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?
Environmental Microbial Genomics Group
Laboratoire Ampère . Ecole Centrale de Lyon . Université de Lyon

Soil
Number of bacterial cells: 2.6x10
29
Number of species ??:
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)

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

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

•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
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

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?

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

• Rare taxa ?
• Inaccessible bacteria, unavailable DNA ?

Not only to determine the extent of the rare biosphere but this of bacterial diversity.

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.
12

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)
13

Soil
Core-metagenome
Core-metapopulation
Rare and very numerous species 14

Everything is everywhere !
Only distribution differs
« everything is everywhere, but, the environment selects » (Bas-Becking)
15

Soil
Core-metagenome
Core-metapopulation
Rare and very numerous species: Do they really matter?
16

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

Optimization of bacterial DNA recovery
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) density
Fraction 4
Fraction 3
Fraction 2
Fraction 1
Cell ring
Y
O
A
R
O
K
P
R
T
E s
Y
O
A
R
E
U
K
T
E
S
Bead beating
Agarose plug
Stringency of the lysis

• Agilent technologies
• Lenght: 20 nucleotides
• 3 186 targets (>20 000 probes)
• Cover all phylogenetic bacterial groups
(8x15K) Agilent

25
20
15
10
5
0
Bacterial genera
Sampling Density gradient
Fraction 4
Fraction 3
Fraction 2
Density
Fraction 1
Cell ring
Number of cells
Lysis
Eukaryotes (density > 1.3) Soft Lyses
DNA size
Undetected with one DNA extraction method

Rothamsted soil phylochip saturation curve
100
90
80
70
60
50
40
30
20
10
0
0
15 DNA extraction methods (about 99% of probes)
Only one DNA extraction method (
~
40% of probes)
2000 4000 6000 8000 10000 12000 14000 16000 18000 20000
Number of probes

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 functions statistically different (Bootstrap)
72.63% 39.83%
4. Cell lysis stringency effect
34.69%

Park Grass: Rothamsted
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)

METAGENOME EXPLOITATION
Domesticated bacterial host
Cloning
Direct
Sequencing
(454)
DNA
Direct or indirect
Extraction
PCR vector
Transformation
Clone Library
Culture in vitro
Cloning and/or sequencing
Cultivable bacteria: less than 1%
Molecular screening
Chemical screening
OMe
CH
3
OMe
O
CH
3
O
O
CH
OH
OMe
OH
3
Biological screening
RISA, T-RFLP, DGGE,
Phylochip
Functional microarrays
Hybridization based gene detection Chemical structure of produced compounds
Direct detection of enzymatic activity
Lombard et al ., 2006

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

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

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

Provide new developing conditions to soil bacterial communities
Bacterial community extracted from soil A
Soil A
80
60
40
20
0
160
140
120
100
1 2 3 4
Diversity in soil A
5 6
160
140
120
100
80
60
40
20
0
1 2 3
160
140
120
100
80
60
40
4
20
0
Sterilized Soil B
6 2 or
4
140
120
100
80
60
40
20
0
5 1 6 2 3 3 4 5 6

CSA
Brévil
Nine soils selected
Talmont St-Hilaire Chinon
Montrond
Martinique Kenya: Embu Congo: Black Point New Caledonia

1.
Extraction of 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)

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

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 »
 « Recipient Soils S7 and S9 »: stronger 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.

Cumulative percentage of newly detected genera (N max
= 1475 = N genera/chip
)
50
45
40
35
60
55
30
25
20
0 1 2 3 4 5 6
Number of soils
T2 = 6 months
7 8 9
CS1
CS2
CS3
CS5

Cumulative percentage of newly detected genera (N max
= 1475 = N genera/chip
)
60
55
50
45
40
35
30
25
20
15
10
T2 = 6 months
0 1 2 3 4
Number of soil communities
5
140
120
100
80
60
40
20
T2 = 6 months
0
0 1 2 3 4
Number of soil communities
5
S1
S2
S4
S7
S9

60
55
50
45
40
35
30
25
20
0 1 2
T2 = 6 months
7 8 9 3 4 5 6
Number of soils
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?

60
55
50
45
40
35
30
25
20
0 1 2
T2 = 6 months
7 8 9 3 4 5 6
Number of soils
Cumulative percentage of newly detected genera (N max
= 1475 = N genera/chip
)
However:
Diversity of conditions offered by the recipient sterilized soils?

Cumulative percentage of genera detected at T0 + T1 + T2
60
55
50
45
40
35
30
25
20
15
10
T2 only
0 1 2 3 4
Number of soil communities
5
70
60
50
40
30
20
T0 + T1 +T2
10
1 2 3 4
Number of soil communities
5
CS: Extracted (and inoculated) community
T0: 1 day
T1: 2 months
T2: 6 months
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
90
All soil communities (n=4)
All sampling times (n=3)
80
70
60
50
40
30
20
1 2 3 4 5 6 7 8 9
Number of soils
Individual communities
1 sampling time (6 months)

Rothamsted soil phylochip saturation curve
100
90
80
70
60
50
40
30
20
10
0
0
15 DNA extraction approaches (about
99% of probes)
One DNA extraction approach (
~
40% of probes)
2000 4000 6000 8000 10000 12000 14000 16000 18000 20000
Number of probes

 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 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 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)

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.

•Collaboration at the international level
•Focus on one « reference » soil

Aurélie Faugier, Sébas tien Cécillon,
Davide Francioli, Tom Delmont ,
Emmanuel Prestat, Jean -Michel
Monier, Timothy M Vogel,