pmoA Sequences Research Articles

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.

Enrichment of Type I Methanotrophs with <i>nirS</i> Genes of Three Emergent Macrophytes in a Eutrophic Wetland in China

The pmoA gene, encoding particulate methane monooxygenase in methanotrophs, and nirS and nirK genes, encoding bacterial nitrite reductases, were examined in the root and rhizosphere sediment of three common emergent macrophytes (Phragmites australis, Typha angustifolia, and Scirpus triqueter) and unvegetated sediment from eutrophic Wuliangsuhai Lake in China. Sequencing analyses indicated that 334 out of 351 cloned pmoA sequences were phylogenetically the most closely related to type I methanotrophs (Gammaproteobacteria), and Methylomonas denitrificans-like organisms accounted for 44.4% of the total community. In addition, 244 out of 250 cloned nirS gene sequences belonged to type I methanotrophs, and 31.2% of nirS genes were the most closely related to paddy rice soil clone SP-2-12 in Methylomonas of the total community. Three genera of type I methanotrophs, Methylomonas, Methylobacter, and Methylovulum, were common in both pmoA and nirS clone libraries in each sample. A quantitative PCR (qPCR) analysis demonstrated that the copy numbers of the nirS and nirK genes were significantly higher in rhizosphere sediments than in unvegetated sediments in P. australis and T. angustifolia plants. In the same sample, the nirS gene copy number was significantly higher than that of nirK. Furthermore, type I methanotrophs were localized in the root tissues according to catalyzed reporter deposition-fluorescence in situ hybridization (CARD-FISH). Thus, nirS-carrying type I methanotrophs were enriched in macrophyte root and rhizosphere sediment and are expected to play important roles in carbon/nitrogen cycles in a eutrophic wetland.

Bacterial Communities in Areas of Oil and Methane Seeps in Pelagic of Lake Baikal.

We have assessed the diversity of bacteria near oil-methane (area I) and methane (area II) seeps in the pelagic zone of Lake Baikal using massive parallel sequencing of 16S rRNA, pmoA, and mxaF gene fragments amplified from total DNA. At depths from the surface to 100m, sequences belonging to Cyanobacteria dominated. In the communities to a depth of 200m of the studied areas, Proteobacteria dominated the deeper layers of the water column. Alphaproteobacteria sequences were predominant in the community near the oil-methane seep, while the community near the methane seep was characterized by the prevalence of Alpha- and Gammaproteobacteria. Among representatives of these classes, type I methanotrophs prevailed in the 16S rRNA gene libraries from the near-bottom area, and type II methanotrophs were detected in minor quantities at different depths. In the analysis of the libraries of the pmoA and mxaF functional genes, we observed the different taxonomic composition of methanotrophic bacteria in the surface and deep layers of the water column. All pmoA sequences from area I were type II methanotrophs and were detected at a depth of 300m, while sequences of type I methanotrophs were the most abundant in deep layers of the water column of area II. All mxaF gene sequences belonged to Methylobacterium representatives. Based on comparative analyses of 16S rRNA, pmoA, and mxaF gene fragment libraries, we suggest that there must be a wider spectrum of functional genes facilitating methane oxidation that were not detected with the primers used.

Influences of Anthropogenic Activities on the Community Structure of N-DAMO Bacteria in the North Canal

Stream ecosystems are the primary receivers of nutrients and organic carbon exported from terrestrial ecosystems and are profoundly influenced by the land of the surrounding landscape. Anthropogenic activities increased the nutrient and organic carbon levels in both stream and benthic biofilms, which are closely related to the differences in the N-DAMO (Nitrate/nitrite-dependent anaerobic methane oxidation) bacterial communities. We studied N-DAMO bacterial communities in streams flowing through anthropogenic land in the city of Beijing. The results showed that anthropogenic activities increase the nutrient and organic carbon levels both midstream and downstream; these are closely related to the difference in the N-DAMO bacterial communities. Phylogenetic analysis revealed that most of the cloned 16S rRNA and pmoA sequences in the North Canal were similar to those recovered from activated sludge and wastewater. The effect of nitrogen on N-DAMO bacteria in the North Canal mainly depended on the main form of available nitrogen and the source of pollutant. Moreover, N-DAMO bacteria present downstream of the North Canal, had a more connected and modular microbial network than those present midstream, where bacterial communities with tightly connected species that were shown to be more vulnerable and sensitive to various disturbances were found.

