Abstract
Denitrification is a critically important biogeochemical pathway that removes fixed nitrogen from ecosystems and thus ultimately controls the rate of primary production in nitrogen-limited systems. We examined the community structure of bacteria containing the nirS gene, a signature gene in the denitrification pathway, from estuarine and salt marsh sediments and from the water column of two of the world’s largest marine oxygen-deficient zones (ODZs). We generated over 125,000 nirS gene sequences, revealing a large degree of genetic diversity including 1,815 unique taxa, the vast majority of which formed clades that contain no cultured representatives. These results underscore how little we know about the genetic diversity of metabolisms underlying this critical biogeochemical pathway. Marine sediments yielded 1,776 unique taxa when clustered at 95 % sequence identity, and there was no single nirS denitrifier that was a competitive dominant; different samples had different highly abundant taxa. By contrast, there were only 39 unique taxa identified in samples from the two ODZs, and 99 % of the sequences belonged to 5 or fewer taxa. The ODZ samples were often dominated by nirS sequences that shared a 92 % sequence identity to a nirS found in the anaerobic ammonium-oxidizing (anammox) genus Scalindua. This sequence was abundant in both ODZs, accounting for 38 and 59 % of all sequences, but it was virtually absent in marine sediments. Our data indicate that ODZs are remarkably depauperate in nirS genes compared to the remarkable genetic richness found in coastal sediments.
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References
Galloway JN, Dentener JF, Capone DG, Boyer EW, Howarth RW, Seitzinger SP et al (2004) Nitrogen cycles: past, present, and future. Biogeochemistry 70:153–226
Thamdrup B (2012) New pathways and processes in the global nitrogen cycle. Ann Rev Ecol Evol Syst 43:407–428
Vitousek PM, Howarth RW (1991) Nitrogen limitation on land and in the sea: how can it occur? Biogeochemistry 13:87–115
Canfield DE, Glazer AN, Falkowski PG (2010) The evolution and future of earth’s nitrogen cycle. Science 330:192–196
Smil V (2002) Nitrogen and food production: proteins for human diets. Ambio 31:126–131
Galloway JN, Aber JD, Erisman JW, Seitzinger SP, Howarth RW, Cowling EB, Crosby BJ (2003) The nitrogen cascade. Bioscience 53:341–356
Bricker SB, Longstaff B, Dennison W, Jones A, Boicourt K, Wicks C et al (2008) Effects of nutrient enrichment in the nation’s estuaries: a decade of change. Harmful Algae 8:21–32
Diaz RJ, Rosenberg R (2008) Spreading dead zones and consequences for marine ecosystems. Science 321:926–929
Zumft W (1997) Cell biology and molecular basis of denitrification. Microbiol Mol Biol Rev 61:533–616
Kuypers MMM, Sliekers AO, Lavik G, Schmid M, Jørgensen BB, Kuenen JG et al (2003) Anaerobic ammonium oxidation by anammox bacteria in the Black Sea. Nature 422:608–611
Kuypers MMM, Lavik G, Woebken D, Schmid M, Fuchs BM, Amann R et al (2005) Massive nitrogen loss from the Benguela upwelling system through anaerobic ammonium oxidation. Proc Natl Acad Sci U S A 102:6239–6240
Lam P, Lavik G, Jensen MM, van de Vossenberg J, Schmid M, Woebken D et al (2009) Revising the nitrogen cycle in the Peruvian oxygen minimum zone. Proc Natl Acad Sci U S A 106:4752–4757
Lam P, Kuypers MMM (2011) Microbial nitrogen cycling processes in oxygen minimum zones. Ann Rev Mar Sci 3:317–345
Codispoti LA, Brandes JA, Christensen JP, Devol AH, Naqvi SWA, Paerl HW et al (2001) The oceanic fixed nitrogen and nitrous oxide budgets: moving targets as we enter the anthropocene? Sci Mar 65:85–105
