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Assessment of prokaryote to eukaryote ratios in environmental samples by SSU rDNA length polymorphism

  • Alexander GuhrEmail author
  • Alfons R. Weig
Original Paper

Abstract

Microbial communities are important regulators of many processes in all ecosystems. Understanding of ecosystem processes requires at least an overview of the involved microorganisms. While in-depth identification of microbial species in environmental samples can be achieved by next generation sequencing, profiling of whole microbial communities can be accomplished via less labour-intensive approaches. Especially automated ribosomal intergenic spacer analysis (ARISA) are of interest as they are highly specific even at fine scales and widely applicable for environmental samples. Yet, established protocols lack the possibility to compare prokaryotic and eukaryotic communities as different primer sets are necessary. However, shifts in the eukaryote to prokaryote ratio can be a useful indicator for ecosystem processes like decomposition or nutrient cycling. We propose a protocol to analyse prokaryotic and eukaryotic communities using a single primer pair based reaction based on a region with variable length (V4, which is about 180 bp shorter in prokaryotes compared to eukaryotes) in the small ribosomal subunit flanked by two highly conservative regions. Shifts in the prokaryotic and eukaryotic ratio between samples can be reliably detected by fragment length polymorphism analysis as well as sequencing of this region. Together with established approaches such as ARISA or 16S and ITS rDNA sequencing, this can provide a more complex insight into microbial community shifts and ecosystem processes.

Keywords

ARISA Environmental samples Metagenome sequencing Microbial communities Prokaryotic to eukaryotic ratios 

Notes

Acknowledgements

We thank M. Hochholzer, A. Kirpal, K. Söllner, U. Hell, C. Knaus and J. Kannieß for help with sample preparation and laboratory work.

Author contributions

AG conceived the project. AG and ARW performed the experiments, analysed the data, and prepared the manuscript.

Funding

This study was supported by Deutsche Forschungsgemeinschaft Grant (DFG-GU 1818/1-1).

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Human and animal rights

This article does not include any studies involving human participants or animals.

Supplementary material

10482_2019_1327_MOESM1_ESM.pdf (211 kb)
Supplementary material 1 (PDF 210 kb)

