Targeted access to the genomes of low-abundance organisms in complex microbial communities.

Current metagenomic approaches to the study of complex microbial consortia provide a glimpse into the community metabolism and occasionally allow genomic assemblies for the most abundant organisms. However, little information is gained for the members of the community present at low frequencies, especially those representing yet-uncultured taxa, which include the bulk of the diversity present in most environments. Here we used phylogenetically directed cell separation by fluorescence in situ hybridization and flow cytometry, followed by amplification and sequencing of a fraction of the genomic DNA of several bacterial cells that belong to the TM7 phylum. Partial genomic assembly allowed, for the first time, a look into the evolution and potential metabolism of a soil representative from this group of organisms for which there are no species in stable laboratory cultures. Genomic reconstruction from targeted cells of uncultured organisms isolated directly from the environment represents a powerful approach to access any specific members of a community and an alternative way to assess the community's metabolic potential.

Environmental whole-genome amplification to access microbial populations in contaminated sediments.

Low-biomass samples from nitrate and heavy metal contaminated soils yield DNA amounts that have limited use for direct, native analysis and screening. Multiple displacement amplification (MDA) using phi29 DNA polymerase was used to amplify whole genomes from environmental, contaminated, subsurface sediments. By first amplifying the genomic DNA (gDNA), biodiversity analysis and gDNA library construction of microbes found in contaminated soils were made possible. The MDA method was validated by analyzing amplified genome coverage from approximately five Escherichia coli cells, resulting in 99.2% genome coverage. The method was further validated by confirming overall representative species coverage and also an amplification bias when amplifying from a mix of eight known bacterial strains. We extracted DNA from samples with extremely low cell densities from a U.S. Department of Energy contaminated site. After amplification, small-subunit rRNA analysis revealed relatively even distribution of species across several major phyla. Clone libraries were constructed from the amplified gDNA, and a small subset of clones was used for shotgun sequencing. BLAST analysis of the library clone sequences showed that 64.9% of the sequences had significant similarities to known proteins, and "clusters of orthologous groups" (COG) analysis revealed that more than half of the sequences from each library contained sequence similarity to known proteins. The libraries can be readily screened for native genes or any target of interest. Whole-genome amplification of metagenomic DNA from very minute microbial sources, while introducing an amplification bias, will allow access to genomic information that was not previously accessible. The reported SSU rRNA sequences and library clone end sequences are listed with their respective GenBank accession numbers, DQ 404590 to DQ 404652, DQ 404654 to DQ 404938, and DX 385314 to DX 389173.