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.

Methylococcus mesophilus sp. nov., the first non-thermotolerant methanotroph of the genus Methylococcus, from a rice field.

Strain 16-5T, a mesophilic methanotroph of the genus Methylococcus, was isolated from rice field soil sampled in Chungcheong Province, Republic of Korea. Strain 16-5T had both particulate and soluble methane monooxygenases and could only grow on methane and methanol as electron donors. Strain 16-5 T cells are Gram-negative, white to light tan in color, non-motile, non-flagellated, diplococcoid to cocci, and have the typical type I intracytoplasmic membrane system. Strain 16-5T grew at 18-38 °C (optimum, 27 °C) and at pH 5.0-8.0 (optimum, pH 6.5-7.0). C16 : 1 ω7c (38.8%), C16 : 1 ω5c (18.8%), C16 : 1 ω6c (16.8%) and C16 : 0 (16.9%) were the major fatty acids, and phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol and an unidentified phospholipid were the major polar lipids. The main respiratory quinone was methylene-ubiquinone-8. Strain 16-5T displayed the highest 16S rRNA gene sequence similarities to other taxonomically recognized members of the genus Methylococcus, i.e. Methylococcus capsulatus TexasT (98.62%) and Methylococcus geothermalis IM1T (98.49 %), which were its closest relatives. It did, however, differ from all other taxonomically described Methylococcus species due to some phenotypic differences, most notably its inability to grow at temperatures above 38 °C, where other Methylococcus species thrive. Its 4.34 Mbp-sized genome has a DNA G+C content of 62.47 mol%, and multiple genome-based properties such as average nucleotide identity and digital DNA-DNA hybridization value distanced it from its closest relatives. Based on the data presented above, this strain represents the first non-thermotolerant species of the genus Methylococcus. The name Methylococcus mesophilus sp. nov. is proposed, and 16-5T (=JCM 35359T=KCTC 82050T) is the type strain.

Methylacidiphilum caldifontis gen. nov., sp. nov., a thermoacidophilic methane-oxidizing bacterium from an acidic geothermal environment, and descriptions of the family Methylacidiphilaceae fam. nov. and order Methylacidiphilales ord. nov.

Strain IT6T, a thermoacidophilic and facultative methane-oxidizing bacterium, was isolated from a mud-water mixture collected from Pisciarelli hot spring in Pozzuoli, Italy. The novel strain is white when grown in liquid or solid media and forms Gram-negative rod-shaped, non-flagellated, non-motile cells. It conserves energy by aerobically oxidizing methane and hydrogen while deriving carbon from carbon dioxide fixation. Strain IT6T had three complete pmoCAB operons encoding particulate methane monooxygenase and genes encoding group 1d and 3b [NiFe] hydrogenases. Simple carbon-carbon substrates such as ethanol, 2-propanol, acetone, acetol and propane-1,2-diol were used as alternative electron donors and carbon sources. Optimal growth occurred at 50-55°C and between pH 2.0-3.0. The major fatty acids were C18 : 0, C15 : 0 anteiso, C14 : 0 iso, C16 : 0 and C14 : 0, and the main polar lipids were phosphatidylethanolamine, aminophospholipid, phosphatidylglycerol, diphosphatidylglycerol, some unidentified phospholipids and glycolipids, and other unknown polar lipids. Strain IT6T has a genome size of 2.19 Mbp and a G+C content of 40.70 mol%. Relative evolutionary divergence using 120 conserved single-copy marker genes (bac120) and phylogenetic analyses based on bac120 and 16S rRNA gene sequences showed that strain IT6T is affiliated with members of the proposed order 'Methylacidiphilales' of the class Verrucomicrobiia in the phylum Verrucomicrobiota. It shared a 16S rRNA gene sequence identity of >96 % with cultivated isolates in the genus 'Methylacidiphilum' of the family 'Methylacidiphilaceae', which are thermoacidophilic methane-oxidizing bacteria. 'Methylacidiphilum sp.' Phi (100 %), 'Methylacidiphilum infernorum' V4 (99.02 %) and 'Methylacidiphilum sp.' RTK17.1 (99.02 %) were its closest relatives. Its physiological and genomic properties were consistent with those of other isolated 'Methylacidiphilum' species. Based on these results, we propose the name Methylacidiphilum caldifontis gen. nov., sp. nov. to accommodate strain IT6T (=KCTC 92103T=JCM 39288T). We also formally propose that the names Methylacidiphilaceae fam. nov. and Methylacidiphilales ord. nov. to accommodate the genus Methylacidiphilum gen. nov.

Methylococcus geothermalis sp. nov., a methanotroph isolated from a geothermal field in the Republic of Korea.