References

  1. Achat DL, Bakker MR, Saur E, Pellerin S, Augusto L, Morel C (2010) Quantifying gross mineralisation of P in dead soil organic matter: testing an isotopic dilution method. Geoderma 158:163–172.  https://doi.org/10.1016/j.geoderma.2010.04.027 CrossRefGoogle Scholar
  2. Bailey VL, Smith JL, Bolton H (2002) Fungal-to-bacterial ratios in soils investigated for enhanced C sequestration. Soil Biol Biochem 34:997–1007.  https://doi.org/10.1016/S0038-0717(02)00033-0 CrossRefGoogle Scholar
  3. Bardgett RD, McAlister E (1999) The measurement of soil fungal: bacterial biomass ratios as an indicator of ecosystem self-regulation in temperate meadow grasslands. Biol Fertil Soils 29:282–290.  https://doi.org/10.1007/s003740050554 CrossRefGoogle Scholar
  4. Bardgett RD, Hobbs PJ, Frostegård Å (1996) Changes in soil fungal: bacterial biomass ratios following reductions in the intensity of management of an upland grassland. Biol Fertil Soils 22:261–264.  https://doi.org/10.1007/BF00382522 CrossRefGoogle Scholar
  5. Blagodatskaya EV, Anderson T-H (1998) Interactive effects of pH and substrate quality on the fungal-to-bacterial ratio and qCO2 of microbial communities in forest soils. Soil Biol Biochem 30:1269–1274.  https://doi.org/10.1016/S0038-0717(98)00050-9 CrossRefGoogle Scholar
  6. Boer Wd, Folman LB, Summerbell RC, Boddy L (2005) Living in a fungal world: impact of fungi on soil bacterial niche development. FEMS Microbiol Rev 29:795–811.  https://doi.org/10.1016/j.femsre.2004.11.005 CrossRefGoogle Scholar
  7. Bolyen E, Rideout JR, Dillon MR, Bokulich NA, Abnet CC, Al-Ghalith GA, Alexander H, Alm EJ, Arumugam M, Asnicar F, Bai Y, Bisanz JE, Bittinger K, Brejnrod A, Brislawn CJ, Brown CT, Callahan BJ, Caraballo-Rodríguez AM, Chase J, Cope EK, Da Silva R, Diener C, Dorrestein PC, Douglas GM, Durall DM, Duvallet C, Edwardson CF, Ernst M, Estaki M, Fouquier J, Gauglitz JM, Gibbons SM, Gibson DL, Gonzalez A, Gorlick K, Guo J, Hillmann B, Holmes S, Holste H, Huttenhower C, Huttley GA, Janssen S, Jarmusch AK, Jiang L, Kaehler BD, Kang KB, Keefe CR, Keim P, Kelley ST, Knights D, Koester I, Kosciolek T, Kreps J, Langille MGI, Lee J, Ley R, Liu Y-X, Loftfield E, Lozupone C, Maher M, Marotz C, Martin BD, McDonald D, McIver LJ, Melnik AV, Metcalf JL, Morgan SC, Morton JT, Naimey AT, Navas-Molina JA, Nothias LF, Orchanian SB, Pearson T, Peoples SL, Petras D, Preuss ML, Pruesse E, Rasmussen LB, Rivers A, Robeson MS, Rosenthal P, Segata N, Shaffer M, Shiffer A, Sinha R, Song SJ, Spear JR, Swafford AD, Thompson LR, Torres PJ, Trinh P, Tripathi A, Turnbaugh PJ, Ul-Hasan S, van der Hooft JJJ, Vargas F, Vázquez-Baeza Y, Vogtmann E, von Hippel M, Walters W, Wan Y, Wang M, Warren J, Weber KC, Williamson CHD, Willis AD, Xu ZZ, Zaneveld JR, Zhang Y, Zhu Q, Knight R, Caporaso JG (2019) Reproducible, interactive, scalable and extensible microbiome data science using QIIME 2. Nat Biotechnol 37:852–857.  https://doi.org/10.1038/s41587-019-0209-9 CrossRefGoogle Scholar
  8. Bünemann EK, Oberson A, Liebisch F, Keller F, Annaheim KE, Huguenin-Elie O, Frossard E (2012) Rapid microbial phosphorus immobilization dominates gross phosphorus fluxes in a grassland soil with low inorganic phosphorus availability. Soil Biol Biochem 51:84–95.  https://doi.org/10.1016/j.soilbio.2012.04.012 CrossRefGoogle Scholar
  9. Cardinale M, Brusetti L, Quatrini P, Borin S, Puglia AM, Rizzi A, Zanardini E, Sorlini C, Corselli C, Daffonchio D (2004) Comparison of different primer sets for use in automated ribosomal intergenic spacer analysis of complex bacterial communities. Appl Environ Microbiol 70:6147–6156.  https://doi.org/10.1128/AEM.70.10.6147-6156.2004 CrossRefGoogle Scholar
