Sulfur and methane oxidation by a single microorganism.

Natural and anthropogenic wetlands are major sources of the atmospheric greenhouse gas methane. Methane emissions from wetlands are mitigated by methanotrophic bacteria at the oxic-anoxic interface, a zone of intense redox cycling of carbon, sulfur, and nitrogen compounds. Here, we report on the isolation of an aerobic methanotrophic bacterium, 'Methylovirgula thiovorans' strain HY1, which possesses metabolic capabilities never before found in any methanotroph. Most notably, strain HY1 is the first bacterium shown to aerobically oxidize both methane and reduced sulfur compounds for growth. Genomic and proteomic analyses showed that soluble methane monooxygenase and XoxF-type alcohol dehydrogenases are responsible for methane and methanol oxidation, respectively. Various pathways for respiratory sulfur oxidation were present, including the Sox-rDsr pathway and the S4I system. Strain HY1 employed the Calvin-Benson-Bassham cycle for CO2 fixation during chemolithoautotrophic growth on reduced sulfur compounds. Proteomic and microrespirometry analyses showed that the metabolic pathways for methane and thiosulfate oxidation were induced in the presence of the respective substrates. Methane and thiosulfate could therefore be independently or simultaneously oxidized. The discovery of this versatile bacterium demonstrates that methanotrophy and thiotrophy are compatible in a single microorganism and underpins the intimate interactions of methane and sulfur cycles in oxic-anoxic interface environments.

Kinetic analysis of a complete nitrifier reveals an oligotrophic lifestyle.

Nitrification, the oxidation of ammonia (NH3) via nitrite (NO2-) to nitrate (NO3-), is a key process of the biogeochemical nitrogen cycle. For decades, ammonia and nitrite oxidation were thought to be separately catalysed by ammonia-oxidizing bacteria (AOB) and archaea (AOA), and by nitrite-oxidizing bacteria (NOB). The recent discovery of complete ammonia oxidizers (comammox) in the NOB genus Nitrospira, which alone convert ammonia to nitrate, raised questions about the ecological niches in which comammox Nitrospira successfully compete with canonical nitrifiers. Here we isolate a pure culture of a comammox bacterium, Nitrospira inopinata, and show that it is adapted to slow growth in oligotrophic and dynamic habitats on the basis of a high affinity for ammonia, low maximum rate of ammonia oxidation, high growth yield compared to canonical nitrifiers, and genomic potential for alternative metabolisms. The nitrification kinetics of four AOA from soil and hot springs were determined for comparison. Their surprisingly poor substrate affinities and lower growth yields reveal that, in contrast to earlier assumptions, AOA are not necessarily the most competitive ammonia oxidizers present in strongly oligotrophic environments and that N. inopinata has the highest substrate affinity of all analysed ammonia oxidizer isolates except the marine AOA Nitrosopumilus maritimus SCM1 (ref. 3). These results suggest a role for comammox organisms in nitrification under oligotrophic and dynamic conditions.

Methane oxidation coupled to nitrate reduction under hypoxia by the Gammaproteobacterium Methylomonas denitrificans, sp. nov. type strain FJG1.

Obligate methanotrophs belonging to the Phyla Proteobacteria and Verrucomicrobia require oxygen for respiration and methane oxidation; nevertheless, aerobic methanotrophs are abundant and active in low oxygen environments. While genomes of some aerobic methanotrophs encode putative nitrogen oxide reductases, it is not understood whether these metabolic modules are used for NOx detoxification, denitrification or other purposes. Here we demonstrate using microsensor measurements that a gammaproteobacterial methanotroph Methylomonas denitrificans sp. nov. strain FJG1(T) couples methane oxidation to nitrate reduction under oxygen limitation, releasing nitrous oxide as a terminal product. Illumina RNA-Seq data revealed differential expression of genes encoding a denitrification pathway previously unknown to methanotrophs as well as the pxmABC operon in M. denitrificans sp. nov. strain FJG1(T) in response to hypoxia. Physiological and transcriptome data indicate that genetic inventory encoding the denitrification pathway is upregulated only upon availability of nitrate under oxygen limitation. In addition, quantitation of ATP levels demonstrates that the denitrification pathway employs inventory such as nitrate reductase NarGH serving M. denitrificans sp. nov. strain FJG1(T) to conserve energy during oxygen limitation. This study unravelled an unexpected metabolic flexibility of aerobic methanotrophs, thereby assigning these bacteria a new role at the metabolic intersection of the carbon and nitrogen cycles.

