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Volume 159, Issue Pt_2

Primers for amplification of nitrous oxide reductase genes associated with Firmicutes and Bacteroidetes in organic-compound-rich soils Free

Abstract

The gene encodes nitrous oxide reductase, a key enzyme in the nitrous oxide reduction that occurs during complete denitrification. Many conventional approaches have used Proteobacteria-based primers to detect in environmental samples. However, these primers often fail to detect in non-Proteobacteria strains, including Firmicutes (Gram-positive) and Bacteroidetes. In this study, newly designed primers successfully amplified this gene from five species (Firmicutes). The primers were used to construct clone libraries from DNA extracted from sludge and domestic animal feedlot soils, all with high organic carbon contents. After DNA sequencing, phylogenetic analysis identified many new sequences with high levels of homology to from Bacteroidetes, probably because of the high sequence similarity of from Firmicutes and Bacteroidetes, and a predominance of Bacteroidetes in feedlot environments. Three sets of new quantitative real-time PCR (qPCR) primers based on our clone library sequences were designed and tested for their specificities. Our data showed that only Bacteroidetes-related sequences were amplified, whereas conventional Proteobacteria-based primers amplified only Proteobacteria-related . Quantitative analysis of with the new qPCR primers recovered ~10 copies per 100 ng DNA. Thus, it appears that amplification with conventional primers is insufficient for developing an understanding of the diversity and abundance of genes in the environment.

  • Received:
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2013-02-01
2024-04-27
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References

