Methylacidiphilum Fumariolicum SolV Research Articles

Assessing Lanthanide-Dependent Methanol Dehydrogenase Activity: The Assay Matters.

Artificial dye-coupled assays have been widely adopted as a rapid and convenient method to assess the activity of methanol dehydrogenases (MDH). Lanthanide(Ln)-dependent XoxF-MDHs are able to incorporate different lanthanides (Lns) in their active site. Dye-coupled assays showed that the earlier Lns exhibit a higher enzyme activity than the late Lns. Despite widespread use, there are limitations: oftentimes a pH of 9 and activators are required for the assay. Moreover, Ln-MDH variants are not obtained by isolation from the cells grown with the respective Ln, but by incubation of an apo-MDH with the Ln. Herein, we report the cultivation of Ln-dependent methanotroph Methylacidiphilum fumariolicum SolV with nine different Lns, the isolation of the respective MDHs and the assessment of the enzyme activity using the dye-coupled assay. We compare these results with a protein-coupled assay using its physiological electron acceptor cytochrome cGJ (cyt cGJ ). Depending on the assay, two distinct trends are observed among the Ln series. The specific enzyme activity of La-, Ce- and Pr-MDH, as measured by the protein-coupled assay, exceeds that measured by the dye-coupled assay. This suggests that early Lns also have a positive effect on the interaction between XoxF-MDH and its cyt cGJ thereby increasing functional efficiency.

Identification and characterisation of a major outer membrane protein from Methylacidiphilum fumariolicum SolV

The outer membrane (OM) protects Gram-negative bacteria against a hostile environment. The proteins embedded in the OM fulfil a number of tasks that are crucial to the bacterial cell. In this study, we identified and characterised a major outer membrane protein (WP_009059494) from Methylacidiphilum fumariolicum SolV. PRED-TMBB and AlphaFold2 predicted this protein to form a porin with a β-barrel structure consisting of ten antiparallel β-sheets and with a small amphipathic N-terminal α-helix in the periplasm. We purified soluble recombinant protein WP_009059494 from E. coli using Tris–HCl buffer with SDS. Antibodies were raised against two peptides in the two large extracellular loops of protein WP_009059494 and immunogold localisation showed this protein to be mainly present in the OM of strain SolV. In addition, this protein is tightly associated with the OM, and is resistant to extraction. Only a small amount can be isolated from the cell envelope using harsh conditions (SDS and boiling). Despite this resistance to extraction, WP_009059494 most likely is an outer membrane protein. A regular lattice could not be detected by negative staining TEM of strain SolV and isolated protein WP_009059494. Considering the specific ecological niche of strain SolV living in a geothermal environment with low pH and high temperatures, this major protein WP_009059494 may act as barrier to resist the extreme conditions found in its natural environment. In addition, we found an absence of the BamB, BamC and BamE proteins of the canonical BAM complex, in Methylacidiphilum and Methylacidimicrobium species. This suggests that these bacteria use a simple BAM complex for folding and transport of OM proteins.

Identification and characterization of an abundant lipoprotein from Methylacidiphilum fumariolicum SolV

Bacterial lipoproteins are characterized by the presence of a conserved N-terminal lipid-modified cysteine residue that allows the hydrophilic protein to anchor into bacterial cell membranes. These lipoproteins play essential roles in a wide variety of physiological processes. Based on transcriptome analysis of the verrucomicrobial methanotroph Methylacidiphilum fumariolicum SolV, we identified a highly expressed lipoprotein, WP_009060351 (139 amino acids), in its genome. The first 86 amino acids are specific for the methanotrophic genera Methylacidiphilum and Methylacidmicrobium, while the last 53 amino acids are present only in lipoproteins of members from the phylum Verrucomicrobiota (Hedlund). Heterologous expression of WP_009060351 in Escherichia coli revealed a 25-kDa dimeric protein and a 60-kDa tetrameric protein. Immunoblotting showed that WP_009060351 was present in the total membrane protein and peptidoglycan fractions of M. fumariolicum SolV. The results suggest an involvement of lipoprotein WP_009060351 in the linkage between the outer membrane and the peptidoglycan.

