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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., With the ubiquitous use of fertilizers in agriculture, nitrate (N[O.sub.3.sup.-]) and nitrite (N[O.sub.2.sup.-]) have become major electron acceptors in freshwater environments (1). The feedback of eutrophication on the atmospheric methane [...]

"Candidatus Methylomirabilis oxyfera" is a newly discovered anaerobic methanotroph that, surprisingly, oxidizes methane through an aerobic methane oxidation pathway. The second step in this aerobic pathway is the oxidation of methanol. In Gramnegative bacteria, the reaction is catalyzed by pyrroloquinoline quinone (PQQ)-dependent methanol dehydrogenase (MDH). The genome of "Ca. Methylomirabilis oxyfera" putatively encodes three different MDHs that are localized in one large gene cluster: one so-called MxaFI-type MDH and two XoxF-type MDHs (XoxF1 and XoxF2). MxaFI MDHs represent the canonical enzymes, which are composed of two PQQ-containing large (α) subunits (MxaF) and two small (β) subunits (MxaI). XoxF MDHs are novel, ecologically widespread, but poorly investigated types of MDHs that can be phylogenetically divided into at least five different clades. The XoxF MDHs described thus far are homodimeric proteins containing a large subunit only. Here, we purified a heterotetrameric MDH from "Ca. Methylomirabilis oxyfera" that consisted of two XoxF and two MxaI subunits. The enzyme was localized in the periplasm of "Ca. Methylomirabilis oxyfera" cells and catalyzed methanol oxidation with appreciable specific activity and affinity (Vmax of 10 μmol min-1 mg-1 protein, Km of 17 μM). PQQ was present as the prosthetic group, which has to be taken up from the environment since the known gene inventory required for the synthesis of this cofactor is lacking. The MDH from "Ca. Methylomirabilis oxyfera" is the first representative of type 1 XoxF proteins to be described. [ABSTRACT FROM AUTHOR]

' Candidatus Methylomirabilis oxyfera'; is a polygon-shaped bacterium that was shown to have the unique ability to couple anaerobic methane oxidation to denitrification, through a newly discovered intra-aerobic pathway. Recently, the complete genome of Methylomirabilis oxyfera was assembled into a 2.7-Mb circular single chromosome by metagenomic sequencing. The genome of M. oxyfera revealed the full potential to perform both methane oxidation and the conversion of nitrite via nitric oxide into oxygen and dinitrogen gas. In this study, we show by immunogold localization that key enzymes from both methane- and nitrite-converting pathways are indeed present in single M. oxyfera cells. Antisera targeting the particulate methane monooxygenase (p MMO) and the cd1 nitrite reductase ( Nir S) were raised and used for immunogold localization in both single- and double-labelling experiments. Our previous studies have shown that M. oxyfera does not develop p MMO-containing intracytoplasmic membranes as is observed in classical proteobacterial methanotrophs. Our results suggest that in M. oxyfera, the p MMO and Nir S enzymes localized to the cytoplasmic membrane and periplasm, respectively. Further, double-labelling showed co-occurrence of p MMO and Nir S in single M. oxyfera cells. [ABSTRACT FROM AUTHOR]

"Candidatus Methylomirabilis oxyfera" is a newly discovered denitrifying methanotroph that is unrelated to previously known methanotrophs. This bacterium is a member of the NC10 phylum and couples methane oxidation to denitrification through a newly discovered intra-aerobic pathway. In the present study, we report the first ultrastructural study of "Ca. Methylomirabilis oxyfera" using scanning electron microscopy, transmission electron microscopy, and electron tomography in combination with different sample preparation methods. We observed that "Ca. Methylomirabilis oxyfera" cells possess an atypical polygonal shape that is distinct from other bacterial shapes described so far. Also, an additional layer was observed as the outermost sheath, which might represent a (glyco)protein surface layer. Further, intracytoplasmic membranes, which are a common feature among proteobacterial methanotrophs, were never observed under the current growth conditions. Our results indicate that "Ca. Methylomirabilis oxyfera" is ultrastructurally distinct from other bacteria by its atypical cell shape and from the classical proteobacterial methanotrophs by its apparent lack of intracytoplasmic membranes. [ABSTRACT FROM AUTHOR]

