Nitrous oxide respiration in acidophilic methanotrophs

Abstract

Aerobic methanotrophic bacteria are considered strict aerobes but are often highly abundant in hypoxic and even anoxic environments. Despite possessing denitrification genes, it remains to be verified whether denitrification contributes to their growth. Here, we show that acidophilic methanotrophs can respire nitrous oxide (N₂O) and grow anaerobically on diverse non-methane substrates, including methanol, C-C substrates, and hydrogen. We study two strains that possess N₂O reductase genes: Methylocella tundrae T4 and Methylacidiphilum caldifontis IT6. We show that N₂O respiration supports growth of Methylacidiphilum caldifontis at an extremely acidic pH of 2.0, exceeding the known physiological pH limits for microbial N₂O consumption. Methylocella tundrae simultaneously consumes N₂O and CH₄ in suboxic conditions, indicating robustness of its N₂O reductase activity in the presence of O₂. Furthermore, in O₂-limiting conditions, the amount of CH₄ oxidized per O₂ reduced increases when N₂O is added, indicating that Methylocella tundrae can direct more O₂ towards methane monooxygenase. Thus, our results demonstrate that some methanotrophs can respire N₂O independently or simultaneously with O₂, which may facilitate their growth and survival in dynamic environments. Such metabolic capability enables these bacteria to simultaneously reduce the release of the key greenhouse gases CO₂, CH_4, and N₂O.

Fig. 1. Maximum-likelihood phylogenetic tree of derived NosZ proteins, with nos operon arrangements in methanotrophic and non-methanotrophic bacterial strains.

The phylogenetic tree was constructed with IQ-TREE (IQ-TREE options: -B 1000 -m LG + F + R5) using aligned NosZ (details in Materials and Methods) and rooted at the mid-point. Bootstrap values ≥ 70% based on 1000 replications are indicated. The scale bar represents a 0.5 change per amino acid position. Organization of the nos operon in methanotrophic strains (labeled in blue text) and closely related non-methanotrophic bacteria are shown. The genes, represented by arrows, are drawn to scale. Homologs are depicted in identical colors. The NosZ amino acid sequences and gene arrangement information were retrieved using the following genome accessions: GCF_017310505.1, Methylacidiphilum caldifontis IT6; GCF_000010785.1, Hydrogenobacter thermophilus TK-6; GCF_011006175.1, Hydrogenobacter sp. T-8; GCF_900215655.1, Hydrogenobacter hydrogenophilus DSM 2913; GCF_000619805.1, Sulfurihydrogenibium subterraneum DSM 15120; GCF_000021565.1, Persephonella marina EX-H1; GCF_000022145.1, Anaeromyxobacter dehalogenans 2CP1; GCF_000013385.1, Anaeromyxobacter dehalogenans 2CP-C; GCF_003054705.1, Opitutus sp. ER46; GCF_000019965.1, Opitutus terrae PB90-1; GCF_901905185.1, Methylocella tundrae PC4; GCA_901905175.1, Methylocella tundrae PC1; CP139089.1, Methylocella tundrae T4; FO000002.1, Methylocystis sp. SC2; GCF_000025965.1, Aromatoleum aromaticum EbN1; GCF_022760775.1, ‘Candidatus Rhodoblastus alkanivorans’ PC3; GCF_000143145.1, Hyphomicrobium denitrificans ATCC 51888; GCF_000344805.1, Bradyrhizobium oligotrophicum S58; GCF_027923385.1, Methylocystis echinoides LMG27198; GCA_003963405.1, Methylocystis sp. AWTPI-1. * indicates that the nosZ genes are truncated due to genome fragmentation. Source Data contains genome annotation information for Methylocella tundrae T4, Methylacidiphilum caldifontis IT6, Methylocystis spp. (strains IM2, IM3, and IM4), and ‘Ca. Methylotropicum kingii’.

Fig. 2. Aerobic and anaerobic growth of N₂OR-containing and N₂OR-lacking Methylocella and Methylacidiphilum strains on methanol.

Methylocella tundrae T4, Methylocella silvestris BL2, Methylacidiphilum caldifontis IT6, and Methylacidiphilum infernorum IT5 cells were grown in LSM medium supplemented with 30 mM methanol as the electron donor and NH₄⁺ as the N-source. Aerobic growth of the 4 strains with O₂ (A, D, G, J), anaerobic growth with N₂O (B, E, H, K), and anaerobic growth without N₂O (C, F, I, L) as the sole terminal electron acceptor were determined by optical density measurements at 600 nm, followed by measurements of O₂ and N₂O consumption in the headspaces of the culture bottles. Note that the trace O₂ present at the start of the incubation in the anaerobic cultures without N₂O did not contribute to obvious growth (C, F, I, L). All experiments were performed in triplicates. Data are presented as mean ± 1 standard deviation (SD), and the error bars are hidden when they are smaller than the width of the symbols. Source data are provided as Source Data file.

Fig. 3. Anaerobic growth of Methylocella strains on methanol or pyruvate as the sole electron donor and NO₃⁻ as the terminal electron acceptor.

