Molecular biological evidence for the occurrence of uncultured members of the actinomycete line of descent in different environments and geographical locations

Abstract

A 16S rDNA based molecular ecological study was performed on a sample taken from a peat bog in Germany. Total DNA was extracted by directly lysing micro-organisms in the peat matrix. The 5' 1400 nucleotides of the bacterial 16S rDNA were amplified using conserved bacterial PCR primers. A clone library was generated by blunt-end cloning and 262 16S rDNA clones were analysed. Of these, 37 were located in the Gram-positive phylum, as determined by hybridization to an oligonucleotide probe specific for Gram-positive bacteria. Analysis of 17 of these clones by sequence analysis and their comparison with published sequences representing all of the main bacterial phyla indicated their membership of the actinomycete line of descent. These peat clones were found to represent three novel lineages, two of which appear to be related to the species Acidimicrobium ferrooxidans, and 'Candidatus Microthrix parvicella'. Clone sequences of the third group are phylogenetically related to Rubrobacter radiotolerans. Comparison with short 16S rDNA clone sequences obtained from DNA isolated from a geothermally heated soil in New Zealand, and from DNA isolated from soil in Australia, Japan and Finland and marine environments from the Atlantic and the Pacific Oceans, suggests that members of these three groups occur in very different environments across the world.