Gruber N, Sarmiento JL (1997) Global patterns of marine nitrogen fixation and denitrification. Global Biogeochem Cy 11:235–266
Hopkinson CS, Giblin AE (2008) Nitrogen dynamics of coastal salt marshes. In: Capone DG, Bronk DA, Mulholland MR, Carpenter EJ (eds) Nitrogen in the Marine Environment, 2nd edn. Elsevier, Amsterdam, pp 991–1036
Koop-Jakobsen K, Giblin AE (2009) Anammox in tidal marsh sediments: the role of salinity, nitrogen loading, and marsh vegetation. Estuar Coasts 32:238–245
Koop-Jakobsen K, Giblin AE (2010) The effect of increased nitrate loading on nitrate reduction via denitrification and DNRA in salt marsh sediments. Limnol Oceanogr 55:789–802
Hamersley MR, Howes BL (2005) Coupled nitrification–denitrification measured in situ in a Spartina alterniflora marsh with a 15NH4 tracer. Mar Ecol Prog Ser 299:123–135
Ward BB, Devol AH, Rich JJ, Chang BX, Bulow SE, Naik H et al (2009) Denitrification as the dominant nitrogen loss process in the Arabian Sea. Nature 461:78–81
Dalsgaard T, Thamdrup B, Farias L, Peter Revsbech NP (2012) Anammox and denitrification in the oxygen minimum zone of the eastern South Pacific. Limnol Oceanogr 57:1331–1346
Ward BB (2013) How nitrogen is lost. Science 341:352–353
Zehr JP, Kudela RM (2011) Nitrogen cycle of the open ocean: from genes to ecosystems. Ann Rev Mar Sci 3:197–225
Jones CM, Hallin S (2010) Ecological and evolutionary factors underlying global and local assembly of denitrifier communities. ISME J 4:633–641
Abell GCJ, Revill AT, Smith C, Bissett AP, Volkman JK, Robert SS (2009) Archaeal ammonia oxidizers and nirS-type denitrifiers dominate sediment nitrifying and denitrifying populations in a subtropical macrotidal estuary. ISME J 4:286–300
Nogales B, Timmis KN, Nedwell DB, Osborne AM (2002) Detection and diversity of expressed denitrification genes in estuarine sediments after reverse transcription-PCR amplification from mRNA. Appl Environ Microbiol 68:5017–5025
Strous M, Pelletier E, Mangenot S, Rattei T, Lehner A, Taylor MW et al (2006) Deciphering the evolution and metabolism of an anammox bacterium from a community genome. Nature 440:790–794
Schmid MC, Maas B, Dapena A, van de Pas-Schoonen K, Van De Vossenberg J, Kartal B et al (2005) Biomarkers for in situ detection of anaerobic ammonium-oxidizing (anammox) bacteria. Appl Environ Microb 71:1677–1684
Li M, Hong Y, Klotz MG, Gu J-D (2010) A comparison of primer sets for detecting 16S rRNA and hydrazine oxidoreductase genes of anaerobic ammonium-oxidizing bacteria in marine sediments. Appl Microbiol Biotechnol 86:781–790
Li M, Ford T, Li X, Gu J-D (2011) Cytochrome cd1-containing nitrite reductase encoding gene nirS as a new functional biomarker for detection of anaerobic ammonium oxidizing (anammox) bacteria. Environ Sci Technol 45:3547–3553
Sogin ML, Morrison HG, Huber JA, Mark Welch D, Huse SM, Neal PR et al (2006) Microbial diversity in the deep sea and the underexplored ‘rare biosphere’. Proc Natl Acad Sci U S A 103:12115–12120
Caporaso JG, Lauber CL, Walters WA, Berg-Lyons D, Lozupone CA, Turnbaugh PJ et al (2011) Global patterns of 16S rRNA diversity at a depth of millions of sequences per sample. Proc Nat Acad Sci USA 108:4516–4522
Canfield DE, Stewart FJ, Thamdrup B, Brabandere LD, Dalsgaard T, Delong EF et al (2010) A cryptic sulfur cycle in oxygen-minimum-zone waters off the Chilean coast. Science 330:1375–1378
Stewart FJ, Ulloa O, Delong EF (2012) Microbial metatranscriptomics in a permanent marine oxygen minimum zone. Environ Microbiol 14:23–40
