All Kinds of Sunny Colors Synthesized from Methane: Genome-Encoded Carotenoid Production by Methylomonas Species.

Carotenoids are secondary metabolites that exhibit antioxidant properties and are characterized by a striking range of colorations from red to yellow. These natural pigments are synthesized by a wide range of eukaryotic and prokaryotic organisms. Among the latter, carotenoid-producing methanotrophic bacteria, which display fast growth on methane or natural gas, are of particular interest as potential producers of a feed protein enriched with carotenoids. Until recently, Methylomonas strain 16a and Methylomonas sp. ZR1 remained the only representatives of the genus for which detailed carotenoid profile was determined. In this study, we analyzed the genome sequences of five strains of Methylomonas species whose pigmentation varied from white and yellow to orange and red, and identified carotenoids produced by these bacteria. Carotenoids synthesized using four pigmented strains included C30 fraction, primarily composed of 4,4'-diaplycopene-4,4'-dioic acid and 4,4'-diaplycopenoic acid, as well as C40 fraction with the major compound represented by 1,1'-dihydroxy-3,4-didehydrolycopene. The genomes of studied Methylomonas strains varied in size between 4.59 and 5.45 Mb and contained 4201-4735 protein-coding genes. These genomes and 35 reference Methylomonas genomes available in the GenBank were examined for the presence of genes encoding carotenoid biosynthesis. Genomes of all pigmented Methylomonas strains harbored genes necessary for the synthesis of 4,4'-diaplycopene-4,4'-dioic acid. Non-pigmented "Methylomonas montana" MW1T lacked the crtN gene required for carotenoid production. Nearly all strains possessed phytoene desaturases, which explained their ability to naturally synthesize lycopene. Thus, members of the genus Methylomonas can potentially be considered as producers of C30 and C40 carotenoids from methane.

One Step Closer to Enigmatic USCα Methanotrophs: Isolation of a Methylocapsa-like Bacterium from a Subarctic Soil.

The scavenging of atmospheric trace gases has been recognized as one of the lifestyle-defining capabilities of microorganisms in terrestrial polar ecosystems. Several metagenome-assembled genomes of as-yet-uncultivated methanotrophic bacteria, which consume atmospheric CH4 in these ecosystems, have been retrieved in cultivation-independent studies. In this study, we isolated and characterized a representative of these methanotrophs, strain D3K7, from a subarctic soil of northern Russia. Strain D3K7 grows on methane and methanol in a wide range of temperatures, between 5 and 30 °C. Weak growth was also observed on acetate. The presence of acetate in the culture medium stimulated growth at low CH4 concentrations (~100 p.p.m.v.). The finished genome sequence of strain D3K7 is 4.15 Mb in size and contains about 3700 protein-encoding genes. According to the result of phylogenomic analysis, this bacterium forms a common clade with metagenome-assembled genomes obtained from the active layer of a permafrost thaw gradient in Stordalen Mire, Abisco, Sweden, and the mineral cryosol at Axel Heiberg Island in the Canadian High Arctic. This clade occupies a phylogenetic position in between characterized Methylocapsa methanotrophs and representatives of the as-yet-uncultivated upland soil cluster alpha (USCα). As shown by the global distribution analysis, D3K7-like methanotrophs are not restricted to polar habitats but inhabit peatlands and soils of various climatic zones.

Growing in Saltwater: Biotechnological Potential of Novel Methylotuvimicrobium- and Methylomarinum-like Methanotrophic Bacteria.

Methanotrophic bacteria that possess a unique ability of using methane as a sole source of carbon and energy have attracted considerable attention as potential producers of a single-cell protein. So far, this biotechnology implied using freshwater methanotrophs, although many regions of the world have limited freshwater resources. This study aimed at searching for novel methanotrophs capable of fast growth in saltwater comparable in composition with seawater. A methane-oxidizing microbial consortium containing Methylomarinum- and Methylotuvimicrobium-like methanotrophs was enriched from sediment from the river Chernavka (water pH 7.5, total salt content 30 g L-1), a tributary river of the hypersaline Lake Elton, southern Russia. This microbial consortium, designated Ch1, demonstrated stable growth on natural gas in a bioreactor in media with a total salt content of 23 to 35.9 g L-1 at a dilution rate of 0.19-0.21 h-1. The highest biomass yield of 5.8 g cell dry weight (CDW)/L with a protein content of 63% was obtained during continuous cultivation of the consortium Ch1 in a medium with a total salt content of 29 g L-1. Isolation attempts resulted in obtaining a pure culture of methanotrophic bacteria, strain Ch1-1. The 16S rRNA gene sequence of strain Ch1-1 displayed 97.09-97.24% similarity to the corresponding gene fragments of characterized representatives of Methylomarinum vadi, methanotrophs isolated from marine habitats. The genome of strain Ch1-1 was 4.8 Mb in size and encoded 3 rRNA operons, and about 4400 proteins. The genome contained the gene cluster coding for ectoine biosynthesis, which explains the ability of strain Ch1-1 to tolerate high salt concentration.

