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

Number of bacterial cells: 2.6x10 29 Soil Torsvik et al., 2002 DNA reassociation method 10 4 different prokaryotic species of equivalent abundances (predicted). Gans et al., 2005 DNA reassociation method 10 7 microbial species per gram of soil (predicted). Roesh et al., 2007 pyrosequencing <10 4 species (detected) Number of species ??:

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

Rare biosphere. 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 Novelty and Uniqueness Patterns of Rare Members of the Soil Biosphere. AEM;74: 5422–542 Ashby et al Serial analysis of rRNA genes and the unexpected dominance of rare members of microbial communities. AEM 73:4532–4542. Pedros-Alio Marine microbial diversity: can it be determined. Trends Microbiol. 14:257–263. Sogin et al Microbial diversity in the deep sea and the underexplored “rare biosphere.” Proc. Natl. Acad. Sci. USA 103:12115–12120 Official definition

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

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

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? The right definition of the « Rare biosphere » in soil ? Rare bacteria or/and inaccessible bacteria or DNA?

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 »

Rare biosphere in soil ? Rare taxa ? Inaccessible bacteria, unavailable DNA ?

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

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

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

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

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

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

Sampling strategies Time of the year Depth Improvement of cell recovery (Nycodenz) Improvement of DNA recovery (sensitivity to lysis treatments) Improvement of DNA recovery (DNA degradation) Stringency of the lysis Bead beating Agarose plug Cell ring density Fraction 1 Fraction 4 Fraction 3 Fraction 2 PROKARYOTEsPROKARYOTEs EUKARYOTESEUKARYOTES Optimization of bacterial DNA recovery

16S rDNA MICROARRAY (8x15K) Agilent targets (> probes) Agilent technologies Cover all phylogenetic bacterial groups Lenght: 20 nucleotides

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

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

1. technological reproducibility2. comparison with an ocean 3. comparison with another soil 11.67% of functions statistically different (Bootstrap) 4. Cell lysis stringency effect 72.63%39.83% 34.69%

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

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

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

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

Extent of the soil bacterial diversity? 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 How to get it?

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

Congo: Black PointKenya: Embu MartiniqueNew Caledonia CSA Brévil Talmont St-Hilaire Chinon Montrond Nine soils selected

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

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

Inoculated CommunityRecipient Soil  « inoculated community » stronger effect than « recipient soil »  « Recipient Soils S7 and S9 »: stronger effect 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.

 A bacterial community inoculated into new (sterilized) soils reveals bacteria genera undetected in the original inoculum  Each inoculated community: Extent of the diversity increases when considering the different recipient soils. Are new taxa detected?

CS5 CS1 CS2 CS3 Cumulative percentage of newly detected genera (N max = 1475 = N genera/chip ) T2 = 6 months

Cumulative percentage of newly detected genera (N max = 1475 = N genera/chip ) S9 S2 S4 S7 S1 T2 = 6 months

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

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

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

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

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

 A bacterial community inoculated into new (sterilized) soils reveals bacteria genera undetected in the original inoculum  Each inoculated community: Extent of the diversity increases when considering the different recipient soils Are new taxa detected? the different incubation times the different extraction techniques… the different DNA analysis methods…

Italian forest soil / Rothamsted soil ( UK) Paolo Nannipieri Maria-Teresa Ceccherini Giacomo Pietramellara Davide FrancioliTom 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)

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.

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

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