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Applied and Environmental Microbiology, January 2009, p. 234-241, Vol. 75, No. 1
0099-2240/09/$08.00+0     doi:10.1128/AEM.01861-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

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 {triangledown} ,{dagger}

Wei E. Huang,1,{ddagger}* Andrew Ferguson,2,3 Andrew C. Singer,1 Kathryn Lawson,3,4 Ian P. Thompson,1 Robert M. Kalin,5 Michael J. Larkin,3,4 Mark J. Bailey,1 and Andrew S. Whiteley1*

Centre for Ecology & Hydrology—Oxford, Mansfield Road, Oxford OX1 3SR, United Kingdom,1 Environmental Engineering Research Centre,2 QUESTOR Centre,3 School of Biological Sciences, The Queen's University of Belfast, Belfast BT7 1NN, United Kingdom,4 Department of Civil Engineering, Strathclyde University, 50 Richmond Street, Glasgow G1 1XN, Scotland5

Received 11 August 2008/ Accepted 30 October 2008

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.

* Corresponding author. Mailing address: CEH—Oxford, Mansfield Road, Oxford OX1 3SR, United Kingdom. Phone: 44 (0)1865 281685. Fax: 44 (0)1865 281696. E-mail for Wei E. Huang: w.huang{at}shef.ac.uk. E-mail for Andrew S. Whiteley: aswhi{at}ceh.ac.uk

{triangledown} Published ahead of print on 7 November 2008.

{dagger} Supplemental material for this article may be found at http://aem.asm.org/.

{ddagger} Present address: Kroto Research Institute, North Campus, University of Sheffield, Broad Lane, Sheffield S3 7HQ, United Kingdom.

Applied and Environmental Microbiology, January 2009, p. 234-241, Vol. 75, No. 1
0099-2240/09/$08.00+0     doi:10.1128/AEM.01861-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.



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