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Applied and Environmental Microbiology, July 2002, p. 3215-3225, Vol. 68, No. 7
0099-2240/02/$04.00+0     DOI: 10.1128/AEM.68.7.3215-3225.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Parallel Characterization of Anaerobic Toluene- and Ethylbenzene-Degrading Microbial Consortia by PCR-Denaturing Gradient Gel Electrophoresis, RNA-DNA Membrane Hybridization, and DNA Microarray Technology

Yoshikazu Koizumi,1 John J. Kelly,2 Tatsunori Nakagawa,1 Hidetoshi Urakawa,3 Saïd El-Fantroussi,3 Saleh Al-Muzaini,4 Manabu Fukui,1* Yoshikuni Urushigawa,5 and David A. Stahl3

Department of Biological Sciences, Graduate School of Science, Tokyo Metropolitan University, 1-1 Minami-Ohsawa, Hachioji, Tokyo 192-0397, Japan,1 Department of Civil Engineering, Northwestern University, Evanston, Illinois 60208-3109,2 Department of Civil and Environmental Engineering, University of Washington, Seattle, Washington 98195-2700,3 Environmental Sciences Department, Kuwait Institute for Scientific Research, 13109 Safat, Kuwait,4 Faculty of System Science and Technology, Akita Prefectural University, 84-4 Tsuchiya-Ebinokuchi, Honjyo, Akita 015-0055, Japan5

Received 24 January 2002/ Accepted 4 April 2002

A mesophilic toluene-degrading consortium (TDC) and an ethylbenzene-degrading consortium (EDC) were established under sulfate-reducing conditions. These consortia were first characterized by denaturing gradient gel electrophoresis (DGGE) fingerprinting of PCR-amplified 16S rRNA gene fragments, followed by sequencing. The sequences of the major bands (T-1 and E-2) belonging to TDC and EDC, respectively, were affiliated with the family Desulfobacteriaceae. Another major band from EDC (E-1) was related to an uncultured non-sulfate-reducing soil bacterium. Oligonucleotide probes specific for the 16S rRNAs of target organisms corresponding to T-1, E-1, and E-2 were designed, and hybridization conditions were optimized for two analytical formats, membrane and DNA microarray hybridization. Both formats were used to characterize the TDC and EDC, and the results of both were consistent with DGGE analysis. In order to assess the utility of the microarray format for analysis of environmental samples, oil-contaminated sediments from the coast of Kuwait were analyzed. The DNA microarray successfully detected bacterial nucleic acids from these samples, but probes targeting specific groups of sulfate-reducing bacteria did not give positive signals. The results of this study demonstrate the limitations and the potential utility of DNA microarrays for microbial community analysis.

* Corresponding author. Mailing address: Department of Biological Sciences, Graduate School of Science, Tokyo Metropolitan University, Minami-ohsawa 1-1, Hachioji, Tokyo 192-0397, Japan. Phone: 81-426-77-2581. Fax: 81-426-77-2559. E-mail: fukui-manabu{at}c.metro-u.ac.jp.

Applied and Environmental Microbiology, July 2002, p. 3215-3225, Vol. 68, No. 7
0099-2240/02/$04.00+0     DOI: 10.1128/AEM.68.7.3215-3225.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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