1 Nature Reviews Microbiology 2005 Vol: 3(6):470-478. DOI: 10.1038/nrmicro1160

The metagenomics of soil

Phylogenetic surveys of soil ecosystems have shown that the number of prokaryotic species found in a single sample exceeds that of known cultured prokaryotes. Soil metagenomics, which comprises isolation of soil DNA and the production and screening of clone libraries, can provide a cultivation-independent assessment of the largely untapped genetic reservoir of soil microbial communities. This approach has already led to the identification of novel biomolecules. However, owing to the complexity and heterogeneity of the biotic and abiotic components of soil ecosystems, the construction and screening of soil-based libraries is difficult and challenging. This review describes how to construct complex libraries from soil samples, and how to use these libraries to unravel functions of soil microbial communities.

Mentions
Figures
FIGURE 1 | Essential steps to explore and exploit the genomic diversity of soil microbial communities by metagenomics.
Shown is a flow diagram of the main steps in the construction of a metagenomic DNA library from a soil sample. Soil DNA is recovered through separation of cells from soil particles followed by cell lysis and DNA recovery, or through direct lysis of cells contained within soil and recovery of DNA. Recovered soil DNA is fragmented and ligated into the linearized cloning vector of choice which might be a plasmid, cosmid, fosmid or BAC (bacterial artificial chromosome). Following the introduction of the recombinant vectors into a suitable bacterial cloning host, screening strategies can be designed to identify those clones which might contain new and useful genes. FIGURE 2 | Examples of activity-based screens.
a | Detection of clones harbouring genes that confer carbonyl formation. Screening is based on the ability of the library-containing Escherichia coli clones to form carbonyls from test substrates, that is, polyols43, 85, during growth on indicator agar. The test substrates are included in the indicator agar, which contains a mixture of pararosaniline and sodium bisulphite (Schiff reagent). The production of carbonyls from test substrates on indicator plates by clones results in formation of a dark red Schiff base. The carbonyl-forming colonies are red and are surrounded by a red zone, whereas colonies failing to form carbonyls from the test substrate remain uncoloured. b | Detection of proteolytic activity. Proteolytic E. coli clones are detected on agar media containing skimmed milk by zones of clearance around the colonies.
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References
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    • . . . Theoretically, the microbial DNA isolated from a soil sample represents the collective DNA of all the indigenous soil microorganisms, and is named the soil metagenome17, 18 . . .
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    • . . . Theoretically, the microbial DNA isolated from a soil sample represents the collective DNA of all the indigenous soil microorganisms, and is named the soil metagenome17, 18 . . .
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    • . . . Many soil DNA extraction protocols have been published, and commercial soil DNA extraction kits are available19, 20, 21, 22, 23, 24, 25, 26, 27 . . .
    • . . . The DNA extraction methods can be divided into two categories: direct lysis of cells contained in the sample matrix followed by separation of the DNA from the matrix and cell debris (pioneered by Ogram et al.19); or separation of the cells from the soil matrix followed by cell lysis (pioneered by Holben et al.20) (Fig. 1) . . .
    • . . . In addition, several methods use mechanical disruption steps such as bead-beating, freeze–thawing or grinding of samples to lyse cells19, 24, 25, 26, 27, 57 . . .
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    • . . . Many protocols for the isolation of soil-derived microbial DNA have been published19, 20, 21, 22, 23, 24, 25, 26, 27. . . .
    • . . . Many soil DNA extraction protocols have been published, and commercial soil DNA extraction kits are available19, 20, 21, 22, 23, 24, 25, 26, 27 . . .
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    • . . . Many protocols for the isolation of soil-derived microbial DNA have been published19, 20, 21, 22, 23, 24, 25, 26, 27. . . .
    • . . . Also, extraction of soil DNA often results in coextraction of humic substances, which interfere with restriction-enzyme digestion and PCR amplification and reduce cloning efficiency, transformation efficiency and the specificity of DNA hybridization21, 54, 55. . . .
    • . . . Many soil DNA extraction protocols have been published, and commercial soil DNA extraction kits are available19, 20, 21, 22, 23, 24, 25, 26, 27 . . .
    • . . . It is usually presumed that the DNA isolated by the direct lysis approach better represents the microbial diversity of a soil sample because this method does not include a cell separation step, so microorganisms that adhere to particles are also lysed21, 60 . . .
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    • . . . Many protocols for the isolation of soil-derived microbial DNA have been published19, 20, 21, 22, 23, 24, 25, 26, 27. . . .
    • . . . Many soil DNA extraction protocols have been published, and commercial soil DNA extraction kits are available19, 20, 21, 22, 23, 24, 25, 26, 27 . . .
    • . . . The separation of microorganisms from the soil matrix is achieved by mild mechanical forces or chemical procedures such as blending, rotating pestle homogenization or the addition of cation-exchange resins, followed by density gradient or differential centrifugation22, 23, 56, 57 . . .
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    • . . . Many protocols for the isolation of soil-derived microbial DNA have been published19, 20, 21, 22, 23, 24, 25, 26, 27. . . .
    • . . . Many soil DNA extraction protocols have been published, and commercial soil DNA extraction kits are available19, 20, 21, 22, 23, 24, 25, 26, 27 . . .
    • . . . The separation of microorganisms from the soil matrix is achieved by mild mechanical forces or chemical procedures such as blending, rotating pestle homogenization or the addition of cation-exchange resins, followed by density gradient or differential centrifugation22, 23, 56, 57 . . .
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    • . . . Many protocols for the isolation of soil-derived microbial DNA have been published19, 20, 21, 22, 23, 24, 25, 26, 27. . . .
    • . . . Many soil DNA extraction protocols have been published, and commercial soil DNA extraction kits are available19, 20, 21, 22, 23, 24, 25, 26, 27 . . .
    • . . . The amounts of DNA isolated from different soil types using a selection of protocols range from less than 1 mg to approximately 500 g of DNA per gram of soil24, 25, 26, 35, 56, 57 . . .
    • . . . In addition, several methods use mechanical disruption steps such as bead-beating, freeze–thawing or grinding of samples to lyse cells19, 24, 25, 26, 27, 57 . . .
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    • . . . Many protocols for the isolation of soil-derived microbial DNA have been published19, 20, 21, 22, 23, 24, 25, 26, 27. . . .
    • . . . As microbial populations are large, sample volumes can be small (500 g in most studies)25, 35, 37, 47, 52 . . .
    • . . . Many soil DNA extraction protocols have been published, and commercial soil DNA extraction kits are available19, 20, 21, 22, 23, 24, 25, 26, 27 . . .
    • . . . In addition, several methods use mechanical disruption steps such as bead-beating, freeze–thawing or grinding of samples to lyse cells19, 24, 25, 26, 27, 57 . . .
  26. Hurt, R. A. et al. Simultaneous recovery of RNA and DNA from soils and sediments. Appl. Environ. Microbiol. 67, 4495-4503 (2001).Development of a method for direct nucleic acid isolation from soils of various compositions , .
    • . . . Many protocols for the isolation of soil-derived microbial DNA have been published19, 20, 21, 22, 23, 24, 25, 26, 27. . . .
    • . . . Many soil DNA extraction protocols have been published, and commercial soil DNA extraction kits are available19, 20, 21, 22, 23, 24, 25, 26, 27 . . .
    • . . . In addition, several methods use mechanical disruption steps such as bead-beating, freeze–thawing or grinding of samples to lyse cells19, 24, 25, 26, 27, 57 . . .
    • . . . An excellent starting point for researchers is the direct lysis method of Hurt et al.26, which allows simultaneous recovery of DNA and RNA from soils of different composition. . . .
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    • . . . Many protocols for the isolation of soil-derived microbial DNA have been published19, 20, 21, 22, 23, 24, 25, 26, 27. . . .
    • . . . These surveys allow cataloguing and comparison of the microbial diversity in different soil habitats, and the comparative analysis of changes in community structure owing to altered environmental factors27, 28, 29, 30, 31, 32 . . .
    • . . . Many soil DNA extraction protocols have been published, and commercial soil DNA extraction kits are available19, 20, 21, 22, 23, 24, 25, 26, 27 . . .