Simultaneous sulfide and methane oxidation by an extremophile

Hydrogen sulfide (H2S) and methane (CH4) are produced in anoxic environments through sulfate reduction and organic matter decomposition. Both gases diffuse upwards into oxic zones where aerobic methanotrophs mitigate CH4 emissions by oxidizing this potent greenhouse gas. Although methanotrophs in myriad environments encounter toxic H2S, it is virtually unknown how they are affected. Here, through extensive chemostat culturing we show that a single microorganism can oxidize CH4 and H2S simultaneously at equally high rates. By oxidizing H2S to elemental sulfur, the thermoacidophilic methanotroph Methylacidiphilum fumariolicum SolV alleviates the inhibitory effects of H2S on methanotrophy. Strain SolV adapts to increasing H2S by expressing a sulfide-insensitive ba3-type terminal oxidase and grows as chemolithoautotroph using H2S as sole energy source. Genomic surveys revealed putative sulfide-oxidizing enzymes in numerous methanotrophs, suggesting that H2S oxidation is much more widespread in methanotrophs than previously assumed, enabling them to connect carbon and sulfur cycles in novel ways.

Minor Actinides Can Replace Essential Lanthanides in Bacterial Life**

AbstractCertain f‐block elements—the lanthanides—have biological relevance in the context of methylotrophic bacteria. The respective strains incorporate these 4 f elements into the active site of one of their key metabolic enzymes, a lanthanide‐dependent methanol dehydrogenase. In this study, we investigated whether actinides, the radioactive 5 f elements, can replace the essential 4 f elements in lanthanide‐dependent bacterial metabolism. Growth studies with Methylacidiphilum fumariolicum SolV and the Methylobacterium extorquens AM1 ΔmxaF mutant demonstrate that americium and curium support growth in the absence of lanthanides. Moreover, strain SolV favors these actinides over late lanthanides when presented with a mixture of equal amounts of lanthanides together with americium and curium. Our combined in vivo and in vitro results establish that methylotrophic bacteria can utilize actinides instead of lanthanides to sustain their one‐carbon metabolism if they possess the correct size and a +III oxidation state.

Minor Actinides Can Replace Essential Lanthanides in Bacterial Life.

Over the last decade, the biological relevance of certain f-elements - the lanthanides - was established in the context of methylotrophic bacteria. The respective strains incorporate these 4f-elements into the active site of one of their key metabolic enzymes, a lanthanide-dependent methanol dehydrogenase. Here, we investigate if actinides, the radioactive 5f-elements, can replace the essential 4f-elements in lanthanide-dependent bacterial metabolism. Growth studies with Methylacidiphilum fumariolicum SolV and the Methylobacterium extorquens AM1 ΔmxaF-mutant demonstrate that americium and curium support growth in the absence of lanthanides. Moreover, strain SolV favors these actinides over late lanthanides, when presented to a mixture of equal amounts of lanthanides together with americium and curium. Bacterial uptake is confirmed by time-resolved laser-induced fluorescence spectroscopy, liquid scintillation counting, and inductively-coupled plasma mass-spectrometry. Additionally, we show catalytic activity of the lanthanide-dependent enzyme upon reconstitution with actinides. Our combined invivo and in vitro results establish that methylotrophic bacteria can utilize actinides instead of lanthanides to sustain their one-carbon metabolism; if they possess the correct size and a +III oxidation state. Furthermore, bacteria capable of utilizing actinides will be able to mobilize these elements in the environment. This may lead to applications for bioremediation and recycling/separation of lanthanides and actinides.

Learning from nature: recovery of rare earth elements by the extremophilic bacterium Methylacidiphilum fumariolicum.

We present the extremophilic bacterium Methylacidiphilum fumariolicum SolV as a platform for the recovery of rare earth elements (REE). Strain SolV is able to selectively extract the light REE from artificial industrial waste sources, natural REE-containing and post-mining waters. Upscaling, different media composition and accumulation over several cycles were successfully implemented, underlining the potential for bio-recovery of REE.

Methanethiol Consumption and Hydrogen Sulfide Production by the Thermoacidophilic Methanotroph Methylacidiphilum fumariolicum SolV.

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.