Ulloa O, Canfield DE, Delong EF, Letelier RM, Stewart FJ (2012) Microbial oceanography of anoxic oxygen minimum zones. Proc Natl Acad Sci U S A 109:15996–16003
Gifford SM, Sharma S, Rinta-Kanto JM, Moran MA (2010) Quantitative analysis of a deeply sequenced marine microbial metatranscriptome. ISME J 5:461–472
Penton CR, Johnson TA, Quensen JF III, Iwai S, Cole JR, Tiedje JM (2013) Functional genes to assess nitrogen cycling and aromatic hydrocarbon degradation: primers and processing matter. Front Microbiol 4:279
Palmer K, Biasi C, Horn MA (2012) Contrasting denitrifier communities relate to contrasting N2O emission pattern from acidic peat soils in arctic tundra. ISME J 6:1058–1077
Dumont MG, Lüke C, Deng Y, Frenzel P (2014) Classification of pmoA amplicon pyrosequences using BLAST and the lowest common ancestor method in MEGAN. Front Microbiol 5:34
Francis CA, O’Mullan GD, Ward BB (2003) Diversity of ammonia monooxygenase (amoA) genes across environmental gradients in Chesapeake Bay sediments. Geobiology 1:129–140
Bulow SE, Francis CA, Jackson GA, Ward BB (2008) Sediment denitrifier community composition and nirS gene expression investigated with functional gene microarrays. Environ Microbiol 10:3057–3069
Valiela I, Teal JM, Persson NY (1976) Production and dynamics of experimentally enriched salt marsh vegetation: belowground biomass. Limnol Oceanogr 21:245–252
Valiela I, Teal JM, Sass WJ (1975) Production and dynamics of salt marsh vegetation and the effects of experimental treatment with sewage sludge: biomass, production and species composition. J Appl Ecol 12:973–981
Bowen JL, Byrnes JEK, Weisman D, Colaneri C (2013) Functional gene pyrosequencing and network analysis: an approach to examine the response of denitrifying bacteria to increased nitrogen supply in salt marsh sediments. Front Microbiol 342:1–12
Jayakumar A, O’Mullan GD, Naqvi SWA, Ward BB (2009) Denitrifying bacterial community composition changes associated with stages of denitrification in oxygen minimum zones. Microb Ecol 58:350–362
Weisman D, Yasuda M, Bowen JL (2013) FunFrame: Functional gene ecological analysis pipeline. Bioinformatics 29:1212–1214
Braker G, Fesefeldt A, Witzel KP (1998) Development of PCR primer systems for amplification of nitrite reductase genes (nirK and nirS) to detect denitrifying bacteria in environmental samples. Appl Environ Microb 64:3769–3775
Quince C, Lanzén A, Curtis TP, Davenport RJ, Hall N, Head IM et al (2009) Accurate determination of microbial diversity from 454 pyrosequencing data. Nat Methods 6:639
Kunin V, Engelbrektson A, Ochman H, Hugenholtz P (2010) Wrinkles in the rare biosphere: pyrosequencing errors can lead to artificial inflation of diversity estimates. Environ Microbiol 12:118–123
Quince C, Lanzén A, Davenport RJ, Turnbaugh PJ (2011) Removing noise from pyrosequenced amplicons. BMC Bioinf 12:38
Schloss PD, Westcott SL (2011) Assessing and improving methods used in operational taxonomic unit-based approaches for 16S rRNA gene sequence analysis. Appl Environ Microbiol 77:3219–3226
Zhang Y, Sun Y (2011) HMM-FRAME: accurate protein domain classification for metagenomic sequences containing frameshift errors. BMC Bioinformatics 12:198
Edgar RC, Haas BJ, Clemente JC, Quince C, Knight R (2011) UCHIME improves sensitivity and speed of chimera detection. Bioinformatics 27:2194–2200
Cock PJA, Antao T, Chang JT, Chapman BA, Cox CJ, Dalke A et al (2009) Biopython: freely available Python tools for computational molecular biology and bioinformatics. Bioinformatics 25:1422–1423
