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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

Huang, W.E. and Ferguson, A. and Singer, A.C. and Lawson, K.A. and Thompson, I.P. and Kalin, R.M. and Larkin, M.J. and Bailey, M.J. and Whiteley, A.S. (2009) 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. Applied and Environmental Microbiology, 75 (1). pp. 234-241. ISSN 0099-2240

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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 (13C)-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 µM. 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.

Item type: Article
ID code: 19367
Notes: Strathprints' policy is to record up to 8 authors per publication, plus any additional authors based at the University of Strathclyde. More authors may be listed on the official publication than appear in the Strathprints' record.
Keywords: genetic functions, microbial populations, in situ, rRNA, mRNA, single-cell raman-fluorescence, Environmental engineering, Microbiology, Ecology, Applied Microbiology and Biotechnology, Food Science, Biotechnology
Subjects: Technology > Engineering (General). Civil engineering (General) > Environmental engineering
Science > Microbiology
Department: Faculty of Engineering > Civil and Environmental Engineering
Faculty of Engineering > Electronic and Electrical Engineering
Depositing user: Strathprints Administrator
Date Deposited: 02 Jun 2010 13:27
Last modified: 05 Sep 2014 02:07

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