High production of ectoine from methane in genetically engineered Methylomicrobium alcaliphilum 20Z by preventing ectoine degradation.

BACKGROUND: Methane is a greenhouse gas with a significant potential to contribute to global warming. The biological conversion of methane to ectoine using methanotrophs represents an environmentally and economically beneficial technology, combining the reduction of methane that would otherwise be combusted and released into the atmosphere with the production of value-added products. RESULTS: In this study, high ectoine production was achieved using genetically engineered Methylomicrobium alcaliphilum 20Z, a methanotrophic ectoine-producing bacterium, by knocking out doeA, which encodes a putative ectoine hydrolase, resulting in complete inhibition of ectoine degradation. Ectoine was confirmed to be degraded by doeA to N-α-acetyl-L-2,4-diaminobutyrate under nitrogen depletion conditions. Optimal copper and nitrogen concentrations enhanced biomass and ectoine production, respectively. Under optimal fed-batch fermentation conditions, ectoine production proportionate with biomass production was achieved, resulting in 1.0 g/L of ectoine with 16 g/L of biomass. Upon applying a hyperosmotic shock after high-cell-density culture, 1.5 g/L of ectoine was obtained without further cell growth from methane. CONCLUSIONS: This study suggests the optimization of a method for the high production of ectoine from methane by preventing ectoine degradation. To our knowledge, the final titer of ectoine obtained by M. alcaliphilum 20ZDP3 was the highest in the ectoine production from methane to date. This is the first study to propose ectoine production from methane applying high cell density culture by preventing ectoine degradation.

Methylomonas defluvii sp. nov., a type I methane-oxidizing bacterium from a secondary sedimentation tank of a wastewater treatment plant.

An aerobic methanotroph was isolated from a secondary sedimentation tank of a wastewater treatment plant and designated strain OY6T. Cells of OY6T were Gram-stain-negative, pink-pigmented, motile rods and contained an intracytoplasmic membrane structure typical of type I methanotrophs. OY6T could grow at a pH range of 4.5-7.5 (optimum pH 6.5) and at temperatures ranging from 20 °C to 37 °C (optimum 30 °C). The major cellular fatty acids were C14 : 0, C16 : 1ω7c/C16 : 1ω6c and C16 : 1ω5c; the predominant respiratory quinone was MQ-8. The genome size was 5.41 Mbp with a DNA G+C content of 51.7 mol%. OY6T represents a member of the family Methylococcaceae of the class Gammaproteobacteria and displayed 95.74-99.64 % 16S rRNA gene sequence similarity to the type strains of species of the genus Methylomonas. Whole-genome comparisons based on average nucleotide identity (ANI) and digital DNA-DNA hybridisation (dDDH) confirmed that OY6T should be classified as representing a novel species. The most closely related type strain was Methylomonas fluvii EbBT, with 16S rRNA gene sequence similarity, ANI by blast (ANIb), ANI by MUMmer (ANIm) and dDDH values of 99.64, 90.46, 91.92 and 44.5 %, respectively. OY6T possessed genes encoding both the particulate methane monooxygenase enzyme and the soluble methane monooxygenase enzyme. It grew only on methane or methanol as carbon sources. On the basis of phenotypic, genetic and phylogenetic data, strain OY6T represents a novel species within the genus Methylomonas for which the name Methylomonas defluvii sp. nov. is proposed, with strain OY6T (=GDMCC 1.4114T=KCTC 8159T=LMG 33371T) as the type strain.

Seasonal dynamics of the microbial methane filter in the water column of a eutrophic coastal basin.

