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Environmental Microbial Genomics Group

Environmental Microbial Genomics Group Laboratoire Ampère . Ecole Centrale de Lyon . Université de Lyon. La biosphère rare du sol, définition, importance, rôle mais comment l’atteindre?. Pascal Simonet. Is there a limit to the extent of the rare (soil) biosphere?

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Environmental Microbial Genomics Group

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  1. Environmental Microbial Genomics Group LaboratoireAmpère . EcoleCentrale de Lyon . Université de Lyon La biosphère rare du sol, définition, importance, rôle mais comment l’atteindre? Pascal Simonet Is there a limit to the extent of the rare (soil) biosphere? Complete sequencing of the soil metagenome: An attainable utopia?

  2. Soil Number of bacterial cells: 2.6x1029 Number of species ??: Torsvik et al., 2002 DNA reassociation method 104 different prokaryotic species of equivalent abundances (predicted). Gans et al., 2005 DNA reassociation method 107 microbial species per gram of soil (predicted). Roesh et al., 2007 pyrosequencing <104 species (detected)

  3. Kessler Farm soil Distribution of various phyla Species distribution Rarefaction curve Novelty and Uniqueness Patterns of Rare Members of the Soil Biosphere. Elshahed et al., 2008 AEM: 74: 5422–5428

  4. Rare biosphere. Official definition Analysis of species distribution patterns usually indicates that while a significant fraction of bacterial biomass belongs to a relatively small number of species, the majority of bacterial species within a complex microbial community are present in extremely low numbers. • Elshahed et al. 2008. Novelty and Uniqueness Patterns of Rare Members of the Soil Biosphere. AEM;74: 5422–542 • Ashby et al 2007. Serial analysis of rRNA genes and the unexpected dominance of rare members of microbial communities. AEM 73:4532–4542. • Pedros-Alio 2006. Marine microbial diversity: can it be determined. Trends Microbiol. 14:257–263. • Sogin et al 2006. Microbial diversity in the deep sea and the underexplored “rare biosphere.” Proc. Natl. Acad. Sci. USA 103:12115–12120

  5. Role of the rare biosphere ? • Genes can be strongly expressed (numerous examples in the literature) • Rare taxa can become dominant when environmental conditions change • Rare taxa are a reservoir of transferable genetic information

  6. DNA microarrays Sequencing metagenome Fingerprints RARE BACTERIA The rare biosphere and sensitivity of techniques Threshold between abundant and rare bacteria ?? Novelty and Uniqueness Patterns of Rare Members of the Soil Biosphere. Elshahed et al., 2008 AEM: 74: 5422–5428

  7. The right definition of the « Rare biosphere » in soil ? Rare bacteria or/and inaccessible bacteria or DNA? Metagenome DNA extraction : • Soil heterogeneity • In situ lysis • Bacteria extraction (Nycodenz) • Cell lysis • DNA adsorption • DNA degradation • Cloning bias • PCR bias • Sequencing bias Rare, protected, lysis recalcitrant bacteria?

  8. Number of colonies increased with the stringency of the lysis treatment!! Recovery of added lambda phage DNA? Max. recovery: 25% Most treatments and soils: less than 10% The clay soil « A black hole »

  9. Rare biosphere in soil ? • Rare taxa ? • Inaccessible bacteria, unavailable DNA ?

  10. What is the rare biosphere ?? DNA extraction: critical bias !!!! Not only to determine the extent of the rare biosphere but this of bacterial diversity.

  11. What is the rare biosphere ?? What can we expect from sequencing? «  METAGENOMICS « 

  12. Genomics: “core-genome” : the genes existing in all strains “dispensable genome” : genes present in two or more strains and genes unique to single strains “pan-genome” : “core-genome” + “dispensable genome” Given that the number of unique genes is vast, the pan-genome of a bacterial species might be orders of magnitude larger than any single genome.

