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Applied and Environmental Microbiology, November 2003, p. 6875-6887, Vol. 69, No. 11
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.11.6875-6887.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

The Isotope Array, a New Tool That Employs Substrate-Mediated Labeling of rRNA for Determination of Microbial Community Structure and Function

Justyna Adamczyk,1 Martin Hesselsoe,2 Niels Iversen,2 Matthias Horn,3 Angelika Lehner,1 Per Halkjaer Nielsen,2 Michael Schloter,4 Peter Roslev,2* and Michael Wagner3

Lehrstuhl für Mikrobiologie, Technische Universität München, D-85350 Freising,1 GSF-Forschungszentrum für Umwelt und Gesundheit GmbH, D-85764 Neuherberg, Germany,4 Section of Environmental Engineering, Aalborg University, DK-9000 Aalborg, Denmark,2 Abteilung für Mikrobielle Ökologie, Institut für Ökologie und Naturschutz, Universität Wien, A-1090 Vienna, Austria3

Received 7 May 2003/ Accepted 14 August 2003

A new microarray method, the isotope array approach, for identifying microorganisms which consume a 14C-labeled substrate within complex microbial communities was developed. Experiments were performed with a small microarray consisting of oligonucleotide probes targeting the 16S rRNA of ammonia-oxidizing bacteria (AOB). Total RNA was extracted from a pure culture of Nitrosomonas eutropha grown in the presence of [14C]bicarbonate. After fluorescence labeling of the RNA and microarray hybridization, scanning of all probe spots for fluorescence and radioactivity revealed that specific signals were obtained and that the incorporation of 14C into rRNA could be detected unambiguously. Subsequently, we were able to demonstrate the suitability of the isotope array approach for monitoring community composition and CO2 fixation activity of AOB in two nitrifying activated-sludge samples which were incubated with [14C]bicarbonate for up to 26 h. AOB community structure in the activated-sludge samples, as predicted by the microarray hybridization pattern, was confirmed by quantitative fluorescence in situ hybridization (FISH) and comparative amoA sequence analyses. CO2 fixation activities of the AOB populations within the complex activated-sludge communities were detectable on the microarray by 14C incorporation and were confirmed independently by combining FISH and microautoradiography. AOB rRNA from activated sludge incubated with radioactive bicarbonate in the presence of allylthiourea as an inhibitor of AOB activity showed no incorporation of 14C and thus was not detectable on the radioactivity scans of the microarray. These results suggest that the isotope array can be used in a PCR-independent manner to exploit the high parallelism and discriminatory power of microarrays for the direct identification of microorganisms which consume a specific substrate in the environment.

* Corresponding author. Mailing address: Section of Environmental Engineering, Department of Life Sciences, Aalborg University, Sohngaardsholmsvej 57, DK-9000 Aalborg, Denmark. Phone: 45 9635 8505. Fax: 45 9635 0558. E-mail: pr{at}bio.auc.dk.

Applied and Environmental Microbiology, November 2003, p. 6875-6887, Vol. 69, No. 11
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.11.6875-6887.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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Copyright © 2003 by the American Society for Microbiology. All rights reserved.