Abstract
Recent metagenomic analyses of Yellowstone National Park (YNP) thermal spring communities suggested the presence of minor archaeal populations that simultaneous PCR-based assays using traditional ‘universal’ 16S rRNA gene primers failed to detect. Here we use metagenomics to identify PCR primers effective at detecting elusive members of the Archaea, assess their efficacy, and describe the diverse and novel archaeal community from a circum-neutral thermal spring from the Bechler region of YNP. We determined that a less commonly used PCR primer, Arch349F, captured more diversity in this spring than the widely used A21F primer. A search of the PCR primers against the RDP 16S rRNA gene database indicated that Arch349F also captured the largest percentage of Archaea, including 41 % more than A21F. Pyrosequencing using the Arch349F primer recovered all of the phylotypes present in the clone-based portion of the study and the metagenome of this spring in addition to several other populations of Archaea, some of which are phylogenetically novel. In contrast to the lack of amplification with traditional 16S rRNA gene primers, our comprehensive analyses suggested a diverse archaeal community in the Bechler spring, with implications for recently discovered groups such as the Geoarchaeota and other undescribed archaeal groups.
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References
Andreotti R, Pérez de León AA, Dowd SE, Guerrero FD, Bendele KG, Scoles GA (2011) Assessment of bacterial diversity in the cattle tick Rhipicephalus (Boophilus) microplus through tag-encoded pyrosequencing. BMC Microbiol 11:6
Baker GC, Smith JJ, Cowan DA (2003) Review and re-analysis of domain-specific 16S primers. J Microbiol Meth 55:541–555
Baker BJ, Comolli LR, Dick GJ, Hauser LJ, Hyatt D, Dill BD, Land ML, VerBerkmoes NC, Hettich RL, Banfield JF (2010) Enigmatic, ultrasmall, uncultivated Archaea. Proc Natl Acad Sci U S A 107:8806–8811
Barns SM, Fundyga RE, Jeffries MW, Pace NR (1994) Remarkable archaeal diversity detected in a Yellowstone National Park hot spring environment. Proc Natl Acad Sci U S A 91:1609–1613
Caporaso JG, Bittinger K, Bushman FD, DeSantis TZ, Andersen GL, Knight R (2010a) PyNAST: a flexible tool for aligning sequences to a template alignment. Bioinformatics 26:266–267
Caporaso JG, Kuczynski J, Stombaugh J, Bittinger K, Bushman FD, Costello EK, Fierer N, Peña AG, Goodrich JK, Gordon JI, Huttley GA, Kelley ST, Knights D, Koenig JE, Ley RE, Lozupone CA, McDonald D, Muegge BD, Pirrung M, Reeder J, Sevinsky JR, Turnbaugh PJ, Walters WA, Widmann J, Yatsunenko T, Zaneveld J, Knight R (2010b) QIIME allows analysis of high-throughput community sequencing data. Nat Methods 7:335–336
Cole JR, Wang Q, Fish JA, Chai B, McGarrell DM, Sun Y, Brown CT, Porras-Alfaro A, Kuske CR, Tiedje JM (2014) Ribosomal Database Project: data and tools for high throughput rRNA analysis. Nucleic Acids Res 42:D633–D642
Costa KC, Navarro JB, Shock EL, Zhang CLL, Soukup D, Hedlund BP (2009) Microbiology and geochemistry of great boiling and mud hot springs in the United States Great Basin. Extremophiles 13:447–459
Darriba D, Taboada GL, Doallo R, Posada D (2012) jModelTest 2: more models, new heuristics and parallel computing. Nat Methods 9:772
DeLong EF (1992) Archaea in coastal marine environments. Proc Natl Acad Sci U S A 89:5685–5689
Dodsworth JA, Hungate BA, Hedlund BP (2011) Ammonia oxidation, denitrification and dissimilatory nitrate reduction to ammonium in two US Great Basin hot springs with abundant ammonia-oxidizing archaea. Environ Microbiol 13:2371–2386
Dowd SE, Wolcott RD, Sun Y, McKeehan T, Smith E, Rhoads D (2008) Polymicrobial nature of chronic diabetic foot ulcer biofilm infections determined using bacterial tag encoded FLX amplicon pyrosequencing (bTEFAP). PLoS ONE 3:e3326
Guy L, Spang A, Saw JH, Ettema TJ (2014) ‘Geoarchaeote NAG1’ is a deeply rooting lineage of the archaeal order Thermoproteales rather than a new phylum. ISME J 8:1353–1357
Hugenholtz P (2002) Exploring prokaryotic diversity in the genomic era. Genome Biol 3: Reviews 0003
Inskeep WP, Jay ZJ, Tringe SG, Herrgård MJ, Rusch DB, YNP Metagenome Project Steering Committee, Working Group Members (2013) The YNP Metagenome Project: Environmental Parameters Responsible for Microbial Distribution in the Yellowstone Geothermal Ecosystem. Front Microbiol 4:67
Inskeep WP, Rusch DB, Jay ZJ, Herrgard MJ, Kozubal MA, Richardson TH, Macur RE, Hamamura N, Jennings R, Fouke BW, Reysenbach AL, Roberto F, Young M, Schwartz A, Boyd ES, Badger JH, Mathur EJ, Ortmann AC, Bateson M, Geesey G, Frazier M (2010) Metagenomes from high-temperature chemotrophic systems reveal geochemical controls on microbial community structure and function. PLoS ONE 5:e9773
Itoh T, Nomura N, Sako Y (2003) Distribution of 16S rRNA introns among the family Thermoproteaceae and their evolutionary implications. Extremophiles 7:229–233
