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PMID: 16672469 Published · ppublish English Evaluation Study Journal Article Research Support, U.S. Gov't, Non-P.H.S.

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

Applied and environmental microbiology ·Vol. 72 ·No. 5 ·2006-05-00 ·Pages 3291-301

Abulencia CB, Wyborski DL, Garcia JA, Podar M, Chen W, Chang SH, Chang HW, Watson D, Brodie EL, Hazen TC, Keller M

Abstract

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.

MeSH Terms
Bacillus Phages Bacteria/classification,genetics Bacterial Proteins/genetics DNA, Bacterial/analysis,isolation & purification Genes, rRNA Genome, Bacterial Geologic Sediments/microbiology Molecular Sequence Data Nucleic Acid Amplification Techniques/methods Phylogeny RNA, Ribosomal/genetics Sequence Analysis, DNA Soil Microbiology Soil Pollutants
Chemicals
Bacterial Proteins DNA, Bacterial RNA, Ribosomal Soil Pollutants
Authors & Affiliations
11 authors, click to expand affiliations / ORCID
Abulencia Carl B
Diversa, 4955 Directors Place, San Diego, CA 92121, USA.
Wyborski Denise L
Garcia Joseph A
Podar Mircea
Chen Wenqiong
Chang Sherman H
Chang Hwai W
Watson David
Brodie Eoin L
Hazen Terry C
Keller Martin
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Article Info
Journal
Applied and environmental microbiology
Abbr.
Appl Environ Microbiol
ISSN
0099-2240
DOI
10.1128/AEM.72.5.3291-3301.2006
Published
2006-05-00
Pages
3291-301
Language
English
Region
United States
NLM ID
7605801
PMCID
PMC1472342
Subset
IM
Databases
GENBANK
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DQ404891, DQ404892, DQ404893, DQ404894, DQ404895, DQ404896, DQ404897, DQ404898, DQ404899, DQ404900, DQ404901, DQ404902, DQ404903, DQ404904, DQ404905, DQ404906, DQ404907, DQ404908, DQ404909, DQ404910, DQ404911, DQ404912, DQ404913, DQ404914, DQ404915, DQ404916, DQ404917, DQ404918, DQ404919, DQ404920, DQ404921, DQ404922, DQ404923, DQ404924, DQ404925, DQ404926, DQ404927, DQ404928, DQ404929, DQ404930, DQ404931, DQ404932, DQ404933, DQ404934, DQ404935, DQ404936, DQ404937, DQ404938, DX385314, DX385315, DX385316, DX385317, DX385318, DX385319, DX385320, DX385321, DX385322, DX385323, DX385324, DX385325, DX385326, DX385327, DX385328, DX385329, DX385330, DX385331, DX385332, DX385333, DX385334, DX385335, DX385336, DX385337, DX385338, DX385339, DX385340, DX385341, DX385342, DX385343, DX385344, DX385345, DX385346, DX385347, DX385348, DX385349, DX385350, DX385351, DX385352, DX385353, DX385354, DX385355, DX385356, DX385357, DX385358, DX385359, DX385360, DX385361, DX385362, DX385363, DX385364, DX385365, DX385366, DX385367, DX385368, DX385369, DX385370, DX385371, DX385372, DX385373, DX385374, DX385375, DX385376, DX385377, DX385378, DX385379, DX385380, DX385381, DX385382, DX385383, DX385384, DX385385, DX385386, DX385387, DX385388, DX385389, DX385390, DX385391, DX385392, DX385393, DX385394, DX385395, DX385396, DX385397, DX385398, DX385399, DX385400, DX385401, DX385402, DX385403, DX385404, DX385405, DX385406, DX385407, DX385408, DX385409, DX385410, DX385411, DX385412, DX385413, DX385414, DX385415, DX385416, DX385417, DX385418, DX385419, DX385420, DX385421, DX385422, DX385423, DX385424, DX385425, DX385426, DX385427, DX385428, DX385429, DX385430, DX385431, DX385432, DX385433, DX385434, DX385435, DX385436, DX385437, DX385438, DX385439, DX385440, DX385441, DX385442, DX385443, DX385444, DX385445, DX385446, DX385447, DX385448, DX385449, DX385450, DX385451, DX385452, DX385453, DX385454, DX385455, DX385456, DX385457, DX385458, DX385459, DX385460, DX385461, DX385462, DX385463, DX385464, DX385465, DX385466, DX385467, DX385468, DX385469, DX385470, DX385471, DX385472, DX385473, DX385474, DX385475, DX385476, DX385477, DX385478, DX385479, DX385480, DX385481, DX385482, DX385483, DX385484, DX385485, DX385486, DX385487, DX385488, DX385489, DX385490, DX385491, DX385492, DX385493, DX385494, DX385495, DX385496, DX385497, DX385498, DX385499, DX385500, DX385501, DX385502, DX385503, DX385504, DX385505, DX385506, DX385507, DX385508, DX385509, DX385510, DX385511, DX385512, DX385513, DX385514, DX385515, DX385516, DX385517, DX385518, DX385519, DX385520, DX385521, DX385522, DX385523, DX385524, DX385525, DX385526, DX385527, DX385528, DX385529, DX385530, DX385531, DX385532, DX385533, DX385534, DX385535, DX385536, DX385537, DX385538, DX385539, DX385540, DX385541, DX385542, DX385543, DX385544, DX385545, DX385546, DX385547, DX385548, DX385549, DX385550, DX385551, DX385552, DX385553, DX385554, DX385555, DX385556, DX385557, DX385558, DX385559, DX385560, DX385561, DX385562, DX385563, DX385564, DX385565, DX385566, DX385567, DX385568, DX385569, DX385570, DX385571, DX385572, DX385573, DX385574, DX385575, DX385576, DX385577, DX385578, DX385579, DX385580, DX385581, DX385582, DX385583, DX385584, DX385585, DX385586, DX385587, DX385588, DX385589, DX385590, DX385591, DX385592, DX385593, DX385594, DX385595, DX385596, DX385597, DX385598, DX385599, DX385600, DX385601, DX385602, DX385603, DX385604, DX385605, DX385606, DX385607, DX385608, DX385609, DX385610, DX385611, DX385612, DX385613, DX385614, DX385615, DX385616, DX385617, DX385618, DX385619, DX385620, DX385621, DX385622, DX385623, DX385624, DX385625, DX385626, DX385627, DX385628, DX385629, DX385630, DX385631, DX385632, DX385633, DX385634, DX385635, DX385636, DX385637, DX385638, DX385639, DX385640, DX385641, DX385642, DX385643, DX385644, DX385645, DX385646, DX385647, DX385648, DX385649, DX385650, DX385651, DX385652, DX385653, DX385654, DX385655, DX385656, DX385657, DX385658, DX385659, DX385660, DX385661, DX385662, DX385663, DX385664
External Links
PubMed source DOI: 10.1128/AEM.72.5.3291-3301.2006 PMC Full Text View journal details
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