Metagenomic Approach to Soil MicrobialMetagenomic Approach to Soil Microbial
Diversity and Functions
Takeshi Fujii, Sho Morimoto, Yuko T. Hoshino, Hiroaki Okada,
Yong Wang, Haiyan Chu*, Bao Zhihua, Matushita Yuko, Seiya TsushimaYong Wang, Haiyan Chu , Bao Zhihua, Matushita Yuko, Seiya Tsushima
National Institute for
Agro-Environmental Sciences
Outline of my presentationOutline of my presentation
A i bi l ld• A microbial world
• What are “metagenome” and “metagenomics”?
• Approaches to the diversity of soil biota using PCR-
DGGE method
• eDNA project
• Approaches to functions of soil microbes using RNA• Approaches to functions of soil microbes using RNA
extracted from soil
• Our Future plan• Our Future plan
A Microbial Planet, the Earth
Microbes surpass human being in mass and numbers. They play important roles in many processes on earth. We live on the earth that have been constructed by microbes.It is important to know the microbial diversity and functions.However, most of them are still untouched and unknown.
Human
5 million years ago
Microorganisms
3 billion years ago
5 million years ago
??
Soil microbes are very small
Soil
Inoculation onto a Petri dish
１μm１cm
Colonies on agar suface
The number of soil microbes are very huge
H b t i
y g
How many bacteria
in soil?
A Russian researcher counted the number of
bacteria in various type of soil from the
tundra in Siberia to top of a mountain in
Russia (Mishustin,1963).
The answer is・・・・・The answer is・・・・・
One gram of soil contained
800,000,000～4,000,000,000 bacteria.
Soil Microbes are decomposers
N2 gas
p
Decomposition
CO2 gas
肥料 CH4 gas
Fertilizer
Decomposition
soilCl
ClnCln
O
Microbes WaterCl
PCB
ダイオキシン
O
Microbes play important roles in
・material cycles on earth.
d d ti f h i l t i l・degradation of chemical materials.
・consumption of fertilizer.
Microbes involves in material cycles on earth y
Total 750 billion ton C
CO255 billion ton C per
year by respiration
Plants fix 110 billion ton C per year
55 billion ton C per year
are produced by soil bacteria.
Pl bi (55 billi C )Plants biomass (55 billion ton C per year)
(Total 1,500 billion ton C)
Bolin, 1981
Soil microbes compete nitrogen fertilizer with plants
NH3Fertiorizer
denitrate
N2
NH3Fertiorizer
nitrogen fixation bacteria
NH3 NO3
Ammonia Nitrification Denitrification
N2O
Ammonia
Nitrifying bacteria Denitrifying bacteria
Some soil microbes cause plant diseasep
+++pathogen ++pathogen +pathogenpathogen pathogen pathogen
It is important for agriculture to find and control the plant pathogen.
Most of Soil microbes are unculturable and unknown
Soil Microbial community
99% =Unculturable and unknown
Known microbes and small animals
Analysis of microbes in the Environment
Nucleic Acids
extracted from soil
S il What microbes (genes) ａｒｅ there?
DNA
Soil
Microbes
PCR-DGGE Analysis
Barcode of Soil Microbial community
What microbes (genes) ａｒｅ there?
DNA clone bank
Hole genes or operons
Microarray Analysis
List of homologue of known genes
Cultivation 100％ of
Metagenomic Analysis
List of existing genes
List of homologue of known genes
tequniques
RNA
Soil Microbe
Real time RT-PCR Analysis
What ａｒｅ microbes (genes) doing?
y
Detection of gene expression in soil
Microarray Analysis
List of expressing known genes
Less than１%
of Soil Microbe
Metatranscriptomic Analysis
List of expressing genes
“Metagenome” and “Metagenomics”
What is Metagenome?g
Metagenome
• DNA extracted from• DNA extracted from
environment
Mi f• Mixture of genome
from various organisms
• Genome of the
Environment
What is Metagenomics?
• Metagenomics is the study of genetic material recovered
directly from environmental samples. Metagenomics is an
emerging field in which the power of genomic analysis (the
analysis of all the DNA in an organism) is applied to entire
communities of microbes bypassing the need to isolatecommunities of microbes, bypassing the need to isolate
and culture individual microbial species.
