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Resolving genetic functions within microbial populations: in situ analyses using rRNA and mRNA stable isotope probing coupled with single-cell raman-fluorescence in situ hybridization.
- Source :
-
Applied and environmental microbiology [Appl Environ Microbiol] 2009 Jan; Vol. 75 (1), pp. 234-41. Date of Electronic Publication: 2008 Nov 07. - Publication Year :
- 2009
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Abstract
- Prokaryotes represent one-half of the living biomass on Earth, with the vast majority remaining elusive to culture and study within the laboratory. As a result, we lack a basic understanding of the functions that many species perform in the natural world. To address this issue, we developed complementary population and single-cell stable isotope ((13)C)-linked analyses to determine microbial identity and function in situ. We demonstrated that the use of rRNA/mRNA stable isotope probing (SIP) recovered the key phylogenetic and functional RNAs. This was followed by single-cell physiological analyses of these populations to determine and quantify in situ functions within an aerobic naphthalene-degrading groundwater microbial community. Using these culture-independent approaches, we identified three prokaryote species capable of naphthalene biodegradation within the groundwater system: two taxa were isolated in the laboratory (Pseudomonas fluorescens and Pseudomonas putida), whereas the third eluded culture (an Acidovorax sp.). Using parallel population and single-cell stable isotope technologies, we were able to identify an unculturable Acidovorax sp. which played the key role in naphthalene biodegradation in situ, rather than the culturable naphthalene-biodegrading Pseudomonas sp. isolated from the same groundwater. The Pseudomonas isolates actively degraded naphthalene only at naphthalene concentrations higher than 30 muM. This study demonstrated that unculturable microorganisms could play important roles in biodegradation in the ecosystem. It also showed that the combined RNA SIP-Raman-fluorescence in situ hybridization approach may be a significant tool in resolving ecology, functionality, and niche specialization within the unculturable fraction of organisms residing in the natural environment.
- Subjects :
- Comamonadaceae isolation & purification
Comamonadaceae metabolism
DNA, Bacterial chemistry
DNA, Bacterial genetics
Molecular Sequence Data
Pseudomonas fluorescens isolation & purification
Pseudomonas fluorescens metabolism
Pseudomonas putida isolation & purification
Pseudomonas putida metabolism
Sequence Analysis, DNA
Water Microbiology
Carbon Isotopes analysis
In Situ Hybridization, Fluorescence methods
Naphthalenes metabolism
RNA, Messenger genetics
RNA, Ribosomal genetics
Staining and Labeling methods
Subjects
Details
- Language :
- English
- ISSN :
- 1098-5336
- Volume :
- 75
- Issue :
- 1
- Database :
- MEDLINE
- Journal :
- Applied and environmental microbiology
- Publication Type :
- Academic Journal
- Accession number :
- 18997025
- Full Text :
- https://doi.org/10.1128/AEM.01861-08