    • . . . In addition, several methods use mechanical disruption steps such as bead-beating, freeze–thawing or grinding of samples to lyse cells19, 24, 25, 26, 27, 57 . . .
    • . . . In addition to the DNA that is recovered from lysed prokaryotes, extracellular DNA and eukaryotic DNA are also recovered27, 57, 58 . . .
  28. Dunbar, J., Takala, S., Barns, S. M., Davis, J. A. & Kuske, C. R. Levels of bacterial community diversity in four arid soils compared by cultivation and 16S rRNA gene cloning. Appl. Environ. Microbiol. 65, 1662-1669 (1999) , .
    • . . . These surveys allow cataloguing and comparison of the microbial diversity in different soil habitats, and the comparative analysis of changes in community structure owing to altered environmental factors27, 28, 29, 30, 31, 32 . . .
    • . . . PCR is most commonly used for sequence-driven screening of soil-based libraries or soil DNA28, 31, 49, 56, 65, 66, 67, 69, 74, 75, 76, 77 . . .
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    • . . . These surveys allow cataloguing and comparison of the microbial diversity in different soil habitats, and the comparative analysis of changes in community structure owing to altered environmental factors27, 28, 29, 30, 31, 32 . . .
  30. Dunbar, J., Barns, S. M., Ticknor, L. O. & Kuske, C. R. Empirical and theoretical bacterial diversity in four Arizona soils. Appl. Environ. Microbiol. 68, 3035-3045 (2002) , .
    • . . . These surveys allow cataloguing and comparison of the microbial diversity in different soil habitats, and the comparative analysis of changes in community structure owing to altered environmental factors27, 28, 29, 30, 31, 32 . . .
  31. Zhou, J. et al. Spatial and resource factors influencing high microbial diversity in soil. Appl. Environ. Microbiol. 68, 326-334 (2002) , .
    • . . . These surveys allow cataloguing and comparison of the microbial diversity in different soil habitats, and the comparative analysis of changes in community structure owing to altered environmental factors27, 28, 29, 30, 31, 32 . . .
    • . . . PCR is most commonly used for sequence-driven screening of soil-based libraries or soil DNA28, 31, 49, 56, 65, 66, 67, 69, 74, 75, 76, 77 . . .
  32. Yeager, C. M. et al. Diazotrophic community structure and function in two successional stages of biological soil crusts from the Colorado plateau and Chihuahuan desert. Appl. Environ. Microbiol. 70, 973-983 (2004) , .
    • . . . These surveys allow cataloguing and comparison of the microbial diversity in different soil habitats, and the comparative analysis of changes in community structure owing to altered environmental factors27, 28, 29, 30, 31, 32 . . .
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    • . . . Other marker genes that are used to monitor microbial diversity include dnaK33 (HSP-70-type molecular chaperone) and amoA34 (ammonia monooxygenase) . . .
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    • . . . Other marker genes that are used to monitor microbial diversity include dnaK33 (HSP-70-type molecular chaperone) and amoA34 (ammonia monooxygenase) . . .
  35. Henne, A., Daniel, R., Schmitz, R. A. & Gottschalk, G. Construction of environmental DNA libraries in Escherichia coli and screening for the presence of genes conferring utilization of 4-hydroxybutyrate. Appl. Environ. Microbiol. 65, 3901-3907 (1999).First report on the isolation of biocatalysts from soil-derived plasmid libraries , .
    • . . . In landmark studies, novel genes that encoded useful enzymes and antibiotics were recovered by direct cloning of soil DNA into plasmid, cosmid or BAC (bacterial artificial chromosome) vectors and screening of the generated libraries35, 36, 37 (for the industrial impact of soil metagenomics see the article by P . . .
    • . . . As microbial populations are large, sample volumes can be small (500 g in most studies)25, 35, 37, 47, 52 . . .
    • . . . The amounts of DNA isolated from different soil types using a selection of protocols range from less than 1 mg to approximately 500 g of DNA per gram of soil24, 25, 26, 35, 56, 57 . . .
    • . . . Direct lysis approaches have been used more frequently than the separation techniques to isolate soil DNA for the construction of libraries35, 36, 37, 39, 42, 43, 45, 47, 61. . . .
    • . . . Small-insert soil-based libraries are useful for the isolation of single genes or small operons encoding new metabolic functions35, 38, 43, 45, 50, 51, 63 . . .
    • . . . Despite these limitations, analysing and screening of libraries has yielded several novel biomolecules35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 61, 63, 70, 71, 72, 73 and provided insights into the genomes of uncultured prokaryotic soil organisms and the ecology of the soil ecosystem58, 65, 66, 69. . . .
    • . . . Most of the screening methods to isolate genes or gene clusters for novel biocatalysts or small molecules are based on detecting activity from library-containing clones35, 36, 37, 38, 39, 40, 41, 42, 43, 45, 46, 47, 48, 49, 50, 51, 61, 63, 64, 72, 73 . . .
    • . . . This strategy has been validated by the isolation of novel genes that encode degradative enzymes35, 37, 38, 39, 41, 43, 45, 63, antibiotic resistance50 and antibiotics36, 46, 47, 48, 49 . . .
  36. Brady, S. F. & Clardy, J. Long-chain N-acyl amino acid antibiotics isolated from heterologously expressed environmental DNA. J. Am. Chem. Soc. 122, 12903-12904 (2000).First isolation of antibiotics from a soil-based cosmid library , .
    • . . . In landmark studies, novel genes that encoded useful enzymes and antibiotics were recovered by direct cloning of soil DNA into plasmid, cosmid or BAC (bacterial artificial chromosome) vectors and screening of the generated libraries35, 36, 37 (for the industrial impact of soil metagenomics see the article by P . . .
    • . . . Direct lysis approaches have been used more frequently than the separation techniques to isolate soil DNA for the construction of libraries35, 36, 37, 39, 42, 43, 45, 47, 61. . . .
    • . . . Large-insert libraries are more appropriate to recover complex pathways that are encoded by large gene clusters or large DNA fragments for the characterization of genomes of uncultured soil microorganisms36, 37, 47, 48, 49, 58, 61, 64, 65, 66, 67 . . .
    • . . . Despite these limitations, analysing and screening of libraries has yielded several novel biomolecules35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 61, 63, 70, 71, 72, 73 and provided insights into the genomes of uncultured prokaryotic soil organisms and the ecology of the soil ecosystem58, 65, 66, 69. . . .
    • . . . Most of the screening methods to isolate genes or gene clusters for novel biocatalysts or small molecules are based on detecting activity from library-containing clones35, 36, 37, 38, 39, 40, 41, 42, 43, 45, 46, 47, 48, 49, 50, 51, 61, 63, 64, 72, 73 . . .
    • . . . This strategy has been validated by the isolation of novel genes that encode degradative enzymes35, 37, 38, 39, 41, 43, 45, 63, antibiotic resistance50 and antibiotics36, 46, 47, 48, 49 . . .
  37. Rondon, M. R. et al. Cloning the soil metagenome: a strategy for accessing the genetic and functional diversity of uncultured microorganisms. Appl. Environ. Microbiol. 66, 2541-2547 (2000).Generation of a large-insert soil-derived BAC library and illustration of the potential of this type of library for soil-based gene discovery , .
    • . . . In landmark studies, novel genes that encoded useful enzymes and antibiotics were recovered by direct cloning of soil DNA into plasmid, cosmid or BAC (bacterial artificial chromosome) vectors and screening of the generated libraries35, 36, 37 (for the industrial impact of soil metagenomics see the article by P . . .
    • . . . As microbial populations are large, sample volumes can be small (500 g in most studies)25, 35, 37, 47, 52 . . .
    • . . . Direct lysis approaches have been used more frequently than the separation techniques to isolate soil DNA for the construction of libraries35, 36, 37, 39, 42, 43, 45, 47, 61. . . .
    • . . . Large-insert libraries are more appropriate to recover complex pathways that are encoded by large gene clusters or large DNA fragments for the characterization of genomes of uncultured soil microorganisms36, 37, 47, 48, 49, 58, 61, 64, 65, 66, 67 . . .
    • . . . Most of the screening methods to isolate genes or gene clusters for novel biocatalysts or small molecules are based on detecting activity from library-containing clones35, 36, 37, 38, 39, 40, 41, 42, 43, 45, 46, 47, 48, 49, 50, 51, 61, 63, 64, 72, 73 . . .
