Complete genome sequence of Klebsiella pneumoniae RX.G5M15, a methanol-metabolizing strain recovered from the sole of a shoe.

The methanol-metabolizing strain Klebsiella pneumoniae RX.G5M15 was isolated from the sole of a shoe in Hong Kong. Its complete genome, a single chromosome and two plasmids totaling 5,381,940 bp (G+C 57.43%), was established through the hybrid assembly.

Genomic Insights into the Landfill Microbial Community: Denitrifying Activity Supporting One-Carbon Utilization.

In spite of the developments in understanding of denitrifying methylotrophy in the recent years, challenges still exist in unravelling the overall biochemistry of nitrate-dependent methane oxidation in novel or poorly characterized/not-yet-cultured bacteria. In the present study, landfill site was mined for novel C1-carbon-metabolizing bacteria which can use nitrate/nitrite as an electron acceptor. A high-throughput rapid plate assay identified three bacterial isolates with eminent ability for nitrate-dependent methane metabolism under anaerobic conditions. Taxonomic identification by whole-genome sequence-based overall genome relatedness indices accurately assigned the isolates AAK_M13, AAK_M29, and AAK_M39 at the species level to Enterobacter cloacae, Bacillus subtilis, and Bacillus halotolerans, respectively. Several genes encoding sub-components involved in alcohol utilization and denitrification pathways, such as adh, fdh, fdo, nar, nir, and nor, were identified in all the genomes. Though no gene clusters encoding MMO/AMO were annotated, sequencing of PCR amplicons revealed similarity with pMMO/AMO gene using translated nucleotide sequence of strains AAK_M29 and AAK_M39, while strain AAK_M13 showed similarity with XRE family transcriptional regulator. This suggests the horizontal gene transfer and/or presence of a truncated version of a housekeeping enzyme encoded by genes exhibiting partial sequence similarity with pMMO genes that mimicked its function at greenhouse gas emission sites. Owing to lack of conclusive evidence for presence of methane metabolism genes in the selected isolates, further experiment was performed to validate their nitrate-dependent methane oxidation capacities. Bacillus subtilis AAK_M29, Bacillus halotolerans AAK_M39, and Enterobacter cloacae AAK_M13 could oxidize 60%, 75%, and 85% of the added methane respectively accompanied by high nitrate reduction (56-62%) thus supporting the correlation between these two activities. The remarkable ability of these isolates for nitrate-dependent methane metabolism has highlighted their role in ecological contribution and biotechnological potential to serve as methane and nitrate sinks in the landfill sites.

Biotechnological potential of psychrotolerant methylobacteria isolated from biotopes of Antarctic oases.

Ten strains of psychrotolerant methylotrophic bacteria were isolated from the samples collected in Larsemann and Bunger Hills (Antarctica). Most of the isolates are assigned to the genus Pseudomonas, representatives of the genera Janthinobacterium, Massilia, Methylotenera and Flavobacterium were also found. Majority of isolates were able to grow on a wide range of sugars, methylamines and other substrates. Optimal growth temperatures for the isolated strains varied from 6 °C to 28 °C. The optimal concentration of NaCl was 0.5-2.0%. The optimal pH values of the medium were 6-7. It was found that three strains synthesized indole-3-acetic acid on a medium with L-tryptophan reaching 11-12 µg/ml. The values of intracellular carbohydrates in several strains exceeded 50 µg/ml. Presence of calcium-dependent and lanthanum-dependent methanol dehydrogenase have been shown for some isolates. Strains xBan7, xBan20, xBan37, xBan49, xPrg27, xPrg48, xPrg51 showed the presence of free amino acids. Bioprospection of Earth cryosphere for such microorganisms has a potential in biotechnology.

Methylotrophic bacteria from rice paddy soils: mineral-nitrogen-utilizing isolates richness in bulk soil and rhizosphere.

