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Applied and Environmental Microbiology, March 2005, p. 1373-1386, Vol. 71, No. 3
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.3.1373-1386.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

16S rRNA Gene-Based Oligonucleotide Microarray for Environmental Monitoring of the Betaproteobacterial Order "Rhodocyclales"

Alexander Loy,1* Claudia Schulz,2 Sebastian Lücker,1 Andreas Schöpfer-Wendels,2,3 Kilian Stoecker,1 Christian Baranyi,1 Angelika Lehner,2,4 and Michael Wagner1

Department of Microbial Ecology, Institute of Ecology and Conservation Biology, University of Vienna, Vienna, Austria,1 Department of Microbiology, Technical University of Munich, Freising,2 National Research Center for Environment and Health, Institute of Epidemiology, Neuherberg, Germany,3 Institut für Lebensmittelsicherheit und Hygiene, Vetsuisse Fakultät, Universität Zürich, Zürich, Switzerland4

Received 23 July 2004/ Accepted 30 September 2004

For simultaneous identification of members of the betaproteobacterial order "Rhodocyclales" in environmental samples, a 16S rRNA gene-targeted oligonucleotide microarray (RHC-PhyloChip) consisting of 79 probes was developed. Probe design was based on phylogenetic analysis of available 16S rRNA sequences from all cultured and as yet uncultured members of the "Rhodocyclales." The multiple nested probe set was evaluated for microarray hybridization with 16S rRNA gene PCR amplicons from 29 reference organisms. Subsequently, the RHC-PhyloChip was successfully used for cultivation-independent "Rhodocyclales" diversity analysis in activated sludge from an industrial wastewater treatment plant. The implementation of a newly designed "Rhodocyclales"-selective PCR amplification system prior to microarray hybridization greatly enhanced the sensitivity of the RHC-PhyloChip and thus enabled the detection of "Rhodocyclales" populations with relative abundances of less than 1% of all bacteria (as determined by fluorescence in situ hybridization) in the activated sludge. The presence of as yet uncultured Zoogloea-, Ferribacterium/Dechloromonas-, and Sterolibacterium-related bacteria in the industrial activated sludge, as indicated by the RHC-PhyloChip analysis, was confirmed by retrieval of their 16S rRNA gene sequences and subsequent phylogenetic analysis, demonstrating the suitability of the RHC-PhyloChip as a novel monitoring tool for environmental microbiology.

* Corresponding author. Mailing address: Abteilung Mikrobielle Ökologie, Institut für Ökologie und Naturschutz, Universität Wien, Althanstr. 14, A-1090 Wien, Austria. Phone: 43 1 4277 54207. Fax: 43 1 4277 54389. E-mail: loy{at}microbial-ecology.net.

Applied and Environmental Microbiology, March 2005, p. 1373-1386, Vol. 71, No. 3
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.3.1373-1386.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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