  13. Soil metagenomics Core-metagenome : genes existing in all soils Core-metapopulation : species found in all soils Specific-metagenome : genes present in two or more soils and genes unique to single soils Specific-metapopulation : species « «« and species «  «  Pan-metagenome : Core-metagenome + Specific metagenome Pan-metapopulation :Core-metapopulation + Specific metapopulation Fundamental questions: The actual ratio Pan/Core (the actual size of specific)

  14. Specific-meta-(genome/pop.) Specific-meta-(genome/pop.) Specific-meta-(genome/pop.) Specific-meta-(genome/pop.)) Soil 3 Soil 1 Soil 2 Soil 5 Soil Core-metagenome Core-metapopulation Rare and very numerous species

  15. Core meta-(genome/population) Core = Pan Pan-meta-(genome/population) Pan-meta-(genome/population) Pan-meta-(genome/population) Soil 3 Soil 1 Soil 2 Everything is everywhere ! Only distribution differs «everything is everywhere, but, the environment selects» (Bas-Becking)

  16. Core meta-(genome/population) Soil Core-metagenome Core-metapopulation Core Pan-meta-(genome/population) Pan-meta-(genome/population) Pan-meta-(genome/population) Pan-meta-(genome/population) Pan-meta-(genome/population) Pan-meta-(genome/population) Pan-meta-(genome/population) Soil 3 Soil 2 Soil 1 Soil 3 Soil 5 Soil 1 Soil 2 Rare and very numerous species: Do they really matter?

  17. Metasoil Project The initial support for Terragenome (complete sequencing of a reference soil metagenome) : • Objective: • Optimization of bacterial DNA recovery. • Metagenomic DNA library construction • Pyrosequencing of directly extracted DNA Park Grass, Rothamsted: an internationally recognized agroecology field experiment for 150 years

  18. Fraction 4 Fraction 3 Fraction 2 Fraction 1 Cell ring density Optimization of bacterial DNA recovery Improvement of DNA recovery (sensitivity to lysis treatments) • Sampling strategies • Time of the year • Depth Improvement of cell recovery (Nycodenz) Improvement of DNA recovery(DNA degradation) P R O K A R Y O T E s E U K A R Y O T E S Bead beating Agarose plug Stringency of the lysis

  19. 16S rDNA MICROARRAY • Agilent technologies • Lenght: 20 nucleotides • 3 186 targets (>20 000 probes) • Cover all phylogenetic bacterial groups (8x15K) Agilent

  20. Density gradient Lysis stringency Sampling DNA size Phylochip probes intensity Bacterial genera Undetected with one DNA extraction method

  21. Rothamsted soil phylochip saturation curve 15 DNA extraction methods (about 99% of probes) Only one DNA extraction method (~40% of probes)

  22. Functional comparison using MG RAST annotation and STAMP statistical analyses 1. technological reproducibility 2. comparison with an ocean 3. comparison with another soil 11.67%of functionsstatisticallydifferent (Bootstrap) 72.63% 39.83% 4. Cell lysis stringency effect 34.69%

  23. Metasoil Project Park Grass: Rothamsted Rare biosphere and pyrosequencing sensitivity ?? Redundancy of sequences in the DNA solution • Metagenomic DNA library construction: 2 000 000 clones • (16 000 equ. bacterial genomes) • Pyrosequencing of metagenome DNA: 60 runs (depth, lysis, season etc.) • 60Gbp (15 000 equ. bacterial genomes) Sufficient effort to reach the rare biosphere???

  24. Cloning DNA Extraction Transformation vector Clone Library PCR Culture in vitro Cloning and/or sequencing RISA, T-RFLP, DGGE, Phylochip Functionalmicroarrays METAGENOME EXPLOITATION Domesticated bacterial host Direct or indirect Direct Sequencing (454) Molecular screening Chemical screening Biological screening Hybridization based gene detection Chemical structure of produced compounds Direct detection of enzymatic activity Cultivable bacteria: less than 1% Lombard et al., 2006

  25. Molecular screening Hybridization screening of metagenomic DNA libraries Metagenomic DNA library construction December 2010: 2 000 000 clones (16 000 equ. bacterial genomes)

  26. SOIL MICROFLORA Abundant/Rare taxa ? The right question ? Extent of the Soil Bacterial Diversity ….independently of the species distribution ?