In coastal waters, methane-oxidizing bacteria (MOB) can form a methane biofilter and mitigate methane emissions. The metabolism of these MOBs is versatile, and the resilience to changing oxygen concentrations is potentially high. It is still unclear how seasonal changes in oxygen availability and water column chemistry affect the functioning of the methane biofilter and MOB community composition. Here, we determined water column methane and oxygen depth profiles, the methanotrophic community structure, methane oxidation potential, and water-air methane fluxes of a eutrophic marine basin during summer stratification and in the mixed water in spring and autumn. In spring, the MOB diversity and relative abundance were low. Yet, MOB formed a methane biofilter with up to 9% relative abundance and vertical niche partitioning during summer stratification. The vertical distribution and potential methane oxidation of MOB did not follow the upward shift of the oxycline during summer, and water-air fluxes remained below 0.6 mmol m-2 d-1. Together, this suggests active methane removal by MOB in the anoxic water. Surprisingly, with a weaker stratification, and therefore potentially increased oxygen supply, methane oxidation rates decreased, and water-air methane fluxes increased. Thus, despite the potential resilience of the MOB community, seasonal water column dynamics significantly influence methane removal.

Diverse type I and type II methanotrophs cultivated from an Indian freshwater wetland habitat.

Wetlands are the main natural sources of methane emissions, which make up a significant portion of greenhouse gas emissions. Such wetland patches serve as rich habitats for aerobic methanotrophs. Limited knowledge of methanotrophs from tropical wetlands widens the scope of study from these habitats. In the present study, a freshwater wetland in a tropical region in India was sampled and serially diluted to obtain methanotrophs in culture. This was followed by the isolation of methanotrophs on agarose-containing plates, incubated under methane: air atmosphere. Methanotrophs are difficult to cultivate, and very few cultures of methanotrophs are available from tropical wetlands. Our current study reports the cultivation of a diverse community of methanotrophs from six genera, namely, Methylomonas, Methylococcus, Methylomagnum, Methylocucumis (type I methanotrophs) along with Methylocystis, Methylosinus (type II methanotrophs). A high abundance of methanotrophs (106-1010 methanotrophs/g fresh weight) was observed in the samples. A Methylococcus strain could represent a putative novel species that was also isolated. Cultures of Methylomagnum and Methylocucumis, two newly described type I methanotrophs exclusively found in rice fields, were obtained. A large number of Methylomonas koyamae strains were cultured. Our study is pioneering in the documentation of culturable methanotrophs from a typical tropical wetland patch. The isolated methanotrophs can act as models for studying methanotroph-based methane mitigation from wetland habitats and can be used for various mitigation and valorization applications.

Versatile methanotrophs form an active methane biofilter in the oxycline of a seasonally stratified coastal basin.

The potential and drivers of microbial methane removal in the water column of seasonally stratified coastal ecosystems and the importance of the methanotrophic community composition for ecosystem functioning are not well explored. Here, we combined depth profiles of oxygen and methane with 16S rRNA gene amplicon sequencing, metagenomics and methane oxidation rates at discrete depths in a stratified coastal marine system (Lake Grevelingen, The Netherlands). Three amplicon sequence variants (ASVs) belonging to different genera of aerobic Methylomonadaceae and the corresponding three methanotrophic metagenome-assembled genomes (MOB-MAGs) were retrieved by 16S rRNA sequencing and metagenomic analysis, respectively. The abundances of the different methanotrophic ASVs and MOB-MAGs peaked at different depths along the methane oxygen counter-gradient and the MOB-MAGs show a quite diverse genomic potential regarding oxygen metabolism, partial denitrification and sulphur metabolism. Moreover, potential aerobic methane oxidation rates indicated high methanotrophic activity throughout the methane oxygen counter-gradient, even at depths with low in situ methane or oxygen concentration. This suggests that niche-partitioning with high genomic versatility of the present Methylomonadaceae might contribute to the functional resilience of the methanotrophic community and ultimately the efficiency of methane removal in the stratified water column of a marine basin.

Characterisation of 'Candidatus Methylobacter titanis' sp. nov., a putative novel species of Methylobacter clade 2 and their distribution in sediments of freshwater lakes in maritime Antarctica.

