Microbial Ecology

, Volume 53, Issue 3, pp 456–470 | Cite as

A nifH-based Oligonucleotide Microarray for Functional Diagnostics of Nitrogen-fixing Microorganisms

  • Lei Zhang
  • Thomas Hurek
  • Barbara Reinhold-HurekEmail author


Nitrogen fixation is an important process in biogeochemical cycles exclusively carried out by prokaryotes, mostly by an evolutionarily conserved nitrogenase protein complex, of which one of the structural genes (nifH) is highly valuable for phylogenetic and diversity analyses. We developed a nifH-based short oligonucleotide microarray (nifH diagnostic microarray) as a rapid tool to effectively monitor nitrogen-fixing diazotrophic populations in a wide range of environments. Taking account of the overwhelming predominance of environmental nifH fragments from uncultivated microorganisms in public databases, our nifH microarray is mainly based on nifH sequences from as yet unidentified prokaryotes. Standard conditions for microarray performance were determined, and criteria for the design of specific oligonucleotides were defined. A primary set of 56 oligonucleotides was validated with fluorescence-labeled single-stranded nifH targets from five reference strains, 26 environmental clones, and artificial mixtures of reference strains. The nifH microarray was applied to analyze the diversity (based on DNA) and activity (based on mRNA) of diazotrophs in roots of wild rice samples from Namibia. Results demonstrated that only a small subset of diazotrophs being present in the sample were actually fixing nitrogen actively. Our data suggest that the developed nifH microarray is a highly reproducible and semiquantitative method for mapping the variability of diazotrophic diversity, allowing rapid comparisons of the relative abundance and activity of diazotrophic prokaryotes in the environment. A further refined nifH microarray comprising of 194 oligonucleotide probes now covers more than 90% of sequences in our nifH database.


Clone Library Wild Rice Terminal Restriction Fragment Length Polymorphism Microarray Hybridization nifH Gene 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.



This work was linked to the CAG supported by the Land Bremen and the German Federal Ministry of Education and Research (BMBF) (grant 0311833 A, TP 14 to B. R.-H.), and was also supported by a grant of the BMBF under the BIOLOG framework to B. R.-H. and T. H. (grant no. 01LC0021). For the sequences of the probe set of the nifH microarray, an application for a patent has been filed by the University of Bremen (PCT/EP2005/008150 and is available at EBI W02006013052).

