Archaea are widespread in extreme and temperate environments, and cultured representatives cover a broad spectrum of metabolic capacities, which sets them up for potentially major roles in the biogeochemistry of their ecosystems. The detection, characterization, and quantification of archaeal functions in mixed communities require Archaea-specific primers or probes for the corresponding metabolic genes. Five pairs of degenerate primers were designed to target archaeal genes encoding key enzymes of nitrogen cycling: nitrite reductases NirA and NirB, nitrous oxide reductase (NosZ), nitrogenase reductase (NifH), and nitrate reductases NapA/NarG. Sensitivity towards their archaeal target gene, phylogenetic specificity, and gene specificity were evaluated in silico and in vitro. Owing to their moderate sensitivity/coverage, the novel nirB-targeted primers are suitable for pure culture studies only. The nirA-targeted primers showed sufficient sensitivity and phylogenetic specificity, but poor gene specificity. The primers designed for amplification of archaeal nosZ performed well in all 3 criteria; their discrimination against bacterial homologs appears to be weakened when Archaea are strongly outnumbered by bacteria in a mixed community. The novel nifH-targeted primers showed high sensitivity and gene specificity, but failed to discriminate against bacterial homologs. Despite limitations, 4 of the new primer pairs are suitable tools in several molecular methods applied in archaeal ecology. 1. Introduction Archaea have been detected in virtually all types of extreme and moderate environments. They play multiple ecological roles, colonizing certain newly emerging habitats [1, 2], interacting with animals such as corals [3, 4], sponges [5, 6], termites [7], or ruminants, forming part of microbe-microbe symbioses [8–10], and driving numerous processes in the biogeochemical C, N, S, and Fe cycles. In addition to relatively well-studied isolates of extremophilic or methanogenic Archaea, uncultured representatives have been detected by their 16S rRNA genes or by metabolic genes that classify their owners into the guilds of sulfate reducers, diazotrophs, ammonia oxidizers, or methanogens. Despite their widespread occurrence, a mere handful of nonmethanogenic Archaea has been isolated from moderate habitats [11–13]. While such isolates are indispensable for insight into archaeal ecophysiology, they have been recalcitrant to cultivation efforts, so that our current ecological research on mesophilic Archaea largely depends on cultivation-independent methods. Molecular
References
[1]
G. W. Nicol, D. Tscherko, T. M. Embley, and J. I. Prosser, “Primary succession of soil Crenarchaeota across a receding glacier foreland,” Environmental Microbiology, vol. 7, no. 3, pp. 337–347, 2005.
[2]
E. A. McCliment, K. M. Voglesonger, P. A. O'Day, E. E. Dunn, J. R. Holloway, and S. C. Cary, “Colonization of nascent, deep-sea hydrothermal vents by a novel Archaeal and Nanoarchaeal assemblage,” Environmental Microbiology, vol. 8, no. 1, pp. 114–125, 2006.
[3]
N. Siboni, E. Ben-Dov, A. Sivan, and A. Kushmaro, “Global distribution and diversity of coral-associated Archaea and their possible role in the coral holobiont nitrogen cycle,” Environmental Microbiology, vol. 10, no. 11, pp. 2979–2990, 2008.
[4]
J. B. Olson and C. A. Kellogg, “Microbial ecology of corals, sponges, and algae in mesophotic coral environments,” FEMS Microbiology Ecology, vol. 73, no. 1, pp. 17–30, 2010.
[5]
C. M. Preston, K. Y. Wu, T. F. Molinski, and E. F. DeLong, “A psychrophilic crenarchaeon inhabits a marine sponge: Cenarchaeum symbiosum gen. nov., sp. nov.,” Proceedings of the National Academy of Sciences of the United States of America, vol. 93, no. 13, pp. 6241–6246, 1996.
[6]
D. Steger, P. Ettinger-Epstein, S. Whalan et al., “Diversity and mode of transmission of ammonia-oxidizing archaea in marine sponges,” Environmental Microbiology, vol. 10, no. 4, pp. 1087–1094, 2008.
[7]
J. H. P. Hackstein and C. K. Stumm, “Methane production in terrestrial arthropods,” Proceedings of the National Academy of Sciences of the United States of America, vol. 91, no. 12, pp. 5441–5445, 1994.
[8]
A. Boetius, K. Ravenschlag, C. J. Schubert et al., “A marine microbial consortium apparently mediating anaerobic oxidation methane,” Nature, vol. 407, no. 6804, pp. 623–626, 2000.