A global analysis on the distribution pattern of the bacteria coupling simultaneous methane oxidation to nitrite reduction

The dominant bacterium Candidatus Methylomirabilis oxyfera (NC10 phylum) was first identified from an enrichment culture and subsequently from various other habitats. To date, the global distribution pattern of this bacterium has not been summarized. This study analyzed the distribution pattern and diversity of nitrite-dependent anaerobic methane oxidation (n-damo) bacteria by retrieving 2343 and 2555 16S rRNA gene and pmoA sequences from public databases, respectively. The phylogenetic trees showed that the distribution of this bacterium in marine notably distinct from other habitats, and the 16S rRNA gene sequences amplified from enrichment cultures established a separate phylogenetic group. The highest richness of their community was detected in marine habitat. Shannon-Wiener index showed that the diversity of them was higher in marine and coastal habitats without distinctive differences between them. In addition, a significant community difference was also found between the marine and freshwater habitats by calculating ANOSIM p values and shared OTUs. PCoA analysis shows that their community composition is habitat specific and shaped by salinity or anthropogenic influence. This analysis provides a synthesis on the global diversity and distribution pattern of this new bacterium with currently available information to date.

The response relationship between nitrogen and aerobic methanotrophs in sediment of urban river, Beijing, China

由于人类活动的影响大量未经处理的废污水汇入城市河流，高浓度的污染物影响了河流中微生物对生物地球化学物质迁移和转化的介导作用.本文选取典型的城市河流——北运河作为研究区域，分析了北运河沉积物中氮素形态以及含量的空间和季节差异性，并结合克隆文库分子生物学的方法，探讨了氮素形态和含量的差异对好氧甲烷氧化菌（aerobic methane-oxidizing microorganisms，MOB）群落特征的影响.结果表明：北运河沉积物中铵态氮（NH₄⁺-N）为氮素的主要存在形态，存在显著的空间差异，其含量在下游显著高于上游，但季节差异不显著.NH₄⁺-N含量的空间差异对MOB的群落结构和群落分布有显著影响，对群落多样性影响不显著.NH₄⁺-N含量的空间分布特征与MOB的群落聚类特征一致，NH₄⁺-N对MOB群落分布的影响显著高于其他形态的氮素，其含量越高，则与MOB群落分布的响应关系越紧密.北运河中NH₄⁺-N的来源影响了沉积物中MOB的主要来源，MOB高同源性菌群的来源与NH₄⁺-N等主要污染物的来源一致.沉积物中MOB物种之间联系的紧密程度依赖于氮素的主要存在形态及其含量水平.NH₄⁺-N含量较高的下游沉积物中微生物彼此之间关系及集聚程度更强，受外界环境变化的干扰程度更强，受人类活动引起环境变化的敏感程度更高.城市河流中氮素的形态和含量差异对甲烷的氧化过程有显著影响.探究城市河流沉积物中高含量的NH₄⁺-N对甲烷产生及消耗的影响过程是控制河流温室气体排放的关键.;Stream ecosystems are the main receivers of nutrient and organic carbon exported from terrestrial ecosystems. Stream ecosystems are profoundly influenced by the land used in the surrounding area. Anthropogenic activities increase the amount of nutrient and organic carbon in both stream and benthic biofilms, which are closely related to differences in the aerobic methane-oxidizing microorganisms (MOB) bacteria microbial communities. We studied MOB bacteria microbial communities in the North Canal, Beijing, China. The results showed that the main form of nitrogen was ammonium nitrogen (NH₄⁺-N) in the North Canal. There are not any significant differences of the NH₄⁺-N between summer and winter, whereas there are significant differences between midstream and downstream. Results of heatmap analysis were closely related to the difference of the NH₄⁺-N in spatial. Phylogenetic analysis indicated that most of the cloned pmoA sequences in the North Canal were similar to those sequences from activated sludge and wastewater. The effect of nitrogen on MOB bacteria in the North Canal mainly depends on the main form of nitrogen and the source of pollutants. Moreover, MOB bacteria in downstream of the North Canal, had a more closer and modular microbial network than midstream, where bacteria communities with tightly connected species had proven to be more vulnerable and sensitive to various disturbances.