  10. Dickie IA, Xu B, Koide RT (2002) Vertical niche differentiation of ectomycorrhizal hyphae in soil as shown by T-RFLP analysis. New Phytol 156:527–535.  https://doi.org/10.1046/j.1469-8137.2002.00535.x CrossRefGoogle Scholar
  11. Dinh M-V, Guhr A, Weig AR, Matzner E (2018) Drying and rewetting of forest floors: dynamics of soluble phosphorus, microbial biomass-phosphorus, and the composition of microbial communities. Biol Fertil Soils 54:761–768.  https://doi.org/10.1007/s00374-018-1300-y CrossRefGoogle Scholar
  12. Dupont AÖC, Griffiths RI, Bell T, Bass D (2016) Differences in soil micro-eukaryotic communities over soil pH gradients are strongly driven by parasites and saprotrophs. Environ Microbiol 18:2010–2024.  https://doi.org/10.1111/1462-2920.13220 CrossRefGoogle Scholar
  13. Fierer N, Jackson JA, Vilgalys R, Jackson RB (2005) Assessment of soil microbial community structure by use of taxon-specific quantitative PCR assays. Appl Environ Microbiol 71:4117–4120.  https://doi.org/10.1128/AEM.71.7.4117-4120.2005 CrossRefGoogle Scholar
  14. Fisher MM, Triplett EW (1999) Automated approach for ribosomal intergenic spacer analysis of microbial diversity and its application to freshwater bacterial communities. Appl Environ Microbiol 65:4630–4636Google Scholar
  15. Gerstberger P, Foken T, Kalbitz K (2004) The Lehstenbach and Steinkreuz catchments in NE Bavaria, Germany. In: Matzner E (ed) Biogeochemistry of forested catchments in a changing environment: a German case study. Springer, Berlin, pp 15–41CrossRefGoogle Scholar
  16. Goberna M, García C, Insam H, Hernández MT, Verdú M (2012) Burning fire-prone Mediterranean shrublands: immediate changes in soil microbial community structure and ecosystem functions. Microb Ecol 64:242–255.  https://doi.org/10.1007/s00248-011-9995-4 CrossRefGoogle Scholar
  17. Hernando-Morales V, Varela MM, Needham DM, Cram J, Fuhrman JA, Teira E (2018) Vertical and seasonal patterns control bacterioplankton communities at two horizontally coherent coastal upwelling sites off Galicia (NW Spain). Microb Ecol 76:866–884.  https://doi.org/10.1007/s00248-018-1179-z CrossRefGoogle Scholar
  18. Högberg MN, Högberg P, Myrold DD (2007) Is microbial community composition in boreal forest soils determined by pH, C-to-N ratio, the trees, or all three? Oecologia 150:590–601.  https://doi.org/10.1007/s00442-006-0562-5 CrossRefGoogle Scholar
  19. Isenbarger TA, Carr CE, Johnson SS, Finney M, Church GM, Gilbert W, Zuber MT, Ruvkun G (2008) The most conserved genome segments for life detection on Earth and other planets. Orig Life Evol Biosph 38:517–533.  https://doi.org/10.1007/s11084-008-9148-z CrossRefGoogle Scholar
  20. Lane DJ, Pace B, Olsen GJ, Stahl DA, Sogin ML, Pace NR (1985) Rapid determination of 16S ribosomal RNA sequences for phylogenetic analyses. Proc Natl Acad Sci USA 82:6955–6959.  https://doi.org/10.1073/pnas.82.20.6955 CrossRefGoogle Scholar
  21. Lauber CL, Hamady M, Knight R, Fierer N (2009) Pyrosequencing-based assessment of soil pH as a predictor of soil bacterial community structure at the continental scale. Appl Environ Microbiol 75:5111–5120.  https://doi.org/10.1128/AEM.00335-09 CrossRefGoogle Scholar
  22. Lee JC, Gutell RR (2012) A comparison of the crystal structures of eukaryotic and bacterial SSU ribosomal RNAs reveals common structural features in the hypervariable regions. PLoS ONE 7:e38203.  https://doi.org/10.1371/journal.pone.0038203 CrossRefGoogle Scholar
  23. Lou J, Yang L, Wang H, Wu L, Xu J (2018) Assessing soil bacterial community and dynamics by integrated high-throughput absolute abundance quantification. PeerJ 6:e4514.  https://doi.org/10.7717/peerj.4514 CrossRefGoogle Scholar
