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Applied and Environmental Microbiology, April 2005, p. 2026-2035, Vol. 71, No. 4
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.4.2026-2035.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Improved Assessment of Denitrifying, N2-Fixing, and Total-Community Bacteria by Terminal Restriction Fragment Length Polymorphism Analysis Using Multiple Restriction Enzymes

Christopher Rösch and Hermann Bothe*

Botanisches Institut, Universität zu Köln, Cologne, Germany

Received 26 August 2004/ Accepted 10 November 2004

A database of terminal restriction fragments (tRFs) of the 16S rRNA gene was set up utilizing 13 restriction enzymes and 17,327 GenBank sequences. A computer program, termed TReFID, was developed to allow identification of any of these 17,327 sequences by means of polygons generated from the specific tRFs of each bacterium. The TReFID program complements and exceeds in its data content the Web-based phylogenetic assignment tool recently described by A. D. Kent, D. J. Smith, B. J. Benson, and E. W. Triplett (Appl. Environ. Microb. 69:6768-6766, 2003). The method to identify bacteria is different, as is the region of the 16S rRNA gene employed in the present program. For the present communication the software of the tRF profiles has also been extended to allow screening for genes coding for N2 fixation (nifH) and denitrification (nosZ) in any bacterium or environmental sample. A number of controls were performed to test the reliability of the TReFID program. Furthermore, the TReFID program has been shown to permit the analysis of the bacterial population structure of bacteria by means of their 16S rRNA, nifH, and nosZ gene content in an environmental habitat, as exemplified for a sample from a forest soil. The use of the TReFID program reveals that noncultured denitrifying and dinitrogen-fixing bacteria might play a more dominant role in soils than believed hitherto.

* Corresponding author. Mailing address: Botanisches Institut, Universität zu Köln, Gyrhofstrasse 15, D-50923 Cologne, Germany. Phone: 49 221 470 2760. Fax: 49 221 470 5039. E-mail: hermann.bothe{at}uni-koeln.de.

Applied and Environmental Microbiology, April 2005, p. 2026-2035, Vol. 71, No. 4
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.4.2026-2035.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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