Metagenomic Profiling: Microarray Analysis of an Environmental Genomic Library

Abstract

Genomic libraries derived from environmental DNA (metagenomic libraries) are useful for characterizing uncultured microorganisms. However, conventional library-screening techniques permit characterization of relatively few environmental clones. Here we describe a novel approach for characterization of a metagenomic library by hybridizing the library with DNA from a set of groundwater isolates, reference strains, and communities. A cosmid library derived from a microcosm of groundwater microorganisms was used to construct a microarray (COSMO) containing ϳ1-kb PCR products amplified from the inserts of 672 cosmids plus a set of 16S ribosomal DNA controls. COSMO was hybridized with Cy5-labeled genomic DNA from each bacterial strain, and the results were compared with the results for a common Cy3-labeled reference DNA sample consisting of a composite of genomic DNA from multiple species. The accuracy of the results was confirmed by the preferential hybridization of each strain to its corresponding rDNA probe. Cosmid clones were identified that hybridized specifically to each of 10 microcosm isolates, and other clones produced positive results with multiple related species, which is indicative of conserved genes. Many clones did not hybridize to any microcosm isolate; however, some of these clones hybridized to community genomic DNA, suggesting that they were derived from microbes that we failed to isolate in pure culture. Based on identification of genes by end sequencing of 17 such clones, DNA could be assigned to functions that have potential ecological importance, including hydrogen oxidation, nitrate reduction, and transposition. Metagenomic profiling offers an effective approach for rapidly characterizing many clones and identifying the clones corresponding to unidentified species of microorganisms.Microorganisms contribute significantly to the earth's biological diversity, yet relatively few of the microorganisms present in nature have been cultured and characterized. It is generally accepted that less than 1% of bacteria and fungi present in most habitats have been cultivated for study in pure culture (2). Although direct analysis of environmental DNA samples by PCR is effective for showing the presence of uncultured microorganisms, biases in primer specificity and amplification of different targets prevent full recognition of microbial diversity (31,37,40,41). Thus, new approaches to examination of community genomes are needed.The use of large-insert genomic libraries is a powerful approach for isolating DNA sequences from complex mixtures of uncultured microorganisms. Direct cloning of DNA from environmental samples makes it possible to avoid some of the biases of cultivation and PCR. In addition, genomic fragments that are Ͼ100 kb long can be obtained, and they provide significant functional and taxonomic information about the organisms from which they were derived. Such metagenomic libraries have been used to identify novel genes from uncultivated species of archaea, bacteria, and viruses that are respons...

Metagenomics: Genomic Analysis of Microbial Communities

“…These methods involve profiling clones with microarrays that identify previously unknown genes in environmental samples (107), subtractive hybridization to eliminate all sequences that hybridize with another environment, or subtractive hybridization to identify differentially expressed genes (35), and genomic sequence tags (28). These methods will enhance the efficiency of screening and aid in identifying minor components in communities and genes that define community uniqueness.…”

Development of a "genome-proxy" microarray for profiling marine microbial communities, and its application to a time series in Monterey Bay, California

“…Environmental genomic libraries have previously been combined with microarray technology, but with the goal of screening the libraries rather than exploring the environment (Sebat et al, 2003, Park et al, 2008. The library inserts themselves serve as probes on a microarray, which is then used to query pure cultures, enriched treatments, and natural communities.…”

“…This requires genomic surveys of the environment of interest (as suggested by Greene and Voordouw, 2003;Zhou, 2003) to provide the native genomic sequence information for probe design. While several arrays have been designed using some sequences derived from the community of interest (for example with environmental isolates' DNA, Greene and Voordouw, 2003;Wu et al, 2004; or 1 kb inserts from a groundwater cosmid library, Sebat et al, 2003), a more comprehensive use of uncultivated genomic data for arrays has not yet been reported.…”

Engineering Complex Phenotypes in Industrial Strains

“…E. coli, Streptomyces liVidans, Pseudomonas putida, and Rhizobium leguminosarium have been used as surrogate hosts for screening metagenomic libraries (93). Recently, high-density DNA microarrays made from genomes of bacterial isolates from the environment, reference strains, and environmental DNA have been used for metagenomic profiling that enables rapid characterization of DNA from metagenomic libraries and DNA from strains that have not been isolated in pure cultures (94).…”