Methanol, the second most abundant volatile organic compound, primarily released from plants, is a major culprit disturbing atmospheric chemistry. Interestingly, ubiquitously found methanol-utilizing bacteria, play a vital role in mitigating atmospheric methanol effects. Despite being extensively characterized, the effect of nitrogen sources on the richness of methanol-utilizers in the bulk soil and rhizosphere is largely unknown. Therefore, the current study was planned to isolate, characterize and explore the richness of cultivable methylotrophs from the bulk soil and rhizosphere of a paddy field using media with varying nitrogen sources. Our data revealed that more genera of methylotrophs, including Methylobacterium, Ancylobacter, Achromobacter, Xanthobacter, Moraxella, and Klebsiella were enriched with the nitrate-based medium compared to only two genera, Hyphomicrobium and Methylobacterium, enriched with the ammonium-based medium. The richness of methylotrophic bacteria also differed substantially in the bulk soil as compared to the rhizosphere. Growth characterization revealed that majority of the newly isolated methanol-utilizing strains in this study exhibited better growth at 37 °C instead of 30 or 45 °C. Moreover, Hyphomicrobium sp. FSA2 was the only strain capable of utilizing methanol even at elevated temperature 45 °C, showing its adaptability to a wide range of temperatures. Differential carbon substrate utilization profiling revealed the facultative nature of all isolated methanol-utilizer strains with Xanthobacter sp. TS3, being an important methanol-utilizer capable of degrading toxic compounds such as acetone and ethylene glycol. Overall, our study suggests the role of nutrients and plant-microbial interaction in shaping the composition of methanol-utilizers in terrestrial environment.

Engineering Terpene Production Pathways in Methylobacterium extorquens AM1.

Terpenes are diverse specialized metabolites naturally found within plants and have important roles in inter-species communication, adaptation and interaction with the environment. Their industrial applications span a broad range, including fragrances, flavors, cosmetics, natural colorants to agrochemicals and therapeutics, yet formal chemical synthesis is economically challenging due to structural complexities. Engineering terpene biosynthesis could represent an alternative in microbial biotechnological workhorses, such as Saccharomyces cerevisiae or Escherichi coli, utilizing sugars or complex media as feedstocks. Host species that metabolize renewable and affordable carbon sources may offer unique sustainable biotechnological alternatives. Methylotrophs are bacteria with the capacity to utilize one-carbon feedstocks, such as methanol or formate. They colonize the phyllosphere (above-ground area) of plants, and many accumulate abundant carotenoid pigments. Methylotrophs have the capacity to take up and use a subset of the rare earth elements known as lanthanides. These metals can enhance one-carbon (methylotrophic) metabolism. Here, we investigated whether manipulating the metabolism enables and enhances terpene production. A carotenoid-deficient mutant potentially liberates carbon, which may contribute to bioproduct accumulation. To test this hypothesis, terpene-producing bacterial strains regulated by two distinct promoters were generated. Wildtype Methylobacterium extorquens, ∆Meta1_3665, a methylotrophic mutant lacking the carotenoid pathway, and an E. coli strain were transformed with an exogenous terpene pathway and grown both in the presence and absence of lanthanides. The extraction, and the comparison of analytical profiles, provided evidence that engineered cultured M. extorquens under control of a native, inducible methylotrophic promoter can yield the sesquiterpene patchoulol when supplemented with lanthanide. In contrast, using a moderate-strength constitutive promoter failed to give production. We demonstrated colonization of the phyllosphere with the engineered strains, supporting the future engineering of selected species of the plant microbiome and with promising implications for the synthetic biology of small molecules.

Industrial and Pharmaceutical Applications of Microbial Diversity of Hypersaline Ecology from Lonar Soda Crater.

