Metagenomic Approach to Soil MicrobialMetagenomic Approach to Soil MicrobialDiversity 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 EarthMicrobes 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.  

Human5 million years ago

Microorganisms3 billion years ago

5 million years ago

??

Soil microbes are very smallSoil

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 bacteriain 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

DecompositionCO2 gas

肥料 CH4 gasFertilizer

Decomposition

soilClClnCln

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 peryear by respiration

Plants fix  110 billion ton C per year

55 billion ton C per yearare 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

NH3Fertiorizerdenitrate

N2

NH3Fertiorizer

nitrogen fixation bacteria

NH3 NO3Ammonia Nitrification Denitrification

N2OAmmonia

Nitrifying bacteria Denitrifying bacteria

Some soil microbes cause plant diseasep

+++pathogen ++pathogen +pathogenpathogen pathogen pathogenIt 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<1%

大腸菌 乳酸菌線虫Nematodes E. coli

放線菌 メタン生成菌糸状菌

Analysis of microbes in the EnvironmentNucleic Acids

extracted from soil

S il What microbes (genes) ａｒｅ there?

DNASoil

MicrobesPCR-DGGE Analysis

Barcode of Soil Microbial community

What microbes (genes) ａｒｅ there?

DNA clone bankHole genes or operons

Microarray AnalysisList of homologue of known genes

Cultivation 100％ of

Metagenomic AnalysisList of existing genes

List of homologue of known genes

tequniques

RNA

Soil Microbe

Real time RT-PCR Analysis

What ａｒｅ microbes (genes) doing?

yDetection of gene expression in soil

Microarray AnalysisList of expressing known genes

Less than１%of Soil Microbe

Metatranscriptomic AnalysisList of expressing genes

“Metagenome” and “Metagenomics”

What is Metagenome?g

Metagenome

• DNA extracted from• DNA extracted from environmentMi 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？

Metagenomeor

・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-pathogensPl t th PGPR PGPF• Plant growth: PGPR, PGPF

• Firtirization: Nitrifying or Denitrifying MicrobesGHG t l A tibit f i b i th i t• GHG control：Actibites of microbes in the environments

• Bioremediation: POPs degrading microbesM 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

PCRAmplification of DNA fragment

Denaturing GradientGel Electrophoreses

S il i b i li d−

ent

−

Soil organisms can be visualized as bands without culture methods

atur

ing

grad

ie

Agarose gel DGGE

＋

dena

＋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 qliquid 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.

PCRSoil

Soil culture

DNA ExtractionPCR

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

Soil3CB x 3 ASS3

ASS7

3-CB Degraders with corresponding 3CBInduced 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

oten

tial

–1 so

il d–1

)

b

cc c

40

60

ficat

ion

poO 3

– -N k

g– b

0

20Nitr

i(m

g N

a a0

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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C OM OMN NPK NP NK PK Manure C OM OMN NPK NP NK PK

Agricultural fields Revealed by PCR DGGE Analysis

16SrDNA DGGE bands amoA DGGE bands

Findings• DGGE targeting 16S rRNA gene detected a specific DGGE band in organic 

manure‐fertilized soils.• Long‐term application of N fertilizers greatly increased AOB community 

Chu et al. 2007 Appl. Environ. Microbiol., 73:485-491, Chu et al. 2007 Soil Biol. Biochem., 39:2971-2976

diversity in soil.• It resulted in the disappearance of some resident AOB species.

Japanese National ProjectDevelopment of soil biodiversity analysisDevelopment of soil biodiversity analysis

system with environmental DNA（ DNA P j ）（eDNA Project）

April 2006～

The Back ground of the project

To achieve sustainable production of high quality crops and vegetables, it is essential to maintain soil fertility and to overcome soil disease such as sickness due to continuous cropping. For this purpose, we have to evaluate not only

h i l d h i l i f il b l bi l i l d ili h i

There is no way to analyze microbial diversity in soil

physical and chemical properties of soils but also biological ones and to utilize their information for soil management.

ay a a y a y

The aim of eDNA ProjectThis project addresses development of analysis methods for soil biodiversity with

environmental DNA (eDNA) which is extracted from soil.

