Marine Biotechnology

, Volume 15, Issue 5, pp 540–551 | Cite as

Phylogenetically Diverse Denitrifying and Ammonia-Oxidizing Bacteria in Corals Alcyonium gracillimum and Tubastraea coccinea

  • Shan Yang
  • Wei Sun
  • Fengli Zhang
  • Zhiyong Li
Original Article


To date, the association of coral–bacteria and the ecological roles of bacterial symbionts in corals remain largely unknown. In particular, little is known about the community components of bacterial symbionts of corals involved in the process of denitrification and ammonia oxidation. In this study, the nitrite reductase (nirS and nirK) and ammonia monooxygenase subunit A (amoA) genes were used as functional markers. Diverse bacteria with the potential to be active as denitrifiers and ammonia-oxidizing bacteria (AOB) were found in two East China Sea corals: stony coral Alcyonium gracillimum and soft coral Tubastraea coccinea. The 16S rRNA gene library analysis demonstrated different communities of bacterial symbionts in these two corals of the same location. Nitrite reductase nirK gene was found only in T. coccinea, while both nirK and nirS genes were detected in A. gracillimum, which might be the result of the presence of different bacterial symbionts in these two corals. AOB rather than ammonia-oxidizing archaea were detected in both corals, suggesting that AOB might play an important role in the ammonia oxidation process of the corals. This study indicates that the coral bacterial symbionts with the potential for nitrite reduction and ammonia oxidation might have multiple ecological roles in the coral holobiont, which promotes our understanding of bacteria-mediated nitrogen cycling in corals. To our knowledge, this study is the first assessment of the community structure and phylogenetic diversity of denitrifying bacteria and AOB in corals based on nirK, nirS, and amoA gene library analysis.


Alcyonium gracillimum Tubastraea coccinea Bacterial symbionts nirniramoDenitrification Ammonia oxidation Phylogenetic diversity 



This work was supported by the National Major Scientific Research Program of China (2013CB956103). The authors are grateful to Prof. Wen Zhang at the Second Military Medical University (China) for the assistance in collecting the coral samples.


