Autotrophic Carbon Dioxide Fixation via the Calvin-Benson-Bassham Cycle by the Denitrifying Methanotroph “Candidatus Methylomirabilis oxyfera”

Abstract

Methane is an important greenhouse gas and the most abundant hydrocarbon in the Earth's atmosphere. Methanotrophic microorganisms can use methane as their sole energy source and play a crucial role in the mitigation of methane emissions in the environment. "Candidatus Methylomirabilis oxyfera" is a recently described intra-aerobic methanotroph that is assumed to use nitric oxide to generate internal oxygen to oxidize methane via the conventional aerobic pathway, including the monooxygenase reaction. Previous genome analysis has suggested that, like the verrucomicrobial methanotrophs, "Ca. Methylomirabilis oxyfera" encodes and transcribes genes for the Calvin-Benson-Bassham (CBB) cycle for carbon assimilation. Here we provide multiple independent lines of evidence for autotrophic carbon dioxide fixation by "Ca. Methylomirabilis oxyfera" via the CBB cycle. The activity of ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO), a key enzyme of the CBB cycle, in cell extracts from an "Ca. Methylomirabilis oxyfera" enrichment culture was shown to account for up to 10% of the total methane oxidation activity. Labeling studies with whole cells in batch incubations supplied with either 13 CH 4 or [ 13 C]bicarbonate revealed that "Ca. Methylomirabilis oxyfera" biomass and lipids became significantly more enriched in 13 C after incubation with 13 C-labeled bicarbonate (and unlabeled methane) than after incubation with 13 C-labeled methane (and unlabeled bicarbonate), providing evidence for autotrophic carbon dioxide fixation. Besides this experimental approach, detailed genomic and transcriptomic analysis demonstrated an operational CBB cycle in "Ca. Methylomirabilis oxyfera." Altogether, these results show that the CBB cycle is active and plays a major role in carbon assimilation by "Ca. Methylomirabilis oxyfera" bacteria. Our results suggest that autotrophy might be more widespread among methanotrophs than was previously assumed and implies that a methanotrophic community in the environment is not necessarily revealed by 13 C-depleted lipids.

High rates of anaerobic methane oxidation in freshwater wetlands reduce potential atmospheric methane emissions

“…10-Methylhexadecanoic acid, a characteristic lipid for 'Methylomirabilis oxyfera', a freshwater bacterium capable of oxidizing methane with nitrite 33 , was either not detected at these sites or found in rather low abundance (o1.5% of all fatty acids; data not shown). This proposed biomarker for n-damo 48 was not sufficiently depleted in 13 C to unambiguously support a relationship with AOM (Supplementary Table 3); a conclusive relationship of this biomarker to AOM is complicated by the evidence for chemoorganoautotrophy of M. oxyfera 49 , in analogy to some ANME archaea 47 , and additional sources of this biomarker such as sulphate-reducing 50 and anammox bacteria 51 . The other proposed diagnostic lipid for M. oxyfera, 10-methyl-hexadec-7-enoic acid 48 , was not detected in any sample.…”

Rare bacteriohopanepolyols as markers for an autotrophic, intra-aerobic methanotroph

“…Two experimental replicates were included for each treatment. Methane and nitrite consumption were monitored over the course of the incubation (24 h) and were similar for all experiments (Rasigraf et al, 2014). The C isotopic enrichments of M. oxyfera hopanoids in the labelling treatments were compared to the C isotope signature of the reference.…”

“…Biomass of enrichment culture Ooij-1 was used for incubation experiments with 13 C-labeled substrates as described elsewhere (Rasigraf et al, 2014). Briefly, M. oxyfera biomass was incubated with either 13 C-labeled CH 4 or 13 C-labeled bicarbonate (99 atom%).…”

“…Some less specific fatty acids (e.g. C 18:0 ; Rasigraf et al, 2014) showed a completely different labeling pattern and these fatty acids are thought to be derived from the co-existing bacteria in the enrichment culture (Rasigraf et al, 2014).…”

“…Furthermore, the labeling experiment revealed that the hopanoids showed the same labeling pattern as the Methylomirabilis spp.-specific fatty acids (cf. Rasigraf et al, 2014). Some less specific fatty acids (e.g.…”