    • . . . This strategy has been validated by the isolation of novel genes that encode degradative enzymes35, 37, 38, 39, 41, 43, 45, 63, antibiotic resistance50 and antibiotics36, 46, 47, 48, 49 . . .
  38. Henne, A., Schmitz, R. A., Bömeke, M., Gottschalk, G. & Daniel, R. Screening of environmental DNA libraries for the presence of genes conferring lipolytic activity on Escherichia coli. Appl. Environ. Microbiol. 66, 3113-3116 (2000) , .
    • . . . The same approach has been used to clone genes from soil communities that code for lipases38, 39, 40, proteases41, 42, oxidoreductases43, amylases44, 45, antibiotics46, 47, 48, 49, antibiotic resistance enzymes50 and membrane proteins51 . . .
    • . . . Small-insert soil-based libraries are useful for the isolation of single genes or small operons encoding new metabolic functions35, 38, 43, 45, 50, 51, 63 . . .
    • . . . Despite these limitations, analysing and screening of libraries has yielded several novel biomolecules35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 61, 63, 70, 71, 72, 73 and provided insights into the genomes of uncultured prokaryotic soil organisms and the ecology of the soil ecosystem58, 65, 66, 69. . . .
    • . . . Most of the screening methods to isolate genes or gene clusters for novel biocatalysts or small molecules are based on detecting activity from library-containing clones35, 36, 37, 38, 39, 40, 41, 42, 43, 45, 46, 47, 48, 49, 50, 51, 61, 63, 64, 72, 73 . . .
  39. Lee, S. -W. et al. Screening for novel lipolytic enzymes from uncultured soil microorganisms. Appl. Microbiol. Biotechnol. 65, 720-726 (2004) , .
    • . . . The same approach has been used to clone genes from soil communities that code for lipases38, 39, 40, proteases41, 42, oxidoreductases43, amylases44, 45, antibiotics46, 47, 48, 49, antibiotic resistance enzymes50 and membrane proteins51 . . .
    • . . . Direct lysis approaches have been used more frequently than the separation techniques to isolate soil DNA for the construction of libraries35, 36, 37, 39, 42, 43, 45, 47, 61. . . .
    • . . . Despite these limitations, analysing and screening of libraries has yielded several novel biomolecules35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 61, 63, 70, 71, 72, 73 and provided insights into the genomes of uncultured prokaryotic soil organisms and the ecology of the soil ecosystem58, 65, 66, 69. . . .
    • . . . Most of the screening methods to isolate genes or gene clusters for novel biocatalysts or small molecules are based on detecting activity from library-containing clones35, 36, 37, 38, 39, 40, 41, 42, 43, 45, 46, 47, 48, 49, 50, 51, 61, 63, 64, 72, 73 . . .
  40. Lorenz, P. & Schleper, C. Metagenome - a challenging source of enzyme discovery. J. Mol. Catal. B Enzym. 19, 13-19 (2002) , .
    • . . . The same approach has been used to clone genes from soil communities that code for lipases38, 39, 40, proteases41, 42, oxidoreductases43, amylases44, 45, antibiotics46, 47, 48, 49, antibiotic resistance enzymes50 and membrane proteins51 . . .
    • . . . Despite these limitations, analysing and screening of libraries has yielded several novel biomolecules35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 61, 63, 70, 71, 72, 73 and provided insights into the genomes of uncultured prokaryotic soil organisms and the ecology of the soil ecosystem58, 65, 66, 69. . . .
    • . . . Most of the screening methods to isolate genes or gene clusters for novel biocatalysts or small molecules are based on detecting activity from library-containing clones35, 36, 37, 38, 39, 40, 41, 42, 43, 45, 46, 47, 48, 49, 50, 51, 61, 63, 64, 72, 73 . . .
    • . . . To improve the representation of rare genomes in a library, normalization procedures such as separating soil DNA based on its AT content might also be used for enrichment40. . . .
  41. Gupta, R., Berg, Q. K. & Lorenz, P. Bacterial alkaline proteases: molecular approaches and industrial applications. Appl. Microbiol. Biotechnol. 59, 15-32 (2002) , .
    • . . . The same approach has been used to clone genes from soil communities that code for lipases38, 39, 40, proteases41, 42, oxidoreductases43, amylases44, 45, antibiotics46, 47, 48, 49, antibiotic resistance enzymes50 and membrane proteins51 . . .
    • . . . Despite these limitations, analysing and screening of libraries has yielded several novel biomolecules35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 61, 63, 70, 71, 72, 73 and provided insights into the genomes of uncultured prokaryotic soil organisms and the ecology of the soil ecosystem58, 65, 66, 69. . . .
    • . . . Most of the screening methods to isolate genes or gene clusters for novel biocatalysts or small molecules are based on detecting activity from library-containing clones35, 36, 37, 38, 39, 40, 41, 42, 43, 45, 46, 47, 48, 49, 50, 51, 61, 63, 64, 72, 73 . . .
    • . . . This strategy has been validated by the isolation of novel genes that encode degradative enzymes35, 37, 38, 39, 41, 43, 45, 63, antibiotic resistance50 and antibiotics36, 46, 47, 48, 49 . . .
    • . . . Another example is the detection of E. coli clones with proteolytic activity on agar plates containing skimmed milk41, 42 (Fig. 2b) . . .
  42. Santosa, D. A. Rapid extraction and purification of environmental DNA for molecular cloning applications and molecular diversity studies. Mol. Biotechnol. 17, 59-64 (2001) , .
    • . . . The same approach has been used to clone genes from soil communities that code for lipases38, 39, 40, proteases41, 42, oxidoreductases43, amylases44, 45, antibiotics46, 47, 48, 49, antibiotic resistance enzymes50 and membrane proteins51 . . .
    • . . . Direct lysis approaches have been used more frequently than the separation techniques to isolate soil DNA for the construction of libraries35, 36, 37, 39, 42, 43, 45, 47, 61. . . .
    • . . . Despite these limitations, analysing and screening of libraries has yielded several novel biomolecules35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 61, 63, 70, 71, 72, 73 and provided insights into the genomes of uncultured prokaryotic soil organisms and the ecology of the soil ecosystem58, 65, 66, 69. . . .
    • . . . Most of the screening methods to isolate genes or gene clusters for novel biocatalysts or small molecules are based on detecting activity from library-containing clones35, 36, 37, 38, 39, 40, 41, 42, 43, 45, 46, 47, 48, 49, 50, 51, 61, 63, 64, 72, 73 . . .
  43. Knietsch, A., Waschkowitz, T., Bowien, S., Henne, A. & Daniel, R. Metagenomes of complex microbial consortia derived from different soils as sources for novel genes conferring formation of carbonyls from short-chain polyols on Escherichia coli. J. Mol. Microbiol. Biotechnol. 5, 46-56 (2003) , .
    • . . . The same approach has been used to clone genes from soil communities that code for lipases38, 39, 40, proteases41, 42, oxidoreductases43, amylases44, 45, antibiotics46, 47, 48, 49, antibiotic resistance enzymes50 and membrane proteins51 . . .
    • . . . Direct lysis approaches have been used more frequently than the separation techniques to isolate soil DNA for the construction of libraries35, 36, 37, 39, 42, 43, 45, 47, 61. . . .
    • . . . Small-insert soil-based libraries are useful for the isolation of single genes or small operons encoding new metabolic functions35, 38, 43, 45, 50, 51, 63 . . .
    • . . . Despite these limitations, analysing and screening of libraries has yielded several novel biomolecules35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 61, 63, 70, 71, 72, 73 and provided insights into the genomes of uncultured prokaryotic soil organisms and the ecology of the soil ecosystem58, 65, 66, 69. . . .
    • . . . Most of the screening methods to isolate genes or gene clusters for novel biocatalysts or small molecules are based on detecting activity from library-containing clones35, 36, 37, 38, 39, 40, 41, 42, 43, 45, 46, 47, 48, 49, 50, 51, 61, 63, 64, 72, 73 . . .
    • . . . This strategy has been validated by the isolation of novel genes that encode degradative enzymes35, 37, 38, 39, 41, 43, 45, 63, antibiotic resistance50 and antibiotics36, 46, 47, 48, 49 . . .