Genome Sequence of a Thermoacidophilic Methanotroph Belonging to the Verrucomicrobiota Phylum from Geothermal Hot Springs in Yellowstone National Park: A Metagenomic Assembly and Reconstruction.

Verrucomicrobiotal methanotrophs are thermoacidophilic methane oxidizers that have been isolated from volcanic and geothermal regions of the world. We used a metagenomic approach that entailed obtaining the whole genome sequence of a verrucomicrobiotal methanotroph from a microbial consortium enriched from samples obtained from Nymph Lake (89.9 °C, pH 2.73) in Yellowstone National Park in the USA. To identify and reconstruct the verrucomicrobiotal genome from Illumina NovaSeq 6000 sequencing data, we constructed a bioinformatic pipeline with various combinations of de novo assembly, alignment, and binning algorithms. Based on the marker gene (pmoA), we identified and assembled the Candidatus Methylacidiphilum sp. YNP IV genome (2.47 Mbp, 2392 ORF, and 41.26% GC content). In a comparison of average nucleotide identity between Ca. Methylacidiphilum sp. YNP IV and Ca. Methylacidiphilum fumariolicum SolV, its closest 16S rRNA gene sequence relative, is lower than 95%, suggesting that Ca. Methylacidiphilum sp. YNP IV can be regarded as a different species. The Ca. Methylacidiphilum sp. YNP IV genome assembly showed most of the key genes for methane metabolism, the CBB pathway for CO2 fixation, nitrogen fixation and assimilation, hydrogenases, and rare earth elements transporter, as well as defense mechanisms. The assembly and reconstruction of a thermoacidophilic methanotroph belonging to the Verrucomicrobiota phylum from a geothermal environment adds further evidence and knowledge concerning the diversity of biological methane oxidation and on the adaptation of this geochemically relevant reaction in extreme environments.

More Than a Methanotroph: A Broader Substrate Spectrum for Methylacidiphilum fumariolicum SolV.

Volcanic areas emit a number of gases including methane and other short chain alkanes, that may serve as energy source for the prevailing microorganisms. The verrucomicrobial methanotroph Methylacidiphilum fumariolicum SolV was isolated from a volcanic mud pot, and is able to grow under thermoacidophilic conditions on different gaseous substrates. Its genome contains three operons encoding a particulate methane monooxygenase (pMMO), the enzyme that converts methane to methanol. The expression of two of these pmo operons is subjected to oxygen-dependent regulation, whereas the expression of the third copy (pmoCAB3) has, so far, never been reported. In this study we investigated the ability of strain SolV to utilize short-chain alkanes and monitored the expression of the pmo operons under different conditions. In batch cultures and in carbon-limited continuous cultures, strain SolV was able to oxidize and grow on C1–C3 compounds. Oxidation of ethane did occur simultaneously with methane, while propane consumption only started once methane and ethane became limited. Butane oxidation was not observed. Transcriptome data showed that pmoCAB1 and pmoCAB3 were induced in the absence of methane and the expression of pmoCAB3 increased upon propane addition. Together the results of our study unprecedently show that a pMMO-containing methanotroph is able to co-metabolize other gaseous hydrocarbons, beside methane. Moreover, it expands the substrate spectrum of verrucomicrobial methanotrophs, supporting their high metabolic flexibility and adaptation to the harsh and dynamic conditions in volcanic ecosystems.

Multiheme hydroxylamine oxidoreductases produce NO during ammonia oxidation in methanotrophs

