硝化微生物富集及其种群结构与基因表达分析
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摘要
对采集的长江口潮滩沉积物进行了硝化微生物的富集培养,通过宏基因组技术分析了富集物中硝化微生物的种群结构.检测到4类硝化微生物(占测序总reads的34.7%),包括新近发现的全程氨氧化微生物(complete ammonia oxidizers, comammox)、严格的亚硝酸盐氧化菌Nitrospira、β-变形菌门氨氧化细菌及氨氧化古菌.其中,comammox占硝化微生物类群的48%.此外,基于宏转录组技术分析了4类硝化菌群的基因表达特征(依据Evolutionary genealogy of genes:Non-supervised Orthologous Groups数据库).研究探讨了潮滩沉积物富集物中硝化微生物的种群结构与基因表达特征,证明了长江口潮滩沉积环境中comammox的存在,并验证了其基因表达活性.研究结果加深了对河口地区硝化微生物的认识,对于未来硝化过程的分子生态学研究有重要启示意义.
Nitrifiers were enriched from the tidal sediments of the Yangtze Estuary. Four kinds of nitrifying groups(accounting for 34.7% of the total reads) were detected based on metagenomics, including the recently discovered complete ammonia oxidizers(comammox),strictly nitrite-oxidizing Nitrospira,/3-proteobacterial ammonia-oxidizing bacteria, and ammonia-oxidizing archaea. Comammox accounted for 48% of the detected nitrifiers. In addition, gene expression patterns of the nitrifier group(according to the classification in Evolutionary genealogy of genes: Non-supervised Orthologous Groupsdatabase) were analyzed based on metatranscriptomics. The present study showed the community structure and gene expression characteristics of nitrifiers enriched from the tidal sediments,especially the newly discovered comammox. Results enriched our knowledge about the nitrifying communities of estuarine environment, which have great significance to the further molecular ecology research of the nitrification processes.
Nitrifiers were enriched from the tidal sediments of the Yangtze Estuary. Four kinds of nitrifying groups(accounting for 34.7% of the total reads) were detected based on metagenomics, including the recently discovered complete ammonia oxidizers(comammox),strictly nitrite-oxidizing Nitrospira,/3-proteobacterial ammonia-oxidizing bacteria, and ammonia-oxidizing archaea. Comammox accounted for 48% of the detected nitrifiers. In addition, gene expression patterns of the nitrifier group(according to the classification in Evolutionary genealogy of genes: Non-supervised Orthologous Groupsdatabase) were analyzed based on metatranscriptomics. The present study showed the community structure and gene expression characteristics of nitrifiers enriched from the tidal sediments,especially the newly discovered comammox. Results enriched our knowledge about the nitrifying communities of estuarine environment, which have great significance to the further molecular ecology research of the nitrification processes.
引文
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[33] BUCHFINK B, XIE C, HUSON D H, Fast and sensitive protein alignment using DIAMOND[J]. Nature Methods,2014, 12(1):59-60.
[34] QIN J, LI R, RAES J, et al. A human gut microbial gene catalogue established by metagenomic sequencing[J].Nature, 2010, 464(7285):59-65.
[35] HUSON D H, MITRA S, RUSCHEWEYH H J, et al. Integrative analysis of environmental sequences using MEGAN4[J]. Genome Research, 2011, 21(9):1552-1560.
[36] PRUESSE E, QUAST C, KNITTEL K, et al. SILVA:A comprehensive online resource for quality checked and aligned ribosomal RNA sequence data compatible with ARB[J]. Nucleic Acids Research, 2007, 35(21):7188-7196.
[37] GRABHERR M G, HAAS B J, YASSOUR M, et al. Full-length transcriptome assembly from RNA-Seq data without a reference genome[J]. Nature Biotechnology, 2011, 29(7):644-652.
[38] TRAPNELL C, WILLIAMS B A, PERTEA G, et al. Transcript assembly and quantification by RNA-Seq reveals unannotated transcripts and isoform switching during cell differentiation[J]. Nature Biotechnology, 2010, 28(5):511-515.
[39] LI B, DEWEY C N. RSEM:Accurate transcript quantification from RNA-Seq data with or without a reference genome[J]. BMC Bioinformatics, 2011, 12(1):323.
[40] PARK B J, PARK S J, YOON D N, et al. Cultivation of autotrophic ammonia-oxidizing archaea from marine sediments in coculture with sulfur-oxidizing bacteria[J]. Appl Environ Microbiol, 2010, 76(22):7575-7587.
[41]金文标,李志鑫,岳洋洋,等.一种污水处理系统中氨氧化古细菌富集培养的方法:中国,CN 103451120[P]. 2013-12-18.
[42] GONZALEZ-CABALEIRO R, CURTIS T P O. Study of the competition between complete nitrification by a single organism and ammonia-and nitrite-oxidizing bacteria[C]//Frontiers in Wastewater Treatment and Modelling. Lecture Notes in Civil Engineering, MANNINA G, Cham, 2017:287-291.
