Transient Oxygen Exposure Causes Profound and Lasting Changes to a Benzene-Degrading Methanogenic Community.

We investigated the impact of oxygen on a strictly anaerobic, methanogenic benzene-degrading enrichment culture derived decades ago from oil-contaminated sediment. The culture includes a benzene fermenter from Deltaproteobacteria candidate clade Sva0485 (referred to as ORM2) and methanogenic archaea. A one-time injection of 0.1 mL air , simulating a small leak into 30 mL batch culture bottle, had no measurable impact on benzene degradation rates, although retrospectively, a tiny enrichment of aerobic taxa was detected. A subsequent 100 times larger injection of air stalled methanogenesis and caused drastic perturbation of the microbial community. A benzene-degrading Pseudomonas became highly enriched and consumed all available oxygen. Anaerobic benzene-degrading ORM2 cell numbers plummeted during this time; re-growth and associated recovery of methanogenic benzene degradation took almost 1 year. These results highlight the oxygen sensitivity of this methanogenic culture and confirm that the mechanism for anaerobic biotransformation of benzene is independent of oxygen, fundamentally different from established aerobic pathways, and is carried out by distinct microbial communities. The study also highlights the importance of including microbial decay in characterizing and modeling mixed microbial communities.

Metagenomic and genomic sequences from a nitrate-reducing benzene-degrading enrichment culture.

Metagenome-assembled genomes (MAGs) were recovered from metagenomic assemblies from a nitrate-reducing benzene-degrading enrichment culture. Ten MAGs of high quality or functional interest to benzene degradation are reported, seven of which are single contig genomes.

Draft genome of an anaerobic nitrate-reducing, benzene-degrading member of the order Thermincolales.

We present a metagenome assembled genome (MAG) of an anaerobic bacterium from a nitrate-reducing, benzene-degrading enrichment culture (NRBC). The draft Thermincolales genome consists of 20 contigs with a total length of 4.09 Mbp and includes putative carboxylase genes likely involved in benzene activation.

Prenylated FMN: Biosynthesis, purification, and Fdc1 activation.

Prenylated flavin mononucleotide (prFMN) is a recently discovered flavin cofactor produced by the UbiX family of FMN prenyltransferases, and is required for the activity of UbiD-like reversible decarboxylases. The latter enzymes are known to be involved in ubiquinone biosynthesis and biotransformation of lignin, aromatic compounds, and unsaturated aliphatic acids. However, exploration of uncharacterized UbiD proteins for biotechnological applications is hindered by our limited knowledge about the biochemistry of prFMN and prFMN-dependent enzymes. Here, we describe experimental protocols and considerations for the biosynthesis of prFMN in vivo and in vitro, in addition to cofactor extraction and application for activation of UbiD proteins.

Biosynthesis and Activity of Prenylated FMN Cofactors.

Prenylated flavin mononucleotide (prFMN) is a recently discovered cofactor required by the UbiD family of reversible decarboxylases involved in ubiquinone biosynthesis, biological decomposition of lignin, and biotransformation of aromatic compounds. This cofactor is synthesized by UbiX-like prenyltransferases catalyzing the transfer of the dimethylallyl moiety of dimethylallyl-monophosphate (DMAP) to FMN. The origin of DMAP for prFMN biosynthesis and the biochemical properties of free prFMN are unknown. We show that in Escherichia coli cells, DMAP can be produced by phosphorylating prenol using ThiM or dephosphorylating DMAPP using Nudix hydrolases. We produced 14 active prenyltransferases whose properties enabled the purification and characterization of protein-free forms of prFMN. In vitro assays revealed that the UbiD-like ferulate decarboxylase (Fdc1) can be activated by free prFMNiminium or C2'-hydroxylated prFMNiminium under both oxidized and reduced conditions. These insights into the biosynthesis and properties of prFMN will facilitate further elucidation of the biochemical diversity of reversible UbiD (de)carboxylases.