Aerobic and nitrite-dependent methanotrophs make a living from oxidizing methane via methanol to carbon dioxide. In addition, these microorganisms cometabolize ammonia due to its structural similarities to methane. The first step in both of these processes is catalyzed by methane monooxygenase, which converts methane or ammonia into methanol or hydroxylamine, respectively. Methanotrophs use methanol for energy conservation, whereas toxic hydroxylamine is a potent inhibitor that needs to be rapidly removed. It is suggested that many methanotrophs encode a hydroxylamine oxidoreductase (mHAO) in their genome to remove hydroxylamine, although biochemical evidence for this is lacking. HAOs also play a crucial role in the metabolism of aerobic and anaerobic ammonia oxidizers by converting hydroxylamine to nitric oxide (NO). Here, we purified an HAO from the thermophilic verrucomicrobial methanotroph Methylacidiphilum fumariolicum SolV and characterized its kinetic properties. This mHAO possesses the characteristic P460 chromophore and is active up to at least 80 °C. It catalyzes the rapid oxidation of hydroxylamine to NO. In methanotrophs, mHAO efficiently removes hydroxylamine, which severely inhibits calcium-dependent, and as we show here, lanthanide-dependent methanol dehydrogenases, which are more prevalent in the environment. Our results indicate that mHAO allows methanotrophs to thrive under high ammonia concentrations in natural and engineered ecosystems, such as those observed in rice paddy fields, landfills, or volcanic mud pots, by preventing the accumulation of inhibitory hydroxylamine. Under oxic conditions, methanotrophs mainly oxidize ammonia to nitrite, whereas in hypoxic and anoxic environments reduction of both ammonia-derived nitrite and NO could lead to nitrous oxide (N2O) production.

How Lanthanide Ions Affect the Addition-Elimination Step of Methanol Dehydrogenases.

The recently discovered methanol dehydrogenase, XoxF, is a widespread enzyme used by methylotrophic bacteria to oxidize methanol for carbon and energy, and requires lanthanide ions for its activity. This enzyme represents an essential component of methanol utilization by both methanol- and methane-utilizing bacteria. The present investigation looks on the electronic, energetic and geometrical behavior of the methanol dehydrogenase from Methylacidiphilum fumariolicum SolV, which is strictly dependent on early lanthanide metals with +3 oxidation states, by examining enzyme-substrate complexes of all the lanthanides. We focus on the catalytic reaction mechanism of two methanol dehydrogenases having as cofactor europium and ytterbium belonging to the mid- and later- series of lanthanides, in comparison with the methanol dehydrogenase containing the cerium, one early lanthanide. Our results provide evidence for the influence of the lanthanide contraction effect in all the elementary steps of the catalytic reaction mechanism. This indication may prove useful for developing new catalytic machineries of enzymes that adopt new-to-nature transformations.

Structure of the 4-hydroxy-tetrahydrodipicolinate synthase from the thermoacidophilic methanotroph Methylacidiphilum fumariolicum SolV and the phylogeny of the aminotransferase pathway.

The enzyme 4-hydroxy-tetrahydrodipicolinate synthase (DapA) is involved in the production of lysine and precursor molecules for peptidoglycan synthesis. In a multistep reaction, DapA converts pyruvate and L-aspartate-4-semialdehyde to 4-hydroxy-2,3,4,5-tetrahydrodipicolinic acid. In many organisms, lysine binds allosterically to DapA, causing negative feedback, thus making the enzyme an important regulatory component of the pathway. Here, the 2.1 Å resolution crystal structure of DapA from the thermoacidophilic methanotroph Methylacidiphilum fumariolicum SolV is reported. The enzyme crystallized as a contaminant of a protein preparation from native biomass. Genome analysis reveals that M. fumariolicum SolV utilizes the recently discovered aminotransferase pathway for lysine biosynthesis. Phylogenetic analyses of the genes involved in this pathway shed new light on the distribution of this pathway across the three domains of life.

Understanding the chemistry of the artificial electron acceptors PES, PMS, DCPIP and Wurster\u2019s Blue in methanol dehydrogenase assays

Methanol dehydrogenases (MDH) have recently taken the spotlight with the discovery that a large portion of these enzymes in nature utilize lanthanides in their active sites. The kinetic parameters of these enzymes are determined with a spectrophotometric assay first described by Anthony and Zatman 55 years ago. This artificial assay uses alkylated phenazines, such as phenazine ethosulfate (PES) or phenazine methosulfate (PMS), as primary electron acceptors (EAs) and the electron transfer is further coupled to a dye. However, many groups have reported problems concerning the bleaching of the assay mixture in the absence of MDH and the reproducibility of those assays. Hence, the comparison of kinetic data among MDH enzymes of different species is often cumbersome. Using mass spectrometry, UV–Vis and electron paramagnetic resonance (EPR) spectroscopy, we show that the side reactions of the assay mixture are mainly due to the degradation of assay components. Light-induced demethylation (yielding formaldehyde and phenazine in the case of PMS) or oxidation of PES or PMS as well as a reaction with assay components (ammonia, cyanide) can occur. We suggest here a protocol to avoid these side reactions. Further, we describe a modified synthesis protocol for obtaining the alternative electron acceptor, Wurster’s blue (WB), which serves both as EA and dye. The investigation of two lanthanide-dependent methanol dehydrogenases from Methylorubrum extorquens AM1 and Methylacidiphilum fumariolicum SolV with WB, along with handling recommendations, is presented.Graphic abstractLanthanide-dependent methanol dehydrogenases. Understanding the chemistry of artificial electron acceptors and redox dyes can yield more reproducible results.

