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 used to study anaerobic benzene degradation. Over half of the cultures bacterial community consists of two closely related Desulfobacterota strains, designated ORM2a and ORM2b, whose mechanisms of benzene activation are unknown. Two new metagenomes, including a complete circularized metagenome-assembled genome (MAG) for ORM2a, enabled a thorough investigation of this cultures proteome. Among the proteins identified were Bam-like subunits of an ATP-independent benzoyl-CoA degradation pathway, as well as downstream {beta}-oxidation proteins yielding acetate. The most abundant proteins identified mapped to two ORM2a gene clusters of unknown function. Homologous and syntenic gene clusters were identified in genomes of ORM2b and a sulfate-reducing Pelotomaculum that also degrades benzene, as well as in nine contigs assembled from hydrothermal vent metagenomes. Extensive homology and structural predictions suggest that the first cluster - termed the "Magic" gene cluster - encodes for enzymes catalyzing the chemically challenging activation of benzene and subsequent transformation steps yielding benzoyl-CoA. The second ("Nanopod") gene cluster encodes a transmembrane complex that may facilitate benzene transport across the cell membrane. Phylogenomic analyses place ORM2a and ORM2b within a novel genus of strict anaerobes specialized for benzene degradation, which we propose naming "Candidatus Anaerobenzenivorax". IMPORTANCEBenzene is a widespread, persistent and toxic pollutant that can accumulate in anoxic environments such as groundwater and sediments. Despite decades of study, the biochemical mechanisms by which benzene is activated under anaerobic conditions remain unproven. This study provides strong genetic and proteomic evidence for a new class of enzymes that initiate anaerobic benzene activation and proposes a preliminary model for their underlying biochemistry. These findings lay a foundation for future biochemical studies and expand our understanding of how microbes carry out extreme redox chemistry in the absence of oxygen.