Dissimilatory (bi-) Sulfite Reductase as a Marker for Phylogenetic and Ecological Studies of Sulfate-Reducing Prokaryotes

All members of the guild of sulfate reducing prokaryotes (SRP) gain energy via dissimilatory sulfate reduction. The phylogenetic inhomogeneity of this group does not allow to target 16S ribosomal RNA genes of all its members with a simple set of oligonucleotide probes or PCR primers. It is further impossible to identify novel lineages of SRP by their ribosomal RNA solely. Thus, the suitability of the dissimilatory (bi-) sulfite reductase (DSR), a key enzyme of the dissimilatory sulfate reduction pathway, as alternative phylogenetic marker molecule for SRP was investigated. For this purpose, the evolutionary history of the DSR was studied using a large collection of pure culture SRP and correspondence with the 16S rRNA gene based phylogeny was inspected. In addition, cultivation independent SRP diversity surveys were carried out based on comparative amino acid sequence analysis of environmentally retrieved dsr clones in order to test whether cultured representatives of SRP adequately represent the natural diversity of this guild. In order to build up an encompassing DSR reference data base, existing primers were optimized to PCR amplify a 1.9 kb dsrAB fragment from 30 pure culture reference strains. These stains represented all lineages of SRP recognized at this time. dsrAB gene fragments were cloned and sequenced. Subsequent comparative phylogenetic sequence analyses of all available DsrAB pure culture sequences and their corresponding 16S rRNA gene sequences lead to the discovery of at least three presumptive lateral dsr gene transfer events from (i) a deltaproteobacterial donor to the genus Thermodesulfobacterium, (ii) a deltaproteobacterial donor related to Desulfobacterium anilini to certain Desulfotomaculum strains, and (iii) a bacterial donor to Archaeoglobus. Although these events complicate the interpretation of dsrAB-based SRP diversity studies, the Dsr-approach represents the best available method for simultaneous detection of recognized and novel SRP in environmental samples. In this thesis, the Dsr-approach was applied to investigate SRP diversity in the water column of Mariager Fjord (Denmark), and Solar Lake (Egypt), as well as tissue material from a marine worm. The sequences originating form these studies were analyzed along with 550 publicly available environmental dsrAB sequences. 13 environmental SRP lineages without closely related isolated or sequenced SRP reference strains were identified suggesting that many environmentally important SRP lineages have not yet been successfully cultured. Further, a detailed inspection of all available environmental dsrAB sequences revealed characteristic SRP lineages in different ecosystems like soil, sediment, marine water, hypersaline water, and in symbiotic relationships. In conclusion, this thesis showed that lateral gene transfer was significantly influencing the evolutionary history of the DSR. The generally accepted opinion of strict vertical transmission of the key enzyme of dissimilatory sulfate reduction has to be adjusted. Nevertheless, it could be demonstrated that the Dsr  approach is a valid tool for investigating the diversity and biogeography of SRPs.