The thermoacidophilic methanotroph Methylacidiphilum fumariolicum SolV oxidizes subatmospheric H2 with a high-affinity, membrane-associated [NiFe] hydrogenase

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.

Electrocatalysis of a Europium-Dependent Bacterial Methanol Dehydrogenase with Its Physiological Electron-Acceptor Cytochrome cGJ.

We report the first electrochemical study of a lanthanoid-dependent methanol dehydrogenase (Eu-MDH) from the acidophilic verrucomicrobial methanotroph Methylacidiphilum fumariolicum SolV with its own physiological cytochrome cGJ electron acceptor. Eu-MDH harbours a redox active 2,7,9-tricarboxypyrroloquinoline quinone (PQQ) cofactor which is non-covalently bound but coordinates trivalent lanthanoid elements including Eu3+ . Eu-MDH and the cytochrome were co-adsorbed with the biopolymer chitosan and cast onto a mercaptoundecanol (MU) monolayer modified Au working electrode. Cyclic voltammetry of cytochrome cGJ reveals a well-defined quasi-reversible FeIII/II redox couple at +255 mV vs. NHE at pH 7.5 and this response is pH independent. The reversible one-electron response of the cytochrome cGJ transforms into a sigmoidal catalytic wave in the presence of Eu-MDH and its substrates (methanol or formaldehyde). The catalytic current was pH-dependent and pH 7.3 was found to be optimal. Kinetic parameters (pH dependence, activation energy) obtained by electrochemistry show the same trends as those obtained from an artificial phenazine ethosulfate/dichlorophenol indophenol assay.

Characterization of a novel cytochrome cGJ as the electron acceptor of XoxF-MDH in the thermoacidophilic methanotroph Methylacidiphilum fumariolicum SolV

Methanotrophs play a prominent role in the global carbon cycle, by oxidizing the potent greenhouse gas methane to CO2. Methane is first converted into methanol by methane monooxygenase. This methanol is subsequently oxidized by either a calcium-dependent MxaF-type or a lanthanide-dependent XoxF-type methanol dehydrogenase (MDH). Electrons from methanol oxidation are shuttled to a cytochrome redox partner, termed cytochrome cL. Here, the cytochrome cL homolog from the thermoacidophilic methanotroph Methylacidiphilum fumariolicum SolV was characterized. SolV cytochrome cGJ is a fusion of a XoxG cytochrome and a periplasmic binding protein XoxJ. Here we show that XoxGJ functions as the direct electron acceptor of its corresponding XoxF-type MDH and can sustain methanol turnover, when a secondary cytochrome is present as final electron acceptor. SolV cytochrome cGJ (XoxGJ) further displays a unique, red-shifted absorbance spectrum, with a Soret and Q bands at 440, 553 and 595 nm in the reduced state, respectively. VTVH-MCD spectroscopy revealed the presence of a low spin iron heme and the data further shows that the heme group exhibits minimal ruffling. The midpoint potential Em,pH7 of +240 mV is similar to other cytochrome cL type proteins but remarkably, the midpoint potential of cytochrome cGJ was not influenced by lowering the pH. Cytochrome cGJ represents the first example of a cytochrome from a strictly lanthanide-dependent methylotrophic microorganism.

Similar but Not the Same: First Kinetic and Structural Analyses of a Methanol Dehydrogenase Containing a Europium Ion in the Active Site.