[9]
U. Jahn, M. Gallenberger, W. Paper et al., “Nanoarchaeum equitans and Ignicoccus hospitalis: new insights into a unique, intimate association of two archaea,” Journal of Bacteriology, vol. 190, no. 5, pp. 1743–1750, 2008.
[10]
C. Moissl-Eichinger and H. Huber, “Archaeal symbionts and parasites,” Current Opinion in Microbiology, vol. 14, no. 3, pp. 364–370, 2011.
[11]
M. K？nneke, A. E. Bernhard, J. R. de la Torre, C. B. Walker, J. B. Waterbury, and D. A. Stahl, “Isolation of an autotrophic ammonia-oxidizing marine archaeon,” Nature, vol. 437, no. 7058, pp. 543–546, 2005.
[12]
B.-J. Park, S.-J. Park, D.-N. Yoon, S. Schouten, J. S. Sinninghe Damsté, and S.-K. Rhee, “Cultivation of autotrophic ammonia-oxidizing archaea from marine sediments in coculture with sulfur-oxidizing bacteria,” Applied and Environmental Microbiology, vol. 76, no. 22, pp. 7575–7587, 2010.
[13]
M. Tourna, M. Stieglmeier, A. Spang et al., “Nitrososphaera viennensis, an ammonia oxidizing archaeon from soil,” Proceedings of the National Academy of Sciences of the United States of America, vol. 108, no. 20, pp. 8420–8425, 2011.
[14]
W. G. Zumft, “Cell biology and molecular basis of denitrification?” Microbiology and Molecular Biology Reviews, vol. 61, no. 4, pp. 533–616, 1997.
[15]
J. P. Amend and E. L. Shock, “Energetics of overall metabolic reactions of thermophilic and hyperthermophilic Archaea and Bacteria,” FEMS Microbiology Reviews, vol. 25, no. 2, pp. 175–243, 2001.
[16]
L. Philippot, “Denitrifying genes in bacterial and Archaeal genomes,” Biochimica et Biophysica Acta, vol. 1577, no. 3, pp. 355–376, 2002.
[17]
P. Cabello, M. D. Roldán, and C. Moreno-Vivián, “Nitrate reduction and the nitrogen cycle in archaea,” Microbiology, vol. 150, no. 11, pp. 3527–3546, 2004.
[18]
C. A. Francis, K. J. Roberts, J. M. Beman, A. E. Santoro, and B. B. Oakley, “Ubiquity and diversity of ammonia-oxidizing archaea in water columns and sediments of the ocean,” Proceedings of the National Academy of Sciences of the United States of America, vol. 102, no. 41, pp. 14683–14688, 2005.
[19]
D. J. Arp and L. Y. Stein, “Metabolism of inorganic N compounds by ammonia-oxidizing bacteria,” Critical Reviews in Biochemistry and Molecular Biology, vol. 38, no. 6, pp. 471–495, 2003.
[20]
U. Purkhold, M. Wagner, G. Timmermann, A. Pommerening-R？ser, and H.-P. Koops, “16S rRNA and amoA-based phylogeny of 12 novel betaproteobacterial ammonia-oxidizing isolates: extension of the dataset and proposal of a new lineage within the nitrosomonads,” International Journal of Systematic and Evolutionary Microbiology, vol. 53, no. 5, pp. 1485–1494, 2003.
[21]
J. R. de la Torre, C. B. Walker, A. E. Ingalls, M. K？nneke, and D. A. Stahl, “Cultivation of a thermophilic ammonia oxidizing archaeon synthesizing crenarchaeol,” Environmental Microbiology, vol. 10, no. 3, pp. 810–818, 2008.
[22]
R. Hatzenpichler, E. V. Lebedeva, E. Spieck et al., “A moderately thermophilic ammonia-oxidizing crenarchaeote from a hot spring,” Proceedings of the National Academy of Sciences of the United States of America, vol. 105, no. 6, pp. 2134–2139, 2008.
[23]
R. Hatzenpichler, “Diversity, physiology and niche differentiation of ammonia-oxidizing archaea,” Applied and Environmental Microbiology, vol. 78, no. 21, pp. 7501–7510, 2012.
[24]
H. Ogata, S. Goto, K. Sato, W. Fujibuchi, H. Bono, and M. Kanehisa, “KEGG: kyoto encyclopedia of genes and genomes,” Nucleic Acids Research, vol. 27, no. 1, pp. 29–34, 1999.