Klindworth A, Pruesse E, Schweer T, Peplies J, Quast C, Horn M, Glöckner FO (2013) Evaluation of general 16S ribosomal RNA gene PCR primers for classical and next-generation sequencing-based diversity studies. Nucleic Acids Res 41:e1
Kozubal MA, Macur RE, Jay ZJ, Beam JP, Malfatti SA, Tringe SG, Kocar BD, Borch T, Inskeep WP (2012) Microbial iron cycling in acidic geothermal springs of Yellowstone National Park: integrating molecular surveys, geochemical processes, and isolation of novel Fe-active microorganisms. Front Microbiol 3:109
Kozubal MA, Romine M, Jennings R, Jay ZJ, Tringe SG, Rusch DB, Beam JP, McCue LA, Inskeep WP (2013) Geoarchaeota: a new candidate phylum in the Archaea from high-temperature acidic iron mats in Yellowstone National Park. ISME J 7:622–634
Kumar PS, Brooker MR, Dowd SE, Camerlengo T (2011) Target region selection is a critical determinant of community fingerprints generated by 16S Pyrosequencing. Plos One 6
Liu Z, Lozupone C, Hamady M, Bushman FD, Knight R (2007) Short pyrosequencing reads suffice for accurate microbial community analysis. Nucleic Acids Res 35:e120
Markowitz VM, Chen IM, Chu K, Szeto E, Palaniappan K, Grechkin Y, Ratner A, Jacob B, Pati A, Huntemann M, Liolios K, Pagani I, Anderson I, Mavromatis K, Ivanova NN, Kyrpides NC (2012) IMG/M: the integrated metagenome data management and comparative analysis system. Nucleic Acids Res 40:D123–D129
Meyer-Dombard DR, Price RE, Pichler T, Amend JP (2012) Prokaryotic populations in Arsenic-Rich Shallow-Sea Hydrothermal Sediments of Ambitle Island, Papua New Guinea. Geomicrobiology J 29:1–17
Meyer-Dombard DR, Amend JP, Osburn MR (2013) Microbial diversity and potential for arsenic and iron biogeochemical cycling at an arsenic rich, shallow-sea hydrothermal vent (Tutum Bay, Papua New Guinea). Chem Geol 348:37–47
Olsen GJ, Lane DJ, Giovannoni SJ, Pace NR, Stahl DA (1986) Microbial ecology and evolution: a ribosomal RNA approach. Annu Rev Microbiol 40:337–365
Quince C, Lanzen A, Davenport RJ, Turnbaugh PJ (2011) Removing noise from pyrosequenced amplicons. Bmc Bioinform 12
Rinke C, Schwientek P, Sczyrba A, Ivanova NN, Anderson IJ, Cheng JF, Darling A, Malfatti S, Swan BK, Gies EA, Dodsworth JA, Hedlund BP, Tsiamis G, Sievert SM, Liu WT, Eisen JA, Hallam SJ, Kyrpides NC, Stepanauskas R, Rubin EM, Hugenholtz P, Woyke T (2013) Insights into the phylogeny and coding potential of microbial dark matter. Nature 499:431–437
Schleper C, Jurgens G, Jonuscheit M (2005) Genomic studies of uncultivated archaea. Nat Rev Microbiol 3:479–488
Schloss PD, Westcott SL, Ryabin T, Hall JR, Hartmann M, Hollister EB, Lesniewski RA, Oakley BB, Parks DH, Robinson CJ, Sahl JW, Stres B, Thallinger GG, Van Horn DJ, Weber CF (2009) Introducing mothur: Open-source, Platform-Independent, Community-supported Software for Describing and Comparing Microbial Communities. Appl Environ Microbiol 75:7537–7541
Suzuki MT, Giovannoni SJ (1996) Bias caused by template annealing in the amplification of mixtures of 16S rRNA genes by PCR. Appl Environ Microbiol 62:625–630
Takacs-Vesbach C, Inskeep WP, Jay ZJ, Herrgard MJ, Rusch DB, Tringe SG, Kozubal MA, Hamamura N, Macur RE, Fouke BW, Reysenbach A-L, McDermott TR, Jennings Rd, Hengartner NW, Xie G (2013) Metagenome sequence analysis of filamentous microbial communities obtained from geochemically distinct geothermal channels reveals specialization of three aquificales lineages. Front Microbiol 4
Takai K, Sako Y (1999) A molecular view of archaeal diversity in marine and terrestrial hot water environments. FEMS Microbiol Ecol 28:177–188
Tamura K, Stecher G, Peterson D, Filipski A, Kumar S (2013) MEGA6: Molecular Evolutionary Genetics Analysis version 6.0. Mol Biol Evol 30:2725–2729
Acknowledgments
This work was funded by National Science Foundation (NSF) DEB0206773 and the Department of Energy-Joint Genome Institute Community Sequencing Program (CSP 787081). DC received additional support from NSF EHR0832947, RT from National Institutes of Health (NIH) R25 GM075149, and KM from the New Mexico Space Grant Consortium. Technical support for sequencing was provided by the University of New Mexico’s Molecular Biology Facility, which is supported by NIH grant P20GM103452 from the Institute Development Award Program of the National Center for Research Resources. We thank W. Inskeep for comments on the manuscript and providing PCR panel success results used in Table S1.
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Communicated by A. Oren.
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Colman, D.R., Thomas, R., Maas, K.R. et al. Detection and analysis of elusive members of a novel and diverse archaeal community within a thermal spring streamer consortium. Extremophiles 19, 307–313 (2015). https://doi.org/10.1007/s00792-014-0715-0
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DOI: https://doi.org/10.1007/s00792-014-0715-0