Methylomonas rapida sp. nov., a novel species of fast-growing, carotenoid-producing obligate methanotrophs with high biotechnological potential.

The genus Methylomonas accommodates strictly aerobic, obligate methanotrophs, with their sole carbon and energy sources restricted to methane and methanol. These bacteria inhabit oxic-anoxic interfaces of various freshwater habitats and have attracted considerable attention as potential producers of a single-cell protein. Here, we characterize two fast-growing representatives of this genus, strains 12 and MP1T, which are phylogenetically distinct from the currently described Methylomonas species (94.0-97.3 % 16S rRNA gene sequence similarity). Strains 12 and MP1T were isolated from freshwater sediments collected in Moscow and Krasnodar regions, respectively. Cells of these strains are Gram-negative, red-pigmented, highly motile thick rods that contain a type I intracytoplasmic membrane system and possess a particulate methane monooxygenase (pMMO) enzyme. These bacteria grow between 8 and 45 °C (optimum 35 °C) in a relatively narrow pH range of 5.5-7.3 (optimum pH 6.6-7.2). Major carotenoids synthesized by these methanotrophs are 4,4'-diaplycopene-4,4'-dioic acid, 1,1'-dihydroxy-3,4-didehydrolycopene and 4,4'-diaplycopenoic acid. High biomass yield, of up to 3.26 g CDW/l, is obtained during continuous cultivation of MP1T on natural gas in a bioreactor at a dilution rate of 0.22 h-1. The complete genome sequence of strain MP1T is 4.59 Mb in size; the DNA G + C content is 52.8 mol%. The genome encodes four rRNA operons, one pMMO operon and 4,216 proteins. The genome sequence displays 82-85 % average nucleotide identity to those of earlier described Methylomonas species. We propose to classify these bacteria as representing a novel species of the genus Methylomonas, M. rapida sp. nov., with the type strain MP1T (=KCTC 92586T = VKM B-3663T).

Complete Genome Sequence of Methylococcus capsulatus MIR, a Methanotroph Capable of Growth on Methanol.

Methylococcus capsulatus MIR is an aerobic methanotroph that was isolated from an activated sludge sample and is capable of growth on methanol. The finished genome of strain MIR is 3.2 Mb in size. It encodes both MxaFI and XoxF methanol dehydrogenases, as well as three different isozymes of formate dehydrogenase.

Expanding Characterized Diversity and the Pool of Complete Genome Sequences of Methylococcus Species, the Bacteria of High Environmental and Biotechnological Relevance.

The bacterial genus Methylococcus, which comprises aerobic thermotolerant methanotrophic cocci, was described half-a-century ago. Over the years, a member of this genus, Methylococcus capsulatus Bath, has become a major model organism to study genomic and metabolic basis of obligate methanotrophy. High biotechnological potential of fast-growing Methylococcus species, mainly as a promising source of feed protein, has also been recognized. Despite this big research attention, the currently cultured Methylococcus diversity is represented by members of the two species, M. capsulatus and M. geothermalis, while finished genome sequences are available only for two strains of these methanotrophs. This study extends the pool of phenotypically characterized Methylococcus strains with good-quality genome sequences by contributing four novel isolates of these bacteria from activated sludge, landfill cover soil, and freshwater sediments. The determined genome sizes of novel isolates varied between 3.2 and 4.0Mb. As revealed by the phylogenomic analysis, strains IO1, BH, and KN2 affiliate with M. capsulatus, while strain Mc7 may potentially represent a novel species. Highest temperature optima (45-50°C) and highest growth rates in bioreactor cultures (up to 0.3h-1) were recorded for strains obtained from activated sludge. The comparative analysis of all complete genomes of Methylococcus species revealed 4,485 gene clusters. Of these, pan-genome core comprised 2,331 genes (on average 51.9% of each genome), with the accessory genome containing 846 and 1,308 genes in the shell and the cloud, respectively. Independently of the isolation source, all strains of M. capsulatus displayed surprisingly high genome synteny and a striking similarity in gene content. Strain Mc7 from a landfill cover soil differed from other isolates by the high content of mobile genetic elements in the genome and a number of genome-encoded features missing in M. capsulatus, such as sucrose biosynthesis and the ability to scavenge phosphorus and sulfur from the environment.

