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MetagenomicApproach to Soil MicrobialApproach ... Soil Microbes PCR-DGGE Analysis Barcode of Soil Microbial community microbes (genes) DNAclonebank Hole genes or operons Microarray

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  • 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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