'Candidatus Methylomirabilis oxyfera'; is a polygon-shaped bacterium that was shown to have the unique ability to couple anaerobic methane oxidation to denitrification, through a newly discovered intra-aerobic pathway. Recently, the complete genome of Methylomirabilis oxyfera was assembled into a 2.7-Mb circular single chromosome by metagenomic sequencing. The genome of M. oxyfera revealed the full potential to perform both methane oxidation and the conversion of nitrite via nitric oxide into oxygen and dinitrogen gas. In this study, we show by immunogold localization that key enzymes from both methane- and nitrite-converting pathways are indeed present in single M. oxyfera cells. Antisera targeting the particulate methane monooxygenase (pMMO) and the cd(1) nitrite reductase (NirS) were raised and used for immunogold localization in both single- and double-labelling experiments. Our previous studies have shown that M. oxyfera does not develop pMMO-containing intracytoplasmic membranes as is observed in classical proteobacterial methanotrophs. Our results suggest that in M. oxyfera, the pMMO and NirS enzymes localized to the cytoplasmic membrane and periplasm, respectively. Further, double-labelling showed co-occurrence of pMMO and NirS in single M. oxyfera cells., (© 2012 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.)

Nitric oxide (NO) and nitrous oxide (N(2)O) are among nature's most powerful electron acceptors. In recent years it became clear that microorganisms can take advantage of the oxidizing power of these compounds to degrade aliphatic and aromatic hydrocarbons. For two unrelated bacterial species, the "NC10" phylum bacterium "Candidatus Methylomirabilis oxyfera" and the γ-proteobacterial strain HdN1 it has been suggested that under anoxic conditions with nitrate and/or nitrite, monooxygenases are used for methane and hexadecane oxidation, respectively. No degradation was observed with nitrous oxide only. Similarly, "aerobic" pathways for hydrocarbon degradation are employed by (per)chlorate-reducing bacteria, which are known to produce oxygen from chlorite [Formula: see text]. In the anaerobic methanotroph M. oxyfera, which lacks identifiable enzymes for nitrogen formation, substrate activation in the presence of nitrite was directly associated with both oxygen and nitrogen formation. These findings strongly argue for the role of NO, or an oxygen species derived from it, in the activation reaction of methane. Although oxygen generation elegantly explains the utilization of "aerobic" pathways under anoxic conditions, the underlying mechanism is still elusive. In this perspective, we review the current knowledge about intra-aerobic pathways, their potential presence in other organisms, and identify candidate enzymes related to quinol-dependent NO reductases (qNORs) that might be involved in the formation of oxygen.

The anaerobic nitrite-reducing methanotroph 'Candidatus Methylomirabilis oxyfera' ('Ca. M. oxyfera') produces oxygen from nitrite by a novel pathway. The major part of the O(2) is used for methane activation and oxidation, which proceeds by the route well known for aerobic methanotrophs. Residual oxygen may serve other purposes, such as respiration. We have found that the genome of 'Ca. M. oxyfera' harbours four sets of genes encoding terminal respiratory oxidases: two cytochrome c oxidases, a third putative bo-type ubiquinol oxidase, and a cyanide-insensitive alternative oxidase. Illumina sequencing of reverse-transcribed total community RNA and quantitative real-time RT-PCR showed that all four sets of genes were transcribed, albeit at low levels. Oxygen-uptake and inhibition experiments, UV-visible absorption spectral characteristics and EPR spectroscopy of solubilized membranes showed that only one of the four oxidases is functionally produced by 'Ca. M. oxyfera', notably the membrane-bound bo-type terminal oxidase. These findings open a new role for terminal respiratory oxidases in anaerobic systems, and are an additional indication of the flexibility of terminal oxidases, of which the distribution among anaerobic micro-organisms may be largely underestimated.

Biological methane oxidation proceeds either through aerobic or anaerobic pathways. The newly discovered bacterium Candidatus 'Methylomirabilis oxyfera' challenges this dichotomy. This bacterium performs anaerobic methane oxidation coupled to denitrification, but does so in a peculiar way. Instead of scavenging oxygen from the environment, like the aerobic methanotrophs, or driving methane oxidation by reverse methanogenesis, like the methanogenic archaea in sulfate-reducing systems, it produces its own supply of oxygen by metabolizing nitrite via nitric oxide into oxygen and dinitrogen gas. The intracellularly produced oxygen is then used for the oxidation of methane by the classical aerobic methane oxidation pathway involving methane mono-oxygenase. The present mini-review summarizes the current knowledge about this process and the micro-organism responsible for it.