Rokubacteria in Northern Peatlands: Habitat Preferences and Diversity Patterns.

Rokubacteria is a phylogenetic clade of as-yet-uncultivated prokaryotes, which are detected in diverse terrestrial habitats and are commonly addressed as members of the rare biosphere. This clade was originally described as a candidate phylum; however, based on the results of comparative genome analysis, was later defined as the order-level lineage, Rokubacteriales, within the phylum Methylomirabilota. The physiology and lifestyles of these bacteria are poorly understood. A dataset of 16S rRNA gene reads retrieved from four boreal raised bogs and six eutrophic fens was examined for the presence of the Rokubacteriales; the latter were detected exclusively in fens. Their relative abundance varied between 0.2 and 4% of all bacteria and was positively correlated with pH, total nitrogen content, and availability of Ca and Mg. To test an earlier published hypothesis regarding the presence of methanotrophic capabilities in Rokubacteria, peat samples were incubated with 10% methane for four weeks. No response to methane availability was detected for the Rokubacteriales, while clear a increase in relative abundance was observed for the conventional Methylococcales methanotrophs. The search for methane monooxygenase encoding genes in 60 currently available Rokubacteriales metagenomes yielded negative results, although copper-containing monooxygenases were encoded by some members of this order. This study suggests that peat-inhabiting Rokubacteriales are neutrophilic non-methanotrophic bacteria that colonize nitrogen-rich wetlands.

Pan-Genome-Based Analysis as a Framework for Demarcating Two Closely Related Methanotroph Genera Methylocystis and Methylosinus.

The Methylocystis and Methylosinus are two of the five genera that were included in the first taxonomic framework of methanotrophic bacteria created half a century ago. Members of both genera are widely distributed in various environments and play a key role in reducing methane fluxes from soils and wetlands. The original separation of these methanotrophs in two distinct genera was based mainly on their differences in cell morphology. Further comparative studies that explored various single-gene-based phylogenies suggested the monophyletic nature of each of these genera. Current availability of genome sequences from members of the Methylocystis/ Methylosinus clade opens the possibility for in-depth comparison of the genomic potentials of these methanotrophs. Here, we report the finished genome sequence of Methylocystis heyeri H2T and compare it to 23 currently available genomes of Methylocystis and Methylosinus species. The phylogenomic analysis confirmed that members of these genera form two separate clades. The Methylocystis/Methylosinus pan-genome core comprised 1,173 genes, with the accessory genome containing 4,941 and 11,192 genes in the shell and the cloud, respectively. Major differences between the genome-encoded environmental traits of these methanotrophs include a variety of enzymes for methane oxidation and dinitrogen fixation as well as genomic determinants for cell motility and photosynthesis.

Thriving in Wetlands: Ecophysiology of the Spiral-Shaped Methanotroph Methylospira mobilis as Revealed by the Complete Genome Sequence.

Candidatus Methylospira mobilis is a recently described spiral-shaped, micro-aerobic methanotroph, which inhabits northern freshwater wetlands and sediments. Due to difficulties of cultivation, it could not be obtained in a pure culture for a long time. Here, we report on the successful isolation of strain Shm1, the first axenic culture of this unique methanotroph. The complete genome sequence obtained for strain Shm1 was 4.7 Mb in size and contained over 4800 potential protein-coding genes. The array of genes encoding C1 metabolic capabilities in strain Shm1 was highly similar to that in the closely related non-motile, moderately thermophilic methanotroph Methylococcus capsulatus Bath. The genomes of both methanotrophs encoded both low- and high-affinity oxidases, which allow their survival in a wide range of oxygen concentrations. The repertoire of signal transduction systems encoded in the genome of strain Shm1, however, by far exceeded that in Methylococcus capsulatus Bath but was comparable to those in other motile gammaproteobacterial methanotrophs. The complete set of motility genes, the presence of both the molybdenum-iron and vanadium-iron nitrogenases, as well as a large number of insertion sequences were also among the features, which define environmental adaptation of Methylospira mobilis to water-saturated, micro-oxic, heterogeneous habitats depleted in available nitrogen.

Draft Genome Sequence of Methylocystis heyeri H2T, a Methanotroph with Habitat-Specific Adaptations, Isolated from a Peatland Ecosystem.

