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Some Definitions

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  1. “Here, we examine the crucial issues and challenges that so far have hindered the metagenomic exploration of soil by drawing on experience from a trans-European project on disease-suppressive soils denoted METACONTROL.”

  2. Some Definitions • organisms requiring highly specific environmental or cultural conditions, slow-growing or rare organisms • phenazine-producing Pseudomonasfluorescens • pyrrolnitrin • polyketides

  3. Notable Soil Metagenomic Studies

  4. Metagenomics 101 Nature Reviews Microbiology 3: 470-478

  5. Soil Metagenomics

  6. Soil Microbial Diversity • Richness: • Evenness: Rank-abundance curves Hughes et al. 2001. Applied and Environmental Microbiology 67:4399–4406 Schloss PD, Handelsman J . 2006. PLoS Comp Biol 2: e92. DOI: 10.1371

  7. Metagenomic Approaches • Unselective, direct or ‘unbiased’ • Environmental shotgun sequencing (ESS) • Pyrosequencing, Solexa (Illumina) • Targeted, indirect or ‘biased’ • Prefractionation • Amplification • Pretreatment

  8. Stable Isotope Probing • Add 13C-labeled substrate to the environment • Separate 13C-incorporated DNA Radajewski et al. 2003. Current Opinion in Biotechnology 14:296–302 Radajewski et al. 2000. Nature 403:646-649

  9. DNA Extraction • Sufficient quantity and quality • HMW DNA often required • Minimum size of 40-100 kb • Pre-separation of bacterial cells from soil • High amount of cells needed (1011)

  10. Library Screening • Molecular • DNA-based approaches (e.g. hybridization, PCR) • Microarrays • Functional • Expression of target genes in a metagenomic host (e.g. E. coli) • Substrate-induced gene expression screening (SIGEX)

  11. Molecular and Functional Screening

  12. SIGEX Overview Fluorescent activated cell sorting Modified from: Microbial Cell Factories 2005, 4:8

  13. FACS Overview http://www.bio.davidson.edu/COURSES/GENOMICS/method/FACS.html

  14. SIGEX Overview Fluorescent activated cell sorting Modified from: Microbial Cell Factories 2005, 4:8

  15. The METACONTROL Project • Collaboration of seven European laboratories • Goal: Explore the biotechnological potential of phytopathogen-suppressive soils • Targets: antibiotics (polyketides) and chitinase biosynthesis • Four suppressive soils + one control soil

  16. METACONTROL Libraries

  17. Conclusions from the METAGENOME Project • “…overall, low numbers of phytopathogen-suppressive clones were found in the four libraries” • <0.05% of clones were positive in functional assays • Molecular screening produced a 0.22% hit rate • Novel PKS sequences found • Further understanding of the production of a leinamycin-like antibiotic • DNA extraction processes were improved • Deliberate bias can improve hit rates

  18. Future Issues • Sampling • DNA fragment size • Library hosts and vectors • Screening • higher throughput cell-sorters • Sequence assembly, sorting and contig annotation