Ammonia-oxidizing archaea possess a wide range of cellular ammonia affinities.

Nitrification, the oxidation of ammonia to nitrate, is an essential process in the biogeochemical nitrogen cycle. The first step of nitrification, ammonia oxidation, is performed by three, often co-occurring guilds of chemolithoautotrophs: ammonia-oxidizing bacteria (AOB), archaea (AOA), and complete ammonia oxidizers (comammox). Substrate kinetics are considered to be a major niche-differentiating factor between these guilds, but few AOA strains have been kinetically characterized. Here, the ammonia oxidation kinetic properties of 12 AOA representing all major cultivated phylogenetic lineages were determined using microrespirometry. Members of the genus Nitrosocosmicus have the lowest affinity for both ammonia and total ammonium of any characterized AOA, and these values are similar to previously determined ammonia and total ammonium affinities of AOB. This contrasts previous assumptions that all AOA possess much higher substrate affinities than their comammox or AOB counterparts. The substrate affinity of ammonia oxidizers correlated with their cell surface area to volume ratios. In addition, kinetic measurements across a range of pH values supports the hypothesis that-like for AOB-ammonia and not ammonium is the substrate for the ammonia monooxygenase enzyme of AOA and comammox. Together, these data will facilitate predictions and interpretation of ammonia oxidizer community structures and provide a robust basis for establishing testable hypotheses on competition between AOB, AOA, and comammox.

Complete genome sequence of the aerobic marine methanotroph Methylomonas methanica MC09.

Methylomonas methanica MC09 is a mesophilic, halotolerant, aerobic, methanotrophic member of the Gammaproteobacteria, isolated from coastal seawater. Here we present the complete genome sequence of this strain, the first available from an aerobic marine methanotroph.

Genome sequence of the methanotrophic alphaproteobacterium Methylocystis sp. strain Rockwell (ATCC 49242).

Methylocystis sp. strain Rockwell (ATCC 49242) is an aerobic methane-oxidizing alphaproteobacterium isolated from an aquifer in southern California. Unlike most methanotrophs in the Methylocystaceae family, this strain has a single pmo operon encoding particulate methane monooxygenase but no evidence of the genes encoding soluble methane monooxygenase. This is the first reported genome sequence of a member of the Methylocystis species of the Methylocystaceae family in the order Rhizobiales.

Low yield and abiotic origin of N2O formed by the complete nitrifier Nitrospira inopinata.

Nitrous oxide (N2O) and nitric oxide (NO) are atmospheric trace gases that contribute to climate change and affect stratospheric and ground-level ozone concentrations. Ammonia oxidizing bacteria (AOB) and archaea (AOA) are key players in the nitrogen cycle and major producers of N2O and NO globally. However, nothing is known about N2O and NO production by the recently discovered and widely distributed complete ammonia oxidizers (comammox). Here, we show that the comammox bacterium Nitrospira inopinata is sensitive to inhibition by an NO scavenger, cannot denitrify to N2O, and emits N2O at levels that are comparable to AOA but much lower than AOB. Furthermore, we demonstrate that N2O formed by N. inopinata formed under varying oxygen regimes originates from abiotic conversion of hydroxylamine. Our findings indicate that comammox microbes may produce less N2O during nitrification than AOB.

Complete Genome Sequence of Methylomonas denitrificans Strain FJG1, an Obligate Aerobic Methanotroph That Can Couple Methane Oxidation with Denitrification.

Methylomonas denitrificans strain FJG1 is a member of the gammaproteobacterial methanotrophs. The sequenced genome of FJG1 reveals the presence of genes that encode methane, methanol, formaldehyde, and formate oxidation. It also contains genes that encode enzymes for nitrate reduction to nitrous oxide, consistent with the ability of FJG1 to couple denitrification with methane oxidation.

Comparison of Nitrogen Oxide Metabolism among Diverse Ammonia-Oxidizing Bacteria.