Methylocella tundrae T4 and Methylocella silvestris BL2 cells were grown in LSM medium supplemented with 30 mM methanol and 2–4 mM NO₃⁻. NH₄⁺ (2 mM) was supplied as the N-source. Anaerobic growth of Methylocella tundrae T4 (A) and Methylocella silvestris BL2 (B) cells on methanol as the sole electron donor with NO₃⁻ as the sole electron acceptor. Anaerobic growth of Methylocella tundrae T4 (C) and Methylocella silvestris BL2 (D) cells on pyruvate as the sole electron donor with NO₃⁻ as the sole electron acceptor. N₂O produced from NO₃⁻ reduction by cells of Methylocella silvestris BL2 grown on methanol or pyruvate is shown as an inset plot within each figure. N₂O production was not observed in strain T4, hence inset plots for N₂O production were not displayed. Lower NO₃⁻ (ca. 2.0 mM) was used in the case of methanol (A) to avoid NO₂⁻ toxicity. Growth was determined by optical density measurements at 600 nm, followed by measurements of NO₃^- and NO₂⁻ concentrations. Data are presented as mean ± 1 SD of triplicate experiments, and the error bars are hidden when they are smaller than the width of the symbols. Source data are provided as Source Data file.

Fig. 4. Microrespirometry-based N₂O and O₂ reduction during methanol oxidation by N₂OR-containing methanotrophs.

N₂O and O₂ reduction by cells of Methylacidiphilum caldifontis IT6 (A) and Methylocella tundrae T4 (B) during methanol oxidation. Filled blue dots represent dissolved N₂O, filled orange dots represent dissolved O₂, and filled black dots represent N₂O reduction rates. Experiments were performed in a microrespirometry (MR) chamber fitted with O₂ and N₂O microsensors. The red arrows mark the addition of 14–33 µM O₂ into the MR chamber. The red- and green-marked numbers close to the red and green lines represent the N₂O reduction rates before and during O₂ reduction (gray-shaded area) in the MR chamber, respectively. Source data are provided as Source Data file.

Fig. 5. Simultaneous N₂O and O₂ reduction by Methylocella tundrae T4 cells during CH₄ oxidation in microrespirometry (MR) and growth experiments.

A MR experiment showing the simultaneous reduction of N₂O and O₂ by Methylocella tundrae T4 cells during CH₄ oxidation. B N₂O and O₂ reduction rates by cells of strain T4 during CH₄ oxidation calculated from (A). The filled orange and blue dots in the upper (A) represent the concentrations of dissolved O₂ and N₂O, respectively. The filled orange and blue dots in the bottom (B) represent the rates of O₂ and N₂O reduction, respectively. Experiments were performed in a MR chamber fitted with O₂ and N₂O microsensors. The red arrow marks the addition of CH₄ (~406 µM) into the MR chamber. The black arrow marks the addition of ~26 µM or ~60 µM O₂ into the MR chamber. The gray-shaded area represents points where N₂O and O₂ are reduced simultaneously. C Growth experiment showing Methylocella tundrae T4 cells reducing N₂O and O₂ simultaneously during CH₄ oxidation. The culture was grown in 2-liter sealed bottles (triplicates) containing 60 mL of LSM medium with 2 mM NH₄⁺ as the N-source. The headspace of the bottles was composed of CH₄ (5%, v/v), O₂ (0.5%, v/v), N₂O (1.4%, v/v), and CO₂ (5%, v/v) and supplemented with additional O₂ (~0.5%, v/v) before its depletion. The incubation period shown in (C) is after the initial 20-day incubation period. After the depletion of O₂, additional O₂ was spiked to observe the simultaneous reduction of O₂ and N₂O during CH₄ oxidation. Data are presented as the mean ± 1 SD of a triplicate experiment, and the error bars are hidden when they are smaller than the width of the symbols. Source data are provided as Source Data file.

Fig. 6. Metabolic reconstruction and transcriptional response of Methylocella tundrae T4 cells to O₂-replete (CH₃OH + O₂) and anoxic (CH₃OH + N₂O) methanol-oxidizing growth conditions.

The genes used to reconstruct the metabolic pathway are listed in Table S5. The gene products are shaded according to the relative fold change (Log₂FC) in gene expression between cells grown under anoxic (CH₃OH + N₂O) and O₂-replete (CH₃OH + O₂) conditions. Genes up-regulated in CH₃OH + N₂O-grown cells are shown in teal green, while those up-regulated in CH₃OH + O₂-grown cells are shown in purple. Note that proteins are not drawn to scale. Methanol oxidation: Methanol is oxidized to formaldehyde in the periplasmic space by the PQQ-dependent methanol dehydrogenase (Xox- and Mxa-type), T4_03519–21, T4_00353–5, and T4_01862–76. The NAD(P)⁺-dependent alcohol dehydrogenase (T4_03199) may also be involved in methanol oxidation to formaldehyde in the cytoplasmic space during anaerobic growth on methanol. Formaldehyde oxidation to formate then proceeds via the tetrahydromethanopterin (H₄MPT) pathway, and C1 incorporation into the serine cycle is mediated by the tetrahydrofolate (H₄F) carbon assimilation pathway. The Calvin-Benson-Bassham pathway is also a possible route for CO₂ fixation. Nitrous oxide reduction: N₂O is reduced to N₂ through the activity of nitrous oxide reductase in the periplasmic space. Electron transfer to NosZ occurs via cytochrome c from the cytochrome bc1 (Qcr) complex^,. Electron transfer to the NosZ may also involve direct interaction with methanol dehydrogenase C-type cytochrome (XoxG, MxaG). The NosR protein may be involved in the transfer of electrons to NosZ (refs. ^,).