  24. Malik AA, Chowdhury S, Schlager V, Oliver A, Puissant J, Vazquez PGM, Jehmlich N, von Bergen M, Griffiths RI, Gleixner G (2016) Soil fungal: bacterial ratios are linked to altered carbon cycling. Front Microbiol 7:1247.  https://doi.org/10.3389/fmicb.2016.01247 CrossRefGoogle Scholar
  25. Popa R, Popa R, Mashall MJ, Nguyen H, Tebo BM, Brauer S (2009) Limitations and benefits of ARISA intra-genomic diversity fingerprinting. J Microbiol Methods 78:111–118.  https://doi.org/10.1016/j.mimet.2009.06.005 CrossRefGoogle Scholar
  26. Ranjard L, Poly F, Lata J-C, Mougel C, Thioulouse J, Nazaret S (2001) Characterization of bacterial and fungal soil communities by automated ribosomal intergenic spacer analysis fingerprints: biological and methodological variability. Appl Environ Microbiol 67:4479–4487.  https://doi.org/10.1128/AEM.67.10.4479-4487.2001 CrossRefGoogle Scholar
  27. Read DJ, Perez-Moreno J (2003) Mycorrhizas and nutrient cycling in ecosystems—a journey towards relevance? New Phytol 157:475–492.  https://doi.org/10.1046/j.1469-8137.2003.00704.x CrossRefGoogle Scholar
  28. Rousk J, Brookes PC, Bååth E (2009) Contrasting soil pH effects on fungal and bacterial growth suggest functional redundancy in carbon mineralization. Appl Environ Microbiol 75:1589–1596.  https://doi.org/10.1128/AEM.02775-08 CrossRefGoogle Scholar
  29. Strickland MS, Rousk J (2010) Considering fungal: bacterial dominance in soils—methods, controls, and ecosystem implications. Soil Biol Biochem 42:1385–1395.  https://doi.org/10.1016/j.soilbio.2010.05.007 CrossRefGoogle Scholar
  30. Turner S, Pryer KM, Miao VPW, Palmer JD (1999) Investigating deep phylogenetic relationships among cyanobacteria and plastids by small subunit rRNA sequence analysis. J Eukaryotic Microbiology 46:327–338.  https://doi.org/10.1111/j.1550-7408.1999.tb04612.x CrossRefGoogle Scholar
  31. van der Heijden MGA, Bakker R, Verwaal J, Scheublin TR, Rutten M, van Logtestijn R, Staehelin C (2006) Symbiotic bacteria as a determinant of plant community structure and plant productivity in dune grassland. FEMS Microbiol Ecol 56:178–187.  https://doi.org/10.1111/j.1574-6941.2006.00086.x CrossRefGoogle Scholar
  32. van der Heijden MGA, Bardgett RD, van Straalen NM (2008) The unseen majority: soil microbes as drivers of plant diversity and productivity in terrestrial ecosystems. Ecol Lett 11:296–310.  https://doi.org/10.1111/j.1461-0248.2007.01139.x CrossRefGoogle Scholar
  33. Voříšková J, Baldrian P (2013) Fungal community on decomposing leaf litter undergoes rapid successional changes. ISME J 7:477–486.  https://doi.org/10.1038/ismej.2012.116 CrossRefGoogle Scholar
  34. Weig AR, Peršoh D, Werner S, Betzlbacher A, Rambold G (2013) Diagnostic assessment of mycodiversity in environmental samples by fungal ITS1 rDNA length polymorphism. Mycol Progress 12:719–725.  https://doi.org/10.1007/s11557-012-0883-1 CrossRefGoogle Scholar
  35. West AW (1986) Improvement of the selective respiratory inhibition technique to measure eukaryote: prokaryote ratios in soils. J Microbiol Methods 5:125–138.  https://doi.org/10.1016/0167-7012(86)90008-4 CrossRefGoogle Scholar
  36. White T, Bruns T, Lee S, Taylor J (1990) Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: Innis MA, Gelfand DH, Sninsky JJ, White TJ (eds) PCR Protocols: a guide to methods and applications. Academic Press, London, pp 315–322Google Scholar

Copyright information

© Springer Nature Switzerland AG 2019

Authors and Affiliations

  1. 1.Department of Soil Ecology, BayCEERUniversity of BayreuthBayreuthGermany
  2. 2.Keylab Genomics and Bioinformatics, BayCEERUniversity of BayreuthBayreuthGermany

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