Global warming has a strong impact on the polar regions, in particular, the Antarctic Peninsula and nearby islands. Methane (CH4) is a major factor in climate change and mitigation of CH4 emissions can be accomplished through microbial oxidation by methanotrophic bacteria. Understanding this biological process is crucial given the shortage of research carried out in this geographical area. The aim of this study was to characterise psychrophilic enrichment cultures of aerobic methanotrophs obtained from lake sediments of the Fildes Peninsula (King George Island, South Shetland Islands) and revealing the distribution of the genus Methylobacter in different lake sediments of the peninsula. Four stable methanotrophic enrichment cultures were obtained and analysed by metagenome-assembled genomes (MAGs). The phylogeny of methanotroph MAGs recovered from these enrichment cultures based on the 16S rRNA gene showed that K-2018 MAG008 and D1-2020 MAG004Ts clustered within the Methylobacter clade 2, with high similarity to Methylobacter tundripaludum SV96T (97.88 and 98.56% respectively). However, the average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values with M. tundripaludum were < 95% (84.8 and 85.0%, respectively) and < 70% (30.2 and 30.3%, respectively), suggesting that they represent a putative novel species for which the name 'Ca. Methylobacter titanis' is proposed. This is the first species of clade 2 of the genus Methylobacter obtained from Antarctica. The bacterial diversity assessed by 16S rRNA gene sequencing of 21 samples of different lakes (water column and sediments) revealed 54 ASVs associated with methanotrophs and the genus Methylobacter as the most abundant. These results suggest that aerobic methanotrophs belonging to the Methylobacter clade 2 would be the main responsible for CH4 oxidation in these sediments.

Methanol bioconversion in Methylotuvimicrobium buryatense 5GB1C through self-cycling fermentation.

Methanol is an abundant and low-cost next-generation carbon source. While many species of methanotrophic bacteria can convert methanol into valuable bioproducts in bioreactors, Methylotuvimicrobium buryatense 5GB1C stands out as one of the most promising strains for industrialization. It has a short doubling time compared to most methanotrophs, remarkable resilience against contamination, and a suite of tools enabling genetic engineering. When approaching industrial applications, growing M. buryatense 5GB1C on methanol using common batch reactor operation has important limitations; for example methanol toxicity leads to mediocre biomass productivity. Advanced bioreactor operation strategies, such as fed-batch and self-cycling fermentation, have the potential to greatly improve the industrial prospects of methanotrophs growing on methanol. Herein, implementation of fed-batch operation led to a 26-fold increase in biomass density, while two different self-cycling fermentation (SCF) strategies led to 3-fold and 10-fold increases in volumetric biomass productivity. Interestingly, while synchronization is a typical trait of microbial populations undergoing SCF, M. buryatense 5GB1C cultures growing under this mode of operation led to stable, reproducible cycles but no significant synchronization.

Type-specific quantification of particulate methane monooxygenase gene of methane-oxidizing bacteria at the oxic-anoxic interface of a surface paddy soil by digital PCR.

Aerobic methane-oxidizing bacteria (MOB) play an important role in mitigating methane emissions from paddy fields. In this study, we developed a differential quantification method for the copy number of pmoA genes of type Ia, Ib, and IIa MOB in paddy field soil using chip-based digital PCR. Three probes specific to the pmoA of type Ia, Ib, and IIa MOB worked well in digital PCR quantification when genomic DNA of MOB isolates and PCR-amplified DNA fragments of pmoA were examined as templates. When pmoA genes in the surface soil layer of a flooded paddy were quantified by digital PCR, the copy numbers of type Ia, Ib, and IIa MOB were 105 -106 , 105 -106 , and 107 copies g-1 dry soil, respectively, with the highest values in the top 0-2-mm soil layer. Especially, the copy numbers of type Ia and Ib MOB increased by 240% and 380% at the top layer after soil flooding, suggesting that the soil circumstances at the oxic-anoxic interfaces were more preferential for growth of type I MOB than type II MOB. Thus, type I MOB likely play an important role in the methane consumption at the surface paddy soil.