The unidentified geochemical and physiochemical characteristics of Soda Lakes across the globe make it a novel reservoir and bring attention to scientific civic for its conceivable industrial and pharmaceutical applications. In India, in the Maharashtra state, Lonar Lake is a naturally created Soda Lake by a meteorite impact. Phylogenetic data from this lake explored a diverse array of microorganisms like haloalkaliphilic bacteria and Archaea. Previously reported studies postulated the major microbial communities present in this lake ecosystem are Proteobacteria, Actinobacteria, Firmicutes, and Cyanobacteria. Furthermore, it also contains Bacteroidetes, Nitrospirae, and Verrucomicrobia. This lake is also rich in phytoplankton, with the predominant presence of the Spirulina plantensis. Unique microbial strains from Lonar Lake ecosystems have fascinated consideration as a source of biological molecules with medicinal, industrial, and biotechnological potential. Recent literature revealed the isolation of antibioticproducing bacteria and alkaline proteases-producing alkaliphilic bacterium, as well as novel species of rare methylotrophs, other bacterial strains involved in producing vital enzymes, and unique actinomycetes are also reported. It indicates that the novel bacterial assemblage not reached hitherto may exist in this modified and unique ecology. This comprehensive review provides information about microbial diversity and its industrial and pharmaceutical interests that exist in Lonar Lake, which could be the future source of bioactive enzymes, biosurfactants, and biofuel and also useful in bioremediation. Furthermore, the novel species of microorganisms isolated from Lonar Lake have applications in the biosynthesis of medicines like antibiotics, antivirals, antifungals, anti-inflammatory agents, and precursors for synthesising valuable products. Data consolidated in the present review will cater to the needs of emerging industrial sectors for their commercial and therapeutic applications.

Methylotrophic Communities Associated with a Greenland Ice Sheet Methane Release Hotspot.

Subglacial environments provide conditions suitable for the microbial production of methane, an important greenhouse gas, which can be released from beneath the ice as a result of glacial melting. High gaseous methane emissions have recently been discovered at Russell Glacier, an outlet of the southwestern margin of the Greenland Ice Sheet, acting not only as a potential climate amplifier but also as a substrate for methane consuming microorganisms. Here, we describe the composition of the microbial assemblage exported in meltwater from the methane release hotspot at Russell Glacier and its changes over the melt season and as it travels downstream. We found that a substantial part (relative abundance 27.2% across the whole dataset) of the exported assemblage was made up of methylotrophs and that the relative abundance of methylotrophs increased as the melt season progressed, likely due to the seasonal development of the glacial drainage system. The methylotrophs were dominated by representatives of type I methanotrophs from the Gammaproteobacteria; however, their relative abundance decreased with increasing distance from the ice margin at the expense of type II methanotrophs and/or methylotrophs from the Alphaproteobacteria and Betaproteobacteria. Our results show that subglacial methane release hotspot sites can be colonized by microorganisms that can potentially reduce methane emissions.

Methylobacterium spp. mitigation of UV stress in mung bean (Vigna radiata L.).

Methylotrophs are a diverse group of bacteria that abundantly colonize the phyllosphere and have great potential to withstand UV irradiation because of their pigmented nature and ability to promote plant growth through various mechanisms. The present study investigated the effects of UVB radiation on plant growth-promoting (PGP) properties of methylotrophic bacteria and the growth of Vigna radiata L. A total of 55 methylotrophic bacteria were isolated from desert plants, and 15 methylotrophs were resistant to UVB radiation for 4 h. All UVB-resistant methylotrophs possess a methyldehydrogenase gene. Identification based on 16S rRNA gene sequencing revealed that all 15 UVB-resistant methylotrophs belonged to the genera Methylorubrum (07), Methylobacterium (07), and Rhodococcus (01). Screening of methylotrophs for PGP activity in the presence and absence of UVB radiation revealed that all isolates showed ACC deaminase activity and growth on a nitrogen-free medium. Furthermore, the production of IAA-like substances ranged from 8.62 to 85.76 µg/mL, siderophore production increased from 3.47 to 65.75% compared to the control. Seed germination assay with V. radiata L. (mung bean) exposed to UVB radiation revealed that methylotrophs improved seed germination, root length, and shoot length compared to the control. The present findings revealed that the isolates SD3, SD2, KD1, KD5, UK1, and UK3 reduced the deleterious effects of UVB radiation on mung bean plants and can be used to protect seedlings from UVB radiation for sustainable agriculture.

