Edwards, Elizabeth A.

ABSTRACTThe Oil Refinery (OR) consortium is a model methanogenic enrichment culture for studying anaerobic benzene degradation. Over 80% of the culture’s bacterial community is comprised of two closely related strains of benzene-fermentingDesulfobacterota(designated ORM2a and ORM2b) whose mechanism of benzene degradation is unknown. Two new metagenomes, including a fully closed metagenome-assembled genome (MAG) for ORM2a, enabled a thorough investigation of this culture’s proteome. Among the proteins identified were Bam-like subunits of an ATP-independent benzoyl-CoA degradation pathway and associated downstream beta-oxidation proteins producing acetyl-CoA. The most abundant proteins identified mapped to two ORM2 gene clusters of unknown function. Syntenic gene clusters were identified in one other known benzene degrader,Pelotomaculumcandidate BPL, as well as a handful of contigs assembled from hydrothermal vent metagenomes. Extensive searches against reference sequence and structural databases indicate that the first (“Magic”) gene cluster likely catalyzes the chemically difficult benzene activation step. The second (“Nanopod”) gene cluster is predicted to code for an efflux system that pumps excess benzene out of cells, mitigating some of its toxigenic effects. Phylogenomic analyses place ORM2a and ORM2b within a novel genus of benzene-degrading specialists which we propose naming “CandidatusBenzenivorax”. We hope to engage the research community to help in confirming the roles of the proteins in the “Magic” and “Nanopod” gene clusters, and to search through their own cultures for these features.

Dehalobacter is a genus of organohalide-respiring bacteria that is recognized for its fastidious growth using reductive dehalogenases (RDases). In the SC05 culture, however, a Dehalobacter population also mineralizes dichloromethane (DCM) produced by chloroform dechlorination using the mec cassette, just downstream of its active RDase. A closed genome of this DCM-mineralizing lineage has previously evaded assembly. Here, we present the genomes of two novel Dehalobacter strains, each of which was assembled from the metagenome of a distinct subculture from SC05. A pangenomic analysis of the Dehalobacter genus, including RDase synteny and phylogenomics, reveals at least five species of Dehalobacter based on average nucleotide identity, RDase and core gene synteny, as well as differential functional genes. An integration hotspot is also pinpointed in the Dehalobacter genome, in which many recombinase islands have accumulated. This nested recombinase island encodes the active RDase and mec cassette in both SC05 Dehalobacter genomes, indicating the transfer of key functional genes between species of Dehalobacter. Horizontal gene transfer between these two novel Dehalobacter strains has implications for the evolutionary history within the SC05 subcultures and of the Dehalobacter genus as a whole, especially regarding adaptation to anthropogenic chemicals.

AbstractThe Candidate Phyla Radiation (CPR, or superphylum Patescibacteria) is a very large group of bacteria with few cultivated representatives first discovered by culture-independent metagenomic analyses. Within the CPR, the candidate phylum Parcubacteria (previously OD1) is prevalent in anoxic lake sediments and groundwater. We identified a bacterium belonging to the Parcubacteria in a methanogenic benzene-degrading enrichment culture originally derived from oil-contaminated sediments. Candidatus Nealsonbacteria DGGOD1a is the only bacterium other than a previously identified benzene-degrading fermenter (Deltaproteobacteria Candidate Sva0485 clade ORM2) consistently and abundantly detected in all active benzene-degrading transfers of this culture. Therefore, we hypothesized that DGGOD1a must serve an important role in sustaining anaerobic benzene metabolism in the consortium. Growth experiments using a variety of possible substrates suggested that it is involved in biomass recycling. Microscopic observations supported by molecular analyses and a closed genome revealed an epibiont lifestyle with very small Ca. Nealsonbacteria DGGOD1a closely associated with much larger Methanosaeta. The images reveal a first example of cross-domain episymbiosis that may apply to other Ca. Nealsonbacteria found in diverse environments.