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Identification of a toluene-degrading bacterium from a soil sample through H218O DNA stable isotope probing

Woods, Angela and Watwood, Maribeth and Schwartz, Egbert (2011) Identification of a toluene-degrading bacterium from a soil sample through H218O DNA stable isotope probing. Applied & Environmental Microbiology, 77 (17). pp. 5995-5999. ISSN 1098-5336


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Publisher’s or external URL: http://dx.doi.org/10.1128/aem.05689-11


DNA stable isotope probing (DNA-SIP) with H218O was used to identify a toluene-degrading bacterium in soil amended with 48 ppm toluene. After quantification of toluene degradation rates in soil, DNA was extracted from soil incubated with H218O, H216O, H216O and 48 ppm toluene, or H218O and 48 ppm toluene. A single DNA band formed along a cesium chloride gradient after isopycnic centrifugation of extracts from soils incubated with H216O. With extracts from soils to which only H218O was added, two distinct DNA bands formed, while three bands formed when DNA extracted from soil incubated with both H218O and toluene was analyzed. We suggest that this third band formed because toluene does not contain any oxygen atoms and toluene-degrading organisms had to transfer oxygen atoms from H218O into metabolic intermediates to form nucleic acids de novo. We extracted the third DNA band and amplified a large fraction of the bacterial 16S rRNA gene. Direct sequencing of the PCR product obtained from the labeled DNA, as well as cloned 16S rRNA amplicons, identified a known toluene degrader, Rhodococcus jostii RHA1. A toluene-degrading bacterial strain was subsequently isolated from soil and shown to be Rhodococcus jostii RHA1. Finally, quantitative real-time PCR analysis showed that the abundance of the 16S rRNA gene of Rhodococcus jostii RHA1 increased in soil after toluene exposure but not in soils from which toluene was withheld. This study indicates that H218O DNA-SIP can be a useful method for identifying pollutant-degrading bacteria in soil.

Item Type: Article
ID number or DOI: 10.1128/AEM.05689-11
Keywords: bacteria; Bacterial genetics; Bacteria -- Metabolism; Bacteriology; Biodegradation; bioreactor; Centrifugation; Centrifugation, Density Gradient; Cesium; Cesium compounds; Chlorine compounds; Cloning; cluster analysis; degradation; diversity; DNA; DNA, Bacterial; DNA, Ribosomal; Gene encoding; insights; Internet protocols; Isotope Labeling; isotopes; Microbial genetics; Microorganisms; Molecular Sequence Data; neighbor-joining method; oxygen; Phenanthrene; phylogenies; Phylogeny; Polymerase chain reaction; residues; Rhodococcus; RNA; RNA, Ribosomal, 16S; Sequence Analysis, DNA; Soil microbiology; soils; Toluene
Subjects: Q Science > QR Microbiology
NAU Depositing Author Academic Status: Faculty/Staff
Department/Unit: College of Engineering, Forestry, and Natural Science > Biological Sciences
Date Deposited: 30 Sep 2015 05:58
URI: http://openknowledge.nau.edu/id/eprint/347

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