Complete genome sequence of Kosakonia pseudosacchari RX.G5M8, a putative methylotroph isolated from Hong Kong soil.

Kosakonia pseudosacchari RX.G5M8, a putative methylotroph, was isolated from garden soil in Hong Kong. Its complete genome, a single chromosome of 4,953,935 bp (GC content 53.91%), was established through hybrid assembly.

Enzymatic properties of alcohol dehydrogenase PedE_M.s. derived from Methylopila sp. M107 and its broad metal selectivity.

As an important metabolic enzyme in methylotrophs, pyrroloquinoline quinone (PQQ)-dependent alcohol dehydrogenases play significant roles in the global carbon and nitrogen cycles. In this article, a calcium (Ca2+)-dependent alcohol dehydrogenase PedE_M.s., derived from the methylotroph Methylopila sp. M107 was inserted into the modified vector pCM80 and heterologously expressed in the host Methylorubrum extorquens AM1. Based on sequence analysis, PedE_M.s., a PQQ-dependent dehydrogenase belonging to a methanol/ethanol family, was successfully extracted and purified. Showing by biochemical results, its enzymatic activity was detected as 0.72 U/mg while the Km value was 0.028 mM while employing ethanol as optimal substrate. The activity of PedE_M.s. could be enhanced by the presence of potassium (K+) and calcium (Ca2+), while acetonitrile and certain common detergents have been found to decrease the activity of PedE_M.s.. In addition, its optimum temperature and pH were 30°C and pH 9.0, respectively. Chiefly, as a type of Ca2+-dependent alcohol dehydrogenase, PedE_M.s. maintained 60-80% activity in the presence of 10 mM lanthanides and displayed high affinity for ethanol compared to other PedE-type enzymes. The 3D structure of PedE_M.s. was predicted by AlphaFold, and it had an 8-bladed propeller-like super-barrel. Meanwhile, we could speculate that PedE_M.s. contained the conserved residues Glu213, Asn300, and Asp350 through multiple sequence alignment by Clustal and ESpript. The analysis of enzymatic properties of PedE_M.s. enriches our knowledge of the methanol/ethanol family PQQ-dependent dehydrogenase. This study provides new ideas to broaden the application of alcohol dehydrogenase in alcohol concentration calculation, biosensor preparation, and other industries.

[Advances in biotransformation of methanol into chemicals].

Methanol has become an attractive substrate for the biomanufacturing industry due to its abundant supply and low cost. The biotransformation of methanol to value-added chemicals using microbial cell factories has the advantages of green process, mild conditions and diversified products. These advantages may expand the product chain based on methanol and alleviate the current problem of biomanufacturing, which is competing with people for food. Elucidating the pathways involving methanol oxidation, formaldehyde assimilation and dissimilation in different natural methylotrophs is essential for subsequent genetic engineering modification, and is more conducive to the construction of novel non-natural methylotrophs. This review discusses the current status of research on methanol metabolic pathways in methylotrophs, and presents recent advances and challenges in natural and synthetic methylotrophs and their applications in methanol bioconversion.

Methanol-based biomanufacturing of fuels and chemicals using native and synthetic methylotrophs.

Methanol has recently gained significant attention as a potential carbon substrate for the production of fuels and chemicals, owing to its high degree of reduction, abundance, and low price. Native methylotrophic yeasts and bacteria have been investigated for the production of fuels and chemicals. Alternatively, synthetic methylotrophic strains are also being developed by reconstructing methanol utilization pathways in model microorganisms, such as Escherichia coli. Owing to the complex metabolic pathways, limited availability of genetic tools, and methanol/formaldehyde toxicity, the high-level production of target products for industrial applications are still under development to satisfy commercial feasibility. This article reviews the production of biofuels and chemicals by native and synthetic methylotrophic microorganisms. It also highlights the advantages and limitations of both types of methylotrophs and provides an overview of ways to improve their efficiency for the production of fuels and chemicals from methanol.