[25]
K. D. Pruitt, T. Tatusova, and D. R. Maglott, “NCBI reference sequences (RefSeq): a curated non-redundant sequence database of genomes, transcripts and proteins,” Nucleic Acids Research, vol. 35, no. 1, pp. D61–D65, 2007.
[26]
D. J. Richardson, B. C. Berks, D. A. Russell, S. Spiro, and C. J. Taylor, “Functional, biochemical and genetic diversity of prokaryotic nitrate reductases,” Cellular and Molecular Life Sciences, vol. 58, no. 2, pp. 165–178, 2001.
[27]
R. W. Ye and S. M. Thomas, “Microbial nitrogen cycles: physiology, genomics and applications,” Current Opinion in Microbiology, vol. 4, no. 3, pp. 307–312, 2001.
[28]
T. Rütting, P. Boeckx, C. Müller, and L. Klemedtsson, “Assessment of the importance of dissimilatory nitrate reduction to ammonium for the terrestrial nitrogen cycle,” Biogeosciences, vol. 8, no. 7, pp. 1779–1791, 2011.
[29]
D. E. Canfield, A. N. Glazer, and P. G. Falkowski, “The evolution and future of Earth's nitrogen cycle,” Science, vol. 330, no. 6001, pp. 192–196, 2010.
[30]
C. M. Jones, B. Stres, M. Rosenquist, and S. Hallin, “Phylogenetic analysis of nitrite, nitric oxide, and nitrous oxide respiratory enzymes reveal a complex evolutionary history for denitrification,” Molecular Biology and Evolution, vol. 25, no. 9, pp. 1955–1966, 2008.
[31]
R. Bartossek, G. W. Nicol, A. Lanzen, H.-P. Klenk, and C. Schleper, “Homologues of nitrite reductases in ammonia-oxidizing archaea: diversity and genomic context,” Environmental Microbiology, vol. 12, no. 4, pp. 1075–1088, 2010.
[32]
I. N. Throb？ck, K. Enwall, ？. Jarvis, and S. Hallin, “Reassessing PCR primers targeting nirS, nirK and nosZ genes for community surveys of denitrifying bacteria with DGGE,” FEMS Microbiology Ecology, vol. 49, no. 3, pp. 401–417, 2004.
[33]
C. J. Smith and A. M. Osborn, “Advantages and limitations of quantitative PCR (Q-PCR)-based approaches in microbial ecology,” FEMS Microbiology Ecology, vol. 67, no. 1, pp. 6–20, 2009.
[34]
J. P. Zehr, B. D. Jenkins, S. M. Short, and G. F. Steward, “Nitrogenase gene diversity and microbial community structure: a cross-system comparison,” Environmental Microbiology, vol. 5, no. 7, pp. 539–554, 2003.
[35]
J. C. Gaby and D. H. Buckley, “A comprehensive evaluation of PCR primers to amplify the nifH gene of nitrogenase,” PLoS ONE, vol. 7, no. 7, Article ID e42149, 2012.
[36]
C. Linhart and R. Shamir, “The degenerate primer design problem,” Bioinformatics, vol. 18, no. 1, pp. S172–S180, 2002.
[37]
P. Rice, I. Longden, and A. Bleasby, “EMBOSS: the European molecular biology open software suite,” Trends in Genetics, vol. 16, no. 6, pp. 276–277, 2000.
[38]
B. P. Keough, T. M. Schmidt, and R. E. Hicks, “Archaeal nucleic acids in picoplankton from Great Lakes on three continents,” Microbial Ecology, vol. 46, no. 2, pp. 238–248, 2003.
[39]
D. A. Pascoe and R. E. Hicks, “Genetic structure and community DNA similarity of picoplankton communities from the Laurentian Great Lakes,” Journal of Great Lakes Research, vol. 30, supplement 1, pp. 185–195, 2004.
[40]
S. F. Altschul, W. Gish, W. Miller, E. W. Myers, and D. J. Lipman, “Basic local alignment search tool,” Journal of Molecular Biology, vol. 215, no. 3, pp. 403–410, 1990.
[41]
A. Marchler-Bauer, S. Lu, J. B. Anderson et al., “CDD: a conserved domain database for the functional annotation of proteins,” Nucleic Acids Research, vol. 39, supplement 1, pp. D225–D229, 2011.
[42]
G. M. Boratyn, A. A. Schaffer, R. Agarwala, S. F. Altschul, D. J. Lipman, and T. L. Madden, “Domain enhanced lookup time accelerated BLAST,” Biology Direct, vol. 7, article 12, 2012.