[43] KITS K D, SEDLACEK C J, LEBEDEVA E V, et al. Kinetic analysis of a complete nitrifier reveals an oligotrophic lifestyle[J]. Nature, 2017, 549(7671):269-272.
[44] COSTA E, PEREZ J, KREFT J. Why is metabolic labour divided in nitrification?[J]. Trends in Microbiology,2006, 14(5):213-219.
[2] KRAFT B, HALINA E T, RITIN S, et al. The environmental controls that govern the end product of bacterial nitrate respiration[J]. Science, 2014, 345(6197):676-679.
[3] VAN KESSEL M A, SPETH D R, ALBERTSEN M, et al. Complete nitrification by a single microorganism[J].Nature, 2015, 528(7583):555-559.
[4] DAIMS H, LEBEDEVA E V, PJEVAC P, et al. Complete nitrification by Nitrospira bacteria[J]. Nature, 2015,528(7583):504-509.
[5] LI J, NEDWELL D B, BEDDOW J, et al. AmoA gene abundances and nitrification potential rates suggest that benthic ammonia-oxidizing bacteria and not archaea dominate N cycling in the Colne Estuary, United Kingdom[J]. Appl Environ Microbiol, 2014, 81(1):159-165.
[6] LAGOSTINA L, GOLDHAMMER T, R(?)Y H, et al. Ammonia-oxidizing bacteria of the Nitrosospira cluster 1dominate over ammonia-oxidizing archaea in oligotrophic surface sediments near the South Atlantic Gyre[J].Environ Microbiol Reports, 2015, 7(3):404-413.
[7] SHEN J, ZHANG L, DI H J, et al. A review of ammonia-oxidizing bacteria and archaea in Chinese soils[J].Frontiers in Microbiology, 2012(3):296.
[8] DAIMS H, L(U|¨)CKER S, WAGNER M. A new perspective on microbes formerly known as nitrite-oxidizing bacteria[J]. Trends in Microbiology, 2016, 24(9):699-712.
[9] PINTO A J, MARCUS D N, IJAZ U Z, et al. Metagenomic evidence for the presence of comammox Nitrospiralike bacteria in a drinking water system[J]. Msphere, 2015(1):e15-e54.
[10] CHAO Y, MAO Y, YU K, et al. Novel nitrifiers and comammox in a full-scale hybrid biofilm and activated sludge reactor revealed by metagenomic approach[J]. Appl Microbiol Biotechnol, 2016, 100(18):8225-8237.
[11] PALOMO A, JANE FOWLER S, GULAY A, et al. Metagenomic analysis of rapid gravity sand filter microbial communities suggests novel physiology of Nitrospira spp.[J]. The ISME Journal, 2016, 10(11):2569-2581.
[12] WANG Y, MA L, MAO Y, et al. Comammox in drinking water systems[J]. Water Research, 2017, 116:332-341.
[13] BARTELME R P, MCLELLAN S L, NEWTON R J. Freshwater recirculating aquaculture system operations drive biofilter bacterial community shifts around a stable nitrifying consortium of ammonia-oxidizing archaea and comammox Nitrospira[J]. Frontiers in Microbiology, 2017(8):101.
[14] WANG J, XIA F, ZELEKE J, et al. An improved protocol with a highly degenerate primer targeting coppercontaining membrane-bound monooxygenase genes for community analysis of methane-and ammonia-oxidizing bacteria[J]. FEMS Microbiol Ecol, 2017, 93(3):w244.
[15] HU H W, HE J Z. Comammox—a newly discovered nitrification process in the terrestrial nitrogen cycle[J].Journal of Soils&Sediments, 2017, 17(12):1-9.
[16] PJEVAC P, SCHAUBERGER C, POGHOSYAN L, et al. AmoA-targeted polymerase chain reaction primers for the specific detection and quantification of comammox Nitrospira in the environment[J]. Frontiers in Microbiology, 2017(8):1508.
[17] BAKER B J, LAZAR C S, TESKE A P, et al. Genomic resolution of linkages in carbon, nitrogen, and sulfur cycling among widespread estuary sediment bacteria[J]. Microbiome, 2015, 3(1):3-14.
[18] MURRAY N J, MA Z, FULLER R A. Tidal flats of the Yellow Sea:A review of ecosystem status and anthropogenic threats[J]. Austral Ecology, 2015, 40(4):472-481.
[19] ZHENG Y, HOU L, NEWELL S, et al. Community dynamics and activity of ammonia-oxidizing prokaryotes in intertidal sediments of the Yangtze Estuary[J]. Appl Environ Microbiol, 2013, 80(1):408-419.
[20] STARR M P, STOLP H, TRUPER H G, et al. The prokaryotes:a handbook on habitats, isolation and identification of bacteria[M].[S.l.]:Springer Science&Business Media, 1981.
[21] HOU L J, LIU M, XU S Y, et al. The effects of semi-lunar spring and neap tidal change on nitrification, denitrification and N20 vertical distribution in the intertidal sediments of the Yangtze estuary, China[J]. Estuarine,Coastal and Shelf Science, 2007, 73(3/4):607-616.