Synergy effects of Methylomonas koyamae and Hyphomicrobium methylovorum under methanethiol stress.

Methanotrophs are able to metabolize volatile organic sulfur compounds (VOSCs), excrete organic carbon during CH4 oxidation, and influence microbial community structure and function of the ecosystem. In return, microbial community structure and environmental factors can affect the growth metabolism of methanotrophs. In this study, Methylomonas koyamae and Hyphomicrobium methylovorum were used for model organisms, and methanethiol (MT) was chosen for a typical VOSC to investigate the synergy effects under VOSC stress. The results showed that when Hyphomicrobium methylovorum was co-cultured with Methylomonas koyamae in the medium with CH4 used as the carbon source, the co-culture had better MT tolerance relative to Methylomonas koyamae and oxidized all CH4 within 120 h, even at the initial MT concentration of 2000 mg m-3. The optimal co-culture ratios of Methylomonas koyamae to Hyphomicrobium methylovorum were 4:1-12:1. Although MT could be converted spontaneously to dimethyl disulfide (DMDS), H2S, and CS2 in air, faster losses of MT, DMDS, H2S, and CS2 were observed in each strain mono-culture and the co-culture. Compared with Hyphomicrobium methylovorum, MT was degraded more quickly in the Methylomonas koyamae culture. During the co-culture, the CH4 oxidation process of Methylomonas koyamae could provide carbon and energy sources for the growth of Hyphomicrobium methylovorum, while Hyphomicrobium methylovorum oxidized MT to help Methylomonas koyamae detoxify. These findings are helpful to understand the synergy effects of Methylomonas koyamae and Hyphomicrobium methylovorum under MT stress and enrich the role of methanotrophs in the sulfur biogeochemical cycle. KEY POINTS: • The co-culture of Methylomonas and Hyphomicrobium has better tolerance to CH3SH. • Methylomonas can provide carbon sources for the growth of Hyphomicrobium. • The co-culture of Methylomonas and Hyphomicrobium enhances the removal of CH4 and CH3SH.

Microbial Community Structure among Honey Samples of Different Pollen Origin.

Honey's antibacterial activity has been recently linked to the inhibitory effects of honey microbiota against a range of foodborne and human pathogens. In the current study, the microbial community structure of honey samples exerting pronounced antimicrobial activity was examined. The honey samples were obtained from different geographical locations in Greece and had diverse pollen origin (fir, cotton, fir-oak, and Arbutus unedo honeys). Identification of honey microbiota was performed by high-throughput amplicon sequencing analysis, detecting 335 distinct taxa in the analyzed samples. Regarding ecological indices, the fir and cotton honeys possessed greater diversity than the fir-oak and Arbutus unedo ones. Lactobacillus kunkeei (basionym of Apilactobacillus kun-keei) was the predominant taxon in the fir honey examined. Lactobacillus spp. appeared to be favored in honey from fir-originated pollen and nectar since lactobacilli were more pronounced in fir compared to fir-oak honey. Pseudomonas, Streptococcus, Lysobacter and Meiothermus were the predominant taxa in cotton honey, whereas Lonsdalea, the causing agent of acute oak decline, and Zymobacter, an osmotolerant facultative anaerobic fermenter, were the dominant taxa in fir-oak honey. Moreover, methylotrophic bacteria represented 1.3-3% of the total relative abundance, independently of the geographical and pollen origin, indicating that methylotrophy plays an important role in honeybee ecology and functionality. A total of 14 taxa were identified in all examined honey samples, including bacilli/anoxybacilli, paracocci, lysobacters, pseudomonads, and sphingomonads. It is concluded that microbial constituents of the honey samples examined were native gut microbiota of melliferous bees and microbiota of their flowering plants, including both beneficial bacteria, such as potential probiotic strains, and animal and plant pathogens, e.g., Staphylococcus spp. and Lonsdalea spp. Further experimentation will elucidate aspects of potential application of microbial bioindicators in identifying the authenticity of honey and honeybee-derived products.