Development of soil biodiversity analysis methods

O t f th j tThe project will provide a new soil diagnosis system based upon soil extracted DNA and will also deliver the challenging new scientific information These will further

Outcome of the project

and will also deliver the challenging new scientific information. These will further contribute to improved soil management for sustainable production.

PCR-DGGE standard method for analysis of soil microbial diversity

細菌 糸状菌 線虫Bacteria Fungi nematodes M M

細菌

M M

糸状菌

M M

線虫g

We developed "DGGE marker" for the three organism groups, sets of DNA fragments of known species, to compare DGGE band patterns across gels. The "marker" has p , p p gbeen already available commercially. With the standardized and optimized DGGE procedures we developed, clear and sharp band patterns can be produced

Sampling Sites in JapanSampling Site：16 (Paddy fields : 3, Farmland :13)SoilType：6

HokkaidoHokkaido

Okinawa

Iwate

AomoriAkita

Fukusima

KyotoGifu Ibaraki

Nagano GunmaKyoto

AichFukuoka Kanagawa

Ibaraki

Mie

Soil Environmental DNA Database (eDDASs)

Advance of DNA Sequencing Technology

Next generationDNA SequencerDNA SequencerCost :1/100, x100 data

＄10,000

＄1,000PersonalGenome

＄10 000 000

＄100,000StandardSample

,Cancer Cell

GenomeResequencing

Resequencing

＄1,000,000,000Human

＄10,000,000Human

GenomeResequensing

Sample Resequencing

GenomeProject1990～2003

Resequensing(Sangar Method) Dr.Watoson genome

2 month

2003 2004 2005 2006 2007 2008 2009 2010 ・・・・・

Dramatic advance of DNA sequencing technology enable metagenome sequencing.

Big Metagenome Sequencing Project

（’08～ 10 Country）International Human Gut Metagenome Project

JapanHattori et al.Human Gut

MetagenomeEurope & USA

Soil Metagenome（TerraGenome）

MetagenomeAnalysis

（05～07年） USAVenter et al.（TerraGenome）

Project（08年～）

Sargasso Sea

Ocean Metagenome

Project

JapaneDNA

Metagenome（04年～）

MetagenomeAnalysis

（?）

Approaches to functions of soil microbes i RNA t t d f ilusing RNA extracted from soil

Analysis of microbes in the EnvironmentNucleic Acids

extracted from soil

S il What microbes (genes) ａｒｅ there?

DNASoil

MicrobesPCR-DGGE Analysis

Barcode of Soil Microbial community

What microbes (genes) ａｒｅ there?

DNA clone bankHole genes or operons

Microarray AnalysisList of homologue of known genes

Cultivation 100％ of

Metagenomic AnalysisList of existing genes

List of homologue of known genes

Real time RT-PCR AnalysisRNA

tequniques Soil MicrobeWhat ａｒｅ microbes (genes) doing?

yDetection of gene expression in soil

Microarray AnalysisList of expressing known genes

Less than１%of Soil Microbe

Metatranscriptomic AnalysisList of expressing genes

Genome-wide Gene Expression Profile of P. putida Incubated in Soil

Substrate (3CB)

With Substrate

RNA

( )

pSL1Tfd operon

plasmid

Without Substrate

ben operon

chromosome

p

P. putida KT2440/pSL1 RNAMicroarray analysis

Strain： P. putida KT2440/pSL1 (3-chlorobenzoate degrader)

O F lOur Future plan

We are now considering improvement of eDDASs ith addition of ne data obtainedeDDASs with addition of new data obtained by new analytical method using microarray technology metagenome sequencing ortechnology, metagenome sequencing or metatranscriptomic approach.

Now we have opened a door to Microbial diversity in soil using metagenomic approaches.in soil using metagenomic approaches.