Thermotolerant methanotrophs belonging to the Methylocaldum genus dominate the methanotroph communities in biogas slurry and cattle dung: A culture-based study from India.

Biogas reactors run on various types of waste, with cattle dung and agricultural wastes being the primary sources in India. As biogas contains 50-60% methane, there is a possibility that the reactors harbour methanotrophs or methane-oxidizing bacteria. We set up serial endpoint dilution enrichments for the cultivation of methanotrophs using slurry from a small biogas reactor and cattle dung samples and obtained cultures of Methylocaldum gracile, a thermotolerant methanotroph. The study was expanded by sampling reactors of another small reactor of 20 L capacity and two 1000 L reactors. Dung samples were obtained from two Indian cattle breeds (Tharparkar and Gir). Pulverized rice straw used for feeding the biogas was also used for experiments. All the enrichment bottles were incubated at 39 °C, the reactors' in-situ temperature, and the rumen gut temperature. Our study isolated four pure cultures most related to Methylocaldum gracile VKM-14LT, two strains from cattle dung samples, and two from reactors. The study also resulted in the cultivation of four additional cultures of Methylocaldum gracile and Methylocaldum tepidum, which were non-axenic and identified by pmoA gene sequencing. Pure cultures Methylocaldum gracile RS-9 and CDP-2 were studied for optimum temperature and oxygen. Both the strains were thermotolerant and grew in the temperature range of 25-45 °C with the optimum between 37 and 45 °C. The cultures could grow with minimal oxygen (0.5%-1%) in the headspace, with growth up to 10% oxygen. To summarize, we report the cultivation and isolation of methanotrophs from biogas slurries and cattle dung samples. Methylocaldum was the dominant methanotroph cultured, probably due to its thermotolerant nature and the ability to grow under variable oxygen conditions. The present study also expands the existing knowledge about habitats known for the genus Methylocaldum. An analysis of the isolated cultures would help us design strategies for methane mitigation from ruminants.

Characterization of methanotrophic community and activity in landfill cover soils under dimethyl sulfide stress.

Landfill cover soil is the environmental interface between landfills and the atmosphere and plays an important role in mitigating CH4 emission from landfills. Here, stable isotope probing microcosms with CH4 or CH4 and dimethyl sulfide (DMS) were carried out to characterize activity and community structure of methanotrophs in landfill cover soils under DMS stress. The CH4 oxidation activity in the landfill cover soils was not obviously influenced at the DMS concentration of 0.05%, while it was inhibited at the DMS concentrations of 0.1% and 0.2%. DMS-S was mainly oxidized to sulfate (SO42-) in the landfill cover soils. In the landfill cover soils, DMS could inhibit the expression of bacteria and decrease the abundances of pmoA and mmoX genes, while it could prompt the expression of pmoA and mmoX genes. γ-Proteobacteria methanotrophs including Methylocaldum, Methylobacter, Crenothrix and unclassified Methylococcaceae and α-Proteobacteria methanotrophs Methylocystis dominated in assimilating CH4 in the landfill cover soils. Of them, Methylobacter and Crenothrix had strong tolerance to DMS or DMS could promote the growth and activity of Methylobacter and Crenothrix, while Methylocaldum had weak tolerance to DMS and showed an inhibitory effect. Metagenomic analyses showed that methanotrophs had the genes of methanethiol oxidation and could metabolize CH4 and methanethiol simultaneously in the landfill cover soils. These findings suggested that methanotrophs might metabolize sulfur compounds in the landfill cover soils, which may provide the potential application in engineering for co-removal of CH4 and sulfur compounds.

Activity and abundance of methanotrophic bacteria in a northern mountainous gradient of wetlands.

