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Applied and Environmental Microbiology, August 2001, p. 3557-3563, Vol. 67, No. 8
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 (
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
and
,
, and
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.
Present address: AMODIA Bioservice GmbH, 38124 Braunschweig, Germany.
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