  27. Extent of the soil bacterial diversity? How to get it? • Genes can be strongly expressed (numerous examples in the literature) • Rare (or unavailable) taxa can become dominant (or accessible) when environmental conditions change • Rare taxa are a reservoir of transferable genetic information

  28. INTRODUCTION Conceptual approach: Provide new developing conditions to soil bacterial communities Bacterial community extracted from soil A Soil A Sterilized Soil B or Diversity in soil A

  29. Nine soils selected Brévil TalmontSt-Hilaire Chinon CSA Montrond Martinique New Caledonia Kenya: Embu Congo: Black Point

  30. CONCEPTUALAPPROACH 1. Extractionof the 9 bacterial communities Nycodenz density gradient 2. Inoculation of each bacterial community into the nine sterilized soils 3. Incubation at RT for 1 day, 2 months, 6 months 4. Monitoring of bacterial community structure evolution (direct DNA extraction, PCR and phylochip)

  31. Two questions: • Are new developing community structures different from the donor ones and from these of the recipient soils? • Are new taxa detected?

  32. Are new developing community structures different from the original donor one and from the one of the recipient soil? Yes: With both a recipient soil and an inoculated community structuring effect. Inoculated Community Recipient Soil • « inoculated community » stronger effect than « recipient soil » • « RecipientSoilsS7 and S9 »: strongereffect

  33. Are new taxa detected? • A bacterialcommunityinoculatedinto new (sterilized) soilsrevealsbacteriageneraundetected in the original inoculum • Eachinoculatedcommunity: Extent of the diversityincreaseswhenconsidering the differentrecipientsoils.

  34. Cumulative percentage of newly detected genera (Nmax = 1475 = Ngenera/chip) CS1 CS2 CS3 CS5 T2 = 6 months

  35. Cumulative percentage of newly detected genera (Nmax = 1475 = Ngenera/chip) S1 S2 S4 S7 S9 T2 = 6 months T2 = 6 months

  36. Cumulative percentage of newly detected genera (Nmax = 1475 = Ngenera/chip) T2 = 6 months 55% (max) of the characterized genera detected (9 soils) Rarefaction curves show a limit Conclusion: Diversity in the rare biosphere very limited?

  37. Cumulative percentage of newly detected genera (Nmax = 1475 = Ngenera/chip) T2 = 6 months However: Diversity of conditions offered by the recipient sterilized soils?

  38. Cumulative percentage of genera detected at T0 + T1 + T2 CS: Extracted (and inoculated) community T0: 1 day T1: 2 months T2: 6 months T0 + T1 +T2 T2 only Genera detected in CS and not later Genera detected at T0, T1, T2 and not in CS Genera detected only at T1

  39. Cumulative percentage of newly detected genera All soil communities (n=4) All sampling times (n=3) Individual communities 1 sampling time (6 months)

  40. Rothamsted soil phylochip saturation curve 15 DNA extraction approaches (about 99% of probes) One DNA extraction approach (~40% of probes)

  41. Are new taxa detected? • A bacterialcommunityinoculatedinto new (sterilized) soilsrevealsbacteriageneraundetected in the original inoculum • Eachinoculatedcommunity: Extent of the diversityincreaseswhenconsideringthe differentrecipientsoils the different incubation times the different extraction techniques… the different DNA analysis methods…

  42. Italian forest soil /Rothamsted soil (UK) Paolo Nannipieri Maria-Teresa Ceccherini Giacomo Pietramellara Davide Francioli Tom Delmont Dipartimento di Scienza del Suolo e Nutrizione della Pianta, Universita` degli Studi di Firenze, Firenze, Italy • Identification of « Italy » and « Rothamsted » specific bacteria. • (Taxonomic microarrays/454/Illumina)

  43. Extent of the bacterial (soil) diversity / extent of the soil (rare) biosphere? Combination of conceptual and methodological approaches. Conceptual approach: Increase the range of conditions offered to developing communities Methodological approach: Phylogenetic microarrays: Limited by the number of probes and specificity /sensitivity of hybridization. Pyrosequencing approaches required.

  44. Conclusion Diversity of Bacteria (rare and abundant) : Huge Attainable if • Collaboration at the international level • Focus on one « reference » soil

  45. Aurélie Faugier, Sébastien Cécillon, Davide Francioli, Tom Delmont, Emmanuel Prestat, Jean-Michel Monier, Timothy M Vogel, Environmental Microbial Genomics www.GenomEnviron.org

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