Ammonia-oxidizing bacteria (AOB) have well characterized genes that encode and express nitrite reductases (NIR) and nitric oxide reductases (NOR). However, the connection between presence or absence of these and other genes for nitrogen transformations with the physiological production of nitric oxide (NO) and nitrous oxide (N2O) has not been tested across AOB isolated from various trophic states, with diverse phylogeny, and with closed genomes. It is therefore unclear if genomic content for nitrogen oxide metabolism is predictive of net N2O production. Instantaneous microrespirometry experiments were utilized to measure NO and N2O emitted by AOB during active oxidation of ammonia (NH3) or hydroxylamine (NH2OH) and through a period of anoxia. This data was used in concert with genomic content and phylogeny to assess whether taxonomic factors were predictive of nitrogen oxide metabolism. Results showed that two oligotrophic AOB strains lacking annotated NOR-encoding genes released large quantities of NO and produced N2O abiologically at the onset of anoxia following NH3-oxidation. Furthermore, high concentrations of N2O were measured during active O2-dependent NH2OH oxidation by the two oligotrophic AOB in contrast to non-oligotrophic strains that only produced N2O at the onset of anoxia. Therefore, complete nitrifier denitrification did not occur in the two oligotrophic strains, but did occur in meso- and eutrophic strains, even in Nitrosomonas communis Nm2 that lacks an annotated NIR-encoding gene. Regardless of mechanism, all AOB strains produced measureable N2O under tested conditions. This work further confirms that AOB require NOR activity to enzymatically reduce NO to N2O in the nitrifier denitrification pathway, and also that abiotic reactions play an important role in N2O formation, in oligotrophic AOB lacking NOR activity.

Complete Genome Sequence of Nitrosomonas ureae Strain Nm10, an Oligotrophic Group 6a Nitrosomonad.

The complete genome of Nitrosomonas ureae strain Nm10, a mesophilic betaproteobacterial ammonia oxidizer isolated from Mediterranean soils in Sardinia, Italy, is reported here. This genome represents a cluster 6a nitrosomonad.

Genome Sequence of Nitrosomonas communis Strain Nm2, a Mesophilic Ammonia-Oxidizing Bacterium Isolated from Mediterranean Soil.

The complete genome sequence of Nitrosomonas communis strain Nm2, a mesophilic betaproteobacterial ammonia oxidizer isolated from Mediterranean soils in Corfu, Greece, is reported here. This is the first genome to describe a cluster 8 Nitrosomonas species and represents an ammonia-oxidizing bacterium commonly found in terrestrial ecosystems.

Diverse electron sources support denitrification under hypoxia in the obligate methanotroph Methylomicrobium album strain BG8.

Aerobic methane-oxidizing bacteria (MOB) are a diverse group of microorganisms that are ubiquitous in natural environments. Along with anaerobic MOB and archaea, aerobic methanotrophs are critical for attenuating emission of methane to the atmosphere. Clearly, nitrogen availability in the form of ammonium and nitrite have strong effects on methanotrophic activity and their natural community structures. Previous findings show that nitrite amendment inhibits the activity of some cultivated methanotrophs; however, the physiological pathways that allow some strains to transform nitrite, expression of gene inventories, as well as the electron sources that support this activity remain largely uncharacterized. Here we show that Methylomicrobium album strain BG8 utilizes methane, methanol, formaldehyde, formate, ethane, ethanol, and ammonia to support denitrification activity under hypoxia only in the presence of nitrite. We also demonstrate that transcript abundance of putative denitrification genes, nirS and one of two norB genes, increased in response to nitrite. Furthermore, we found that transcript abundance of pxmA, encoding the alpha subunit of a putative copper-containing monooxygenase, increased in response to both nitrite and hypoxia. Our results suggest that expression of denitrification genes, found widely within genomes of aerobic methanotrophs, allow the coupling of substrate oxidation to the reduction of nitrogen oxide terminal electron acceptors under oxygen limitation. The present study expands current knowledge of the metabolic flexibility of methanotrophs by revealing that a diverse array of electron donors support nitrite reduction to nitrous oxide under hypoxia.

Draft genomes of gammaproteobacterial methanotrophs isolated from terrestrial ecosystems.

Genome sequences of Methylobacter luteus, Methylobacter whittenburyi, Methylosarcina fibrata, Methylomicrobium agile, and Methylovulum miyakonense were generated. The strains represent aerobic methanotrophs typically isolated from various terrestrial ecosystems.

Genome Sequence of the Obligate Gammaproteobacterial Methanotroph Methylomicrobium album Strain BG8.

The complete genome sequence of Methylomicrobium album strain BG8, a methane-oxidizing gammaproteobacterium isolated from freshwater, is reported. Aside from a conserved inventory of genes for growth on single-carbon compounds, M. album BG8 carries a range of gene inventories for additional carbon and nitrogen transformations but no genes for growth on multicarbon substrates or for N fixation.