Isolierung von DNA und Konstruktion einer Metagenombank aus dem Sediment des Flusses Leine: partielle Sequenzierung und Annotation des Metagenoms sowie Analyse der mikrobiellen Diversität

Abstract

Isolation of environmental DNA, subsequent construction of metagenomic libraries, or amplification and cloning of 16S rRNA genes, and final sequencing of cloned DNA allow phylogenetic and physiological characterization of habitats without enrichment of microorganism.To gain insight into the complex microbial population of the River Leine sediment (near Göttingen, Germany) a sample was taken from the upper 15 cm layer. The isolated DNA was employed for construction of plasmid and cosmid libraries. 12 cosmids were chosen for complete sequencing of their inserts by a shot-gun approach. After ORF finding and annotation different gene clusters have been identified. In some cases, the annotated functions have been experimentally confirmed. Approximately 50 % of all amino acid sequences deduced from the identified open reading frames lack similarities to sequences of proteins with known functions or to any other known protein sequence. To initially characterize the complex microbial population present in this microbial niche 16S rRNA gene analysis was performed by PCR using primer pairs directed against Bacteria or Archaea. PCR products were cloned and sequenced. The generated 16S rRNA gene sequences were compared with sequences of sequence databases. Subsequently, phylogenetic trees were created. Approximately 50 % of the 16S rRNA gene sequences obtained show the highest similarities to 16S rRNA genes derived from uncharacterized microorganisms. The sequence sl1394 shows a high identity (97 %) to the 16S rRNA gene sequence of the uncultured clone S1G12. This clone seems to be a member of the seldom detected candidate division TM7. Members of 11 different bacterial phyla were detected. In most cases, the phylogenetic analyses of archaeal 16S rRNA genes revealed their affiliation to 16S rRNA genes from members of the phylum Euryarchaeota. Finally, the entirely sequenced plasmid pEry1 harboring a soil DNA insert was studied. This plasmid was known to confer growth of Escherichia coli on mineral medium with erythritol as sole energy and carbon source. The open reading frame responsible for this ability was identified. Molecular weight and specific activity of this enzyme were analysed. Growth parameters of E. coli JM109 carrying pEry1 in mineral medium with erythritol as sole energy and carbon source were determined.