Only three biological pathways are known to produce oxygen: photosynthesis, chlorate respiration and the detoxification of reactive oxygen species. Here we present evidence for a fourth pathway, possibly of considerable geochemical and evolutionary importance. The pathway was discovered after metagenomic sequencing of an enrichment culture that couples anaerobic oxidation of methane with the reduction of nitrite to dinitrogen. The complete genome of the dominant bacterium, named 'Candidatus Methylomirabilis oxyfera', was assembled. This apparently anaerobic, denitrifying bacterium encoded, transcribed and expressed the well-established aerobic pathway for methane oxidation, whereas it lacked known genes for dinitrogen production. Subsequent isotopic labelling indicated that 'M. oxyfera' bypassed the denitrification intermediate nitrous oxide by the conversion of two nitric oxide molecules to dinitrogen and oxygen, which was used to oxidize methane. These results extend our understanding of hydrocarbon degradation under anoxic conditions and explain the biochemical mechanism of a poorly understood freshwater methane sink. Because nitrogen oxides were already present on early Earth, our finding opens up the possibility that oxygen was available to microbial metabolism before the evolution of oxygenic photosynthesis.

Anaerobic methane oxidation coupled to denitrification was recently assigned to bacteria belonging to the uncultured phylum NC10. In this study, we incubated sediment from a eutrophic ditch harboring a diverse community of NC10 bacteria in a bioreactor with a constant supply of methane and nitrite. After 6 months, fluorescence in situ hybridization showed that NC10 bacteria dominated the resulting population. The enrichment culture oxidized methane and reduced nitrite to dinitrogen gas. We assessed NC10 phylum diversity in the inoculum and the enrichment culture, compiled the sequences currently available for this bacterial phylum, and showed that of the initial diversity, only members of one subgroup had been enriched. The growth of this subgroup was monitored by quantitative PCR and correlated to nitritereducing activity and the total biomass of the culture. Together, the results indicate that the enriched subgroup of NC10 bacteria is responsible for anaerobic methane oxidation coupled to nitrite reduction. Due to methodological limitations (a strong bias against NC10 bacteria in 16S rRNA gene clone libraries and inhibition by commonly used stopper material) the environmental distribution and importance of these bacteria could be largely underestimated at present.Atmospheric concentrations of methane have risen 2.6-fold since preindustrial times (10). After several years of stagnation, there was again a clear increase in the methane concentration in 2007 (29). Currently, it is uncertain whether an increase in the number of sources and production or a decrease in the number of sinks and consumption is responsible for this reversal of the trend.Freshwater habitats like natural wetlands and rice fields are a major source (38% [9]) of atmospheric methane. In the absence of other documented electron donors, aerobic methane oxidation is assumed to be the most important sink in these habitats, but the role of alternative electron donors is not well understood (19,30). Anaerobic methane oxidation coupled to denitrification is energetically favorable, but evidence that it occurs is scarce. In marine, methane-containing sediments, nitrate and nitrite are usually not quantitatively important electron acceptors; in freshwater sediments the denitrifying and aerobic zones are in close proximity (3,22,35), possibly masking the process from detection. To our knowledge, concomitant methane and nitrate profiles of sediments have never been published.So far, methane oxidation coupled to denitrification has received the most attention in the field of hydrogeology. In groundwater, contamination with nitrate and nitrite occurs frequently, whereas electron donors are limiting. Methane plumes often form around landfills, and their attenuation has sometimes been attributed to denitrification (2, 37). So far, a single previous study unambiguously demonstrated anaerobic oxidation of methane coupled to denitrification in a contaminated freshwater aquifer (32). The first in vitro observation of anaerobic methane oxidation coupled to denit...

Cellulosomes are large, multiprotein complexes that tether plant biomass-degrading enzymes together for improved hydrolysis. These complexes were first described in anaerobic bacteria, where species-specific dockerin domains mediate the assembly of enzymes onto cohesin motifs interspersed within protein scaffolds. The versatile protein assembly mechanism conferred by the bacterial cohesin-dockerin interaction is now a standard design principle for synthetic biology. For decades, analogous structures have been reported in anaerobic fungi, which are known to assemble by sequence-divergent non-catalytic dockerin domains (NCDDs). However, the components, modular assembly mechanism and functional role of fungal cellulosomes remain unknown. Here, we describe a comprehensive set of proteins critical to fungal cellulosome assembly, including conserved scaffolding proteins unique to the Neocallimastigomycota. High-quality genomes of the anaerobic fungi Anaeromyces robustus, Neocallimastix californiae and Piromyces finnis were assembled with long-read, single-molecule technology. Genomic analysis coupled with proteomic validation revealed an average of 312 NCDD-containing proteins per fungal strain, which were overwhelmingly carbohydrate active enzymes (CAZymes), with 95 large fungal scaffoldins identified across four genera that bind to NCDDs. Fungal dockerin and scaffoldin domains have no similarity to their bacterial counterparts, yet several catalytic domains originated via horizontal gene transfer with gut bacteria. However, the biocatalytic activity of anaerobic fungal cellulosomes is expanded by the inclusion of GH3, GH6 and GH45 enzymes. These findings suggest that the fungal cellulosome is an evolutionarily chimaeric structure-an independently evolved fungal complex that co-opted useful activities from bacterial neighbours within the gut microbiome.

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Anaerobic ammonium-oxidizing (anammox) bacteria play an important role in the biogeochemical cycling of nitrogen. They derive their energy for growth from the conversion of ammonium and nitrite into dinitrogen gas in the complete absence of oxygen. Several methods have been used to detect the presence and activity of anammox bacteria in the environment, including 16S rRNA gene-based approaches. The use of the 16S rRNA gene to study biodiversity has the disadvantage that it is not directly related to the physiology of the target organism and that current primers do not completely capture the anammox diversity. Here we report the development of PCR primer sets targeting a subunit of the hydrazine synthase (hzsA), which represents a unique phylogenetic marker for anammox bacteria. The tested primers were able to retrieve hzsA gene sequences from anammox enrichment cultures, full-scale anammox wastewater treatment systems, and a variety of freshwater and marine environmental samples, covering all known anammox genera.