Methylocystis heyeri H2T is an aerobic facultative methanotroph which was isolated from an acidic Sphagnum peat bog lake and is a common inhabitant of peatland ecosystems. This bacterium possesses two particulate methane monooxygenases with low and high affinity to methane and a number of genomic adaptations to acidic conditions.

Fatty Acid and Hopanoid Adaption to Cold in the Methanotroph Methylovulum psychrotolerans.

Three strains of aerobic psychrotolerant methanotrophic bacteria Methylovulum psychrotolerans, isolated from geographically remote low-temperature environments in Northern Russia, were grown at three different growth temperatures, 20, 10 and 4°C and were found to be capable of oxidizing methane at all temperatures. The three M. psychrotolerans strains adapted their membranes to decreasing growth temperature by increasing the percent of unsaturated fatty acid (FAs), both for the bulk and intact polar lipid (IPL)-bound FAs. Furthermore, the ratio of ßOH-C16:0 to n-C16:0 increased as growth temperature decreased. The IPL head group composition did not change as an adaption to temperature. The most notable hopanoid temperature adaptation of M. psychrotolerans was an increase in unsaturated hopanols with decreasing temperature. As the growth temperature decreased from 20 to 4°C, the percent of unsaturated M. psychrotolerans bulk-FAs increased from 79 to 89 % while the total percent of unsaturated hopanoids increased from 27 to 49 %. While increased FA unsaturation in response to decreased temperature is a commonly observed response in order to maintain the liquid-crystalline character of bacterial membranes, hopanoid unsaturation upon cold exposition has not previously been described. In order to investigate the mechanisms of both FA and hopanoid cold-adaption in M. psychrotolerans we identified genes in the genome of M. psychrotolerans that potentially code for FA and hopanoid desaturases. The unsaturation of hopanoids represents a novel membrane adaption to maintain homeostasis upon cold adaptation.

Granulicella sibirica sp. nov., a psychrotolerant acidobacterium isolated from an organic soil layer in forested tundra, West Siberia.

An isolate of strictly aerobic, pale-pink pigmented bacteria, strain AF10T, was obtained from an organic soil layer in forested tundra, Nadym region, West Siberia. Cells of strain AF10T were Gram-negative, non-motile rods that produced an amorphous extracellular polysaccharide-like substance and formed large cell aggregates in old cultures. These bacteria were chemoorganotrophic, mildly acidophilic and psychrotolerant, and grew between pH 3.5 and 7.0 (optimum, pH 4.5-5.0) and at temperatures between 2 and 30 °C. The preferred growth substrates were sugars and some polysaccharides. The major fatty acids were iso-C15 : 0, C16 : 0, C16 : 1∆9 c and 13,16-dimethyl octacosanedioic acid. The genome of strain AF10T was 6.14 Mbp in size and encoded a wide repertoire of carbohydrate active enzymes. The genomic DNA G+C content was 59.8 mol%. Phylogenetic analysis indicated that strain AF10T is a member of the genus Granulicella, family Acidobacteriaceae, but displays 94.4-98.0 % 16S rRNA gene sequence similarity to currently described members of this genus. On the basis of phenotypic, chemotaxonomic, phylogenetic and genomic analyses, we propose to classify this bacterium as representing a novel species of the genus Granulicella, Granulicellasibirica sp. nov. Strain AF10T (=DSM 104461T=VKM B-3276T) is the type strain.

Draft Genome Sequence of Methylovulum psychrotolerans Sph1T, an Obligate Methanotroph from Low-Temperature Environments.

Methylovulum psychrotolerans Sph1T is an aerobic, obligate methanotroph, which was isolated from cold methane seeps in West Siberia. This bacterium possesses only a particulate methane monooxygenase and is widely distributed in low-temperature environments. Strain Sph1T has the genomic potential for biosynthesis of hopanoids required for the maintenance of intracytoplasmic membranes.

Methylovulum psychrotolerans sp. nov., a cold-adapted methanotroph from low-temperature terrestrial environments, and emended description of the genus Methylovulum.