    • . . . An example is the screening of soil-based libraries for genes conferring polyol oxidoreductase activity43, 85, which was based on the ability of the recombinant E. coli strains to form carbonyls from polyols (Fig. 2a) . . .
    • . . . Screening is based on the ability of the library-containing Escherichia coli clones to form carbonyls from test substrates, that is, polyols43, 85, during growth on indicator agar . . .
  44. Richardson, T. H. et al. A novel, high performance enzyme for starch liquefaction. Discovery and optimization of a low pH, thermostable -amylase. J. Biol. Chem. 277, 26501-26507 (2002) , .
    • . . . The same approach has been used to clone genes from soil communities that code for lipases38, 39, 40, proteases41, 42, oxidoreductases43, amylases44, 45, antibiotics46, 47, 48, 49, antibiotic resistance enzymes50 and membrane proteins51 . . .
    • . . . Despite these limitations, analysing and screening of libraries has yielded several novel biomolecules35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 61, 63, 70, 71, 72, 73 and provided insights into the genomes of uncultured prokaryotic soil organisms and the ecology of the soil ecosystem58, 65, 66, 69. . . .
  45. Yun, J. et al. Characterization of a novel amylolytic enzyme encoded by a gene from a soil-derived metagenomic library. Appl. Environ. Microbiol. 70, 7229-7235 (2004) , .
    • . . . The same approach has been used to clone genes from soil communities that code for lipases38, 39, 40, proteases41, 42, oxidoreductases43, amylases44, 45, antibiotics46, 47, 48, 49, antibiotic resistance enzymes50 and membrane proteins51 . . .
    • . . . Direct lysis approaches have been used more frequently than the separation techniques to isolate soil DNA for the construction of libraries35, 36, 37, 39, 42, 43, 45, 47, 61. . . .
    • . . . Small-insert soil-based libraries are useful for the isolation of single genes or small operons encoding new metabolic functions35, 38, 43, 45, 50, 51, 63 . . .
    • . . . Despite these limitations, analysing and screening of libraries has yielded several novel biomolecules35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 61, 63, 70, 71, 72, 73 and provided insights into the genomes of uncultured prokaryotic soil organisms and the ecology of the soil ecosystem58, 65, 66, 69. . . .
    • . . . Most of the screening methods to isolate genes or gene clusters for novel biocatalysts or small molecules are based on detecting activity from library-containing clones35, 36, 37, 38, 39, 40, 41, 42, 43, 45, 46, 47, 48, 49, 50, 51, 61, 63, 64, 72, 73 . . .
  46. Wang, G. Y. et al. Novel natural products from soil DNA libraries in a streptomycete host. Org. Lett. 2, 2401-2404 (2000).First report on the use of a non-E. coli host for screening of metagenomic libraries , .
    • . . . The same approach has been used to clone genes from soil communities that code for lipases38, 39, 40, proteases41, 42, oxidoreductases43, amylases44, 45, antibiotics46, 47, 48, 49, antibiotic resistance enzymes50 and membrane proteins51 . . .
    • . . . Despite these limitations, analysing and screening of libraries has yielded several novel biomolecules35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 61, 63, 70, 71, 72, 73 and provided insights into the genomes of uncultured prokaryotic soil organisms and the ecology of the soil ecosystem58, 65, 66, 69. . . .
    • . . . Most of the screening methods to isolate genes or gene clusters for novel biocatalysts or small molecules are based on detecting activity from library-containing clones35, 36, 37, 38, 39, 40, 41, 42, 43, 45, 46, 47, 48, 49, 50, 51, 61, 63, 64, 72, 73 . . .
    • . . . This strategy has been validated by the isolation of novel genes that encode degradative enzymes35, 37, 38, 39, 41, 43, 45, 63, antibiotic resistance50 and antibiotics36, 46, 47, 48, 49 . . .
    • . . . Recently, other bacterial hosts such as Streptomyces or Pseudomonas strains have been used to expand the range of soil-derived genes which can be detected during functional screens46, 49, 62 . . .
  47. MacNeil, I. A. et al. Expression and isolation of antimicrobial small molecules from soil DNA libraries. J. Mol. Microbiol. Biotechnol. 3, 301-308 (2001) , .
    • . . . The same approach has been used to clone genes from soil communities that code for lipases38, 39, 40, proteases41, 42, oxidoreductases43, amylases44, 45, antibiotics46, 47, 48, 49, antibiotic resistance enzymes50 and membrane proteins51 . . .
    • . . . As microbial populations are large, sample volumes can be small (500 g in most studies)25, 35, 37, 47, 52 . . .
    • . . . Direct lysis approaches have been used more frequently than the separation techniques to isolate soil DNA for the construction of libraries35, 36, 37, 39, 42, 43, 45, 47, 61. . . .
    • . . . Large-insert libraries are more appropriate to recover complex pathways that are encoded by large gene clusters or large DNA fragments for the characterization of genomes of uncultured soil microorganisms36, 37, 47, 48, 49, 58, 61, 64, 65, 66, 67 . . .
    • . . . Despite these limitations, analysing and screening of libraries has yielded several novel biomolecules35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 61, 63, 70, 71, 72, 73 and provided insights into the genomes of uncultured prokaryotic soil organisms and the ecology of the soil ecosystem58, 65, 66, 69. . . .
    • . . . Most of the screening methods to isolate genes or gene clusters for novel biocatalysts or small molecules are based on detecting activity from library-containing clones35, 36, 37, 38, 39, 40, 41, 42, 43, 45, 46, 47, 48, 49, 50, 51, 61, 63, 64, 72, 73 . . .
  48. Gillespie, D. E. et al. Isolation of antibiotics turbomycin A and B from a metagenomic library of soil microbial DNA. Appl. Environ. Microbiol. 68, 4310-4306 (2002) , .
    • . . . The same approach has been used to clone genes from soil communities that code for lipases38, 39, 40, proteases41, 42, oxidoreductases43, amylases44, 45, antibiotics46, 47, 48, 49, antibiotic resistance enzymes50 and membrane proteins51 . . .
    • . . . Large-insert libraries are more appropriate to recover complex pathways that are encoded by large gene clusters or large DNA fragments for the characterization of genomes of uncultured soil microorganisms36, 37, 47, 48, 49, 58, 61, 64, 65, 66, 67 . . .
    • . . . Most of the screening methods to isolate genes or gene clusters for novel biocatalysts or small molecules are based on detecting activity from library-containing clones35, 36, 37, 38, 39, 40, 41, 42, 43, 45, 46, 47, 48, 49, 50, 51, 61, 63, 64, 72, 73 . . .
  49. Courtois, S. et al. Recombinant environmental libraries provide access to microbial diversity for drug discovery from natural products. Appl. Environ. Microbiol. 69, 49-55 (2003).Strategy for increasing the screening efficiency of large-insert metagenomic libraries by using a cosmid shuttle vector , .
    • . . . The same approach has been used to clone genes from soil communities that code for lipases38, 39, 40, proteases41, 42, oxidoreductases43, amylases44, 45, antibiotics46, 47, 48, 49, antibiotic resistance enzymes50 and membrane proteins51 . . .
    • . . . Shuttle cosmid or BAC vectors can be used to transfer libraries that are produced in E. coli to other hosts such as Streptomyces or Pseudomonas species49, 62 . . .
    • . . . Large-insert libraries are more appropriate to recover complex pathways that are encoded by large gene clusters or large DNA fragments for the characterization of genomes of uncultured soil microorganisms36, 37, 47, 48, 49, 58, 61, 64, 65, 66, 67 . . .
    • . . . PCR is most commonly used for sequence-driven screening of soil-based libraries or soil DNA28, 31, 49, 56, 65, 66, 67, 69, 74, 75, 76, 77 . . .
    • . . . This approach has been used to identify phylogenetic anchors such as 16S rRNA genes65, 66, 69 and genes encoding enzymes with highly conserved domains such as polyketide synthases49, 67, 75 gluconic acid reductases76 and nitrile hydratases77 . . .
    • . . . Most of the screening methods to isolate genes or gene clusters for novel biocatalysts or small molecules are based on detecting activity from library-containing clones35, 36, 37, 38, 39, 40, 41, 42, 43, 45, 46, 47, 48, 49, 50, 51, 61, 63, 64, 72, 73 . . .
