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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: 2.6x1029
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)
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
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
RARE BACTERIA
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 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 «
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
Core = Pan
Everything is everywhere !
Only distribution differs
«everything is everywhere, but, the environment selects» (Bas-Becking)
15
Soil
Core-metagenome
Core-metapopulation
Core
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
Improvement of
cell recovery
(Nycodenz)
Improvement
of DNA recovery
(sensitivity to lysis
treatments)
•Depth
Fraction 4
P
R
O
Fraction 3
K
A
R
Fraction 2
Y
O
Fraction 1
Cell ring
T
E
s
E
U
K
A
Bead beating
R
Y
O
Agarose plug
T
density
E
S
Stringency of the lysis
Improvement
of DNA recovery
(DNA degradation)
16S rDNA MICROARRAY
• Agilent technologies
• Lenght: 20 nucleotides
• 3 186 targets (>20 000 probes)
• Cover all phylogenetic bacterial
groups
(8x15K) Agilent
Bacterial genera
Dietzia
Coxiella
Clostridium
Achromatium
Henriciella
Schlegelella
Butyricimonas
Tetrasphaera
Sinorhizobium
Microbacterium
Rhodanobacter
Megasphaera
Actinocorallia
Methylomonas
Rhodobacter
Desulfonauticus
Azorhizobium
Candidatus Cardinium
Salmonella
Caldanaerobacter
Phormidium
Methylovorus
Fervidococcus
Chromohalobacter
Staphylococcus
Edaphobacter
Actinopolyspora
Aquabacterium
Caldithrix
Chloroflexus
Demequina
Flavobacterium
Halonatronum
Hyphomicrobium
Lactobacillus
Methanoculleus
Mycobacterium
Nonomuraea
Paucisalibacillus
Pseudoxanthomonas
Sandarakinotalea
Sphingobium
Suttonella
Thioreductor
Yersinia
Phylochip probes
intensity
Mesorhizobium
15
Density
gradient
Fraction 2
20
Fraction 1
Density
Cell ring
Number
of cells
Eukaryotes (density > 1.3)
DNA quality
25
Yield
Sampling
Lysis
stringency
Stringent Lyses
DNA size
Fraction 4
Fraction 3
Soft Lyses
10
5
0
Undetected with one DNA extraction method
Rothamsted soil phylochip saturation curve
% of positive phylogenetic probes
100
90
80
70
15 DNA extraction methods (about 99% of
probes)
60
50
40
30
Only one DNA extraction method (~40%
of probes)
20
10
0
0
2000
4000
6000
8000
10000
12000
14000
Number of probes
16000
18000
20000
Functional comparison using MG RAST annotation and STAMP statistical
analyses
1. technological reproducibility
11.67% of functions statistically
different (Bootstrap)
2. comparison with an ocean
72.63%
4. Cell lysis stringency effect
34.69%
3. comparison with another soil
39.83%
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???
METAGENOME EXPLOITATION
Domesticated bacterial host
Cloning
DNA
Transformation
Direct or indirect
Extraction
PCR
vector
Direct
Clone Library
Sequencing
(454)
Molecular
screening
Chemical
screening
Biological
screening
OMe
CH3
OMe
CH3
O
O
CH3
O
OH
OH
OMe
Culture
in vitro
Cloning and/or
sequencing
Cultivable bacteria: less than 1%
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
Molecular screening
Hybridization screening of metagenomic DNA libraries
Metagenomic DNA library construction
December 2010: 2 000 000 clones
(16 000 equ. bacterial genomes)
25
SOIL MICROFLORA
Abundant/Rare taxa
?
The right question ?
Extent of the Soil Bacterial Diversity
….independently of the species distribution ?
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
INTRODUCTION
Conceptual approach:
Provide new developing conditions to soil bacterial communities
Bacterial community extracted from soil A
Soil A
Sterilized Soil B
160
160
160
140
140
140
140
120
120
120
120
100
100
100
80
80
80
60
60
60
40
40
40
20
20
20
0
2
3
4
Diversity in soil A
5
6
80
or
60
40
20
0
0
1
100
1
2
3
4
0
51
62
3
4
5 1
62
3
4
5
6
Nine soils selected
Brévil
Talmont St-Hilaire
CSA
Martinique
Chinon
Montrond
Kenya: Embu
Congo: Black Point
New Caledonia
CONCEPTUAL APPROACH
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
Are new taxa detected?
 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 (Nmax = 1475 = Ngenera/chip)
Cumulative percentage of newly detected
genera (Nmax=1475)
60
55
50
45
CS1
CS2
CS3
CS5
40
35
30
25
T2 = 6 months
20
0
1
2
3
4
5
6
Number of soils
7
8
9
Cumulative percentage of newly detected genera (Nmax = 1475 = Ngenera/chip)
140
55
S1
S2
S4
S7
S9
120
50
Increase in detected genera
(cumulative %)
Cumulative percentage of newly detected
genera (Nmax=1475)
60
45
40
35
30
25
20
100
80
60
40
20
15
T2 = 6 months
10
0
1
2
3
4
5
Number of soil communities
T2 = 6 months
0
0
1
2
3
4
5
Number of soil communities
Cumulative percentage of newly detected
genera (Nmax=1475)
60
55
50
Cumulative percentage of newly detected genera (Nmax = 1475 = Ngenera/chip)
45
40
35
30
25
T2 = 6 months
20
0
1
2
3
4
5
6
7
8
9
Number of soils
55% (max) of the characterized genera detected (9 soils)
Rarefaction curves show a limit
Conclusion: Diversity in the rare biosphere very limited?
Cumulative percentage of newly detected
genera (Nmax=1475)
60
55
50
Cumulative percentage of newly detected genera (Nmax = 1475 = Ngenera/chip)
45
40
35
30
25
T2 = 6 months
20
0
1
2
3
4
5
6
7
8
9
Number of soils
However:
Diversity of conditions offered by the recipient sterilized soils?
Cumulative percentage of genera detected at T0 + T1 + T2
70
Cumulative percentage of newly detected
genera (Nmax=1475)
Cumulative percentage of newly detected
genera (Nmax=1475)
60
55
50
45
40
35
30
25
20
15
T2 only
10
0
1
2
3
60
CS: Extracted (and
inoculated) community
T0: 1 day
T1: 2 months
T2: 6 months
50
40
30
20
T0 + T1 +T2
10
4
5
Number of soil communities
1
2
3
4
5
Number of soil communities
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
(Nmax=1475)
Cumulative percentage of newly detected
genera
90
All soil communities (n=4)
All sampling times (n=3)
80
70
60
Individual communities
1 sampling time (6 months)
50
40
30
20
1
2
3
4
5
6
Number of soils
7
8
9
Rothamsted soil phylochip saturation curve
% of positive phylogenetic probes
100
90
80
70
15 DNA extraction approaches (about
99% of probes)
60
50
40
30
One DNA extraction approach (~40% of
probes)
20
10
0
0
2000
4000
6000
8000
10000
12000
14000
Number of probes
16000
18000
20000
Are new taxa detected?
 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.
Conclusion
Diversity of Bacteria (rare and abundant) : Huge
Attainable if
•Collaboration at the international level
•Focus on one « reference » soil
Aurélie Faugier, Sébastien Cécillon,
Davide Francioli, Tom Delmont,
Emmanuel Prestat, Jean-Michel
Monier, Timothy M Vogel,
Environmental
Microbial
Genomics
www.GenomEnviron.org