Methane oxidation and diversity of aerobic methanotrophs in forest and agricultural soddy–podzolic soils

Soddy-podzolic soils are widely distributed in European Russia, but their role as a sink for atmospheric methane is poorly documented and there is no information on the methanotroph diversity. We analysed the potential CH4-oxidation rates in soil samples and showed that the rate was significantly higher in forest soil than in arable soil, 1.21 and 0.40ng CH4g soil −1day−1, respectively. PCR-DGGE and clone library analysis indicated the distinct methanotrophic communities in these soils. The pmoA sequences associated with uncultured soil methanotrophs, referred to as NUSC, dominated forest soil, while in agricultural soil, type I (Methylobacter, Methylocaldum) and type II (Methylocystis, Methylosinus) methanotrophs were dominant. A newly developed primer set was applied in qPCR analysis and revealed that the copy number of pmoA genes of NUSC methanotrophs in forest soil was (9.2±0.87)×104g soil−1, whereas the transcript number was (1.33±0.31)×106g soil −1. We concluded that differences between the CH4 oxidation rates between forest and agriculture soils were driven by the structure of the methane-oxidizing community and that a novel group of methanotrophs may be an active participant in this process.

Relative Abundance and Diversity of Bacterial Methanotrophs at the Oxic-Anoxic Interface of the Congo Deep-Sea Fan.

Sitting at ∼5,000 m water depth on the Congo-Angola margin and ∼760 km offshore of the West African coast, the recent lobe complex of the Congo deep-sea fan receives large amounts of fluvial sediments (3–5% organic carbon). This organic-rich sedimentation area harbors habitats with chemosynthetic communities similar to those of cold seeps. In this study, we investigated relative abundance, diversity and distribution of aerobic methane-oxidizing bacteria (MOB) communities at the oxic–anoxic interface of sedimentary habitats by using fluorescence in situ hybridization and comparative sequence analysis of particulate mono-oxygenase (pmoA) genes. Our findings revealed that sedimentary habitats of the recent lobe complex hosted type I and type II MOB cells and comparisons of pmoA community compositions showed variations among the different organic-rich habitats. Furthermore, the pmoA lineages were taxonomically more diverse compared to methane seep environments and were related to those found at cold seeps. Surprisingly, MOB phylogenetic lineages typical of terrestrial environments were observed at such water depth. In contrast, MOB cells or pmoA sequences were not detected at the previous lobe complex that is disconnected from the Congo River inputs.

Spatial change of reservoir nitrite-dependent methane-oxidizing microorganisms

Nitrite-dependent anaerobic methane oxidation (n-damo), catalyzed by microorganisms affiliated with bacterial phylum NC10, can have an important contribution to the reduction of the methane emission from anoxic freshwater sediment to the atmosphere. However, information on the variation of sediment n-damo organisms in reservoirs is still lacking. The present study monitored the spatial change of sediment n-damo organisms in the oligotrophic freshwater Xinfengjiang Reservoir (South China). Sediment samples were obtained from six different sampling locations and two sediment depths (0–5 cm, 5–10 cm). Sediment n-damo bacterial abundance was found to vary with sampling location and layer depth, which was likely influenced by pH and nitrogen level. The presence of the n-damo pmoA gene was found in all these samples. A remarkable shift occurred in the diversity and composition of sediment n-damo pmoA gene sequences. A variety of distinctively different n-damo pmoA clusters existed in reservoir sediments. The pmoA sequences affiliated with Candidatus Methylomirabilis oxyfera formed the largest group, while a significant proportion of the obtained n-damo pmoA gene sequences showed no close relationship to those from any known NC10 species. In addition, the present n-damo process was found in reservoir sediment, which could be enhanced by nitrite nitrogen amendment.