Two isolates of aerobic methanotrophic bacteria, strains Sph1T and Sph2, were obtained from cold methane seeps in a floodplain of the river Mukhrinskaya, Irtysh basin, West Siberia. Another morphologically and phenotypically similar methanotroph, strain OZ2, was isolated from a sediment of a subarctic freshwater lake, Archangelsk region, northern Russia. Cells of these three strains were Gram-stain-negative, light-pink-pigmented, non-motile, encapsulated, large cocci that contained an intracytoplasmic membrane system typical of type I methanotrophs. They possessed a particulate methane monooxygenase enzyme and utilized only methane and methanol. Strains Sph1T, Sph2 and OZ2 were able to grow at a pH range of 4.0-8.9 (optimum at pH 6.0-7.0) and at temperatures between 2 and 36 °C. Although their temperature optimum was at 20-25 °C, these methanotrophs grew well at lower temperatures, down to 4 °C. The major cellular fatty acids were C16 : 1ω5c, C16 : 1ω6c, C16 : 1ω7c, C16 : 1ω8c, C16 : 0 and C14 : 0; the DNA G+C content was 51.4-51.9 mol%. Strains Sph1T, Sph2 and OZ2 displayed nearly identical (99.1-99.7 % similarity) 16S rRNA gene sequences and belonged to the family Methylococcaceae of the class Gammaproteobacteria. The most closely related organism was Methylovulum miyakonense HT12T (96.0-96.5 % 16S rRNA gene sequence similarity and 90 % pmoA sequence similarity). The novel isolates, however, differed from Methylovulum miyakonense HT12T by cell morphology, pigmentation, absence of soluble methane monooxygenase, more active growth at low temperatures, growth over a broader pH range and higher DNA G+C content. On the basis of these differences, we propose a novel species, Methylovulum psychrotolerans sp. nov., to accommodate these methanotrophs. Strain Sph1T (=LMG 29227T=VKM B-3018T) is the type strain.

Draft genome sequences of gammaproteobacterial methanotrophs isolated from lake washington sediment.

The genomes of Methylosarcina lacus LW14(T) (=ATCC BAA-1047(T) = JCM 13284(T)), Methylobacter sp. strain 21/22, Methylobacter sp. strain 31/32, Methylomonas sp. strain LW13, Methylomonas sp. strain MK1, and Methylomonas sp. strain 11b were sequenced and are reported here. All the strains are obligately methanotrophic bacteria isolated from the sediment of Lake Washington.

Methane-fed microbial microcosms show differential community dynamics and pinpoint taxa involved in communal response.

We report observations on the dynamics of bacterial communities in response to methane stimulus in laboratory microcosm incubations prepared with lake sediment samples. We first measured taxonomic compositions of long-term enrichment cultures and determined that, although dominated by Methylococcaceae types, these cultures also contained accompanying types belonging to a limited number of bacterial taxa, methylotrophs and non-methylotrophs. We then followed the short-term community dynamics, in two oxygen tension regimens (150 µM and 15 µM), observing rapid loss of species diversity. In all microcosms, a single type of Methylobacter represented the major methane-oxidizing partner. The accompanying members of the communities revealed different trajectories in response to different oxygen tensions, with Methylotenera species being the early responders to methane stimulus under both conditions. The communities in both conditions were convergent in terms of their assemblage, suggesting selection for specific taxa. Our results support prior observations from metagenomics on distribution of carbon from methane among diverse bacterial populations and further suggest that communities are likely responsible for methane cycling, rather than a single type of microbe.

Gammaproteobacterial methanotrophs dominate cold methane seeps in floodplains of West Siberian rivers.

A complex system of muddy fluid-discharging and methane (CH4)-releasing seeps was discovered in a valley of the river Mukhrinskaya, one of the small rivers of the Irtysh Basin, West Siberia. CH4 flux from most (90%) of these gas ebullition sites did not exceed 1.45 g CH4 h(-1), while some seeps emitted up to 5.54 g CH4 h(-1). The δ(13)C value of methane released from these seeps varied between -71.1 and -71.3‰, suggesting its biogenic origin. Although the seeps were characterized by low in situ temperatures (3.5 to 5°C), relatively high rates of methane oxidation (15.5 to 15.9 nmol CH4 ml(-1) day(-1)) were measured in mud samples. Fluorescence in situ hybridization detected 10(7) methanotrophic bacteria (MB) per g of mud (dry weight), which accounted for up to 20.5% of total bacterial cell counts. Most (95.8 to 99.3%) methanotroph cells were type I (gammaproteobacterial) MB. The diversity of methanotrophs in this habitat was further assessed by pyrosequencing of pmoA genes, encoding particulate methane monooxygenase. A total of 53,828 pmoA gene sequences of seep-inhabiting methanotrophs were retrieved and analyzed. Nearly all of these sequences affiliated with type I MB, including the Methylobacter-Methylovulum-Methylosoma group, lake cluster 2, and several as-yet-uncharacterized methanotroph clades. Apparently, microbial communities attenuating methane fluxes from these local but strong CH4 sources in floodplains of high-latitude rivers have a large proportion of potentially novel, psychrotolerant methanotrophs, thereby providing a challenge for future isolation studies.