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Applied and Environmental Microbiology, May 2006, p. 3291-3301, Vol. 72, No. 5
0099-2240/06/$08.00+0     doi:10.1128/AEM.72.5.3291-3301.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Environmental Whole-Genome Amplification To Access Microbial Populations in Contaminated Sediments

Carl B. Abulencia,1,2 Denise L. Wyborski,1,2 Joseph A. Garcia,1 Mircea Podar,1 Wenqiong Chen,1,2 Sherman H. Chang,1 Hwai W. Chang,1 David Watson,3 Eoin L. Brodie,2,4 Terry C. Hazen,2,4 and Martin Keller1,2*

Diversa, San Diego, California 92121,1 Oak Ridge National Laboratory, Oak Ridge, Tennessee 37831,3 Lawrence Berkeley National Laboratory, Berkeley, California 94720,4 Virtual Institute for Microbial Stress and Survival{dagger},{dagger}

Received 10 December 2005/ Accepted 26 February 2006

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 {phi}29 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.

* Corresponding author. Mailing address: Diversa, 4955 Directors Place, San Diego, CA 92121. Phone: (858) 526-5162. Fax: (858) 526-5662. E-mail: mkeller{at}diversa.com.

http://vimss.lbl.gov.

Applied and Environmental Microbiology, May 2006, p. 3291-3301, Vol. 72, No. 5
0099-2240/06/$08.00+0     doi:10.1128/AEM.72.5.3291-3301.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



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