A pmoA-based study reveals dominance of yet uncultured Type I methanotrophs in rhizospheres of an organically fertilized rice field in India

Rice fields are one of the important sources of anthropogenic methane. Methanotrophs can oxidize up to 30 % of the produced methane and thus have a pivotal environmental role in methane mitigation. India occupies the largest region under rice cultivation; however, most of the studies done on methanotrophic communities have focused on the Northern region. We studied methanotrophic community of a flooded, organically fertilized rice field using pmoA clone library approach. Organic rice fields impose a more serious threat as they produce more methane. pmoA gene is the main functional gene which is primarily used for taxonomical analysis of methanotrophs. Our results showed that the pmoA clone libraries from two growth stages of rice were dominated by pmoA sequences which were very distant from cultivated Type Ia methanotrophic genera (80–82 % nucleotide similarity) indicative of the presence of a putatively novel genus. We designated this group of clones as ‘rice field clones’ as this also includes many pmoA sequences originating from other rice fields. Thus, our current knowledge of methanotroph diversity from Indian rice fields has been expanded revealing that a substantial portion of methanotrophic diversity is unexplored.Electronic supplementary materialThe online version of this article (doi:10.1007/s13205-016-0453-3) contains supplementary material, which is available to authorized users.

A new cell morphotype among methane oxidizers: a spiral-shaped obligately microaerophilic methanotroph from northern low-oxygen environments.

Although representatives with spiral-shaped cells are described for many functional groups of bacteria, this cell morphotype has never been observed among methanotrophs. Here, we show that spiral-shaped methanotrophic bacteria do exist in nature but elude isolation by conventional approaches due to the preference for growth under micro-oxic conditions. The helical cell shape may enable rapid motility of these bacteria in water-saturated, heterogeneous environments with high microbial biofilm content, therefore offering an advantage of fast cell positioning under desired high methane/low oxygen conditions. The pmoA genes encoding a subunit of particulate methane monooxygenase from these methanotrophs form a new genus-level lineage within the family Methylococcaceae, type Ib methanotrophs. Application of a pmoA-based microarray detected these bacteria in a variety of high-latitude freshwater environments including wetlands and lake sediments. As revealed by the environmental pmoA distribution analysis, type Ib methanotrophs tend to live very near the methane source, where oxygen is scarce. The former perception of type Ib methanotrophs as being typical for thermal habitats appears to be incorrect because only a minor proportion of pmoA sequences from these bacteria originated from environments with elevated temperatures.

Diversity of Methane-Oxidizing Bacteria in Soils from "Hot Lands of Medolla" (Italy) Featured by Anomalous High-Temperatures and Biogenic CO2 Emission.

“Terre Calde di Medolla” (TCM) (literally, “Hot Lands of Medolla”) refers to a farming area in Italy with anomalously high temperatures and diffuse emissions of biogenic CO2, which has been linked to CH4 oxidation processes from a depth of 0.7 m to the surface. We herein assessed the composition of the total bacterial community and diversity of methane-oxidizing bacteria (MOB) in soil samples collected at a depth at which the peak temperature was detected (0.6 m). Cultivation-independent methods were used, such as: i) a clone library analysis of the 16S rRNA gene and pmoA (coding for the α-subunit of the particulate methane monooxygenase) gene, and ii) Terminal Restriction Fragment Length Polymorphism (T-RFLP) fingerprinting. The 16S rRNA gene analysis assessed the predominance of Actinobacteria, Acidobacteria, Proteobacteria, and Bacillus in TCM samples collected at a depth of 0.6 m along with the presence of methanotrophs (Methylocaldum and Methylobacter) and methylotrophs (Methylobacillus). The phylogenetic analysis of pmoA sequences showed the presence of MOB affiliated with Methylomonas, Methylocystis, Methylococcus, and Methylocaldum in addition to as yet uncultivated and uncharacterized methanotrophs. Jaccard’s analysis of T-RFLP profiles at different ground depths revealed a similar MOB composition in soil samples at depths of 0.6 m and 0.7 m, while this similarity was weaker between these samples and those taken at a depth of 2.5 m, in which the genus Methylocaldum was absent. These results correlate the anomalously high temperatures of the farming area of “Terre Calde di Medolla” with the presence of microbial methane-oxidizing bacteria.

