Identification of a Highly Expressed Gene Cluster Likely Coding for Benzene Activation Enzymes in a Methanogenic Enrichment Culture

The Oil Refinery (OR) consortium is a model methanogenic enrichment culture for studying anaerobic benzene degradation. Over 50% of the culture′s bacterial community is comprised of two closely related strains of benzene-fermenting Desulfobacterota (designated ORM2a and ORM2b) whose mechanism of benzene degradation is unknown. Two new metagenomes, including a complete circularized 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 ORM2a gene clusters of unknown function. Syntenic gene clusters were identified in two other known benzene degrader, ORM2b and Pelotomaculum candidate BPL, and in nine 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 may pump excess benzene out of cells to mitigate toxicity. Phylogenomic analyses place ORM2a and ORM2b within a novel genus of benzene-degrading specialists which we propose naming ″Candidatus Benzenivorax″.