Influence of commonly used primer systems on automated ribosomal intergenic spacer analysis of bacterial communities in environmental samples.
Due to the high diversity of bacteria in many ecosystems, their slow generation times, specific but mostly unknown nutrient requirements and syntrophic interactions, isolation based approaches in microbial ecology mostly fail to describe microbial community structure. Thus, cultivation independent techniques, which rely on directly extracted nucleic acids from the environment, are a well-used alternative. For example, bacterial automated ribosomal intergenic spacer analysis (B-ARISA) is one of the widely used methods for fingerprinting bacterial communities after PCR-based amplification of selected regions of the operon coding for rRNA genes using community DNA. However, B-ARISA alone does not provide any taxonomic information and the results may be severely biased in relation to the primer set selection. Furthermore, amplified DNA stemming from mitochondrial or chloroplast templates might strongly bias the obtained fingerprints. In this study, we determined the applicability of three different B-ARISA primer sets to the study of bacterial communities. The results from in silico analysis harnessing publicly available sequence databases showed that all three primer sets tested are specific to bacteria but only two primers sets assure high bacterial taxa coverage (1406f/23Sr and ITSF/ITSReub). Considering the study of bacteria in a plant interface, the primer set ITSF/ITSReub was found to amplify (in silico) sequences of some important crop species such as Sorghum bicolor and Zea mays. Bacterial genera and plant species potentially amplified by different primer sets are given. These data were confirmed when DNA extracted from soil and plant samples were analyzed. The presented information could be useful when interpreting existing B-ARISA results and planning B-ARISA experiments, especially when plant DNA can be expected.
UFZ-Helmholtz Centre for Environmental Research, Department of Soil Ecology, Halle (Saale), Germany; Technical University of Munich, Chair for Soil Science, Oberschleissheim, Germany.
Witoon
W
Purahong
Helmholtz Zentrum München, Research Unit for Environmental Genomics, Oberschleissheim, Germany.
Barbara
B
Stempfhuber
UFZ-Helmholtz Centre for Environmental Research, Department of Soil Ecology, Halle (Saale), Germany; University of Leipzig, Institute of Biology, Leipzig, Germany.
Guillaume
G
Lentendu
UFZ-Helmholtz Centre for Environmental Research, Department of Soil Ecology, Halle (Saale), Germany.
Davide
D
Francioli
UFZ-Helmholtz Centre for Environmental Research, Department of Soil Ecology, Halle (Saale), Germany.
Thomas
T
Reitz
UFZ-Helmholtz Centre for Environmental Research, Department of Soil Ecology, Halle (Saale), Germany; University of Leipzig, Institute of Biology, Leipzig, Germany; German Centre for Integrative Biodiversity Research (iDiv), Leipzig, Germany.
François
F
Buscot
Technical University of Munich, Chair for Soil Science, Oberschleissheim, Germany; Helmholtz Zentrum München, Research Unit for Environmental Genomics, Oberschleissheim, Germany.
Michael
M
Schloter
UFZ-Helmholtz Centre for Environmental Research, Department of Soil Ecology, Halle (Saale), Germany.
Dirk
D
Krüger
D001419:Bacteria / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D044822:Biodiversity; D017931:DNA Primers; D005798:Genes, Bacterial; D016133:Polymerase Chain Reaction; D012335:RNA, Ribosomal / Q000235:genetics; D012984:Software; D012988:Soil Microbiology
D017931:DNA Primers; D012335:RNA, Ribosomal