Methane uptake and diversity of methanotrophic bacteria was investigated across six hydrologically connected wetlands in a mountainous forest landscape upstream of lake Langtjern, southern Norway. From floodplain through shrubs, forest and sedges to a Sphagnum covered site, growing season CH4 production was insufficiently consumed to balance release into the atmosphere. Emission increased by soil moisture ranging 0.6-6.8 mg CH4 m-2  h-1 . Top soils of all sites consumed CH4 including at the lowest 78 ppmv CH4 supplied, thus potentially oxidizing 17-51 nmol CH4 g-1 dw h-1 , with highest Vmax 440 nmol g-1 dw h-1 under Sphagnum and lowest Km 559 nM under hummocked Carex. Nine genera and several less understood type I and type II methanotrophs were detected by the key functional gene pmoA involved in methane oxidation. Microarray signal intensities from all sites revealed Methylococcus, the affiliated Lake Washington cluster, Methylocaldum, a Japanese rice cluster, Methylosinus, Methylocystis and the affiliated Peat264 cluster. Notably enriched by site was a floodplain Methylomonas and a Methylocapsa-affiliated watershed cluster in the Sphagnum site. The climate sensitive water table was shown to be a strong controlling factor highlighting its link with the CH4 cycle in elevated wetlands.

[Engineering application of aerobic methane oxidizing bacteria (methanotrophs): a review].

Aerobic methane oxidizing bacteria (methanotrophs) can use methane as carbon source and energy source, eliminating 10%-20% of global methane. Methanotrophs can also effectively synthesize valuable methane-derived products. This article introduced the methane oxidizing mechanism of methanotrophs, and summarized the practical application and research hotspots of methanotrophs in the field of methane emission reduction in the landfill, ventilation air methane mitigation in coal mines, valuable chemicals biosynthesis, as well as oil and gas reservoir exploration. Main factors influencing the pollutant removal and the biosynthesis efficiency in various applications were also discussed. Based on the study of large-scale cultivation of methanotrophs, some measures to benefit the application and promotion of aerobic methane oxidizing biotechnology were proposed. This includes investigating the effect of intermediate metabolites on methanotrophs activity and population structure, and exploiting economical and efficient alternative culture media and culture techniques.

Environmental Factors Affecting the Community of Methane-oxidizing Bacteria.

Methane-oxidizing bacteria (MOB) are ubiquitous and play an important role in the mitigation of global warming by reducing methane. MOB are commonly classified into Type I and Type II, belonging to Gammaproteobacteria and Alphaproteobacteria, respectively, and the diversity of MOB has been examined. However, limited information is currently available on favorable environments for the respective MOB. To investigate the environmental factors affecting the dominant type in the MOB community, we performed MOB enrichment using down-flow hanging sponge reactors under 38 different environmental conditions with a wide range of methane (0.01-80%) and ammonium concentrations (0.001-2,000| |mg N L-1) and pH 4-7. Enrichment results revealed that pH was a crucial factor influencing the MOB type enriched. Type II was dominantly enriched at low pH (4-5), whereas Type I was dominant around neutral pH (6-7). However, there were some unusual cultivated biomass samples. Even though high methane oxidation activity was observed, very few or zero conventional MOB were detected using common FISH probes and primer sets for the 16S rRNA gene and pmoA gene amplification. Mycobacterium mostly dominated the microbial community in the biomass cultivated at very high NH4+ concentrations, strongly implying that it exhibits methane oxidation activity. Collectively, the present results revealed the presence of many unknown phylogenetic groups with the capacity for methane oxidation other than the reported MOB.

[Spatial and temporal characteristics of community structure of nitrite-dependent anaerobic methane-oxidizing bacteria in paddy soil]. / ç¨»ç”°åœŸå£¤äºšç¡é ¸ç›åž‹ç”²çƒ·åŽŒæ°§æ°§åŒ–èŒç¾¤è½ç»“æž„çš„æ—¶ç©ºç‰¹å¾.

