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Applied and Environmental Microbiology, August 2001, p. 3557-3563, Vol. 67, No. 8
0099-2240/01/$04.00+0   DOI: 10.1128/AEM.67.8.3557-3563.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Effect of Primers Hybridizing to Different Evolutionarily Conserved Regions of the Small-Subunit rRNA Gene in PCR-Based Microbial Community Analyses and Genetic Profiling

Achim Schmalenberger, Frank Schwieger,dagger and Christoph C. Tebbe*

Institut für Agrarökologie, Bundesforschungsanstalt für Landwirtschaft (FAL), 38116 Braunschweig, Germany

Received 26 February 2001/Accepted 15 May 2001

Genetic profiling techniques of microbial communities based on PCR-amplified signature genes, such as denaturing gradient gel electrophoresis or single-strand-conformation polymorphism (SSCP) analysis, are normally done with PCR products of less than 500-bp. The most common target for diversity analysis, the small-subunit rRNA genes, however, are larger, and thus, only partial sequences can be analyzed. Here, we compared the results obtained by PCR targeting different variable (V) regions (V2 and V3, V4 and V5, and V6 to V8) of the bacterial 16S rRNA gene with primers hybridizing to evolutionarily conserved flanking regions. SSCP analysis of single-stranded PCR products generated from 13 different bacterial species showed fewer bands with products containing V4-V5 (average, 1.7 bands per organism) than with V2-V3 (2.2 bands) and V6-V8 (2.3 bands). We found that the additional bands (>1 per organism) were caused by intraspecies operon heterogeneities or by more than one conformation of the same sequence. Community profiles, generated by PCR-SSCP from bacterial-cell consortia extracted from rhizospheres of field-grown maize (Zea mays), were analyzed by cloning and sequencing of the dominant bands. A total of 48 sequences could be attributed to 34 different strains from 10 taxonomical groups. Independent of the primer pairs, we found proteobacteria (alpha , beta , and gamma  subgroups) and members of the genus Paenibacillus (low G+C gram-positive) to be the dominant organisms. Other groups, however, were only detected with single primer pairs. This study gives an example of how much the selection of different variable regions combined with different specificities of the flanking "universal" primers can affect a PCR-based microbial community analysis.

* Corresponding author. Mailing address: FAL-Institut für Agrarökologie, Bundesallee 50, 38116 Braunschweig, Germany. Phone: 49 (531) 596 2553. Fax: 49 (531) 596 2599. E-mail: christoph.tebbe{at}fal.de.

dagger Present address: AMODIA Bioservice GmbH, 38124 Braunschweig, Germany.

Applied and Environmental Microbiology, August 2001, p. 3557-3563, Vol. 67, No. 8
0099-2240/01/$04.00+0   DOI: 10.1128/AEM.67.8.3557-3563.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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