Supplementary material


  1. 1.
    Bodrossy, L, Sessitsch, A (2004) Oligonucleotide microarrays in microbial diagnostics. Curr Opin Microbiol 7: 246–255CrossRefGoogle Scholar
  2. 2.
    Bodrossy, L, Stralis-Pavese, N, Konrad-Koszler, M, Weilharter, A, Reichenauer, TG, Schofer, D, Sessitsch, A (2006) mRNA-based parallel detection of active methanotroph populations by use of a diagnostic microarray. Appl Environ Microbiol 72: 1672–1676PubMedCrossRefGoogle Scholar
  3. 3.
    Bodrossy, L, Stralis-Pavese, N, Murrell, JC, Radajewski, S, Weilharter, A, Sessitsch, A (2003) Development and validation of a diagnostic microbial microarray for methanotrophs. Environ Microbiol 5: 566–582PubMedCrossRefGoogle Scholar
  4. 4.
    Chang, S, Puryear, J, Cairney, J (1993) A simple and efficient method for isolating RNA from pine trees. Plant Mol Biol Reporter 11: 113–116CrossRefGoogle Scholar
  5. 5.
    Chien, YT, Zinder, SH (1994) Cloning, DNA sequencing, and characterization of a nifD-homologous gene from the archaeon Methanosarcina barkeri 227 which resembles nifD1 from the eubacterium Clostridium pasteurianum. J Bacteriol 176: 6590–6598PubMedGoogle Scholar
  6. 6.
    Denef, VJ, Park, J, Rodrigues, JL, Tsoi, TV, Hashsham, SA, Tiedje, JM (2003) Validation of a more sensitive method for using spotted oligonucleotide DNA microarrays for functional genomics studies on bacterial communities. Environ Microbiol 5: 933–943PubMedCrossRefGoogle Scholar
  7. 7.
    Engelhard, M, Hurek, T, Reinhold-Hurek, B (2000) Preferential occurrence of diazotrophic endophytes, Azoarcus spp., in wild rice species and land races of Oryza sativa in comparison with modern races. Environ Microbiol 2: 131–141PubMedCrossRefGoogle Scholar
  8. 8.
    Fotin, AV, Drobyshev, AL, Proudnikov, DY, Perov, AN, Mirzabekov, AD (1998) Parallel thermodynamic analysis of duplexes on oligodeoxyribonucleotide microchips. Nucleic Acids Res 26: 1515–1521PubMedCrossRefGoogle Scholar
  9. 9.
    Gibson, G (2002) Microarrays in ecology and evolution: A preview. Mol Ecol 11: 17–24PubMedCrossRefGoogle Scholar
  10. 10.
    Hurek, T, Reinhold-Hurek, B (2005) Molecular ecology of N2-fixing microbes associated with graminaceous plants. In: Werner, D, Newton, WE (Eds.) Agriculture, Forestry, Ecology and the Environment. Kluwer Academic Publishers, Dordrecht, The Netherlands, pp. 173–198CrossRefGoogle Scholar
  11. 11.
    Hurek, T, Egener, T, Reinhold-Hurek, B (1997) Divergence in nitrogenases of Azoarcus spp., Proteobacteria of the β-subclass. J Bacteriol 179: 4172–4178PubMedGoogle Scholar
  12. 12.
    Hurek, T, Burggraf, S, Woese, CR, Reinhold-Hurek, B (1993) 16S rRNA-targeted polymerase chain reaction and oligonucleotide hybridization to screen for Azoarcus spp., grass-associated diazotrophs. Appl Environ Microbiol 59: 3816–3824PubMedGoogle Scholar
  13. 13.
    Hurek, T, Handley, L, Reinhold-Hurek, B, Piché, Y (2002) Azoarcus grass endophytes contribute fixed nitrogen to the plant in an unculturable state. Mol Plant–Microbe Interact 15: 233–242PubMedGoogle Scholar
  14. 14.
    Jenkins, BD, Steward, GF, Short, SM, Ward, BB, Zehr, JP (2004) Fingerprinting diazotroph communities in the Chesapeake Bay by using a DNA macroarray. Appl Environ Microbiol 70: 1767–1776PubMedCrossRefGoogle Scholar
  15. 15.
    Knauth, S, Hurek, T, Brar, D, Reinhold-Hurek, B (2005) Influence of different Oryza cultivars on expression of nifH gene pools in roots of rice. Environ Microbiol 7: 1725–1733PubMedCrossRefGoogle Scholar
  16. 16.