Paddy fields are one of the most important methane sources, which have great impacts on climate change. The nitrite-dependent anaerobic methane oxidation, by NC10 phylum bacteria-Candidatus Methylomirabilis oxyfera (M. oxyfera)-like bacteria, is a new process regulating methane emission from paddy fields. However, little is known about the spatial and temporal variations of M. oxyfera-like bacterial communities and the regulating factors in paddy soils. We investigated the community composition, diversity, and abundance of M. oxyfera-like bacteria in 0-40 cm depth of paddy soils at key growth stages of rice, including tillering, jointing, flowering, and milky stages. Results of high-throughput sequencing showed that community composition of M. oxyfera-like bacteria differed significantly among different soil layers, while no significant variation was observed among different rice growth stages. The diversity of M. oxyfera-like bacteria increased with soil depth. Real-time quantitative PCR showed that the 16S rRNA gene abundance of M. oxyfera-like bacteria ranged from 5.73×106 to 2.56×107 copies·g-1 (dry weight), with the highest gene abundance in the 10-20 cm layer. Further, the abundance of these bacteria showed a decreasing trend with rice growth. Soil organic carbon content and soil pH were correlated with the M. oxyfera-like bacterial community structures and abundance. In all, our results suggested a certain degree of heterogeneity of spatial and temporal distribution of M. oxyfera-like bacterial communities in paddy soils, which was largely influenced by soil organic carbon and soil pH.

Multispecies Populations of Methanotrophic Methyloprofundus and Cultivation of a Likely Dominant Species from the Iheya North Deep-Sea Hydrothermal Field.

The Methyloprofundus clade is represented by uncultivated methanotrophic bacterial endosymbionts of deep-sea bathymodiolin mussels, but only a single free-living species has been cultivated to date. This study reveals the existence of free-living Methyloprofundus variants in the Iheya North deep-sea hydrothermal field in the mid-Okinawa Trough. A clade-targeted amplicon analysis of the particulate methane monooxygenase gene (pmoA) detected 647 amplicon sequence variants (ASVs) of the Methyloprofundus clade in microbial communities newly formed in in situ colonization systems. Such systems were deployed at colonies of bathymodiolin mussels and a galatheoid crab in diffuse-flow areas. These ASVs were classified into 161 species-like groups. The proportion of the species-like groups representing endosymbionts of mussels was unexpectedly low. A methanotrophic bacterium designated INp10, a likely dominant species in the Methyloprofundus population in this field, was enriched in a biofilm formed in a methane-fed cultivation system operated at 10°C. Genomic characterization with the gene transcription data set of INp10 from the biofilm suggested traits advantageous to niche competition in environments, such as mobility, chemotaxis, biofilm formation, offensive and defensive systems, and hypoxia tolerance. The notable metabolic traits that INp10 shares with some Methyloprofundus members are the use of lanthanide-dependent XoxF as the sole methanol dehydrogenase due to the absence of the canonical MxaFI, the glycolytic pathway using fructose-6-phosphate aldolase instead of fructose-1,6-bisphosphate aldolase, and the potential to perform partial denitrification from nitrate under oxygen-limited conditions. These findings help us better understand the ecological strategies of this possibly widespread marine-specific methanotrophic clade. IMPORTANCE The Iheya North deep-sea hydrothermal field in the mid-Okinawa Trough is characterized by abundant methane derived from organic-rich sediments and diverse chemosynthetic animal species, including those harboring methanotrophic bacterial symbionts, such as bathymodiolin mussels Bathymodiolus japonicus and "Bathymodiolus" platifrons and a galatheoid crab, Shinkaia crosnieri. Symbiotic methanotrophs have attracted significant attention, and yet free-living methanotrophs in this environment have not been studied in detail. We focused on the free-living Methyloprofundus spp. that thrive in this hydrothermal field and identified an unexpectedly large number of species-like groups in this clade. Moreover, we enriched and characterized a methanotroph whose genome sequence indicated that it corresponds to a new species in the genus Methyloprofundus. This species might be a dominant member of the indigenous Methyloprofundus population. New information on free-living Methyloprofundus populations suggests that the hydrothermal field is a promising locale at which to investigate the adaptive capacity and associated genetic diversity of Methyloprofundus spp.

