title: Stable-isotope probing with multiple growth substrates to determine substrate specificity of uncultivated bacteria.
abstract: Stable-isotope probing (SIP) has been used to determine which microorganisms in a complex environmental sample are capable of metabolizing a labeled substrate. We hypothesized that DNA-based stable-isotope probing with a combination of a (13)C-labeled carbon source and a second, unlabeled carbon source could be combined with analyses of the entire gradient of separated DNA to provide information concerning the utilization of a mixture of environmentally relevant compounds by uncultivated organisms. As a test of the method, we evaluated the response of a microbial community in a laboratory bioreactor treating contaminated soil to two polycyclic aromatic hydrocarbons (PAH). The compounds were added either individually as [U-(13)C]phenanthrene or [U-(13)C]pyrene, or as a mixture in which one was labeled with (13)C and the other was unlabeled. After ultracentrifugation of DNA extracted from a given incubation, fractions containing DNA enriched with varying levels of (13)C were examined by denaturing-gradient gel electrophoresis (DGGE) and by real-time quantitative PCR (qPCR) for 16S rRNA genes belonging to organisms in groups of bacteria previously associated with PAH degradation by single-compound SIP. Four groups of bacteria (three uncultivated) were followed in this study. Two of the uncultivated groups showed evidence for simultaneous or sequential utilization of both compounds while the remaining two appeared to assimilate carbon from only one of the compounds. DNA-based SIP therefore appears to be useful to evaluate the selectivity among substrates in a mixture by uncultivated microbes.
authors.affiliation: Department of Environmental Sciences and Engineering, School of Public Health, University of North Carolina, Chapel Hill, North Carolina, 27599-7431, USA. drsingle@email.unc.edu
authors.forename: David R
authors.initials: DR
authors.lastname: Singleton
authors.forename: Mark
authors.initials: M
authors.lastname: Hunt
authors.forename: Sabrina N
authors.initials: SN
authors.lastname: Powell
authors.forename: Roberto
authors.initials: R
authors.lastname: Frontera-Suau
authors.forename: Michael D
authors.initials: MD
authors.lastname: Aitken
mesh_terms: D001673:Biodegradation, Environmental; D019149:Bioreactors; D002247:Carbon Isotopes; D042621:Comamonadaceae / Q000235:genetics / Q000254:growth & development* / Q000378:metabolism; D004269:DNA, Bacterial / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D011084:Polycyclic Aromatic Hydrocarbons / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D012336:RNA, Ribosomal, 16S / Q000378:metabolism; D012988:Soil Microbiology; D013379:Substrate Specificity
chemical_list: D002247:Carbon Isotopes; D004269:DNA, Bacterial; D011084:Polycyclic Aromatic Hydrocarbons; D012336:RNA, Ribosomal, 16S