beeson K,Tran B,Smith H,Baden-Tillson H,Stewart C,Thorpe J,Freeman J,Andrews-Pfannkoch C,Venter JE,Li K,Kravitz S,Heidelberg JF,Utterback T,Rogers YH,Falcón LI,Souza V,Bonilla-Rosso G,Eguiarte LE,Karl DM,Sathyendranath S,Platt T,Bermingham E,Gallardo V,Tamayo-Castillo G,Ferrari MR,Strausberg RL,Nealson K,Friedman R,Frazier M,Venter JC.The sorcerer II global ocean sampling expedition:northwest Atlantic through eastern tropical Pacific.PLo S Biology,2007,5(3):e77.
[11]Howard EC,Sun SL,Biers EJ,Moran MA.Abundant and diverse bacteria involved in DMSP degradation in marine surface waters.Environmental Microbiology,2008,10(9):2397-2410.
[12]Cai XL,Luo JF,Lin WT,Tian GL.Microbial community in nitrogen cycle of aquaculture water of the Pearl River Delta.Acta Microbiologica Sinica,2012,52(5):645-653.(in Chinese)蔡小龙,罗剑飞,林炜铁,田国梁.珠三角养殖水体中参与氮循环的微生物群落结构.微生物学报,2012,52(5):645-653.
[13]Nurizzo D,CutruzzolàF,Arese M,Bourgeois D,Brunori M,Cambillau C,Tegoni M.Conformational changes occurring upon reduction and NO binding in nitrite reductase from Pseudomonas aeruginosa.Biochemistry,1998,37(40):13987-13996.
[14]Nurizzo D,Silvestrini MC,Mathieu M,CutruzzolàF,Bourgeois D,Fül?p V,Hajdu J,Brunori M,Tegoni M,Cambillau C.N-terminal arm exchange is observed in the 2.15?crystal structure of oxidized nitrite reductase from Pseudomonas aeruginosa.Structure,1997,5(9):1157-1171.
[15]Antonyuk SV,Strange RW,Sawers G,Eady RR,Hasnain SS.Atomic resolution structures of resting-state,substrate-and product-complexed Cu-nitrite reductase provide insight into catalytic mechanism.Proceedings of the National Academy of Sciences of the United States of America,2005,102(34):12041-12046.
[16]Adman ET,Godden JW,Turley S.The structure of copper-nitrite reductase from Achromobacter cycloclastes at five p H values,with NO2-bound and with type II copper depleted.The Journal of Biological Chemistry,1995,270(46):27458-27474.
[17]Li Y,Hodak M,Bernholc J.Enzymatic mechanism of copper-containing nitrite reductase.Biochemistry,2015,54(5):1233-1242.
[18]Martiny AC,Kathuria S,Berube PM.Widespread metabolic potential for nitrite and nitrate assimilation among Prochlorococcus ecotypes.Proceedings of the National Academy of Sciences of the United States of America,2009,106(26):10787-10792.
[19]Zeng W,Li L,Yang YY,Zhang Y,Wang SY.Short-cut nitrification and denitrification in A2O process treating domestic wastewater.China Environmental Science,2010,30(5):625-632.(in Chinese)曾薇,李磊,杨莹莹,张悦,王淑莹.A2O工艺处理生活污水短程硝化反硝化的研究.中国环境科学,2010,30(5):625-632.
[20]Ma Y,Wang SY,Zeng W,Peng YZ,Zhou L.A/O pilot-scale nitrogen removal process treating domestic wastewaterⅠ.The study of short-cut nitrification and denitrification.Acta Scientiae Circumstantiae,2006,26(5):703-709.(in Chinese)马勇,王淑莹,曾薇,彭永臻,周利.A/O生物脱氮工艺处理生活污水中试(一)短程硝化反硝化的研究.环境科学学报,2006,26(5):703-709.
[21]Zhang L,Cui LH.The denitrification status and its research progress of construction wetland.Chinese Agricultural Science Bulletin,2012,28(5):268-272.(in Chinese)张玲,崔理华.人工湿地脱氮现状与研究进展.中国农学通报,2012,28(5):268-272.
[22]Wu YF,LüXW,Zhong ZP,Shi J,Xu W.Influencing factors of nitrogen removal from stream water using special riverine ecosystem.Journal of Central South University(Science and Technology),2011,42(2):539-545.(in Chinese)吴义锋,吕锡武,仲兆平,史静,徐微.河渠岸坡特定生态系统的脱氮效率及影响因素.中南大学学报(自然科学版),2011,42(2):539-545.
[23]Fu RB,Yang HZ,Gu GW,Zhang Z.Nitrogen removal from rural sewage by subsurface horizontal-flow in artificial wetlands.Technology of Water Treatment,2006,32(1):18-22.(in Chinese)付融冰,杨海真,顾国维,张政.潜流人工湿地对农村生活污水氮去除的研究.水处理技术,2006,32(1):18-22.
[24]Peng YZ,Sun HW,Yang Q.The biochemical reaction mechanism and kinetics of partial nitrification.Acta Scientiae Circumstantiae,2008,28(5):817-824.(in Chinese)彭永臻,孙洪伟,杨庆.短程硝化的生化机理及其动力学.环境科学学报,2008,28(5):817-824.
[25]Qu Y,Zhang PY,Yu DS,Guo SS,Yang RX.Bioaugmentation for shortcut nitrification in SBR treating for sewage containing sea water by nitrification-aerobic denitrification bacteria.Environmental Science,2010,31(10):2376-2384.(in Chinese)曲洋,张培玉,于德爽,郭沙沙,杨瑞霞.异养硝化/好氧反硝化菌生物强化含海水污水的SBR短程硝化系统初探.环境科学,2010,31(10):2376-2384.
[26]Mu LP,Huang J,Gou S.Denitrification by heterotrophic nitrification microbial consortium and aerobic granular sludge.Chinese Journal of Applied&Environmental Biology,2009,15(3):356-360.(in Chinese)牟丽娉,黄钧,苟莎.异养硝化微生物菌剂及其好氧颗粒污泥的脱氮试验.应用与环境生物学报,2009,15(3):356-360.
[27]Zhou SL,Huang TL,Bai SY,He XX.Nitrogen removal characteristics of mixed aerobic denitrification bacteria under in-situ biological inoculation.Acta Microbiologica Sinica,2016,56(4):590-602.(in Chinese)周石磊,黄廷林,白士远,何秀秀.贫营养好氧反硝化菌群的水库原位投菌脱氮特性.微生物学报,2016,56(4):590-602.