Methanotrophs aerobically oxidize methane to carbon dioxide to make a living and are known to degrade various other short chain carbon compounds as well. Volatile organic sulfur compounds such as methanethiol (CH3SH) are important intermediates in the sulfur cycle. Although volatile organic sulfur compounds co-occur with methane in various environments, little is known about how these compounds affect methanotrophy. The enzyme methanethiol oxidase catalyzing the oxidation of methanethiol has been known for decades, but only recently the mtoX gene encoding this enzyme was identified in a methylotrophic bacterium. The presence of a homologous gene in verrucomicrobial methanotrophs prompted us to examine how methanotrophs cope with methanethiol. Here, we show that the verrucomicrobial methanotroph Methylacidiphilum fumariolicum SolV consumes methanethiol and produces H2S, which is concurrently oxidized. Consumption of methanethiol is required since methanethiol inhibits methane oxidation. Cells incubated with ∼15 μM methanethiol from the start clearly showed inhibition of growth. After depletion of methanethiol, growth resumed within 1 day. Genes encoding a putative methanethiol oxidase were found in a variety of methanotrophs. Therefore, we hypothesize that methanethiol degradation is a widespread detoxification mechanism in methanotrophs in a range of environments.

Methanotrophs are an important group of microorganisms that counteract methane emissions to the atmosphere. Methane-oxidising bacteria of the Alpha- and Gammaproteobacteria have been studied for over a century, while methanotrophs of the phylum Verrucomicrobia are a more recent discovery. Verrucomicrobial methanotrophs are extremophiles that live in very acidic geothermal ecosystems. Currently, more than a dozen strains have been isolated, belonging to the genera Methylacidiphilum and Methylacidimicrobium. Initially, these methanotrophs were thought to be metabolically confined. However, genomic analyses and physiological and biochemical experiments over the past years revealed that verrucomicrobial methanotrophs, as well as proteobacterial methanotrophs, are much more metabolically versatile than previously assumed. Several inorganic gases and other molecules present in acidic geothermal ecosystems can be utilised, such as methane, hydrogen gas, carbon dioxide, ammonium, nitrogen gas and perhaps also hydrogen sulfide. Verrucomicrobial methanotrophs could therefore represent key players in multiple volcanic nutrient cycles and in the mitigation of greenhouse gas emissions from geothermal ecosystems. Here, we summarise the current knowledge on verrucomicrobial methanotrophs with respect to their metabolic versatility and discuss the factors that determine their diversity in their natural environment. In addition, key metabolic, morphological and ecological characteristics of verrucomicrobial and proteobacterial methanotrophs are reviewed.

The hydroxylamine oxidoreductase (HAO) family consists of octaheme proteins that harbor seven bis-His ligated electron-transferring hemes and one 5-coordinate catalytic heme with His axial ligation. Oxidative HAOs have a homotrimeric configuration with the monomers covalently attached to each other via a unique double cross-link between a Tyr residue and the catalytic heme moiety of an adjacent subunit. This cross-linked active site heme, termed the P460 cofactor, has been hypothesized to modulate enzyme reactivity toward oxidative catalysis. Conversely, the absence of this cross-link is predicted to favor reductive catalysis. However, this prediction has not been directly tested. In this study, an HAO homolog that lacks the heme-Tyr cross-link (HAOr) was purified to homogeneity from the nitrite-dependent anaerobic ammonium-oxidizing (anammox) bacterium Kuenenia stuttgartiensis, and its catalytic and spectroscopic properties were assessed. We show that HAOr reduced nitrite to nitric oxide and also reduced nitric oxide and hydroxylamine as nonphysiological substrates. In contrast, HAOr was not able to oxidize hydroxylamine or hydrazine supporting the notion that cross-link-deficient HAO enzymes are reductases. Compared with oxidative HAOs, we found that HAOr harbors an active site heme with a higher (at least 80 mV) midpoint potential and a much lower degree of porphyrin ruffling. Based on the physiology of anammox bacteria and our results, we propose that HAOr reduces nitrite to nitric oxide in vivo, providing anammox bacteria with NO, which they use to activate ammonium in the absence of oxygen.

Methylomirabilis bacteria perform anaerobic methane oxidation coupled to nitrite reduction via an intra-aerobic pathway, producing carbon dioxide and dinitrogen gas. These diderm bacteria possess an unusual polygonal cell shape with sharp ridges that run along the cell body. Previously, a putative surface protein layer (S-layer) was observed as the outermost cell layer of these bacteria. We hypothesized that this S-layer is the determining factor for their polygonal cell shape. Therefore, we enriched the S-layer from M. lanthanidiphila cells and through LC-MS/MS identified a 31 kDa candidate S-layer protein, mela_00855, which had no homology to any other known protein. Antibodies were generated against a synthesized peptide derived from the mela_00855 protein sequence and used in immunogold localization to verify its identity and location. Both on thin sections of M. lanthanidiphila cells and in negative-stained enriched S-layer patches, the immunogold localization identified mela_00855 as the S-layer protein. Using electron cryo-tomography and sub-tomogram averaging of S-layer patches, we observed that the S-layer has a hexagonal symmetry. Cryo-tomography of whole cells showed that the S-layer and the outer membrane, but not the peptidoglycan layer and the cytoplasmic membrane, exhibited the polygonal shape. Moreover, the S-layer consisted of multiple rigid sheets that partially overlapped, most likely giving rise to the unique polygonal cell shape. These characteristics make the S-layer of M. lanthanidiphila a distinctive and intriguing case to study.