    • . . . Recently, other bacterial hosts such as Streptomyces or Pseudomonas strains have been used to expand the range of soil-derived genes which can be detected during functional screens46, 49, 62 . . .
  50. Riesenfeld, C. S., Goodman, R. M. & Handelsman, J. Uncultured soil bacteria are a reservoir of new antibiotic resistance genes. Environ. Microbiol. 6, 981-989 (2004).References 50 and 51 illustrate the immense power of activity-based screening strategies using host strains or mutants of host strains that require heterologous complementation for growth under selective conditions , .
    • . . . The same approach has been used to clone genes from soil communities that code for lipases38, 39, 40, proteases41, 42, oxidoreductases43, amylases44, 45, antibiotics46, 47, 48, 49, antibiotic resistance enzymes50 and membrane proteins51 . . .
    • . . . Small-insert soil-based libraries are useful for the isolation of single genes or small operons encoding new metabolic functions35, 38, 43, 45, 50, 51, 63 . . .
    • . . . Despite these limitations, analysing and screening of libraries has yielded several novel biomolecules35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 61, 63, 70, 71, 72, 73 and provided insights into the genomes of uncultured prokaryotic soil organisms and the ecology of the soil ecosystem58, 65, 66, 69. . . .
    • . . . Most of the screening methods to isolate genes or gene clusters for novel biocatalysts or small molecules are based on detecting activity from library-containing clones35, 36, 37, 38, 39, 40, 41, 42, 43, 45, 46, 47, 48, 49, 50, 51, 61, 63, 64, 72, 73 . . .
    • . . . This strategy has been validated by the isolation of novel genes that encode degradative enzymes35, 37, 38, 39, 41, 43, 45, 63, antibiotic resistance50 and antibiotics36, 46, 47, 48, 49 . . .
  51. Majernik, A., Gottschalk, G. & Daniel, R. Screening of environmental DNA libraries for the presence of genes conferring Na+(Li+)/H+ antiporter activity on Escherichia coli:characterization of the recovered genes and the corresponding gene products. J. Bacteriol. 183, 6645-6653 (2001) , .
    • . . . The same approach has been used to clone genes from soil communities that code for lipases38, 39, 40, proteases41, 42, oxidoreductases43, amylases44, 45, antibiotics46, 47, 48, 49, antibiotic resistance enzymes50 and membrane proteins51 . . .
    • . . . Small-insert soil-based libraries are useful for the isolation of single genes or small operons encoding new metabolic functions35, 38, 43, 45, 50, 51, 63 . . .
    • . . . Most of the screening methods to isolate genes or gene clusters for novel biocatalysts or small molecules are based on detecting activity from library-containing clones35, 36, 37, 38, 39, 40, 41, 42, 43, 45, 46, 47, 48, 49, 50, 51, 61, 63, 64, 72, 73 . . .
    • . . . An example is complementation of a Na+/H+ antiporter-deficient E. coli strain with soil-derived libraries, which led to the identification of two new genes that encode Na+/H+ antiporters from a soil library consisting of 1,480,000 clones51 . . .
  52. Liesack, W. & Stackebrandt, E. Occurrence of novel groups of the domain Bacteria as revealed by analysis of genetic material isolated from an Australian terrestrial environment. J. Bacteriol. 174, 5072-5078 (1992) , .
    • . . . As microbial populations are large, sample volumes can be small (500 g in most studies)25, 35, 37, 47, 52 . . .
  53. Martin-Laurent, F. et al. DNA extractions from soils: old bias for new microbial diversity analysis methods. Appl. Environ. Microbiol. 67, 2354-2359 (2001) , .
    • . . . Because of the heterogeneity of soils, the extent of microbial diversity and the adherence of microorganisms to soil particles, DNA extraction is particularly challenging53 . . .
  54. Tebbe, C. C. & Vahjen, W. Interference of humic acids and DNA extracted directly from soil in detection and transformation of recombinant DNA from bacteria and yeast. Appl. Environ. Microbiol. 59, 2657-2665 (1993) , .
    • . . . Also, extraction of soil DNA often results in coextraction of humic substances, which interfere with restriction-enzyme digestion and PCR amplification and reduce cloning efficiency, transformation efficiency and the specificity of DNA hybridization21, 54, 55. . . .
  55. Tsai, Y. -L., & Olson, B. H. Detection of low numbers of bacterial cells in soils and sediments. Appl. Environ. Microbiol. 58, 754-757 (1992) , .
    • . . . Also, extraction of soil DNA often results in coextraction of humic substances, which interfere with restriction-enzyme digestion and PCR amplification and reduce cloning efficiency, transformation efficiency and the specificity of DNA hybridization21, 54, 55. . . .
  56. Courtois, S. et al. Quantification of bacterial subgroups in soil: comparison of DNA extracted directly from soil or from cells previously released by density gradient centrifugation. Environ. Microbiol. 3, 431-439 (2001) , .
    • . . . The amounts of DNA isolated from different soil types using a selection of protocols range from less than 1 mg to approximately 500 g of DNA per gram of soil24, 25, 26, 35, 56, 57 . . .
    • . . . The separation of microorganisms from the soil matrix is achieved by mild mechanical forces or chemical procedures such as blending, rotating pestle homogenization or the addition of cation-exchange resins, followed by density gradient or differential centrifugation22, 23, 56, 57 . . .
    • . . . In addition, the average size of the isolated DNA is larger than that typically obtained by the direct lysis approach56 and is therefore more suitable for the generation of large-insert libraries. . . .
    • . . . As different soil microorganisms have different susceptibilities to cell lysis methods, the sequences present in the isolated DNA and the libraries is dependent on the extraction method56, 57, 59. . . .
    • . . . However, Courtois et al.56 found no significant difference in the spectrum of bacterial diversity during a comparison of DNA extracted directly from soil with DNA that was isolated from cells that were separated from the soil matrix . . .
    • . . . PCR is most commonly used for sequence-driven screening of soil-based libraries or soil DNA28, 31, 49, 56, 65, 66, 67, 69, 74, 75, 76, 77 . . .
  57. Gabor, E. M., de Vries, E. J. & Janssen, D. B. Efficient recovery of environmental DNA for expression cloning by indirect methods. FEMS Microbiol. Ecol. 44, 153-163 (2003).References 57 and 58 compare direct cell lysis with cell separation approaches with respect to species representation and content of eukaryotic DNA in the isolated soil DNA , .
    • . . . The amounts of DNA isolated from different soil types using a selection of protocols range from less than 1 mg to approximately 500 g of DNA per gram of soil24, 25, 26, 35, 56, 57 . . .
    • . . . More DNA is recovered using the direct lysis approaches, for example, Gabor et al.57 recorded a 10 to 100-fold reduction in the DNA yield using the cell separation approach compared with the direct lysis approach. . . .
    • . . . In addition, several methods use mechanical disruption steps such as bead-beating, freeze–thawing or grinding of samples to lyse cells19, 24, 25, 26, 27, 57 . . .
    • . . . In addition to the DNA that is recovered from lysed prokaryotes, extracellular DNA and eukaryotic DNA are also recovered27, 57, 58 . . .
    • . . . The separation of microorganisms from the soil matrix is achieved by mild mechanical forces or chemical procedures such as blending, rotating pestle homogenization or the addition of cation-exchange resins, followed by density gradient or differential centrifugation22, 23, 56, 57 . . .
    • . . . As different soil microorganisms have different susceptibilities to cell lysis methods, the sequences present in the isolated DNA and the libraries is dependent on the extraction method56, 57, 59. . . .
    • . . . As expression in bacterial hosts is usually limited to prokaryotic genes and soil DNA can, depending on the isolation method, contain an important amount of eukaryotic DNA57, using eukaryotic hosts could also be useful for function-driven screens of soil-based libraries. . . .
  58. Treusch, A. H. et al. Characterization of large-insert DNA libraries from soil for environmental genomic studies of Archaea. Environ Microbiol. 6, 970-980 (2004) , .
    • . . . In addition to the DNA that is recovered from lysed prokaryotes, extracellular DNA and eukaryotic DNA are also recovered27, 57, 58 . . .
