Metagenom-Analysen zum PVC-Superphylum im anoxischen Reisfeldboden

In this project metagenomic libraries of the rice field soil were constructed which are now available for novel comprehensive questions. The metagenomic libraries were screened to retrieve genomic fragments affiliated with members of the Planctomycetes-Verrucomicrobia-Chlamydiae (PVC) superphylum. In addition to fosmid clones of the candidate divisions BRC1 (Bacterial Rice Cluster 1), OP3 (Obsidian Pool 3) and NBL-UPA2 (novel bacterial lineage of uncertain phylogenetic affiliation 2) we obtained representatives of the Verrucomicrobia and Planctomycetes in particular of the genus Pirellula. Four genomic fragments belonging to the candidate divisions BRC1, OP3 and NBL-UPA2 were selected for sequencing. Phylogenies of the 16S and 23S rRNA genes showed a monophyletic ancestor of members of the PVC superphylum and confirmed the affiliation of OP3 and NBL-UPA2 to this superphylum. The results suggested that candidate divisions BRC1 and WS3 (Wurthmith aquifer 3) might also belong to the PVC superphylum. However, this must be support by further analysis. The rRNA operon of the OP3 related genomic fragments possess a conventional structure following the order 16S, 23S and 5S rRNA genes and the tRNA genes for alanine and isoleucine in the intergenic spacer region between the 16S and 23S rRNA. Annotation and comparative sequence analysis provided no informative basis to compose specific enrichment strategies for these yet uncultured microorganisms. Microbial cells of OP3 possess most likely a classical Gram negative cell wall structure. A hight number of predicted open reading frames of the BRC1 genomic fragment showed in BlastP analysis highest similarities with homologs of members of the Planctomycetes. To our surprise, a relatively large proportion of protein coding genes on OP3 related genomic fragments IV32F6, IV180F6 and IV226A8 exhibited closest similarities to homologs affiliated with members of the Deltaproteobacteria in particular with the Desulfuromonadales, Syntrophobacterales and Desulfovibrio sp. known to develop syntrophic interactions. The taxonomic assignment of predicted coding genes on the OP3 genomic fragments raised the hypothesis that yet uncultivated OP3 microorganisms might exist in syntrophic association. On the basis of these results enrichment cultures were established with the aim to retrieve OP3 and BRC1 related genomic fragments in higher frequency in metagenomic libraries. Unfortunately, these microorganisms were not detected in the enrichment cultures with and without the addition of the potential syntrophic partner Methanobacterium formicicum DSM 1535T. In contrast, mesophilic members of the archaeal lineages Rice Cluster (RC)-1 and RC-III were detected. However, the relative abundance of these lineages in the enriched consortia could not be enhanced by novel enrichment strategies.