The methane-oxidizing bacterium Methylacidimicrobium thermophilum AP8 thrives in acidic geothermal ecosystems that are characterized by high degassing of methane (CH4), H2, H2S, and by relatively high lanthanide concentrations. Lanthanides (atomic numbers 57 to 71) are essential in a variety of high-tech devices, including mobile phones. Remarkably, the same elements are actively taken up by methanotrophs/methylotrophs in a range of environments, since their XoxF-type methanol dehydrogenases require lanthanides as a metal cofactor. Lanthanide-dependent enzymes seem to prefer the lighter lanthanides (lanthanum, cerium, praseodymium, and neodymium), as slower methanotrophic/methylotrophic growth is observed in medium supplemented with only heavier lanthanides. Here, we purified XoxF1 from the thermoacidophilic methanotroph Methylacidimicrobium thermophilum AP8, which was grown in medium supplemented with neodymium as the sole lanthanide. The neodymium occupancy of the enzyme is 94.5% ± 2.0%, and through X-ray crystallography, we reveal that the structure of the active site shows interesting differences from the active sites of other methanol dehydrogenases, such as an additional aspartate residue in close proximity to the lanthanide. Nd-XoxF1 oxidizes methanol at a maximum rate of metabolism (Vmax) of 0.15 ± 0.01 μmol · min−1 · mg protein−1 and an affinity constant (Km) of 1.4 ± 0.6 μM. The structural analysis of this neodymium-containing XoxF1-type methanol dehydrogenase will expand our knowledge in the exciting new field of lanthanide biochemistry.

Nitrite-oxidizing bacteria belonging to the genus Nitrospira mediate a key step in nitrification and play important roles in the biogeochemical nitrogen cycle and wastewater treatment. While these organisms have recently been shown to exhibit metabolic flexibility beyond their chemolithoautotrophic lifestyle, including the use of simple organic compounds to fuel their energy metabolism, the metabolic networks controlling their autotrophic and mixotrophic growth remain poorly understood. Here, we reconstructed a genome-scale metabolic model for Nitrospira moscoviensis (iNmo686) and used flux balance analysis to evaluate the metabolic networks controlling autotrophic and formatotrophic growth on nitrite and formate, respectively. Subsequently, proteomic analysis and [13C]bicarbonate and [13C]formate tracer experiments coupled to metabolomic analysis were performed to experimentally validate model predictions. Our findings corroborate that N. moscoviensis uses the reductive tricarboxylic acid cycle for CO2 fixation, and we also show that N. moscoviensis can indirectly use formate as a carbon source by oxidizing it first to CO2 followed by reassimilation, rather than direct incorporation via the reductive glycine pathway. Our study offers the first measurements of Nitrospira's in vivo central carbon metabolism and provides a quantitative tool that can be used for understanding and predicting their metabolic processes. IMPORTANCE Nitrospira spp. are globally abundant nitrifying bacteria in soil and aquatic ecosystems and in wastewater treatment plants, where they control the oxidation of nitrite to nitrate. Despite their critical contribution to nitrogen cycling across diverse environments, detailed understanding of their metabolic network and prediction of their function under different environmental conditions remains a major challenge. Here, we provide the first constraint-based metabolic model of Nitrospira moscoviensis representing the ubiquitous Nitrospira lineage II and subsequently validate this model using proteomics and 13C-tracers combined with intracellular metabolomic analysis. The resulting genome-scale model will serve as a knowledge base of Nitrospira metabolism and lays the foundation for quantitative systems biology studies of these globally important nitrite-oxidizing bacteria.

Anaerobic ammonium-oxidizing (anammox) bacteria mediate a key step in the biogeochemical nitrogen cycle and have been applied worldwide for the energy-efficient removal of nitrogen from wastewater. However, outside their core energy metabolism, little is known about the metabolic networks driving anammox bacterial anabolism and use of different carbon and energy substrates beyond genome-based predictions. Here, we experimentally resolved the central carbon metabolism of the anammox bacterium Candidatus ‘Kuenenia stuttgartiensis’ using time-series ¹³C and ²H isotope tracing, metabolomics, and isotopically nonstationary metabolic flux analysis. Our findings confirm predicted metabolic pathways used for CO₂ fixation, central metabolism, and amino acid biosynthesis in K. stuttgartiensis, and reveal several instances where genomic predictions are not supported by in vivo metabolic fluxes. This includes the use of the oxidative branch of an incomplete tricarboxylic acid cycle for alpha-ketoglutarate biosynthesis, despite the genome not having an annotated citrate synthase. We also demonstrate that K. stuttgartiensis is able to directly assimilate extracellular formate via the Wood–Ljungdahl pathway instead of oxidizing it completely to CO₂ followed by reassimilation. In contrast, our data suggest that K. stuttgartiensis is not capable of using acetate as a carbon or energy source in situ and that acetate oxidation occurred via the metabolic activity of a low-abundance microorganism in the bioreactor’s side population. Together, these findings provide a foundation for understanding the carbon metabolism of anammox bacteria at a systems-level and will inform future studies aimed at elucidating factors governing their function and niche differentiation in natural and engineered ecosystems.