    Kumar, S, Tamura, K, Nei, M (2004) MEGA3: Integrated software for molecular evolutionary genetics analysis and sequence alignment. Brief Bioinform 5: 150–163PubMedCrossRefGoogle Scholar
  17. 17.
    Loy, A, Lehner, A, Lee, N, Adamczyk, J, Meier, H, Ernst, J et al. (2002) Oligonucleotide microarray for 16S rRNA gene-based detection of all recognized lineages of sulfate-reducing prokaryotes in the environment. Appl Environ Microbiol 68: 5064–5081PubMedCrossRefGoogle Scholar
  18. 18.
    Ludwig, W, Strunk, O, Westram, R, Richter, L, Meier, H, Yadhukumar et al. (2004) ARB: A software environment for sequence data. Nucleic Acids Res 32: 1363–1371PubMedCrossRefGoogle Scholar
  19. 19.
    Nicholas, KB, Nicholas, HBJ (1997) GeneDoc: a tool for editing and annotating multiple sequence alignments.
  20. 20.
    Niemeyer, CM, Boldt, L, Ceyhan, B, Blohm, D (1999) Evaluation of single-stranded nucleic acids as carriers in the DNA-directed assembly of macromolecules. J Biomol Struct Dyn 17: 527–538PubMedGoogle Scholar
  21. 21.
    Noda, S, Ohkuma, M, Usami, R, Horikoshi, K, Kudo, T (1999) Culture-independent characterization of a gene responsible for nitrogen fixation in the symbiotic microbial community in the gut of the termite Neotermes koshunensis. Appl Environ Microbiol 65: 4935–4942PubMedGoogle Scholar
  22. 22.
    Ohkuma, M, Noda, S, Kudo, T (1999) Phylogenetic diversity of nitrogen fixation genes in the symbiotic microbial community in the gut of diverse termites. Appl Environ Microbiol 65: 4926–4934PubMedGoogle Scholar
  23. 23.
    Peplies, J, Glöckner, FO, Amann, R (2003) Optimization strategies for DNA microarray-based detection of bacteria with 16S rRNA-targeting oligonucleotide probes. Appl Environ Microbiol 69: 1397–1407PubMedCrossRefGoogle Scholar
  24. 24.
    Raymond, J, Siefert, JL, Staples, CR, Blankenship, RE (2004) The natural history of nitrogen fixation. Mol Biol Evol 21: 541–554PubMedCrossRefGoogle Scholar
  25. 25.
    Reinhold-Hurek, B, Hurek, T (2000) Reassessment of the taxonomic structure of the diazotrophic genus Azoarcus sensu lato and description of three new genera and species, Azovibrio restrictus gen. nov., sp. nov., Azospira oryzae gen. nov., sp. nov, and Azonexus funguphilus gen. nov., sp. nov. Int J Syst Evol Microbiol 50: 649–659PubMedGoogle Scholar
  26. 26.
    Reinhold-Hurek, B, Hurek, T, Gillis, M, Hoste, B, Vancanneyt, M, Kersters, K, De Ley, J (1993) Azoarcus gen. nov., nitrogen-fixing proteobacteria associated with roots of Kallar grass (Leptochloa fusca (L.) Kunth) and description of two species Azoarcus indigens sp. nov. and Azoarcus communis sp. nov. Int J Syst Bacteriol 43: 574–584CrossRefGoogle Scholar
  27. 27.
    Relógio, A, Schwager, C, Richter, A, Ansorge, W, Valcarcel, J (2002) Optimization of oligonucleotide-based DNA microarrays. Nucleic Acids Res 30: e51PubMedCrossRefGoogle Scholar
  28. 28.
    Rozen, S, Skaletsky, H (2000) Primer3 on the WWW for general users and for biologist programmers. Methods Mol Biol 132: 365–386PubMedGoogle Scholar
  29. 29.
    Schütz, E, von Ahsen, N (1999) Spreadsheet software for thermodynamic melting point prediction of oligonucleotide hybridization with and without mismatches. BioTechniques 27: 1218–1222PubMedGoogle Scholar
  30. 30.
    Shchepinov, MS, Case-Green, SC, Southern, EM (1997) Steric factors influencing hybridisation of nucleic acids to oligonucleotide arrays. Nucleic Acids Res 25: 1155–1161PubMedCrossRefGoogle Scholar
  31. 31.
    Shi, SJ, Scheffer, A, Bjeldanes, E, Reynolds, MA, Arnold, LJ (2001) DNA exhibits multi-stranded binding recognition on glass microarrays. Nucleic Acids Res 29: 4251–4256PubMedCrossRefGoogle Scholar