Methanotroph community structure and processes in an inland river affected by natural gas macro-seeps.

Methane availability in freshwaters is usually associated with spatial-temporal variation in methanogenesis. Unusually, however, natural gas macro-seeps occur along the Condamine River in eastern Australia which elevate ambient water-column methane concentrations more than 3,000 times. We quantified the spatial-temporal variation in methane oxidation rates and the total microbial and methanotroph community composition (through the amplification and sequencing of 16S rRNA and particulate methane monooxygenase (pmoA) genes), and the factors mediating this variation, in reaches with and without macro-seeps. Sediment methane oxidation rates were, on average, 29 times greater, and the abundance of methanotrophs significantly higher, in the vicinity of methane macro-seeps compared to non-seep sites. Methylocystis was the most abundant methanotroph group at all sites, but type Ib methanotrophs showed the steepest increase in abundance at seep sites. pmoA gene analysis identified these as clade 501, while 16S rRNA gene analysis identified these as the closely related genus Methylocaldum. Sediment methane oxidation rates and the relative abundance and composition of benthic microbial communities were primarily influenced by methane availability which was in turn related to variation in river discharge. Methane-derived carbon may be an important energy source for the aquatic food webs in reaches affected by natural gas macro-seeps.

A Computational Framework for Identifying Promoter Sequences in Nonmodel Organisms Using RNA-seq Data Sets.

Engineering microorganisms into biological factories that convert renewable feedstocks into valuable materials is a major goal of synthetic biology; however, for many nonmodel organisms, we do not yet have the genetic tools, such as suites of strong promoters, necessary to effectively engineer them. In this work, we developed a computational framework that can leverage standard RNA-seq data sets to identify sets of constitutive, strongly expressed genes and predict strong promoter signals within their upstream regions. The framework was applied to a diverse collection of RNA-seq data measured for the methanotroph Methylotuvimicrobium buryatense 5GB1 and identified 25 genes that were constitutively, strongly expressed across 12 experimental conditions. For each gene, the framework predicted short (27-30 nucleotide) sequences as candidate promoters and derived -35 and -10 consensus promoter motifs (TTGACA and TATAAT, respectively) for strong expression in M. buryatense. This consensus closely matches the canonical E. coli sigma-70 motif and was found to be enriched in promoter regions of the genome. A subset of promoter predictions was experimentally validated in a XylE reporter assay, including the consensus promoter, which showed high expression. The pmoC, pqqA, and ssrA promoter predictions were additionally screened in an experiment that scrambled the -35 and -10 signal sequences, confirming that transcription initiation was disrupted when these specific regions of the predicted sequence were altered. These results indicate that the computational framework can make biologically meaningful promoter predictions and identify key pieces of regulatory systems that can serve as foundational tools for engineering diverse microorganisms for biomolecule production.

Transcriptomic and Metabolomic Responses to Carbon and Nitrogen Sources in Methylomicrobium album BG8.