The trace amounts (0.53 ppmv) of atmospheric hydrogen gas (H2) can be utilized by microorganisms to persist during dormancy. This process is catalyzed by certain Actinobacteria, Acidobacteria, and Chloroflexi, and is estimated to convert 75 × 1012 g H2 annually, which is half of the total atmospheric H2. This rapid atmospheric H2 turnover is hypothesized to be catalyzed by high-affinity [NiFe] hydrogenases. However, apparent high-affinity H2 oxidation has only been shown in whole cells, rather than for the purified enzyme. Here, we show that the membrane-associated hydrogenase from the thermoacidophilic methanotroph Methylacidiphilum fumariolicum SolV possesses a high apparent affinity (Km(app) = 140 nM) for H2 and that methanotrophs can oxidize subatmospheric H2. Our findings add to the evidence that the group 1h [NiFe] hydrogenase is accountable for atmospheric H2 oxidation and that it therefore could be a strong controlling factor in the global H2 cycle. We show that the isolated enzyme possesses a lower affinity (Km = 300 nM) for H2 than the membrane-associated enzyme. Hence, the membrane association seems essential for a high affinity for H2. The enzyme is extremely thermostable and remains folded up to 95 °C. Strain SolV is the only known organism in which the group 1h [NiFe] hydrogenase is responsible for rapid growth on H2 as sole energy source as well as oxidation of subatmospheric H2. The ability to conserve energy from H2 could increase fitness of verrucomicrobial methanotrophs in geothermal ecosystems with varying CH4 fluxes. We propose that H2 oxidation can enhance growth of methanotrophs in aerated methane-driven ecosystems. Group 1h [NiFe] hydrogenases could therefore contribute to mitigation of global warming, since CH4 is an important and extremely potent greenhouse gas.

Emissions of the strong greenhouse gas methane (CH4) to the atmosphere are mitigated by methanotrophic microorganisms. Methanotrophs found in extremely acidic geothermal systems belong to the phylum Verrucomicrobia. Thermophilic verrucomicrobial methanotrophs from the genus Methylacidiphilum can grow autotrophically on hydrogen gas (H2), but it is unknown whether this also holds for their mesophilic counterparts from the genus Methylacidimicrobium. To determine this, we examined H2 consumption and CO2 fixation by the mesophilic verrucomicrobial methanotroph Methylacidimicrobium tartarophylax 4AC. We found that strain 4AC grows autotrophically on H2 with a maximum growth rate of 0.0048 h–1 and a yield of 2.1 g dry weight⋅mol H2–1, which is about 12 and 41% compared to the growth rate and yield on methane, respectively. The genome of strain 4AC only encodes for an oxygen-sensitive group 1b [NiFe] hydrogenase and H2 is respired only when oxygen concentrations are below 40 μM. Phylogenetic analysis and genomic comparison of methanotrophs revealed diverse [NiFe] hydrogenases, presumably with varying oxygen sensitivity and affinity for H2, which could drive niche differentiation. Our results show that both thermophilic and mesophilic verrucomicrobial methanotrophs can grow as autotrophs on H2 as a sole energy source. Our results suggest that verrucomicrobial methanotrophs are particularly well-equipped to thrive in hostile volcanic ecosystems, since they can consume H2 as additional energy source.

A new method has been developed to synthesize compact yttriumtrihydride by making use of a thin film technique. For electrical measurements yttrium films of typically 500 nm thickness are covered under UHV conditions by a 5 nm thick palladium overlayer which consists of electrically disconnected islands. Loading of these films with hydrogen up to the trihydride phase can then be done ex-situ in a reasonably short time (around 20-40 h) by applying gas pressures of about 60 × 10⁵ Pa. For a thicker Pd layer (above 20 nm) this time can be considerably shorter (t ∼ 125 s). The film morphology stays intact during the loading process although the film thickness increases by approximately 11% and the crystal structure changes from h.c.p. to f.c.c. and back to h.c.p. These samples are, therefore, very well suited for an investigation of the remarkable electrical and optical properties of trihydrides, as recently reported by Huiberts et al. (Nature, 380, 1996, 231). In this article we give evidence for the island structure of the palladium overlayer and make a comparison of a number of physical properties of yttrium and its related hydrides as thin films with literature values for the same material in bulk form. These properties include lattice parameters for the different hydride phases, electrical resistivity for yttrium and its dihydride and Hall coefficient for yttrium. The characteristics of the yttriumhydride thin films are very similar to those of bulk material. Furthermore, we performed concentration measurements and resistivity measurements during hydrogen loading. It is shown that the resistivity rises three orders of magnitude when yttrium is loaded up to the trihydride phase at 60 × 10⁵ Pa.