好氧甲烷氧化菌的生理生态特征及其在自然湿地中的群落多样性研究进展

好氧甲烷氧化菌的生理生态特征及其在自然湿地中的群落多样性研究进展

Uncultivated Methylocystis Species in Paddy Soil Include Facultative Methanotrophs that Utilize Acetate.

Methanotrophs are crucial in regulating methane emission from rice field systems. Type II methanotrophs in particular are often observed in high abundance in paddy soil. Some cultivated species of Methylocystis are able to grow on acetate in the absence of methane. We hypothesize that the dominant type II methanotrophs in paddy soil might facultatively utilize acetate for growth, which we evaluate in the present study. The measurement of methane oxidation rates showed that the methanotrophic activity in paddy soil was inhibited by the addition of acetate compared to the continuous supplementation of methane, but the paddy soil maintained the methane oxidation capacity and recovered following methane supplementation. Terminal restriction fragment length polymorphism analysis (T-RFLP) combined with cloning and sequencing of pmoA genes showed that Methylocystis was enriched after incubation with added acetate, while the type I methanotrophs Methylocaldum/Methylococcus and Methylobacter were enriched by methane supplementation. A comparison of pmoA sequences obtained in this study with those in the public database indicated that they were globally widespread in paddy soils or in associated with rice roots. Furthermore, we performed stable isotope probing (SIP) of pmoA messenger RNA (mRNA) to investigate the assimilation of (13)C-acetate by paddy soil methanotrophs. RNA-SIP revealed that Methylocystis-related methanotrophs which shared the same genotype of the above enriched species were significantly labelled. It indicates that these methanotrophs actively assimilated the labelled acetate in paddy soil. Altogether, these results suggested that uncultivated Methylocystis species are facultative methanotrophs utilizing acetate as a secondary carbon source in paddy soil.

Distribution and diversity of Verrucomicrobia methanotrophs in geothermal and acidic environments

Recently, methanotrophic members of the phylum Verrucomicrobia have been described, but little is known about their distribution in nature. We surveyed methanotrophic bacteria in geothermal springs and acidic wetlands via pyrosequencing of 16S rRNA gene amplicons. Putative methanotrophic Verrucomicrobia were found in samples covering a broad temperature range (22.5-81.6°C), but only in acidic conditions (pH 1.8-5.0) and only in geothermal environments, not in acidic bogs or fens. Phylogenetically, three 16S rRNA gene sequence clusters of putative methanotrophic Verrucomicrobia were observed. Those detected in high-temperature geothermal samples (44.1-81.6°C) grouped with known thermoacidiphilic 'Methylacidiphilum' isolates. A second group dominated in moderate-temperature geothermal samples (22.5-40.1°C) and a representative mesophilic methanotroph from this group was isolated (strain LP2A). Genome sequencing verified that strain LP2A possessed particulate methane monooxygenase, but its 16S rRNA gene sequence identity to 'Methylacidiphilum infernorum' strain V4 was only 90.6%. A third group clustered distantly with known methanotrophic Verrucomicrobia. Using pmoA-gene targeted quantitative polymerase chain reaction, two geothermal soil profiles showed a dominance of LP2A-like pmoA sequences in the cooler surface layers and 'Methylacidiphilum'-like pmoA sequences in deeper, hotter layers. Based on these results, there appears to be a thermophilic group and a mesophilic group of methanotrophic Verrucomicrobia. However, both were detected only in acidic geothermal environments.

Classification of pmoA amplicon pyrosequences using BLAST and the lowest common ancestor method in MEGAN