  32. 32.
    Southern, E, Mir, K, Shchepinov, M (1999) Molecular interactions on microarrays. Nat Genet 21: 5–9PubMedCrossRefGoogle Scholar
  33. 33.
    Steward, GF, Jenkins, BD, Ward, BB, Zehr, JP (2004) Development and testing of a DNA macroarray to assess nitrogenase (nifH) gene diversity. Appl Environ Microbiol 70: 1455–1465PubMedCrossRefGoogle Scholar
  34. 34.
    Sugimoto, N, Nakano, M, Nakano, S (2000) Thermodynamics–structure relationship of single mismatches in RNA/DNA duplexes. Biochemistry 39: 11270–11281PubMedCrossRefGoogle Scholar
  35. 35.
    Tan, Z, Hurek, T, Reinhold-Hurek, B (2003) Effect of N-fertilization, plant genotype and environmental conditions on nifH gene pools in roots of rice. Environ Microbiol 5: 1009–1015PubMedCrossRefGoogle Scholar
  36. 36.
    Taroncher-Oldenburg, G, Griner, EM, Francis, CA, Ward, BB (2003) Oligonucleotide microarray for the study of functional gene diversity in the nitrogen cycle in the environment. Appl Environ Microbiol 69: 1159–1171PubMedCrossRefGoogle Scholar
  37. 37.
    Thompson, JD, Gibson, TJ, Plewniak, F, Jeanmougin, F, Higgins, DG (1997) The CLUSTAL_X windows interface: Flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res 25: 4876–4882PubMedCrossRefGoogle Scholar
  38. 38.
    Urakawa, H, Noble, PA, El Fantroussi, S, Kelly, JJ, Stahl, DA (2002) Single-base-pair discrimination of terminal mismatches by using oligonucleotide microarrays and neural network analyses. Appl Environ Microbiol 68: 235–244PubMedCrossRefGoogle Scholar
  39. 39.
    Wilson, KH, Wilson, WJ, Radosevich, JL, DeSantis, TZ, Viswanathan, VS, Kuczmarski, TA, Andersen, GL (2002) High-density microarray of small-subunit ribosomal DNA probes. Appl Environ Microbiol 68: 2535–2541PubMedCrossRefGoogle Scholar
  40. 40.
    Zani, S, Mellon, MT, Collier, JL, Zehr, JP (2000) Expression of nifH genes in natural microbial assemblages in Lake George, New York, detected by reverse transcriptase PCR. Appl Environ Microbiol 66: 3119–3124PubMedCrossRefGoogle Scholar
  41. 41.
    Zehr, JP, McReynolds, LA (1989) Use of degenerate oligonucleotides for amplification of the nifH gene from the marine cyanobacterium Trichodesmium thiebautii. Appl Environ Microbiol 55: 2522–2526PubMedGoogle Scholar
  42. 42.
    Zehr, JP, Capone, DG (1996) Problems and promises of assaying the genetic potential for nitrogen fixation in the marine environment. Microb Ecol 32: 263–281PubMedCrossRefGoogle Scholar
  43. 43.
    Zehr, JP, Mellon, MT, Zani, S (1998) New nitrogen-fixing microorganisms detected in oligotrophic oceans by amplification of nitrogenase (nifH) genes. Appl Environ Microbiol 64: 3444–3450PubMedGoogle Scholar
  44. 44.
    Zehr, JP, Jenkins, BD, Short, SM, Steward, GF (2003) Nitrogenase gene diversity and microbial community structure: A cross-system comparison. Environ Microbiol 5: 539–554PubMedCrossRefGoogle Scholar
  45. 45.
    Zhang, L, Hurek, T, Reinhold-Hurek, B (2005) Position of the fluorescent label is a crucial factor determining signal intensity in microarray hybridisations. Nucleic Acids Res 33: e166PubMedCrossRefGoogle Scholar
  46. 46.
    Zhou, JZ (2003) Microarrays for bacterial detection and microbial community analysis. Curr Opin Microbiol 6: 288–294PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, Inc. 2006

Authors and Affiliations

  • Lei Zhang
    • 1
  • Thomas Hurek
    • 1
  • Barbara Reinhold-Hurek
    • 1
    Email author
  1. 1.Laboratory of General Microbiology, Center of Applied Gene Sensoric (CAG)University of BremenBremenGermany

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