Since the discovery of the biological relevance of rare earth elements (REEs) for numerous different bacteria, questions concerning the advantages of REEs in the active sites of methanol dehydrogenases (MDHs) over calcium(II) and of why bacteria prefer light REEs have been a subject of debate. Here we report the cultivation and purification of the strictly REE‐dependent methanotrophic bacterium Methylacidiphilum fumariolicum SolV with europium(III), as well as structural and kinetic analyses of the first methanol dehydrogenase incorporating Eu in the active site. Crystal structure determination of the Eu‐MDH demonstrated that overall no major structural changes were induced by conversion to this REE. Circular dichroism (CD) measurements were used to determine optimal conditions for kinetic assays, whereas inductively coupled plasma mass spectrometry (ICP‐MS) showed 70 % incorporation of Eu in the enzyme. Our studies explain why bacterial growth of SolV in the presence of Eu3+ is significantly slower than in the presence of La3+/Ce3+/Pr3+: Eu‐MDH possesses a decreased catalytic efficiency. Although REEs have similar properties, the differences in ionic radii and coordination numbers across the series significantly impact MDH efficiency.

Impact of the lanthanide contraction on the activity of a lanthanide-dependent methanol dehydrogenase - a kinetic and DFT study.

Interest in the bioinorganic chemistry of lanthanides is growing rapidly as more and more lanthanide-dependent bacteria are being discovered. Especially the earlier lanthanides have been shown to be preferentially utilized by bacteria that need these Lewis acids as cofactors in their alcohol dehydrogenase enzymes. Here, we investigate the impact of the lanthanide ions lanthanum(iii) to lutetium(iii) (excluding Pm) on the catalytic parameters (vmax, KM, kcat/KM) of a methanol dehydrogenase (MDH) isolated from Methylacidiphilum fumariolicum SolV. Kinetic experiments and DFT calculations were used to discuss why only the earlier lanthanides (La-Gd) promote high MDH activity. Impact of Lewis acidity, coordination number preferences, stability constants and other properties that are a direct result of the lanthanide contraction are discussed in light of the two proposed mechanisms for MDH.

Ammonia Oxidation and Nitrite Reduction in the Verrucomicrobial Methanotroph Methylacidiphilum fumariolicum SolV.

The Solfatara volcano near Naples (Italy), the origin of the recently discovered verrucomicrobial methanotroph Methylacidiphilum fumariolicum SolV was shown to contain ammonium () at concentrations ranging from 1 to 28 mM. Ammonia (NH3) can be converted to toxic hydroxylamine (NH2OH) by the particulate methane monooxygenase (pMMO), the first enzyme of the methane (CH4) oxidation pathway. Methanotrophs rapidly detoxify the intermediate NH2OH. Here, we show that strain SolV performs ammonium oxidation to nitrite at a rate of 48.2 nmol .h−1.mg DW−1 under O2 limitation in a continuous culture grown on hydrogen (H2) as an electron donor. In addition, strain SolV carries out nitrite reduction at a rate of 74.4 nmol .h−1.mg DW−1 under anoxic condition at pH 5–6. This range of pH was selected to minimize the chemical conversion of nitrite () potentially occurring at more acidic pH values. Furthermore, at pH 6, we showed that the affinity constants (Ks) of the cells for NH3 vary from 5 to 270 μM in the batch incubations with 0.5–8% (v/v) CH4, respectively. Detailed kinetic analysis showed competitive substrate inhibition between CH4 and NH3. Using transcriptome analysis, we showed up-regulation of the gene encoding hydroxylamine dehydrogenase (haoA) cells grown on H2/ compared to the cells grown on CH4/ which do not have to cope with reactive N-compounds. The denitrifying genes nirk and norC showed high expression in H2/ and CH4/ grown cells compared to cells growing at μmax (with no limitation) while the norB gene showed downregulation in CH4/ grown cells. These cells showed a strong upregulation of the genes in nitrate/nitrite assimilation. Our results demonstrate that strain SolV can perform ammonium oxidation producing nitrite. At high concentrations of ammonium this may results in toxic effects. However, at low oxygen concentrations strain SolV is able to reduce nitrite to N2O to cope with this toxicity.