The classification of high-throughput sequencing data of protein-encoding genes is not as well established as for 16S rRNA. The objective of this work was to develop a simple and accurate method of classifying large datasets of pmoA sequences, a common marker for methanotrophic bacteria. A taxonomic system for pmoA was developed based on a phylogenetic analysis of available sequences. The taxonomy incorporates the known diversity of pmoA present in public databases, including both sequences from cultivated and uncultivated organisms. Representative sequences from closely related genes, such as those encoding the bacterial ammonia monooxygenase, were also included in the pmoA taxonomy. In total, 53 low-level taxa (genus-level) are included. Using previously published datasets of high-throughput pmoA amplicon sequence data, we tested two approaches for classifying pmoA: a naïve Bayesian classifier and BLAST. Classification of pmoA sequences based on BLAST analyses was performed using the lowest common ancestor (LCA) algorithm in MEGAN, a software program commonly used for the analysis of metagenomic data. Both the naïve Bayesian and BLAST methods were able to classify pmoA sequences and provided similar classifications; however, the naïve Bayesian classifier was prone to misclassifying contaminant sequences present in the datasets. Another advantage of the BLAST/LCA method was that it provided a user-interpretable output and enabled novelty detection at various levels, from highly divergent pmoA sequences to genus-level novelty.

Abundance and diversity of methanotrophic Gammaproteobacteria in northern wetlands

Numeric abundance, identity and pH preferences of methanotrophic Gammaproteobacteria (type I methanotrophs) inhabiting the northern acidic wetlands were studied. The rates of methane oxidation by peat samples from six-wetlands of European Northern Russia (pH 3.9-4.7) varied from 0.04 to 0.60 μg CH4 g(-1) peat h(-1). The number of cells revealed by hybridization with fluorochrome-labeled probes M84 + M705 specific for type I methanotrophs was 0.05-2.16 x 10(5) cells g(-1) dry peat, i.e. 0.4-12.5% of the total number of methanotrophs and 0.004-0.39% of the total number of bacteria. Analysis of the fragments of the pmoA gene encoding particulate methane monooxygenase revealed predominance of the genus Methylocystis (92% of the clones) in the studied sample of acidic peat, while the proportion of the pmoA sequences of type I methanotrophs was insignificant (8%). PCR amplification of the 16S rRNA gene fragments of type I methanotrophs with TypeIF-Type IR primers had low specificity, since only three sequences out of 53 analyzed belonged to methanotrophs and exhibited 93-99% similarity to those of Methylovulum, Methylomonas, and Methylobacter species. Isolates of type I methanotrophs obtained from peat (strains SH10 and 83A5) were identified as members of the species Methylomonaspaludis and Methylovulum miyakonense, respectively. Only Methylomonaspaludum SH10 was capable of growth in acidic media (pH range for growth 3.8-7.2 with the optimum at pH 5.8-6.2), while Methylovulum miyakonense 83A5 exhibited the typical growth characteristics of neutrophilic methanotrophs (pH range for growth 5.5-8.0 with the optimum at pH 6.5-7.5).

Methylomarinovum caldicuralii gen. nov., sp. nov., a moderately thermophilic methanotroph isolated from a shallow submarine hydrothermal system, and proposal of the family Methylothermaceae fam. nov.

A novel methane-oxidizing bacterium, strain IT-9(T), was isolated from a shallow submarine hydrothermal system occurring in a coral reef in Japan. Strain IT-9(T) was a Gram-negative, aerobic, motile, coccoid or oval-shaped bacterium with the distinctive intracytoplasmic membrane arrangement of a type I methanotroph. Strain IT-9(T) was a moderately thermophilic, obligate methanotroph that grew on methane and methanol at 30-55 °C (optimum 45-50 °C). The strain possessed the particulate methane monooxygenase (pMMO). The ribulose monophosphate pathway was operative for carbon assimilation. NaCl was required for growth within a concentration range of 1-5 % (optimum 3 %). The hao gene encoding hydroxylamine oxidoreductase (HAO) involved in nitrification was detected by a PCR experiment. The major phospholipid fatty acids were C16 : 0 and C18 : 1ω7c. The major isoprenoid quinone was Q-8. The DNA G+C content was 66.0 mol%. The 16S rRNA gene sequence of strain IT-9(T) was only moderately related to the sequences of members of the closest genera Methylohalobius (94.1 % similarity) and Methylothermus (91.7-91.9 % similarity); however, those sequences formed a deeply branching monophyletic group within the order Methylococcales. Phylogenies based on 16S rRNA gene sequences, deduced partial PmoA sequences and deduced partial Hao sequences and physiological and chemotaxonomic characteristics revealed that strain IT-9(T) represents a novel species of a new genus, for which the name Methylomarinovum caldicuralii gen. nov., sp. nov. is proposed. The type strain of Methylomarinovum caldicuralii is IT-9(T) ( = JCM 13666(T) = DSM 19749(T)). In addition, we propose a new family, Methylothermaceae fam. nov., in the order Methylococcales, to accommodate the genera Methylothermus, Methylohalobius and Methylomarinovum. The genera Methylothermus and Methylohalobius have been recognized as being distinct from other genera in the methane-oxidizing order Methylococcales in the class Gammaproteobacteria. These genera form a distinctive monophyletic lineage within the order on the basis of 16S rRNA gene sequence phylogeny. This seems consistent with their distinctive physiological traits; the genus Methylothermus includes the most thermophilic species, and the genus Methylohalobius includes the most halophilic species, within the order. Although these two genera include only three species at the time of writing, similar sequences of 16S rRNA genes and pmoA genes encoding pMMO have been detected in a geothermal area or deep-sea hydrothermal vent fields by studies using culture-independent techniques. This suggests that unknown methanotrophs of this lineage inhabit various extreme environments.

