Keltjens, Jan T.

ABSTRACT “ 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 Gram-negative 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 ( V max of 10 μmol min −1 mg −1 protein, K m 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.

Summary ‘ Candidatus Methylomirabilis oxyfera’ is a denitrifying methanotroph that performs nitrite‐dependent anaerobic methane oxidation through a newly discovered intra‐aerobic pathway. In this study, we investigated the response of a M. oxyfera enrichment culture to oxygen. Addition of either 2% or 8% oxygen resulted in an instant decrease of methane and nitrite conversion rates. Oxygen exposure also led to a deviation in the nitrite to methane oxidation stoichiometry. Oxygen‐uptake and inhibition studies with cell‐free extracts displayed a change from cytochrome c to quinol as electron donor after exposure to oxygen. The change in global gene expression was monitored by deep sequencing of cDNA using Illumina technology. After 24 h of oxygen exposure, transcription levels of 1109 (out of 2303) genes changed significantly when compared with the anoxic period. Most of the genes encoding enzymes of the methane oxidation pathway were constitutively expressed. Genes from the denitrification pathway, with exception of one of the putative nitric oxide reductases, norZ2 , were severely downregulated. The majority of known genes involved in the vital cellular functions, such as nucleic acid and protein biosynthesis and cell division processes, were downregulated. The alkyl hydroperoxide reductase, ahpC , and genes involved in the synthesis/repair of the iron–sulfur clusters were among the few upregulated genes. Further, transcription of the pmoCAB genes of aerobic methanotrophs present in the non‐ M. oxyfera community were triggered by the presence of oxygen. Our results show that oxygen‐exposed cells of M. oxyfera were under oxidative stress and that in spite of its oxygenic capacity, exposure to microoxic conditions has an overall detrimental effect.

The anaerobic nitrite-reducing methanotroph ‘CandidatusMethylomirabilis oxyfera’ (‘Ca.M. oxyfera’) produces oxygen from nitrite by a novel pathway. The major part of the O2is 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 cytochromecoxidases, a third putativebo-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-boundbo-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.