    • . . . Large-insert libraries are more appropriate to recover complex pathways that are encoded by large gene clusters or large DNA fragments for the characterization of genomes of uncultured soil microorganisms36, 37, 47, 48, 49, 58, 61, 64, 65, 66, 67 . . .
    • . . . Despite these limitations, analysing and screening of libraries has yielded several novel biomolecules35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 61, 63, 70, 71, 72, 73 and provided insights into the genomes of uncultured prokaryotic soil organisms and the ecology of the soil ecosystem58, 65, 66, 69. . . .
    • . . . This approach has been successfully used in the characterization of uncultivated members of the Acidobacteria phylum, which are abundant in soil but about which little is known66, 69, and to access the genomes of uncultivated Archaea in soil58, 65 . . .
  59. Stach, J. E. M., Bathe, S., Clapp, J. P. & Burns, R. G. PCR-SSCP comparison of 16S rDNA sequence diversity in soil DNA obtained using different isolation and purification methods. FEMS Microbiol. Ecol. 36, 139-151 (2001) , .
    • . . . As different soil microorganisms have different susceptibilities to cell lysis methods, the sequences present in the isolated DNA and the libraries is dependent on the extraction method56, 57, 59. . . .
  60. Leff, L. G., Dana, J. R., McArthur, J. V. & Shimkets, L. J. Comparison of methods of DNA extraction from stream sediments. Appl. Environ. Microbiol. 61, 1141-1143 (1995) , .
    • . . . It is usually presumed that the DNA isolated by the direct lysis approach better represents the microbial diversity of a soil sample because this method does not include a cell separation step, so microorganisms that adhere to particles are also lysed21, 60 . . .
  61. Brady, S. F., Chao, C. J. & Clardy, J. Long-chain N-acyltyrosine synthases from environmental DNA. Appl. Environ. Microbiol. 70, 46865-46870 (2004) , .
    • . . . Direct lysis approaches have been used more frequently than the separation techniques to isolate soil DNA for the construction of libraries35, 36, 37, 39, 42, 43, 45, 47, 61. . . .
    • . . . Large-insert libraries are more appropriate to recover complex pathways that are encoded by large gene clusters or large DNA fragments for the characterization of genomes of uncultured soil microorganisms36, 37, 47, 48, 49, 58, 61, 64, 65, 66, 67 . . .
    • . . . Despite these limitations, analysing and screening of libraries has yielded several novel biomolecules35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 61, 63, 70, 71, 72, 73 and provided insights into the genomes of uncultured prokaryotic soil organisms and the ecology of the soil ecosystem58, 65, 66, 69. . . .
    • . . . Most of the screening methods to isolate genes or gene clusters for novel biocatalysts or small molecules are based on detecting activity from library-containing clones35, 36, 37, 38, 39, 40, 41, 42, 43, 45, 46, 47, 48, 49, 50, 51, 61, 63, 64, 72, 73 . . .
  62. Martinez, A. et al. Genetically modified bacterial strains and novel bacterial artificial chromosome shuttle vectors for constructing environmental libraries and detecting heterologous natural products in multiple expression hosts. Appl. Environ. Microbiol. 70, 2452-2463 (2004) , .
    • . . . Shuttle cosmid or BAC vectors can be used to transfer libraries that are produced in E. coli to other hosts such as Streptomyces or Pseudomonas species49, 62 . . .
    • . . . Recently, other bacterial hosts such as Streptomyces or Pseudomonas strains have been used to expand the range of soil-derived genes which can be detected during functional screens46, 49, 62 . . .
  63. Gabor, E. M., de Vries, E. J. & Janssen, D. B. Construction, characterization, and use of small-insert gene banks of DNA isolated from soil and enrichment cultures for the recovery of novel amidases. Environ. Microbiol. 6, 948-958 (2004) , .
    • . . . Small-insert soil-based libraries are useful for the isolation of single genes or small operons encoding new metabolic functions35, 38, 43, 45, 50, 51, 63 . . .
    • . . . Despite these limitations, analysing and screening of libraries has yielded several novel biomolecules35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 61, 63, 70, 71, 72, 73 and provided insights into the genomes of uncultured prokaryotic soil organisms and the ecology of the soil ecosystem58, 65, 66, 69. . . .
    • . . . Most of the screening methods to isolate genes or gene clusters for novel biocatalysts or small molecules are based on detecting activity from library-containing clones35, 36, 37, 38, 39, 40, 41, 42, 43, 45, 46, 47, 48, 49, 50, 51, 61, 63, 64, 72, 73 . . .
    • . . . This strategy has been validated by the isolation of novel genes that encode degradative enzymes35, 37, 38, 39, 41, 43, 45, 63, antibiotic resistance50 and antibiotics36, 46, 47, 48, 49 . . .
    • . . . To increase this frequency, enrichment steps for microorganisms harbouring the desired traits have been used prior to library construction63, 78, 85, 86, 87 . . .
    • . . . This strategy has been successfully used to isolate biotechnologically useful gene products, including alcohol oxidoreductases85, coenzyme B12-dependent dehydratases78, amidases63, agarases87 and genes involved in biotin synthesis86 . . .
  64. Brady, S. F., Chao, C. J., Handelsman, J. & Clardy, J. Cloning and heterologous expression of a natural product biosynthetic gene cluster from eDNA. Org. Lett. 3, 1981-1984 (2001) , .
    • . . . Large-insert libraries are more appropriate to recover complex pathways that are encoded by large gene clusters or large DNA fragments for the characterization of genomes of uncultured soil microorganisms36, 37, 47, 48, 49, 58, 61, 64, 65, 66, 67 . . .
    • . . . Most of the screening methods to isolate genes or gene clusters for novel biocatalysts or small molecules are based on detecting activity from library-containing clones35, 36, 37, 38, 39, 40, 41, 42, 43, 45, 46, 47, 48, 49, 50, 51, 61, 63, 64, 72, 73 . . .
  65. Quaiser, A. et al. First insight into the genome of an uncultivated crenarchaeota from soil. Environ. Microbiol. 4, 603-611 (2002) , .
    • . . . Large-insert libraries are more appropriate to recover complex pathways that are encoded by large gene clusters or large DNA fragments for the characterization of genomes of uncultured soil microorganisms36, 37, 47, 48, 49, 58, 61, 64, 65, 66, 67 . . .
    • . . . Despite these limitations, analysing and screening of libraries has yielded several novel biomolecules35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 61, 63, 70, 71, 72, 73 and provided insights into the genomes of uncultured prokaryotic soil organisms and the ecology of the soil ecosystem58, 65, 66, 69. . . .
    • . . . PCR is most commonly used for sequence-driven screening of soil-based libraries or soil DNA28, 31, 49, 56, 65, 66, 67, 69, 74, 75, 76, 77 . . .
    • . . . This approach has been used to identify phylogenetic anchors such as 16S rRNA genes65, 66, 69 and genes encoding enzymes with highly conserved domains such as polyketide synthases49, 67, 75 gluconic acid reductases76 and nitrile hydratases77 . . .
    • . . . This approach has been successfully used in the characterization of uncultivated members of the Acidobacteria phylum, which are abundant in soil but about which little is known66, 69, and to access the genomes of uncultivated Archaea in soil58, 65 . . .
  66. Quaiser, A. et al. Acidobacteria form a coherent but highly diverse group within the bacterial domain: evidence from environmental genomics. Mol. Microbiol. 50, 563-575 (2003).Partial genomic characterization of uncultivated Acidobacteria , .
    • . . . Large-insert libraries are more appropriate to recover complex pathways that are encoded by large gene clusters or large DNA fragments for the characterization of genomes of uncultured soil microorganisms36, 37, 47, 48, 49, 58, 61, 64, 65, 66, 67 . . .
    • . . . PCR is most commonly used for sequence-driven screening of soil-based libraries or soil DNA28, 31, 49, 56, 65, 66, 67, 69, 74, 75, 76, 77 . . .
    • . . . This approach has been successfully used in the characterization of uncultivated members of the Acidobacteria phylum, which are abundant in soil but about which little is known66, 69, and to access the genomes of uncultivated Archaea in soil58, 65 . . .