Methane oxidation activity and diversity of aerobic methanotrophs in pH-neutral and semi-neutral thermal springs of the Kunashir Island, Russian Far East

Aerobic methane oxidation has been mostly studied in environments with moderate to low temperatures. However, the process also occurs in terrestrial thermal springs, where little research on the subject has been done to date. The potential activity of methane oxidation and diversity of aerobic methanotrophic bacteria were studied in sediments of thermal springs with various chemical and physical properties, sampled across the Kunashir Island, the Kuriles archipelago. Activity was measured by means of the radioisotope tracer technique utilizing (14)C-labeled methane. Biodiversity assessments were based on the particulate methane monooxygenase (pmoA) gene, which is found in all known thermophilic and thermotolerant methanotrophs. We demonstrated the possibility of methane oxidation in springs with temperature exceeding 74°C, and the most intensive methane uptake was shown in springs with temperatures about 46°C. PmoA was detected in 19 out of 30 springs investigated and the number of pmoA gene copies varied between 10(4) and 10(6) copies per ml of sediment. Phylogenetic analysis of PmoA sequences revealed the presence of methanotrophs from both the Alpha- and Gammaproteobacteria. Our results suggest that methanotrophs inhabiting thermal springs with temperature exceeding 50°C may represent novel thermophilic and thermotolerant species of the genera Methylocystis and Methylothermus, as well as previously undescribed Gammaproteobacteria.

Methylomarinum vadi gen. nov., sp. nov., a methanotroph isolated from two distinct marine environments

Two aerobic methane-oxidizing bacterial strains were isolated from distinct marine environments in Japan. Strains IT-4(T) and T2-1 were Gram-stain-negative, aerobic, motile, plump short rods or oval-shaped bacteria with a single polar flagellum and type I intracytoplasmic membranes. They were obligate methanotrophs that grew only on methane or methanol. Each strain possessed the particulate methane monooxygenase (pMMO). The ribulose monophosphate pathway was operative for carbon assimilation. The strains grew best at 37 °C, and did not grow at 45 °C. NaCl was required for growth within a concentration range of 1-8 % (w/v). The major phospholipid fatty acids were C16 : 0, C16 : 1ω7c, and C16 : 1ω5t. The major isoprenoid quinone was MQ-8. The DNA G+C content was 50.9-51.7 mol%. The 16S rRNA gene sequences of the strains showed 99.4 % similarity to each other, and DNA-DNA hybridization analysis indicated that the strains were representatives of the same species. The 16S rRNA gene sequences were highly similar to some marine environmental sequences (94.0-97.7 % similarity), but did not show similarities more than 94 % with sequences of members of other related genera, such as Methylomicrobium, Methylobacter, Methylomonas and Methylosarcina. Phylogenies based on 16S rRNA gene sequences and deduced partial PmoA sequences, and the physiological and chemotaxonomic characteristics revealed that strains IT-4(T) and T2-1 represent a novel species of a new genus in the family Methylococcaceae, for which the name Methylomarinum vadi gen. nov., sp. nov. is proposed. The type strain is IT-4(T) ( = JCM 13665(T) = DSM 18976(T)).