  1. Basaglia M., Toffanin A., Baldan E., Bottegal M., Shapleigh J. P., Casella S.( 2007). Selenite-reducing capacity of the copper-containing nitrite reductase of Rhizobium sullae. FEMS Microbiol Lett 269:124–130 [View Article][PubMed]
     [Google Scholar]
  2. Chen Z., Liu J., Wu M., Xie X., Wu J., Wei W.( 2012). Differentiated response of denitrifying communities to fertilization regime in paddy soil. Microb Ecol 63:446–459 [View Article][PubMed]
     [Google Scholar]
  3. Chèneby D., Hartmann A., Henault C., Topp E., Germon J. C.( 1998). Diversity of denitrifying microflora and ability to reduce N2O in 2 soils. Biol Fertil Soils 28:19–26 [View Article]
     [Google Scholar]
  4. Coelho C., González P. J., Moura J. G., Moura I., Trincão J., João Romão M.( 2011). The crystal structure of Cupriavidus necator nitrate reductase in oxidized and partially reduced states. J Mol Biol 408:932–948 [View Article][PubMed]
     [Google Scholar]
  5. Durso L. M., Harhay G. P., Smith T. P., Bono J. L., DeSantis T. Z., Clawson M. L.( 2011). Bacterial community analysis of beef cattle feedlots reveals that pen surface is distinct from feces. Foodborne Pathog Dis 8:647–649 [View Article][PubMed]
     [Google Scholar]
  6. Felske A., Wolterink A., Van Lis R., Akkermans A. D.( 1998). Phylogeny of the main bacterial 16S rRNA sequences in Drentse A grassland soils (The Netherlands). Appl Environ Microbiol 64:871–879[PubMed]
     [Google Scholar]
  7. Feng L., Wang W., Cheng J., Ren Y., Zhao G., Gao C., Tang Y., Liu X., Han W.& other authors ( 2007). Genome and proteome of long-chain alkane degrading Geobacillus thermodenitrificans NG80-2 isolated from a deep-subsurface oil reservoir. Proc Natl Acad Sci U S A 104:5602–5607 [View Article][PubMed]
     [Google Scholar]
  8. Freney J. R., Trevitt A. C. F., De Datta S. K., Obcemea W. N., Real J. G.( 1990). The interdependence of ammonia volatilization and denitrification as nitrogen loss processes in flooded rice fields in the Philippines. Biol Fertil Soils 9:31–36 [View Article]
     [Google Scholar]
  9. Green S. J., Prakash O., Gihring T. M., Akob D. M., Jasrotia P., Jardine P. M., Watson D. B., Brown S. D., Palumbo A. V., Kostka J. E.( 2010). Denitrifying bacteria isolated from terrestrial subsurface sediments exposed to mixed-waste contamination. Appl Environ Microbiol 76:3244–3254 [View Article][PubMed]
     [Google Scholar]
  10. Guo G. X., Deng H., Qiao M., Mu Y. J., Zhu Y. G.( 2011). Effect of pyrene on denitrification activity and abundance and composition of denitrifying community in an agricultural soil. Environ Pollut 159:1886–1895 [View Article][PubMed]
     [Google Scholar]
  11. Hallin S., Lindgren P. E.( 1999). PCR detection of genes encoding nitrite reductase in denitrifying bacteria. Appl Environ Microbiol 65:1652–1657[PubMed]
     [Google Scholar]
  12. Henry S., Bru D., Stres B., Hallet S., Philippot L.( 2006). Quantitative detection of the nosZ gene, encoding nitrous oxide reductase, and comparison of the abundances of 16S rRNA, narG, nirK, and nosZ genes in soils. Appl Environ Microbiol 72:5181–5189 [View Article][PubMed]
     [Google Scholar]
  13. Heylen K., Vanparys B., Wittebolle L., Verstraete W., Boon N., De Vos P.( 2006). Cultivation of denitrifying bacteria: optimization of isolation condition and diversity study. Appl Environ Microbiol 72:2637–2643 [View Article]
     [Google Scholar]
  14. Janssen P. H.( 2006). Identifying the dominant soil bacterial taxa in libraries of 16S rRNA and 16S rRNA genes. Appl Environ Microbiol 72:1719–1728 [View Article][PubMed]
     [Google Scholar]
  15. Jones C. M., Welsh A., Throbäck I. N., Dörsch P., Bakken L. R., Hallin S.( 2011). Phenotypic and genotypic heterogeneity among closely related soil-borne N2- and N2O-producing Bacillus isolates harboring the nosZ gene. FEMS Microbiol Ecol 76:541–552 [View Article][PubMed]
     [Google Scholar]
  16. Jung J., Yeom J., Kim J., Han J., Lim H. S., Park H., Hyun S., Park W.( 2011). Change in gene abundance in the nitrogen biogeochemical cycle with temperature and nitrogen addition in Antarctic soils. Res Microbiol 162:1018–1026 [View Article][PubMed]
     [Google Scholar]
  17. Kloos K., Mergel A., Rosch C., Bothe H.( 2001). Denitrification within the genus Azospirillum and other associative bacteria. Aust J Plant Physiol 28:991–998
     [Google Scholar]
  18. Liu X., Gao C., Zhang A., Jin P., Wang L., Feng L.( 2008). The nos gene cluster from Gram-positive bacterium Geobacillus thermodenitrificans NG80-2 and functional characterization of the recombinant NosZ. FEMS Microbiol Lett 289:46–52 [View Article][PubMed]
     [Google Scholar]
  19. Miñana-Galbis D., Pinzón D. L., Lorén J. G., Manresa A., Oliart-Ros R. M.( 2010). Reclassification of Geobacillus pallidus (Scholz et al. 1988) Banat et al. 2004 as Aeribacillus pallidus gen. nov., comb. nov. Int J Syst Evol Microbiol 60:1600–1604 [View Article][PubMed]
     [Google Scholar]
  20. Nogales B., Timmis K. N., Nedwell D. B., Osborn A. M.( 2002). Detection and diversity of expressed denitrification genes in estuarine sediments after reverse transcription-PCR amplification from mRNA. Appl Environ Microbiol 68:5017–5025 [View Article][PubMed]
     [Google Scholar]
  21. Nõlvak H., Truu M., Truu J.( 2012). Evaluation of quantitative real-time PCR workflow modifications on 16S rRNA and tetA gene quantification in environmental samples. Sci Total Environ 426:351–358 [View Article][PubMed]
     [Google Scholar]
  22. Pujol Pereira E. I., Chung H., Scow K., Sadowsky M. J., van Kessel C., Six J.( 2011). Soil nitrogen transformations under elevated atmospheric CO2 and O3 during the soybean growing season. Environ Pollut 159:401–407 [View Article][PubMed]
     [Google Scholar]
  23. Ravishankara A. R., Daniel J. S., Portmann R. W.( 2009). Nitrous oxide (N2O): the dominant ozone-depleting substance emitted in the 21st century. Science 326:123–125 [View Article][PubMed]
     [Google Scholar]
  24. Scala D. J., Kerkhof L. J.( 1998). Nitrous oxide reductase (nosZ) gene-specific PCR primers for detection of denitrifiers and three nosZ genes from marine sediments. FEMS Microbiol Lett 162:61–68 [View Article][PubMed]
     [Google Scholar]
  25. Singh B. K., Bardgett R. D., Smith P., Reay D. S.( 2010). Microorganisms and climate change: terrestrial feedbacks and mitigation options. Nat Rev Microbiol 8:779–790 [View Article][PubMed]
     [Google Scholar]
  26. Smith R. L., Ceazan M. L., Brooks M. H.( 1994). Autotrophic, hydrogen-oxidizing, denitrifying bacteria in groundwater, potential agents for bioremediation of nitrate contamination. Appl Environ Microbiol 60:1949–1955[PubMed]
     [Google Scholar]
  27. Suharti, de Vries S.( 2005). Membrane-bound denitrification in the Gram-positive bacterium Bacillus azotoformans. Biochem Soc Trans 33:130–133 [View Article][PubMed]
     [Google Scholar]
  28. Throbäck I. N., Enwall K., Jarvis Å., Hallin S.( 2004). Reassessing PCR primers targeting nirS, nirK and nosZ genes for community surveys of denitrifying bacteria with DGGE. FEMS Microbiol Ecol 49:401–417 [View Article][PubMed]
     [Google Scholar]
  29. Zumft W. G.( 1997). Cell biology and molecular basis of denitrification. Microbiol Mol Biol Rev 61:533–616[PubMed]
     [Google Scholar]
  30. Zumft W. G., Bothe H.( 2007). Nitrous oxides reductases. Biology of the Nitrogen Cycle67–81 Bothe H., Ferguson S. J., Newton W. E. Amsterdam: Elsevier; [View Article]
     [Google Scholar]
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Primers for amplification of nitrous oxide reductase genes associated with Firmicutes and Bacteroidetes in organic-compound-rich soils
Microbiology 159, 307 (2013); https://doi.org/10.1099/mic.0.060194-0
/content/journal/micro/10.1099/mic.0.060194-0
/content/journal/micro/10.1099/mic.0.060194-0
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