Methyl-Based Methanogenesis: an Ecological and Genomic Review.

Methyl-based methanogenesis is one of three broad categories of archaeal anaerobic methanogenesis, including both the methyl dismutation (methylotrophic) pathway and the methyl-reducing (also known as hydrogen-dependent methylotrophic) pathway. Methyl-based methanogenesis is increasingly recognized as an important source of methane in a variety of environments. Here, we provide an overview of methyl-based methanogenesis research, including the conditions under which methyl-based methanogenesis can be a dominant source of methane emissions, experimental methods for distinguishing different pathways of methane production, molecular details of the biochemical pathways involved, and the genes and organisms involved in these processes. We also identify the current gaps in knowledge and present a genomic and metagenomic survey of methyl-based methanogenesis genes, highlighting the diversity of methyl-based methanogens at multiple taxonomic levels and the widespread distribution of known methyl-based methanogenesis genes and families across different environments.

Secondary metabolites from methylotrophic bacteria: their role in improving plant growth under a stressed environment.

Climate change is considered a natural disaster that causes the ecosystem to fluctuate and increase temperature, as well as the amount of UV radiation (UV-A and UV-B) on the Earth's surface. Consequently, greenhouse gases such as chlorofluorocarbons, methane, nitrogen oxide, and carbon dioxide have become obstacles to the development of sustainable agriculture. To overcome environmental stress such as phytopathogens, drought, salinity, heavy metals, and high-low temperatures, the utilization of microorganisms is a viable option. The synthesis of secondary metabolites by methylotrophic bacteria improves plant metabolism, enhances tolerance, and facilitates growth. The genus Methylobacterium is a pink-pigmented facultative methylotrophs which abundantly colonizes plants, especially young leaves, owing to the availability of methanol. Secondary metabolites such as amino acids, carotenoids, hormones, antimicrobial compounds, and other compounds produced by methylotrophic bacteria enhance plant metabolism under stress conditions. Therefore, in this review, we discuss the role of secondary metabolites produced by methylotrophic bacteria and their role in promoting plant growth under stress.

Graphene as Thinnest Coating on Copper Electrodes in Microbial Methanol Fuel Cells.

Dehydrogenation of methanol (CH3OH) into direct current (DC) in fuel cells can be a potential energy conversion technology. However, their development is currently hampered by the high cost of electrocatalysts based on platinum and palladium, slow kinetics, the formation of carbon monoxide intermediates, and the requirement for high temperatures. Here, we report the use of graphene layers (GL) for generating DC electricity from microbially driven methanol dehydrogenation on underlying copper (Cu) surfaces. Genetically tractable Rhodobacter sphaeroides 2.4.1 (Rsp), a nonarchetypical methylotroph, was used for dehydrogenating methanol at the GL-Cu surfaces. We use electrochemical methods, microscopy, and spectroscopy methods to assess the effects of GL on methanol dehydrogenation by Rsp cells. The GL-Cu offers a 5-fold higher power density and 4-fold higher current density compared to bare Cu. The GL lowers charge transfer resistance to methanol dehydrogenation by 4 orders of magnitude by mitigating issues related to pitting corrosion of underlying Cu surfaces. The presented approach for catalyst-free methanol dehydrogenation on copper electrodes can improve the overall sustainability of fuel cell technologies.