  1. Ainsworth TD, Thurber RV, Gates RD (2010) The future of coral reefs: a microbial perspective. Trends Ecol Evol 25:233–240PubMedCrossRefGoogle Scholar
  2. Amann RI, Ludwig W, Schleifer KH (1995) Phylogenetic identification and in situ detection of individual microbial cells without cultivation. Microbiol Mol Biol R 59:143–169Google Scholar
  3. Baker AC (2004) Symbiont diversity on coral reefs and its relationship to bleaching resistance and resilience. In: Rosenberg E, Loya Y (eds) Coral heath and disease. Springer, Berlin, pp 177–194CrossRefGoogle Scholar
  4. Beman JM, Popp BN, Francis CA (2008) Molecular and biogeochemical evidence for ammonia oxidation by marine Crenarchaeota in the Gulf of California. ISME J 2:429–441PubMedCrossRefGoogle Scholar
  5. Beman JM, Roberts KJ, Wegley L, Rohwer F, Francis CA (2007) Distribution and diversity of archaeal ammonia monooxygenase genes associated with corals. Appl Environ Microbiol 73:5642–5647PubMedCrossRefGoogle Scholar
  6. Bourne DG, Munn CB (2005) Diversity of bacteria associated with the coral Pocillopora damicornis from the Great Barrier Reef. Environ Microbiol 7:1162–1174PubMedCrossRefGoogle Scholar
  7. 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 Microbiol 64:3769–3775PubMedGoogle Scholar
  8. Braker G, Zhou J, Wu L, Devol AH, Tiedje JM (2000) Nitrite reductase genes (nirK and nirS) as functional markers to investigate diversity of denitrifying bacteria in Pacific Northwest marine sediment communities. Appl Environ Microbiol 66:2096–2104PubMedCrossRefGoogle Scholar
  9. Capone D, Dunham S, Horrigan S, Duguay L (1992) Microbial nitrogen transformations in unconsolidated coral reef sediments. Mar Ecol - Prog Ser 80:75–88CrossRefGoogle Scholar
  10. Curtis TP, Sloan WT, Scannell JW (2002) Estimating prokaryotic diversity and its limits. Proc Natl Acad Sci USA 99:10494–10499PubMedCrossRefGoogle Scholar
  11. Delong EF (1992) Archaea in coastal marine environment. Proc Natl Acad Sci USA 89:5685–5689PubMedCrossRefGoogle Scholar
  12. DiSalvo L, Gundersen K (1971) Regenerative functions and microbial ecology of coral reefs. I. Assays for microbial population. Can J Microbiol 17:1081–1089PubMedCrossRefGoogle Scholar
  13. Fiore CL, Jarett JK, Olson ND, Lesser MP (2010) Nitrogen fixation and nitrogen transformations in marine symbioses. Trends Microbiol 18:455–463PubMedCrossRefGoogle Scholar
  14. Heylen K, Gevers D, Vanparys B, Wittebolle L, Geets J, Boon N, De Vos P (2006) The incidence of nirS and nirK and their genetic heterogeneity in cultivated denitrifiers. Environ Microbiol 8:2012–21PubMedCrossRefGoogle Scholar
  15. Hoffmann F, Radax R, Woebken D, Holtappels M, Lavik G et al (2009) Complex nitrogen cycling in the sponge Geodia barretti. Environ Microbiol 11:2228–2243PubMedCrossRefGoogle Scholar
  16. Huber T (2004) Bellerophon: a program to detect chimeric sequences in multiple sequence alignments. Bioinformatics 20:2317–2319PubMedCrossRefGoogle Scholar
  17. 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 34:69–78CrossRefGoogle Scholar
  18. Jones CM, Stres B, Rosenquist M, Hallin S (2008) Phylogenetic analysis of nitrite, nitric oxide, and nitrous oxide respiratory enzymes reveal a complex evolutionary history for denitrification. Mol Biol Evol 25:1955–1966PubMedCrossRefGoogle Scholar
  19. Kellogg CA (2004) Tropical archaea: diversity associated with the surface microlayer of corals. Mar Ecol-Prog Ser 273:81–88CrossRefGoogle Scholar
  20. Kimes NE, Van Nostrand JD, Weil E, Zhou J, Morris PJ (2010) Microbial functional structure of Montastraea faveolata, an important Caribbean reef-building coral, differs between healthy and yellow-band diseased colonies. Environ Microbiol 12:541–556PubMedCrossRefGoogle Scholar
  21. Knowlton N, Rohwer F (2003) Multispecies microbial mutualisms on coral reefs: the host as a habitat. The American Naturalist 162:S51–S62PubMedCrossRefGoogle Scholar
  22. Kooperman N, Ben-Dov E, Kramarsky-Winter E, Barak Z, Kushmaro A (2007) Coral mucus-associated bacterial communities from natural and aquarium environments. FEMS Microbiol Lett 276:106–113PubMedCrossRefGoogle Scholar
  23. Koren O, Rosenberg E (2006) Bacteria associated with mucus and tissues of the coral Oculina patagonica in summer and winter. Appl Environ Microbiol 72:5254–5259PubMedCrossRefGoogle Scholar
  24. Lane DJ (1991) 16S/23S rRNA sequencing. In: Stackebrandt E, Goodfellow M (eds) Nucleic acid techniques in bacterial systematics. Wiley, Chichester, pp 115–175Google Scholar
  25. Lesser MP, Mazel CH, Gorbunov MY, Falkowski PG (2004) Discovery of symbiotic nitrogen-fixing cyanobacteria in corals. Science 305:997–1000PubMedCrossRefGoogle Scholar
  26. Lins-de-Barros MM, Vieira RP, Cardoso AM, Monteiro VA, Turque AS et al (2010) Archaea, bacteria, and algal plastid associated with the reef-building corals Siderastrea stellata and Mussismilia hispida from Buzios, South Atlantic Ocean, Brazil. Microb Ecol 59:523–532PubMedCrossRefGoogle Scholar
  27. Markell DA, Trench RK (1993) Macromolecules exuded bysymbiotic dinoflagellates in culture: amino acid and sugar composition. J Phycol 29:64–68CrossRefGoogle Scholar
  28. Mohamed NM, Colman AS, Tal Y, Hill RT (2008) Diversity and expression of nitrogen fixation genes in bacterial symbionts of marine sponges. Environ Microbiol 10:2910–2921PubMedCrossRefGoogle Scholar