Methanotrophs use methane as their sole carbon and energy source and represent an attractive platform for converting single-carbon feedstocks into value-added compounds. Optimizing these species for biotechnological applications involves choosing an optimal growth substrate based on an understanding of cellular responses to different nutrients. Although many studies of methanotrophs have examined growth rate, yield, and central carbon flux in cultures grown with different carbon and nitrogen sources, few studies have examined more global cellular responses to different media. Here, we evaluated global transcriptomic and metabolomic profiles of Methylomicrobium album BG8 when grown with methane or methanol as the carbon source and nitrate or ammonium as the nitrogen source. We identified five key physiological changes during growth on methanol: M. album BG8 cultures upregulated transcripts for the Entner-Doudoroff and pentose phosphate pathways for sugar catabolism, produced more ribosomes, remodeled the phospholipid membrane, activated various stress response systems, and upregulated glutathione-dependent formaldehyde detoxification. When using ammonium, M. album BG8 upregulated hydroxylamine dehydrogenase (haoAB) and overall central metabolic activity, whereas when using nitrate, cultures upregulated genes for nitrate assimilation and conversion. Overall, we identified several nutrient source-specific responses that could provide a valuable basis for future research on the biotechnological optimization of these species. IMPORTANCE Methanotrophs are gaining increasing interest for their biotechnological potential to convert single-carbon compounds into value-added products such as industrial chemicals, fuels, and bioplastics. Optimizing these species for biotechnological applications requires a detailed understanding of how cellular activity and metabolism vary across different growth substrates. Although each of the two most commonly used carbon sources (methane or methanol) and nitrogen sources (ammonium or nitrate) in methanotroph growth media have well-described advantages and disadvantages in an industrial context, their effects on global cellular activity remain poorly characterized. Here, we comprehensively describe the transcriptomic and metabolomic changes that characterize the growth of an industrially promising methanotroph strain on multiple combinations of carbon and nitrogen sources. Our results represent a more holistic evaluation of cellular activity than previous studies of core metabolic pathways and provide a valuable basis for the future biotechnological optimization of these species.

Seasonal Dynamics of Abundance, Structure, and Diversity of Methanogens and Methanotrophs in Lake Sediments.

Understanding temporal and spatial microbial community abundance and diversity variations is necessary to assess the functional roles played by microbial actors in the environment. In this study, we investigated spatial variability and temporal dynamics of two functional microbial sediment communities, methanogenic Archaea and methanotrophic bacteria, in Lake Bourget, France. Microbial communities were studied from 3 sites sampled 4 times over a year, with one core sampled at each site and date, and 5 sediment layers per core were considered. Microbial abundance in the sediment were determined using flow cytometry. Methanogens and methanotrophs community structures, diversity, and abundance were assessed using T-RFLP, sequencing, and real-time PCR targeting mcrA and pmoA genes, respectively. Changes both in structure and abundance were detected mainly at the water-sediment interface in relation to the lake seasonal oxygenation dynamics. Methanogen diversity was dominated by Methanomicrobiales (mainly Methanoregula) members, followed by Methanosarcinales and Methanobacteriales. For methanotrophs, diversity was dominated by Methylobacter in the deeper area and by Methylococcus in the shallow area. Organic matter appeared to be the main environmental parameter controlling methanogens, while oxygen availability influenced both the structure and abundance of the methanotrophic community.

Draft genome of a novel methanotrophic Methylobacter sp. from the volcanic soils of Pantelleria Island.

The genus Methylobacter is considered an important and often dominant group of aerobic methane-oxidizing bacteria in many oxic ecosystems, where members of this genus contribute to the reduction of CH4 emissions. Metagenomic studies of the upper oxic layers of geothermal soils of the Favara Grande, Pantelleria, Italy, revealed the presence of various methane-oxidizing bacteria, and resulted in a near complete metagenome assembled genome (MAG) of an aerobic methanotroph, which was classified as a Methylobacter species. In this study, the Methylobacter sp. B2 MAG was used to investigate its metabolic potential and phylogenetic affiliation. The MAG has a size of 4,086,539 bp, consists of 134 contigs and 3955 genes were found, of which 3902 were protein coding genes. All genes for CH4 oxidation to CO2 were detected, including pmoCAB encoding particulate methane monooxygenase (pMMO) and xoxF encoding a methanol dehydrogenase. No gene encoding a formaldehyde dehydrogenase was present and the formaldehyde to formate conversion follows the tetrahydromethanopterin (H4MPT) pathway. "Ca. Methylobacter favarea" B2 uses the Ribulose-Mono-Phosphate (RuMP) pathway for carbon fixation. Analysis of the MAG indicates that Na+/H+ antiporters and the urease system might be important in the maintenance of pH homeostasis of this strain to cope with acidic conditions. So far, thermoacidophilic Methylobacter species have not been isolated, however this study indicates that members of the genus Methylobacter can be found in distinct ecosystems and their presence is not restricted to freshwater or marine sediments.