Analysis of actinomycete communities by specific amplification of genes encoding 16S rRNA and gel-electrophoretic separation in denaturing gradients

Abstract

A group-specific primer, F243 (positions 226 to 243, Escherichia coli numbering), was developed by comparison of sequences of genes encoding 16S rRNA (16S rDNA) for the detection of actinomycetes in the environment with PCR and temperature or denaturing gradient gel electrophoresis (TGGE or DGGE, respectively). The specificity of the forward primer in combination with different reverse ones was tested with genomic DNA from a variety of bacterial strains. Most actinomycetes investigated could be separated by TGGE and DGGE, with both techniques giving similar results. Two strategies were employed to study natural microbial communities. First, we used the selective amplification of actinomycete sequences (E. coli positions 226 to 528) for direct analysis of the products in denaturing gradients. Second, a nested PCR providing actinomycete-specific fragments (E. coli positions 226 to 1401) was used which served as template for a PCR when conserved primers were used. The products (E. coli positions 968 to 1401) of this indirect approach were then separated by use of gradient gels. Both approaches allowed detection of actinomycete communities in soil. The second strategy allowed the estimation of the relative abundance of actinomycetes within the bacterial community. Mixtures of PCR-derived 16S rDNA fragments were used as model communities consisting of five actinomycetes and five other bacterial species. Actinomycete products were obtained over a 100-fold dilution range of the actinomycete DNA in the model community by specific PCR; detection of the diluted actinomycete DNA was not possible when conserved primers were used. The methods tested for detection were applied to monitor actinomycete community changes in potato rhizosphere and to investigate actinomycete diversity in different soils.