  29. Mouchka ME, Hewson I, Harvell CD (2010) Coral-associated bacterial assemblages: current knowledge and the potential for climate-driven impacts. Integr Comp Biol 50:662–674PubMedCrossRefGoogle Scholar
  30. Neulinger SC, Gartner A, Jarnegren J, Ludvigsen M, Lochte K et al (2009) Tissue-associated “Candidatus Mycoplasma corallicola” and filamentous bacteria on the cold-water coral Lophelia pertusa (Scleractinia). Appl Environ Microbiol 75:1437–1444PubMedCrossRefGoogle Scholar
  31. Oakley BB, Francis CA, Roberts KJ, Fuchsman CA, Srinivasan S et al (2007) Analysis of nitrite reductase (nirK and nirS) genes and cultivation reveal depauperate community of denitrifying bacteria in the Black Sea suboxic zone. Environ Microbiol 9:118–130PubMedCrossRefGoogle Scholar
  32. Olson ND, Ainsworth TD, Gates RD, Takabayashi M (2009) Diazotrophic bacteria associated with Hawaiian Montipora corals: diversity and abundance in correlation with symbiotic dinoflagellates. J Exp Mar Biol Ecol 371:140–146CrossRefGoogle Scholar
  33. Ritchie KB (2006) Regulation of microbial populations by coral surface mucus and mucus-associated bacteria. Mar Ecol-Prog Ser 322:1–14CrossRefGoogle Scholar
  34. Rohwer F, Seguritan V, Azam F, Knowlton N (2002) Diversity and distribution of coral-associated bacteria. Mar Ecol-Prog Ser 243:1–10CrossRefGoogle Scholar
  35. Rosenberg E, Koren O, Reshef L, Efrony R, Zilber-Rosenberg I (2007) The role of microorganisms in coral health, disease and evolution. Nat Rev Microbiol 5:355–362PubMedCrossRefGoogle Scholar
  36. Rotthauwe J, Boer W, Liesack W (1995) Comparative analysis of gene sequences encoding ammonia monooxygenase of Nitrosospira sp. AHB1 and Nitrosolobus multiformis C–71. FEMS Microbiol Lett 133:131–135PubMedCrossRefGoogle Scholar
  37. Rotthauwe JH, Witzel KP, Liesack W (1997) The ammonia monooxygenase structural gene amoA as a functional marker: molecular fine-scale analysis of natural ammonia-oxidizing populations. Appl Environ Microbiol 63:4704–4712PubMedGoogle Scholar
  38. Schloss PD, Handelsman J (2005) Introducing DOTUR, a computer program for defining operational taxonomic units and estimating species richness. Appl Environ Microbiol 71:1501–1506PubMedCrossRefGoogle Scholar
  39. Shnit-Orland M, Kushmaro A (2009) Coral mucus-associated bacteria: a possible first line of defense. FEMS Microbiol Ecol 67:371–380PubMedCrossRefGoogle Scholar
  40. Siboni N, Ben-Dov E, Sivan A, Kushmaro A (2008) Global distribution and diversity of coral-associated archaea and their possible role in the coral holobiont nitrogen cycle. Environ Microbiol 10:2979–2990PubMedCrossRefGoogle Scholar
  41. Sorokin YI (1973) Trophical role of bacteria in the ecosystem of the coral reef. Nature 242:415–417CrossRefGoogle Scholar
  42. Spain AM, Peacock AD, Istok JD, Elshahed MS, Najar FZ et al (2007) Identification and isolation of a Castellaniella species important during biostimulation of an acidic nitrate- and uranium-contaminated aquifer. Appl Environ Microbiol 73:4892–4904PubMedCrossRefGoogle Scholar
  43. Steger D, Ettinger-Epstein P, Whalan S, Hentschel U, De Nys R et al (2008) Diversity and mode of transmission of ammonia-oxidizing archaea in marine sponges. Environ Microbiol 10:1087–1094PubMedCrossRefGoogle Scholar
  44. Wegley L, Edwards R, Rodriguez-Brito B, Liu H, Rohwer F (2007) Metagenomic analysis of the microbial community associated with the coral Porites astreoides. Environ Microbiol 9:2707–2719PubMedCrossRefGoogle Scholar
  45. Williams WM (1987) Nitrogen fixation (acetylene reduction) associated with the living coral Acropora variabili. Mar Biol 94:531–535CrossRefGoogle Scholar
  46. Yan T, Fields MW, Wu L, Zu Y, Tiedje JM, Zhou J (2003) Molecular diversity and characterization of nitrite reductase gene fragments (nirK and nirS) from nitrate– and uranium–contaminated groundwater. Environ Microbiol 5:13–24PubMedCrossRefGoogle Scholar
  47. Yellowlees D, Rees TAV, Fitt WK (1994) Effect of ammonium-supplemented seawater on glutamine synthetase and glutamate dehydrogenase activities in host tissue and zooxanthellae of Pocillopora damicornis and on ammonium uptake rates of the zooxanthellae. Pac Sci 48:291–295Google Scholar
  48. Yokouchi H, Fukuoka Y, Mukoyama D, Calugay R, Takeyama H, Matsunaga T (2006) Whole-metagenome amplification of a microbial community associated with scleractinian coral by multiple displacement amplification using ϕ29 polymerase. Environ Microbiol 8:1155–1163PubMedCrossRefGoogle Scholar
  49. Yonge C (1929) Progress of the Great Barrier Reef expedition. Nature 123:89–99CrossRefGoogle Scholar
  50. Zehr JP, Kudela RM (2011) Nitrogen cycle of the open ocean: from genes to ecosystems. Annu Rev Mar Sci 3:197–225CrossRefGoogle Scholar
  51. Zumft WG (1997) Cell biology and molecular basis of denitrification. Microbiol Mol Biol R 61:533–616Google Scholar

Copyright information

© Springer Science+Business Media New York 2013

Authors and Affiliations

  • Shan Yang
    • 1
    • 2
  • Wei Sun
    • 1
    • 2
  • Fengli Zhang
    • 1
    • 2
  • Zhiyong Li
    • 1
    • 2
  1. 1.Marine Biotechnology Laboratory, State Key Laboratory of Microbial MetabolismShanghai Jiao Tong UniversityShanghaiPeople’s Republic of China
  2. 2.School of Life Sciences and BiotechnologyShanghai Jiao Tong UniversityShanghaiPeople’s Republic of China

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