Francis CA, O’Mullan GD, Cornwell JC, Ward BB (2013) Transitions in nirS-type denitrifier diversity, community composition, and biogeochemical activity along the Chesapeake Bay estuary. Front Microbiol 237:1–10
Cai Y, Sun Y (2011) ESPRIT-Tree: hierarchical clustering analysis of millions of 16S rRNA pyrosequences in quasilinear computational time. Nucleic Acids Res 39:e95–e95
Caporaso JG, Kuczynski J, Stombaugh J, Bittinger K, Bushman FD, Costello EK et al (2010) QIIME allows analysis of high-throughput community sequencing data. Nat Methods 7:335–336
Gihring TM, Green SJ, Schadt CW (2011) Massively parallel rRNA gene sequencing exacerbates the potential for biased community diversity comparisons due to variable library sizes. Environ Microbiol 14:285–290
Oksanen J, Blanchet FG, Kindt R, Legendre P, Minchin, O’Hara RB, et al (2013) Vegan: community ecology package. R package version 2.0-7. http://cran.r-project.org/web/packages/vegan/
R. Core Team (2014) A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria
Caporaso JG, Bittinger K, Bushman FD, Desantis TZ, Andersen GL, Knight R (2010) PyNAST: a flexible tool for aligning sequences to a template alignment. Bioinformatics 26:266–267
Price MN, Dehal PS, Arkin AP (2010) FastTree 2: approximately maximum-likelihood trees for large alignments. PLoS One 5:e9490
Letunic I, Bork P (2011) Interactive tree of life v2: online annotation and display of phylogenetic trees made easy. Nucleic Acids Res 39:W475–W478
Stevens H, Ulloa O (2008) Bacterial diversity in the oxygen minimum zone of the eastern tropical South Pacific. Environ Microbiol 10:1244–1259
Bryant JA, Stewart FJ, Eppley JM, Delong EF (2012) Microbial community phylogenetic and trait diversity declines with depth in a marine oxygen minimum zone. Ecology 93:1659–1673
Lozupone CA, Knight R (2007) Global patterns in bacterial diversity. Proc Natl Acad Sci U S A 104:11436–11440
Bowen JL, Morrison HG, Hobbie JE, Sogin ML (2012) Salt marsh sediment diversity: a test of the variability of the rare biosphere among environmental replicates. ISME J 6:2014–2023
Kondo R, Purdy KJ, Silva SQ, Nedwell DB (2007) Spatial dynamics of sulphate-reducing bacterial compositions in sediment along a salinity gradient in a UK Estuary. Microbes Environ 22:11–19
Klepac-Ceraj V, Bahr M, Crump BC, Teske AP, Hobbie JE, Polz MF (2004) High overall diversity and dominance of microdiverse relationships in salt marsh sulphate-reducing bacteria. Environ Microbiol 6:686–698
Bahr M, Crump BC, Klepac-Ceraj V, Teske A, Sogin ML, Hobbie JE (2005) Molecular characterization of sulfate-reducing bacteria in a New England salt marsh. Environ Microbiol 7:1175–1185
Beman JM, Francis CA (2006) Diversity of ammonia-oxidizing archaea and bacteria in the sediments of a hypernutrified subtropical estuary: Bahia del Tobari, Mexico. Appl Environ Microbiol 72:7767–7777
Mosier AC, Francis CA (2008) Relative abundance and diversity of ammonia-oxidizing archaea and bacteria in the San Francisco Bay estuary. Environ Microbiol 10:3002–3016
Moin NS, Nelson KA, Bush A, Bernhard AE (2009) Distribution and diversity of archaeal and bacterial ammonia oxidizers in salt marsh sediments. Appl Environ Microb 75:7461–7468
Lovell CR, Friez MJ, Longshore JW, Bagwell CE (2001) Recovery and phylogenetic analysis of nifH sequences from diazotrophic bacteria associated with dead aboveground biomass of Spartina alterniflora. Appl Environ Microbiol 67:5308–5314
