IP5: High-resolution Microbial Community Structure

People: Christa Schleper (PI), Vigdis Torsvik (co-PI), Tim Urich (co-PI), Antje Gittel, Ricardo Alves

Background

It is known that soil ecosystems harbour the most complex microbial ecosystems on Earth, but very little is known about their composition and structure which is particularly true for permafrost soils. The flexibility of the microbial community in permafrost to react on temperature fluctuation and other physical and chemical changes will determine their reaction to cryoturbation. Thus, the genetic potential of the microorganisms present in permafrost is likely to determine whether permafrost environments will become net sources or sinks of greenhouse gases following warming.

In-depth description of the abundance, diversity, distribution and activity of microbial communities in different transects of cryoturbated Siberian permafrost (Cherskyi, Taymyr, Vorkuta) will therefore provide comparative data to dissect the major group of organisms active in carbon turnover. It will also shed light on organisms that are highly adapted/sensitive to temperature and climate changes or to secondary effects imposed by cryoturbation.

Aims and objectives

  • To provide a comprehensive, and detailed picture of the microbial community structure (diversity & abundance) in permafrost soils of different geographic regions and physical settings
  • To dissect differences in the community structure of cryoturbated versus unturbated soils (including organic surface horizons, cryoturbated soil, adjacent mineral horizons and upper permafrost soil) and identify the dominant groups involved in carbon and nitrogen cycling
  • To enlighten the role of Thaumarchaeota in carbon and nitrogen cycling in Arctic soils
  • To gain deeper insights into the abundance, diversity and activity of methanogens and methanotrophs in Arctic Soil
  • To analyse changes of microbial communities during laboratory incubations (experiment E1) and during an in situ cryoturbation experiment (E2).

Methodology

Comprehensive in-depth community profiling

The microbial community structure will be analysed using a variant of the barcoding strategy used for 454 sequencing and the Illumina GAIIx platform (Hamady et al. Nature Methods 2008; Caporaso et al. PNAS 2010). In detail, PCR products from the V4 hypervariable region of rRNA will be obtained from purified DNA extracts, pooled prior to sequencing and individual sequences from the different samples will be subsequently binned based on artificial, sample-specific barcode sequences.

Despite the limitiation in read length (single reads 75-100 bp, paired reads 150-200 bp), it has recently been shown that this approach is sufficient for large-scale comparisons among microbial communities at different scales (Liu et al. Nucl Acid Res 2007, Caporaso et al. PNAS 2010). Together with the tremendous amount of sequence reads per sample, we aim for unprecedented spatial resolution capturing different geographic regions and physical settings. Together with IP4, which will contribute diversity and abundance data for fungi, we will obtain integrated data for all microbial groups relevant in SOM degradation.

This sequencing effort will be part of and in close collaberation with the Earth Microbiome Project (http://www.earthmicrobiome.org/) whose initiators aim to collect and sequence up to 200,000 environmental samples to produce a global Gene Atlas and thereby charaterize the global microbial taxonomic and functional diversity of the Earth’s microbiome.

Quantification of ribosomal RNA genes and functional genes involved in carbon and nitrogen cycling

To complement the data obtained from the amplicon sequencing approach and to gain further information about the community structure,  SybrGreen I-based quantitative PCR (qPCR) targeting 16S rRNA of total Bacteria and total Archaea will be applied to samples from all transects. Furthermore, genes involved in carbon and nitrogen cycling (e.g., methane production and oxidation, nitrification) will be specifically detected and quantified.

Metatranscriptomics

The analysis of the total RNA pool of a community allows to simultaneously characterize the taxonomic community composition, metabolic gene content and gain information about gene expression. Thus, metatranscriptomic analyses offer the opportunity to reach beyond the community’s genomic potential as assessed in DNA-based methods, towards its in situ activity. The total RNA pool is naturally enriched not only in functionally, but also taxonomically relevant molecules, i.e. messenger RNA (mRNA) and ribosomal RNA (rRNA), respectively.
We have previously established appropriate experimental and analytical procedures (the “double-RNA” approach, Urich et al., 2008) for in-depth characterization of microbial communities by studying mRNA and rRNA molecules simultaneously from the same sample. In PCR-independent community profiling of all three domains of life can thus be linked to information on the active state of these communities.

This approach will be applied to (i) selected samples of the transect sampling (4 different soil horizons: upper organic soil layer, a representative cryoturbated layer, an adjacent mineral soil layer, and a sample from the upper permafrost) and (ii) samples form the two experimental set-ups (decomposition experiment, E1 and cryoturbation experiment, E2).

Cooperations

Earth Microbiome Project (EMP): Jack Gilbert and Janet Jansson (Berkeley Lab Earth Science Division) (Argonne National Labs)

References

Caporaso, J. G., Lauber, C. L., Walters, W. A., Berg-Lyons, D., Lozupone, C. A., Turnbaugh, P. J., Fierer, N., and Knight, R. (2010). Global patterns of 16S rRNA diversity at a depth of millions of sequences per sample. Proc Nat Acad Sci. doi: 10.1073/pnas.1000080107.

Hamady, M., Walker, J. J., Harris, J. K., Gold, N. J., and Knight, R. (2008). Error-correcting barcoded primers for pyrosequencing hundreds of samples in multiplex. Nature Methods 5: 235-237.

Liu, Z., Lozupone, C. A., Hamady, M., Bushman, F. D. and Knight, R. (2007). Short pyrosequencing reads suffice for accurate microbial community analysis. Nucl Acids Res. 35: e120, 1-10.

Urich, T., Lanzén, A., Qi, J., Huson, D. H., Schleper, C., and Schuster, S. C. (2008). Simultaneous Assessment of Soil Microbial Community Structure and Function through Analysis of the Meta-Transcriptome. PLoS one 3: e2527