A Gram-stain-negative, aerobic, non-motile and coccoid methanotroph, strain IM1T, was isolated from hot spring soil. Cells of strain IM1T were catalase-negative, oxidase-positive and displayed a characteristic intracytoplasmic membrane arrangement of type I methanotrophs. The strain possessed genes encoding both membrane-bound and soluble methane monooxygenases and grew only on methane or methanol. The strain was capable of growth at temperatures between 15 and 48 °C (optimum, 30-45 °C) and pH values between pH 4.8 and 8.2 (optimum, pH 6.2-7.0). Based on phylogenetic analysis of 16S rRNA gene and PmoA sequences, strain IM1T was demonstrated to be affiliated to the genus Methylococcus. The 16S rRNA gene sequence of this strain was most closely related to the sequences of an uncultured bacterium clone FD09 (100 %) and a partially described cultured Methylococcus sp. GDS2.4 (99.78 %). The most closely related taxonomically described strains were Methylococcus capsulatus TexasT (97.92 %), Methylococcus capsulatus Bath (97.86 %) and Methyloterricola oryzae 73aT (94.21 %). Strain IM1T shared average nucleotide identity values of 85.93 and 85.62 % with Methylococcus capsulatus strains TexasT and Bath, respectively. The digital DNA-DNA hybridization value with the closest type strain was 29.90 %. The DNA G+C content of strain IM1T was 63.3 mol% and the major cellular fatty acids were C16 : 0 (39.0 %), C16 : 1 ω7c (24.0 %), C16 : 1 ω6c (13.6 %) and C16 : 1 ω5c (12.0 %). The major ubiquinone was methylene-ubiquinone-8. On the basis of phenotypic, genetic and phylogenetic data, strain IM1T represents a novel species of the genus Methylococcus for which the name Methylococcus geothermalis sp. nov. is proposed, with strain IM1T (=JCM 33941T=KCTC 72677T) as the type strain.

Nitrous oxide respiration in acidophilic methanotrophs.

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, CH4, and N2O.

Transcriptomic Insights into Archaeal Nitrification in the Amundsen Sea Polynya, Antarctica.

Antarctic polynyas have the highest Southern Ocean summer primary productivity, and due to anthropogenic climate change, these areas have formed faster recently. Ammonia-oxidizing archaea (AOA) are among the most ubiquitous and abundant microorganisms in the ocean and play a primary role in the global nitrogen cycle. We utilized metagenomics and metatranscriptomics to gain insights into the physiology and metabolism of AOA in polar oceans, which are associated with ecosystem functioning. A polar-specific ecotype of AOA, from the "Candidatus Nitrosomarinus"-like group, was observed to be dominant in the Amundsen Sea Polynya (ASP), West Antarctica, during a succession of summer phytoplankton blooms. AOA had the highest transcriptional activity among prokaryotes during the bloom decline phase (DC). Metatranscriptomic analysis of key genes involved in ammonia oxidation, carbon fixation, transport, and cell division indicated that this polar AOA ecotype was actively involved in nitrification in the bloom DC in the ASP. This study revealed the physiological and metabolic traits of this key polar-type AOA in response to phytoplankton blooms in the ASP and provided insights into AOA functions in polar oceans.

Ammonia-oxidizing archaea in biological interactions.

The third domain Archaea was known to thrive in extreme or anoxic environments based on cultivation studies. Recent metagenomics-based approaches revealed a widespread abundance of archaea, including ammonia-oxidizing archaea (AOA) of Thaumarchaeota in non-extreme and oxic environments. AOA alter nitrogen species availability by mediating the first step of chemolithoautotrophic nitrification, ammonia oxidation to nitrite, and are important primary producers in ecosystems, which affects the distribution and activity of other organisms in ecosystems. Thus, information on the interactions of AOA with other cohabiting organisms is a crucial element in understanding nitrogen and carbon cycles in ecosystems as well as the functioning of whole ecosystems. AOA are self-nourishing, and thus interactions of AOA with other organisms can often be indirect and broad. Besides, there are possibilities of specific and obligate interactions. Mechanisms of interaction are often not clearly identified but only inferred due to limited knowledge on the interaction factors analyzed by current technologies. Here, we overviewed different types of AOA interactions with other cohabiting organisms, which contribute to understanding AOA functions in ecosystems.

Verrucomicrobial methanotrophs grow on diverse C3 compounds and use a homolog of particulate methane monooxygenase to oxidize acetone.

Short-chain alkanes (SCA; C2-C4) emitted from geological sources contribute to photochemical pollution and ozone production in the atmosphere. Microorganisms that oxidize SCA and thereby mitigate their release from geothermal environments have rarely been studied. In this study, propane-oxidizing cultures could not be grown from acidic geothermal samples by enrichment on propane alone, but instead required methane addition, indicating that propane was co-oxidized by methanotrophs. "Methylacidiphilum" isolates from these enrichments did not grow on propane as a sole energy source but unexpectedly did grow on C3 compounds such as 2-propanol, acetone, and acetol. A gene cluster encoding the pathway of 2-propanol oxidation to pyruvate via acetol was upregulated during growth on 2-propanol. Surprisingly, this cluster included one of three genomic operons (pmoCAB3) encoding particulate methane monooxygenase (PMO), and several physiological tests indicated that the encoded PMO3 enzyme mediates the oxidation of acetone to acetol. Acetone-grown resting cells oxidized acetone and butanone but not methane or propane, implicating a strict substrate specificity of PMO3 to ketones instead of alkanes. Another PMO-encoding operon, pmoCAB2, was induced only in methane-grown cells, and the encoded PMO2 could be responsible for co-metabolic oxidation of propane to 2-propanol. In nature, propane probably serves primarily as a supplemental growth substrate for these bacteria when growing on methane.