  67. Ginolhac, A. et al. Phylogenetic analysis of polyketide synthase I domains from soil metagenomic libraries allows selection of promising clones. Appl. Environ. Microbiol. 70, 5522-5527 (2004) , .
    • . . . Large-insert libraries are more appropriate to recover complex pathways that are encoded by large gene clusters or large DNA fragments for the characterization of genomes of uncultured soil microorganisms36, 37, 47, 48, 49, 58, 61, 64, 65, 66, 67 . . .
    • . . . PCR is most commonly used for sequence-driven screening of soil-based libraries or soil DNA28, 31, 49, 56, 65, 66, 67, 69, 74, 75, 76, 77 . . .
    • . . . This approach has been used to identify phylogenetic anchors such as 16S rRNA genes65, 66, 69 and genes encoding enzymes with highly conserved domains such as polyketide synthases49, 67, 75 gluconic acid reductases76 and nitrile hydratases77 . . .
  68. Riesenfeld, C. S., Schloss, P. D. & Handelsman, J. Metagenomics: genomic analysis of microbial communities. Annu. Rev. Genet. 38, 525-552 (2004) , .
    • . . . To achieve substantial representation of the genomes from rare members (less than 1%) of the soil community, it has been calculated that libraries containing 10,000 Gb of soil DNA (1011 BAC clones) might be required68 . . .
  69. Liles, M. R., Manske, B. F., Bintrim, S. B., Handelsman, J. & Goodman, R. M. A census of rRNA genes and linked genomic sequences within a soil metagenomic library. Appl. Environ. Microbiol. 69, 2684-2691 (2003) , .
    • . . . In addition, a comparison of the 16S rRNA genes in a BAC library with a collection of DNA fragments that were generated by direct PCR amplification and cloning of the 16S rRNA genes from the same soil sample indicated that the representation of certain bacterial groups in the library was different from that present in the soil sample69 . . .
    • . . . Despite these limitations, analysing and screening of libraries has yielded several novel biomolecules35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 61, 63, 70, 71, 72, 73 and provided insights into the genomes of uncultured prokaryotic soil organisms and the ecology of the soil ecosystem58, 65, 66, 69. . . .
    • . . . PCR is most commonly used for sequence-driven screening of soil-based libraries or soil DNA28, 31, 49, 56, 65, 66, 67, 69, 74, 75, 76, 77 . . .
    • . . . This approach has been used to identify phylogenetic anchors such as 16S rRNA genes65, 66, 69 and genes encoding enzymes with highly conserved domains such as polyketide synthases49, 67, 75 gluconic acid reductases76 and nitrile hydratases77 . . .
    • . . . This approach has been successfully used in the characterization of uncultivated members of the Acidobacteria phylum, which are abundant in soil but about which little is known66, 69, and to access the genomes of uncultivated Archaea in soil58, 65 . . .
  70. Robertson, D. E. et al. Exploring nitrilase sequence for enantioselective catalysis. Appl. Environ. Microbiol. 70, 2429-2436 (2004) , .
    • . . . Despite these limitations, analysing and screening of libraries has yielded several novel biomolecules35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 61, 63, 70, 71, 72, 73 and provided insights into the genomes of uncultured prokaryotic soil organisms and the ecology of the soil ecosystem58, 65, 66, 69. . . .
  71. Gray, K. A., Richardson, T. H., Robertson, D. E., Swanson, P. E. & Subramanian, M. V. Soil-based gene discovery: a new technology to accelerate and broaden biocatalytic applications. Adv. Appl. Microbiol. 52, 1-27 (2003) , .
    • . . . Despite these limitations, analysing and screening of libraries has yielded several novel biomolecules35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 61, 63, 70, 71, 72, 73 and provided insights into the genomes of uncultured prokaryotic soil organisms and the ecology of the soil ecosystem58, 65, 66, 69. . . .
  72. Brady, S. F. & Clardy, J. Synthesis of long-chain fatty acid enol esters isolated from an environmental DNA clone. Org. Lett. 5, 121-124 (2003) , .
    • . . . Despite these limitations, analysing and screening of libraries has yielded several novel biomolecules35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 61, 63, 70, 71, 72, 73 and provided insights into the genomes of uncultured prokaryotic soil organisms and the ecology of the soil ecosystem58, 65, 66, 69. . . .
    • . . . Most of the screening methods to isolate genes or gene clusters for novel biocatalysts or small molecules are based on detecting activity from library-containing clones35, 36, 37, 38, 39, 40, 41, 42, 43, 45, 46, 47, 48, 49, 50, 51, 61, 63, 64, 72, 73 . . .
  73. Brady, S. F., Chao, C. J. & Clardy, J. New natural product families from an environmental DNA (eDNA) gene cluster. J. Am. Chem. Soc. 124, 9968-9969 (2002) , .
    • . . . Despite these limitations, analysing and screening of libraries has yielded several novel biomolecules35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 61, 63, 70, 71, 72, 73 and provided insights into the genomes of uncultured prokaryotic soil organisms and the ecology of the soil ecosystem58, 65, 66, 69. . . .
    • . . . Most of the screening methods to isolate genes or gene clusters for novel biocatalysts or small molecules are based on detecting activity from library-containing clones35, 36, 37, 38, 39, 40, 41, 42, 43, 45, 46, 47, 48, 49, 50, 51, 61, 63, 64, 72, 73 . . .
  74. Borneman, J. & Triplett, E. W. Molecular microbial diversity in soils from eastern Amazonia: evidence for unusual microorganisms and microbial population shifts associated with deforestation. Appl. Environ. Microbiol. 63, 2647-2653 (1997) , .
    • . . . PCR is most commonly used for sequence-driven screening of soil-based libraries or soil DNA28, 31, 49, 56, 65, 66, 67, 69, 74, 75, 76, 77 . . .
  75. Seow, K. -T. et al. A study of iterative type II polyketide synthases, using bacterial genes cloned from soil DNA: a means to access and use genes from uncultured microorganisms. J. Bacteriol. 179, 7360-7368 (1997) , .
    • . . . PCR is most commonly used for sequence-driven screening of soil-based libraries or soil DNA28, 31, 49, 56, 65, 66, 67, 69, 74, 75, 76, 77 . . .
    • . . . This approach has been used to identify phylogenetic anchors such as 16S rRNA genes65, 66, 69 and genes encoding enzymes with highly conserved domains such as polyketide synthases49, 67, 75 gluconic acid reductases76 and nitrile hydratases77 . . .
  76. Eschenfeldt, W. H. et al. DNA from uncultured organisms as a source of 2,5-diketo-D-gluconic acid reductases. Appl. Environ. Microbiol. 67, 4206-4214 (2001) , .
    • . . . PCR is most commonly used for sequence-driven screening of soil-based libraries or soil DNA28, 31, 49, 56, 65, 66, 67, 69, 74, 75, 76, 77 . . .
    • . . . This approach has been used to identify phylogenetic anchors such as 16S rRNA genes65, 66, 69 and genes encoding enzymes with highly conserved domains such as polyketide synthases49, 67, 75 gluconic acid reductases76 and nitrile hydratases77 . . .
  77. Precigou, S., Goulas, P. & Duran, R. Rapid and specific identification of nitrile hydratase (Nhase)-encoding genes. FEMS Microbiol. Lett. 204, 155-161 (2001) , .
    • . . . PCR is most commonly used for sequence-driven screening of soil-based libraries or soil DNA28, 31, 49, 56, 65, 66, 67, 69, 74, 75, 76, 77 . . .
    • . . . This approach has been used to identify phylogenetic anchors such as 16S rRNA genes65, 66, 69 and genes encoding enzymes with highly conserved domains such as polyketide synthases49, 67, 75 gluconic acid reductases76 and nitrile hydratases77 . . .
  78. Knietsch, A., Bowien, S., Whited, G., Gottschalk, G. & Daniel, R. Identification and characterization of genes encoding coenzyme B12-dependent glycerol and diol dehydratases from metagenomic DNA libraries derived from enrichment cultures. Appl. Environ. Microbiol. 69, 3048-3060 (2003) , .
    • . . . Hybridization using target-specific probes has also been used to screen soil-based libraries78 . . .
    • . . . To increase this frequency, enrichment steps for microorganisms harbouring the desired traits have been used prior to library construction63, 78, 85, 86, 87 . . .