[22] ROTTHAUWE J H, WITZEL K P, LIESACK W. The ammonia monooxygenase structural gene amoa as a functional marker:Molecular fine-scale analysis of natural ammonia-oxidizing populations[J]. Appl Environ Microbiol, 1997, 63(12):4704-4712.
[23] FRANCIS C A, ROBERTS K J, BEMAN J M, et al. Ubiquity and diversity of ammonia-oxidizing archaea in water columns and sediments of the ocean.[J]. Proc Natl Acad Sci USA, 2005, 102(41):14683-14688.
[24] LI D, LIU C, LUO R, et al. MEGAHIT:An ultra-fast single-node solution for large and complex metagenomics assembly via succinct de Bruijn graph[J]. Bioinformatics, 2015, 31(10):1674-1676.
[25] DR M, WALLER A, SUNAGAWA S, et al. Assessment of metagenomic assembly using simulated next generation sequencing data[J]. PLoS ONE, 2012, 7(2):e31386.
[26] NIELSEN H B, ALMEIDA M, JUNCKER A S, et al. Identification and assembly of genomes and genetic elements in complex metagenomic samples without using reference genomes[J]. Nature Biotechnology, 2014,32(8):822-828.
[27] KARLSSON F H, FAK F, NOOKAEW I, et al. Symptomatic atherosclerosis is associated with an altered gut metagenome[J]. Nature Communications, 2012, 3(4):1245.
[28] ZELLER G, TAP J, VOIGT A Y, et al. Potential of fecal microbiota for early-stage detection of colorectal cancer[J]. Mol Syst Biol, 2014, 10(11):766.
[29] LI J, JIA H, CAI X, et al. An integrated catalog of reference genes in the human gut microbiome[J]. Nature Biotechnology, 2014, 32(8):834-841.
[30] GU S, FANG L, XU X. UNIT 11.11 using SOAPaligner for short reads alignment[J]. Current Protocols in Bioinformatics, 2013, 44:11.
[31] FU L, NIU B, ZHU Z, et al. CD-HIT:Accelerated for clustering the next-generation sequencing data[J].Bioinformatics, 2012, 28(23):3150-3152.
[32] COTILLARD A, KENNEDY S P, KONG L C, et al. Dietary intervention impact on gut microbial gene richness[J]. Nature, 2013, 500(7464):585-588.
[33] BUCHFINK B, XIE C, HUSON D H, Fast and sensitive protein alignment using DIAMOND[J]. Nature Methods,2014, 12(1):59-60.
[34] QIN J, LI R, RAES J, et al. A human gut microbial gene catalogue established by metagenomic sequencing[J].Nature, 2010, 464(7285):59-65.
[35] HUSON D H, MITRA S, RUSCHEWEYH H J, et al. Integrative analysis of environmental sequences using MEGAN4[J]. Genome Research, 2011, 21(9):1552-1560.
[36] PRUESSE E, QUAST C, KNITTEL K, et al. SILVA:A comprehensive online resource for quality checked and aligned ribosomal RNA sequence data compatible with ARB[J]. Nucleic Acids Research, 2007, 35(21):7188-7196.
[37] GRABHERR M G, HAAS B J, YASSOUR M, et al. Full-length transcriptome assembly from RNA-Seq data without a reference genome[J]. Nature Biotechnology, 2011, 29(7):644-652.
[38] TRAPNELL C, WILLIAMS B A, PERTEA G, et al. Transcript assembly and quantification by RNA-Seq reveals unannotated transcripts and isoform switching during cell differentiation[J]. Nature Biotechnology, 2010, 28(5):511-515.
[39] LI B, DEWEY C N. RSEM:Accurate transcript quantification from RNA-Seq data with or without a reference genome[J]. BMC Bioinformatics, 2011, 12(1):323.
[40] PARK B J, PARK S J, YOON D N, et al. Cultivation of autotrophic ammonia-oxidizing archaea from marine sediments in coculture with sulfur-oxidizing bacteria[J]. Appl Environ Microbiol, 2010, 76(22):7575-7587.
[41]金文标,李志鑫,岳洋洋,等.一种污水处理系统中氨氧化古细菌富集培养的方法:中国,CN 103451120[P]. 2013-12-18.
[42] GONZALEZ-CABALEIRO R, CURTIS T P O. Study of the competition between complete nitrification by a single organism and ammonia-and nitrite-oxidizing bacteria[C]//Frontiers in Wastewater Treatment and Modelling. Lecture Notes in Civil Engineering, MANNINA G, Cham, 2017:287-291.
[43] KITS K D, SEDLACEK C J, LEBEDEVA E V, et al. Kinetic analysis of a complete nitrifier reveals an oligotrophic lifestyle[J]. Nature, 2017, 549(7671):269-272.
[44] COSTA E, PEREZ J, KREFT J. Why is metabolic labour divided in nitrification?[J]. Trends in Microbiology,2006, 14(5):213-219.