Assessing the Genetic Potential of Uncultivated Sulfate Reducing Bacteria

Abstract

The anaerobic oxidation of methane with sulfate (AOM) removes more than 90% of the methane produced in marine sediments. The process is mediated by consortia of anaerobic methanotrophic archaea (ANME) and sulfate-reducing bacteria (SRB). Previous studies focusing on the archaeal part of ANME/SRB consortia yielded as yet only a fragmentary understanding of this process. Additionally, whereas ANME clades have been repeatedly studied with respect to phylogeny, key genes, and genomic capabilities, little is known about their sulfate-reducing partner. Thus, in order to change this situation, this thesis focused on SRB associated with AOM.In the first part of this thesis, SRB associated with Archaea from the ANME-2 clade were investigated. Sequences of bacterial 16S rRNA genes retrieved from ANME-2/SRB enrichment cultures supported a previous hypothesis that ANME-2 associated SRB belong to the SEEP-SRB1 group within the deltaproteobacterial Desulfosarcina/Desulfococcus (DSS) group. Using fluorescence in situ hybridization (FISH) and probes for newly defined SEEP-SRB1 subgroups (a-f), bacteria from the SEEP-SRB1a subgroup were identified as the dominant sulfatereducing partners in ANME-2 consortia in samples from six different AOM sites. In contrast to their abundance as ANME-2 partners, single SEEP-SRB1a cells were very rare (<1%) in all but one of the examined samples. This suggested a highly adapted if not even obligate syntrophic lifestyle of the SEEP-SRB1a group in ANME-2 consortia. Additionally, SEEP-SRB1a was also detected as an alternative partner of archaea of the ANME-3 clade which was previously described to be predominantly associated with SRB of the Desulfobulbus group.In the second part of this thesis, the diversity of SRB in AOM habitats was investigated using aprA and dsrAB, key genes of sulfate-reduction, as functional markers. AprA and DsrAB diversity in different samples from methanotrophic microbial mats from the Black Sea as well as in two enrichment cultures from sediment above gas hydrates at Hydrate Ridge was lower compared to not enriched Hydrate Ridge sediment. Clone libraries were dominated by sequences affiliated with Desulfobacteraceae. Sequences within this group featured a considerable diversity. Most of the retrieved sequences affiliated with clusters that possessed no cultured representative. One AprA cluster was identified to represent SEEP-SRB1a by using a combination of FISH and fluorescence-activated cell sorting.In the third part of this thesis, it was attempted to obtain knowledge about the genetic potential of SEEP-SRB1a. Since no pure cultures of SEEP-SRB1a existed, a metagenomic approach was used. For this, DNA from an enrichment culture dominated by ANME-2 and SEEP-SRB1a was used for constructing a large-insert fosmid library and for performing next-generation pyrosequencing. Altogether, 570 Mbp of sequence data was thus generated which could be assembled into longer contigs. In total, 9,075 contigs could be mapped onto the genome of Desulfococcus oleovorans Hxd3, the closest fully sequenced relative of SEEPSRB1a, and thereby could be assigned to SEEP-SRB1a. Two contigs carrying putative SEEP-SRB1a apr and dsr genes, provided a first glimpse of the genetic potential of these bacteria.