C iti ？
Metagenomics
DNA or RNA
・Communities？
・Functions？
・New Genes？
Metagenome
or
・New Medicine？
・
・
・or
Metatranscriptome
The targets of Metagenomics
Soil Microbial community
99% U lt bl d k99% =Unculturable and unknown
Metagenome analysis
• Plant desease: Pathogens, Anti-pathogens
Pl t th PGPR PGPF• Plant growth: PGPR, PGPF
• Firtirization: Nitrifying or Denitrifying Microbes
GHG t l A tibit f i b i th i t• GHG control：Actibites of microbes in the environments
• Bioremediation: POPs degrading microbes
M di i A tibi ti d i i b• Medicines: Antibiotics produsing microbes
• Etc.
Approaches to the diversity of soil biota
using PCR DGGE methodusing PCR-DGGE method
Improved DNA extraction methods from soilp
skim milk
+: skim milk added; - : skim milk not added. Without skim milk, DNA was extracted
successfully only from soils 2 and 4.
Yuko T. Hoshino and Naoyuki Matsumoto (2004) Microbes and Environment 19: 13-19
Skim milk dramatically improves extraction efficiency of DNA from soil.
Identification of Microbes by PCR-DGGE
PCR
y
DNA
DGGE
PCR
Amplification of
DNA fragment
Denaturing Gradient
Gel Electrophoreses
S il i b i li d
−
en
t
−
Soil organisms can be visualized as
bands without culture methods
at
ur
in
g
gr
ad
ie
Agarose gel DGGE
＋
de
na
＋Bar codes of soil microbial
community structure
DNA fragments with the
same size and different
sequences make one band.
DNA fragments different
sequences are separated.
community structure
A Problem in the Isolation of Effective Degraders in Soilg
Screening of some degraders by relying only on cultivation techniques such as g g y y g y q
liquid enrichment often fail to isolate the actual degraders in the environment.
Substrate
Soil
Substrate
Liquid Enriched culture
No degradation
Why?
g
Community Analyses of 3-Chlorobenzoate (3CB)
Degraders in Soil or liquid culture by PCR DGGEDegraders in Soil or liquid culture by PCR-DGGE
To get an answer for the question raised in the previous slide the communityTo get an answer for the question raised in the previous slide , the community
structure of 3CB-degraders in soil and liquid culture were compared by PCR
DGGE analysis targeting 3-Chlorobenzoate (3CB) degradation genes.
PCR
Soil
Soil culture
DNA Extraction
PCR
Substrate (3CB)Substrate (3CB)
Target geneSoil
Liquid Enriched culture
(benA,16SrDNA)
Soil
DGGE Analysis
Identification of 3-CB Degraders by PCR-DGGE
3CB
3CB-amended soil culture Liquid culture
3CB
0 2 4 6 16 23 30 1 2 3 4 5 day
PCR-DGGE patterns targeting benzoate 1,2-dioxygenase alpha subunit genes (benA)
Isolation of 3-CB Degraders by PCR-DGGE
Soil
3CB x 3 ASS3
ASS7
3-CB Degraders with
corresponding 3CB
Induced DGGE bandsSoil
Soil culture
3CB
ASS7
ASS8
ASS11
Induced DGGE bands
Soil extract
3CB agar plate
3CBASS11
ASS14
16S rRNA benA
・Morimoto. S et al. (2008) Microbes. Environ., 23:285-292
Yes, degraded!!
3CB 3CB 3CB 3CB 3CB 3CB
Community Structure of Ammonia-oxidizing Bacteria in
Agricultural fields Revealed by PCR-DGGE Analysis
Control OM ½ OMN NPK NP PK
Agricultural fields Revealed by PCR-DGGE Analysis
No add ition Organic manure Mineral fertilizer
120 d
A long-term fertilizer experiment, which has been carried out for 16 years in China
80
100
ot
en
tia
l
–1
so
il
d–
1 )
b
cc c
40
60
fic
at
io
n
po
O 3
– -
N
k
g– b
0
20N
itr
i
(m
g
N
a a
0
control OM 1/2OMN NPK NP NK PK
Long-term fertilization of N fertilizers greatly increased nitrification potential.
Community Structure of Ammonia-oxidizing Bacteria in
Agricultural fields Revealed by PCR-DGGE Analysis
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