Advances in biotransformation of methanol into chemicals / 生物工程学报

Methanol has become an attractive substrate for the biomanufacturing industry due to its abundant supply and low cost. The biotransformation of methanol to value-added chemicals using microbial cell factories has the advantages of green process, mild conditions and diversified products. These advantages may expand the product chain based on methanol and alleviate the current problem of biomanufacturing, which is competing with people for food. Elucidating the pathways involving methanol oxidation, formaldehyde assimilation and dissimilation in different natural methylotrophs is essential for subsequent genetic engineering modification, and is more conducive to the construction of novel non-natural methylotrophs. This review discusses the current status of research on methanol metabolic pathways in methylotrophs, and presents recent advances and challenges in natural and synthetic methylotrophs and their applications in methanol bioconversion.

Developing methylotrophic microbial platforms for a methanol-based bioindustry.

Methanol, a relatively cheap and renewable single-carbon feedstock, has gained considerable attention as a substrate for the bio-production of commodity chemicals. Conventionally produced from syngas, along with emerging possibilities of generation from methane and CO2, this C1 substrate can serve as a pool for sequestering greenhouse gases while supporting a sustainable bio-economy. Methylotrophic organisms, with the inherent ability to use methanol as the sole carbon and energy source, are competent candidates as platform organisms. Accordingly, methanol bioconversion pathways have been an attractive target for biotechnological and bioengineering interventions in developing microbial cell factories. This review summarizes the recent advances in methanol-based production of various bulk and value-added chemicals exploiting the native and synthetic methylotrophic organisms. Finally, the current challenges and prospects of streamlining these methylotrophic platforms are discussed.

Methanol utilizers of the rhizosphere and phyllosphere of a common grass and forb host species.

BACKGROUND: Managed grasslands are global sources of atmospheric methanol, which is one of the most abundant volatile organic compounds in the atmosphere and promotes oxidative capacity for tropospheric and stratospheric ozone depletion. The phyllosphere is a favoured habitat of plant-colonizing methanol-utilizing bacteria. These bacteria also occur in the rhizosphere, but their relevance for methanol consumption and ecosystem fluxes is unclear. Methanol utilizers of the plant-associated microbiota are key for the mitigation of methanol emission through consumption. However, information about grassland plant microbiota members, their biodiversity and metabolic traits, and thus key actors in the global methanol budget is largely lacking. RESULTS: We investigated the methanol utilization and consumption potentials of two common plant species (Festuca arundinacea and Taraxacum officinale) in a temperate grassland. The selected grassland exhibited methanol formation. The detection of 13C derived from 13C-methanol in 16S rRNA of the plant microbiota by stable isotope probing (SIP) revealed distinct methanol utilizer communities in the phyllosphere, roots and rhizosphere but not between plant host species. The phyllosphere was colonized by members of Gamma- and Betaproteobacteria. In the rhizosphere, 13C-labelled Bacteria were affiliated with Deltaproteobacteria, Gemmatimonadates, and Verrucomicrobiae. Less-abundant 13C-labelled Bacteria were affiliated with well-known methylotrophs of Alpha-, Gamma-, and Betaproteobacteria. Additional metagenome analyses of both plants were consistent with the SIP results and revealed Bacteria with methanol dehydrogenases (e.g., MxaF1 and XoxF1-5) of known but also unusual genera (i.e., Methylomirabilis, Methylooceanibacter, Gemmatimonas, Verminephrobacter). 14C-methanol tracing of alive plant material revealed divergent potential methanol consumption rates in both plant species but similarly high rates in the rhizosphere and phyllosphere. CONCLUSIONS: Our study revealed the rhizosphere as an overlooked hotspot for methanol consumption in temperate grasslands. We further identified unusual new but potentially relevant methanol utilizers besides well-known methylotrophs in the phyllosphere and rhizosphere. We did not observe a plant host-specific methanol utilizer community. Our results suggest that our approach using quantitative SIP and metagenomics may be useful in future field studies to link gross methanol consumption rates with the rhizosphere and phyllosphere microbiome.