Bagwell CE, Dantzler M, Bergholz PW, Lovell CR (2001) Host-specific ecotype diversity of rhizoplane diazotrophs of the perennial glasswort Salicornia virginica and selected salt marsh grasses. Aquat Microb Ecol 23:293–300
Zehr JP, Jenkins BD, Short SM, Steward GF (2003) Nitrogenase gene diversity and microbial community structure: a cross-system comparison. Environ Microbiol 5:539–554
Lehmann J, Solomon D, Kinyangi J, Dathe L, Wirick S, Jacobsen C (2008) Spatial complexity of soil organic matter forms at nanometre scales. Nat Geosci 1:238–242
Howes BL, Howarth RW, Teal JM, Valiela I (1981) Oxidation–reduction potentials in a salt marsh: spatial patterns and interactions with primary production. Limnol Oceanogr 26:350–360
Crump BC, Hopkinson CS, Sogin ML, Hobbie JE (2004) Microbial biogeography along an estuarine salinity gradient: combined influences of bacterial growth and residence time. Appl Environ Microbiol 70:1494–1505
Woebken D, Lam P, Kuypers MMM, Naqvi SWA, Kartal B et al (2008) A microdiversity study of anammox bacteria reveals a novel Candidatus Scalindua phylotype in marine oxygen minimum zones. Environ Microbiol 10:3106–3119
Jayakumar A, Naqvi SWA, Ward BB (2009) Distribution and relative quantification of key genes involved in fixed nitrogen loss from the Arabian Sea oxygen minimum zone. In: Wiggert JD, Hood RR, Naqvi SWA, Brink KH, Smith SL (eds) Indian ocean biogeochemical processes and ecological variability. American Geophysical Union, Washington, DC, pp 187–203
Jayakumar DA, Francis CA, Naqvi SWA, Ward BB (2004) Diversity of nitrite reductase genes (nirS) in the denitrifying water column of the coastal Arabian Sea. Aquat Microb Ecol 34:69–78
Lozupone C, Knight R (2005) UniFrac: a new phylogenetic method for comparing microbial communities. Appl Environ Microbiol 71:8228–8235
Lozupone C, Lladser ME, Knights D, Stombaugh J, Knight R (2011) UniFrac: an effective distance metric for microbial community comparison. ISME J 5:169–172
Jayakumar A, Peng X, Ward BB (2013) Community composition of bacteria involved in fixed nitrogen loss in the water column of two major oxygen minimum zones in the ocean. Aquat Microb Ecol 70:245–259
Thamdrup B, Dalsgaard T, Jensen MM, Ulloa O, Farias L, Escribano R (2006) Anaerobic ammonium oxidation in the oxygen-deficient waters off northern Chile. Limnol Oceanogr 51:2145–2156
Bulow SE, Rich JJ, Naik HS, Pratihary AK, Ward BB (2010) Denitrification exceeds anammox as a nitrogen loss pathway in the Arabian Sea oxygen minimum zone. Deep-Sea Res I 57:384–393
Acknowledgments
Funding for this work came from multiple NSF grants to BBW and from the following NSF grants: DEB-0717155 (to John Hobbie) and DBI-0400819 (to Jennifer L. Bowen). Additional support was provided via start-up funds from University of Massachusetts Boston to JLB. We would like to acknowledge the Salt Pond Sanctuaries and Dr. E. F. X. Hughes and family for allowing us to have access to the Great Sippewissett marsh plots, and Ivan Valiela for providing support and maintenance of the plots. Christopher Jones and Sara Hallin provided us with their curated nirS alignment. Comments from anonymous reviewers greatly improved the quality of this manuscript.
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Bowen, J.L., Weisman, D., Yasuda, M. et al. Marine Oxygen-Deficient Zones Harbor Depauperate Denitrifying Communities Compared to Novel Genetic Diversity in Coastal Sediments. Microb Ecol 70, 311–321 (2015). https://doi.org/10.1007/s00248-015-0582-y
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DOI: https://doi.org/10.1007/s00248-015-0582-y