    • . . . This strategy has been successfully used to isolate biotechnologically useful gene products, including alcohol oxidoreductases85, coenzyme B12-dependent dehydratases78, amidases63, agarases87 and genes involved in biotin synthesis86 . . .
  79. Stokes, H. W. et al. Gene cassette PCR: sequence-independent recovery of entire genes from environmental DNA. Appl. Environ. Microbiol. 67, 5240-5246 (2001).Library-independent approach for recovery of novel genes from environmental samples , .
    • . . . Stokes et al.79 described a different PCR-based approach that uses primers that target a 59-bp recombination site . . .
  80. Wu, L. et al. Development and evaluation of functional gene arrays for detection of selected genes in the environment. Appl. Environ. Microbiol. 67, 5780-5790 (2001).One of the first reports on using DNA microarray technology for assessing functional gene diversity in soil microbial communities , .
    • . . . Microarray technology could be useful for analysing the soil metagenome and profiling metagenomic libraries80, 81, 82, 83, 84 . . .
    • . . . For example, genes encoding key reactions in the nitrogen cycle were detected using microarrays from samples that were collected from soil, and provided information on the composition and activity of the complex soil microbial community80 . . .
  81. Zhou, J. & Thompson, D. K. Challenges in applying microarrays to environmental studies. Curr. Opin. Biotechnol. 13, 204-207 (2002) , .
    • . . . Microarray technology could be useful for analysing the soil metagenome and profiling metagenomic libraries80, 81, 82, 83, 84 . . .
    • . . . However, microarray methods for gene detection show a 100 to 10,000-fold lower sensitivity than PCR81 . . .
  82. Cho, J. -C. & Tiedje, J. M. Quantitative detection of microbial genes by using DNA microarrays. Appl. Environ. Microbiol. 68, 1425-1430 (2002) , .
    • . . . Microarray technology could be useful for analysing the soil metagenome and profiling metagenomic libraries80, 81, 82, 83, 84 . . .
  83. Denef, V. J. et al. Validation of a more sensitive method for using spotted oligonucleotide DNA microarrays for functional genomics studies on bacterial communities. Environ. Microbiol. 5, 933-943 (2003) , .
    • . . . Microarray technology could be useful for analysing the soil metagenome and profiling metagenomic libraries80, 81, 82, 83, 84 . . .
  84. Sebat, J. L., Colwell, F. S. & Crawford, R. L. Metagenomic profiling: microarray analysis of an environmental genomic library. Appl. Environ. Microbiol. 69, 4927-4934 (2003) , .
    • . . . Microarray technology could be useful for analysing the soil metagenome and profiling metagenomic libraries80, 81, 82, 83, 84 . . .
  85. Knietsch, A., Waschkowitz, T., Bowien, S., Henne, A. & Daniel, R. Construction and screening of metagenomic libraries derived from enrichment cultures: generation of a gene bank for genes conferring alcohol oxidoreductase activity on Escherichia coli. Appl. Environ. Microbiol. 69, 1408-1416 (2003) , .
    • . . . An example is the screening of soil-based libraries for genes conferring polyol oxidoreductase activity43, 85, which was based on the ability of the recombinant E. coli strains to form carbonyls from polyols (Fig. 2a) . . .
    • . . . Screening is based on the ability of the library-containing Escherichia coli clones to form carbonyls from test substrates, that is, polyols43, 85, during growth on indicator agar . . .
    • . . . To increase this frequency, enrichment steps for microorganisms harbouring the desired traits have been used prior to library construction63, 78, 85, 86, 87 . . .
    • . . . Nevertheless, a combination of traditional enrichment and metagenomic technologies is an efficient tool to increase the amount of positive clones in a screen and to isolate novel biomolecules when samples from complex habitats such as soil are used as starting material and non-vigorous enrichment steps are carried out85, 88 . . .
    • . . . This strategy has been successfully used to isolate biotechnologically useful gene products, including alcohol oxidoreductases85, coenzyme B12-dependent dehydratases78, amidases63, agarases87 and genes involved in biotin synthesis86 . . .
  86. Entcheva, P., Liebl, W., Johann, A., Hartsch, T. & Streit, W. Direct cloning from enrichment cultures, a reliable strategy for isolation of complete operons and genes from microbial consortia. Appl. Environ. Microbiol. 67, 89-99 (2001) , .
    • . . . To increase this frequency, enrichment steps for microorganisms harbouring the desired traits have been used prior to library construction63, 78, 85, 86, 87 . . .
    • . . . This strategy has been successfully used to isolate biotechnologically useful gene products, including alcohol oxidoreductases85, coenzyme B12-dependent dehydratases78, amidases63, agarases87 and genes involved in biotin synthesis86 . . .
  87. Voget, S. et al. Prospecting for novel biocatalysts in a soil metagenome. Appl. Environ. Microbiol. 69, 6235-6242 (2003).The library derived from an agarolytic microbial consortium enriched from soil harbours a large number of genes encoding industrially relevant biocatalysts , .
    • . . . To increase this frequency, enrichment steps for microorganisms harbouring the desired traits have been used prior to library construction63, 78, 85, 86, 87 . . .
    • . . . This strategy has been successfully used to isolate biotechnologically useful gene products, including alcohol oxidoreductases85, coenzyme B12-dependent dehydratases78, amidases63, agarases87 and genes involved in biotin synthesis86 . . .
  88. Daniel, R. The soil metagenome - a rich resource for the discovery of novel natural products. Curr. Opin. Microbiol. 15, 199-204 (2004) , .
    • . . . Nevertheless, a combination of traditional enrichment and metagenomic technologies is an efficient tool to increase the amount of positive clones in a screen and to isolate novel biomolecules when samples from complex habitats such as soil are used as starting material and non-vigorous enrichment steps are carried out85, 88 . . .
  89. Radajewski, S., McDonald, I. R. & Murrell, J. C. Stable-isotope probing of nucleic acids: a window to the function of uncultured microorganisms. Curr. Opin. Biotechnol. 14, 296-302 (2003) , .
    • . . . Other potential methods that could be used to enrich genomes from metabolically active members of the soil microbial community prior to library construction are stable isotope probing89, 90 (see also the article by M . . .
  90. Wellington, E. M. H., Berry, A. & Krsek, M. Resolving functional diversity in relation to microbial community structure in soil: exploiting genomics and stable isotope probing. Curr. Opin. Microbiol. 6, 295-301 (2003) , .
    • . . . Other potential methods that could be used to enrich genomes from metabolically active members of the soil microbial community prior to library construction are stable isotope probing89, 90 (see also the article by M . . .
  91. Yin, B., Crowley, D., Sparovek, G., De Melo, W. J. & Borneman, J. Bacterial functional redundancy along a soil reclamation gradient. Appl. Environ. Microbiol. 66, 4361-4365 (2000) , .
    • . . . Murrell in this issue) and enrichment with bromodeoxyuridine in the presence of selective substrates91 . . .
  92. Schloss, P. D., Larget, B. R. & Handelsman, J. Integration of microbial ecology and statistics: a test to compare gene libraries. Appl. Environ. Microbiol. 70, 5485-5492 (2004).A statistical approach for comparing gene libraries is described , .
    • . . . Programs such as –LIBSHUFF92, which has been employed for comparison of 16S rRNA gene libraries, might be useful for this purpose after further development . . .
  93. Uchiyama, T., Abe, T., Ikemura, T. & Watanabe, K. Substrate-induced gene-expression screening of environmental metagenome libraries for isolation of catabolic genes. Nature Biotechnol. 23, 88-93 (2005).This strategy for screening of metagenomic libraries has enormous potential for soil-based gene discovery , .
    • . . . Recently, a third high-throughput screening strategy, which is termed substrate-induced gene expression cloning (SIGEX) has been introduced for the identification and recovery of genes that encode catabolic pathways93 . . .
  94. Venter, J. C. et al. Environmental shotgun sequencing of the Sargasso Sea. Science 304, 66-74 (2004) , .
    • . . . Although considerable progress has been made in the characterization of microbial communities by random sequencing94, a further improvement of sequencing technologies and bioinformatic tools for analysing the enormous amount of data produced, combined with a reduction in sequencing costs, is required to apply this technique to the soil metagenome . . .
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