A molecular approach to search for diversity among bacteria in the environment

Abstract

Molecular 16S rDNA-based techniques were applied to a peat sample from northern Germany in order to investigate the bacterial diversity present and compare the clone sequences with those obtained from similar studies on other terrestrial samples. Genomic DNA was extracted from the peat matrix by a direct lysis procedure. 16S rRNA genes were amplified using PCR primers targeting conserved regions of bacterial 16S rDNA. 16S rDNA fragments were blunt end cloned into a plasmid vector and the resulting clone library of 262 sequences was screened by hybridization with different oligonucleotide probes and sequence analysis of randomly selected clones. The 16S rDNA insert of 76 clones was partially sequenced. Clones identified either by hybridization or by sequence analysis fell into three phyla. As judged by hybridization with a specific oligonucleotide probe, 42% of the clones represented members of the alpha subclass of Proteobacteria. Twenty-five of these clones were selected randomly for sequence analysis; none could be assigned to any of the known genera of this subclass. The second largest clone group comprises 15% of the clones and clusters aroundAcidimicrobium ferrooxidans andRubrobacter radiotolerans, both of which are remotely related to members of the order Actinomycetales. The third major clone cluster (10%) was moderately to remotely related to theAcidobacterium capsulatum phylum. Of the additional clones sequenced, a few could be assigned to other subclasses ofProteobacteria, theVerrucomicrobium phylum and the phylum of spirochetes. Comparison of the results presented here with those from other environments reveals a significant number of common clone clusters. As the vast majority of sequences retrieved from any of the marine and terrestrial samples investigated so far by molecular methods indicate the presence of novel bacterial species it can be assumed that a huge, as yet untapped biotechnological potential is present in the environment.