Identification of a toluene-degrading bacterium from a soil sample through H 2 18O DNA stable isotope probing

Research output: Contribution to journalArticle

15 Citations (Scopus)

Abstract

DNA stable isotope probing (DNA-SIP) with H 2 18O 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 H 2 18O, H 2 16O, H 2 16O and 48 ppm toluene, or H 2 18O and 48 ppm toluene. A single DNA band formed along a cesium chloride gradient after isopycnic centrifugation of extracts from soils incubated with H 2 16O. With extracts from soils to which only H 2 18O was added, two distinct DNA bands formed, while three bands formed when DNA extracted from soil incubated with both H 2 18O 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 H 2 18O 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 H 2 18O DNA-SIP can be a useful method for identifying pollutant-degrading bacteria in soil.

Original languageEnglish (US)
Pages (from-to)5995-5999
Number of pages5
JournalApplied and Environmental Microbiology
Volume77
Issue number17
DOIs
StatePublished - Sep 1 2011

Fingerprint

toluene
Toluene
Isotopes
stable isotopes
stable isotope
Soil
soil sampling
Bacteria
DNA
bacterium
bacteria
soil
Rhodococcus (bacteria)
Rhodococcus
ribosomal RNA
soil bacteria
rRNA Genes
Isopycnic Centrifugation
Oxygen
oxygen

ASJC Scopus subject areas

  • Applied Microbiology and Biotechnology
  • Food Science
  • Biotechnology
  • Ecology

Cite this

@article{183dec521ca7472296a786188df3e4cc,
title = "Identification of a toluene-degrading bacterium from a soil sample through H 2 18O DNA stable isotope probing",
abstract = "DNA stable isotope probing (DNA-SIP) with H 2 18O 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 H 2 18O, H 2 16O, H 2 16O and 48 ppm toluene, or H 2 18O and 48 ppm toluene. A single DNA band formed along a cesium chloride gradient after isopycnic centrifugation of extracts from soils incubated with H 2 16O. With extracts from soils to which only H 2 18O was added, two distinct DNA bands formed, while three bands formed when DNA extracted from soil incubated with both H 2 18O 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 H 2 18O 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 H 2 18O DNA-SIP can be a useful method for identifying pollutant-degrading bacteria in soil.",
author = "Angela Woods and Watwood, {Maribeth E} and Egbert Schwartz",
year = "2011",
month = "9",
day = "1",
doi = "10.1128/AEM.05689-11",
language = "English (US)",
volume = "77",
pages = "5995--5999",
journal = "Applied and Environmental Microbiology",
issn = "0099-2240",
publisher = "American Society for Microbiology",
number = "17",

}

TY - JOUR

T1 - Identification of a toluene-degrading bacterium from a soil sample through H 2 18O DNA stable isotope probing

AU - Woods, Angela

AU - Watwood, Maribeth E

AU - Schwartz, Egbert

PY - 2011/9/1

Y1 - 2011/9/1

N2 - DNA stable isotope probing (DNA-SIP) with H 2 18O 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 H 2 18O, H 2 16O, H 2 16O and 48 ppm toluene, or H 2 18O and 48 ppm toluene. A single DNA band formed along a cesium chloride gradient after isopycnic centrifugation of extracts from soils incubated with H 2 16O. With extracts from soils to which only H 2 18O was added, two distinct DNA bands formed, while three bands formed when DNA extracted from soil incubated with both H 2 18O 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 H 2 18O 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 H 2 18O DNA-SIP can be a useful method for identifying pollutant-degrading bacteria in soil.

AB - DNA stable isotope probing (DNA-SIP) with H 2 18O 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 H 2 18O, H 2 16O, H 2 16O and 48 ppm toluene, or H 2 18O and 48 ppm toluene. A single DNA band formed along a cesium chloride gradient after isopycnic centrifugation of extracts from soils incubated with H 2 16O. With extracts from soils to which only H 2 18O was added, two distinct DNA bands formed, while three bands formed when DNA extracted from soil incubated with both H 2 18O 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 H 2 18O 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 H 2 18O DNA-SIP can be a useful method for identifying pollutant-degrading bacteria in soil.

UR - http://www.scopus.com/inward/record.url?scp=80052720880&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=80052720880&partnerID=8YFLogxK

U2 - 10.1128/AEM.05689-11

DO - 10.1128/AEM.05689-11

M3 - Article

C2 - 21742928

AN - SCOPUS:80052720880

VL - 77

SP - 5995

EP - 5999

JO - Applied and Environmental Microbiology

JF - Applied and Environmental Microbiology

SN - 0099-2240

IS - 17

ER -