Microorganisms maintain metabolic activity in clouds, with recognized impacts on the chemistry of small organic compounds, radicals, and their precursors. However, how microbial activity is modulated by cloud environmental variables remains unknown. Here we explored the metabolic response of an assemblage of representative microbial isolates from cloud water, composed of a basidiomycetous yeast (Dioszegia hungarica) and three bacterial strains (Rhodococcus enclensis, Pseudomonas syringae, and Pseudomonas graminis), in synthetic cloud water exposed to contrasted conditions of temperature (5°C vs 17°C), light (dark vs artificial solar light) and oxidants (0 μM vs 250 μM H2O2), to mimic typical cloud conditions during winter night and summer day. Metabolomics and metatranscriptomics allowed the identification of 25 differentially abundant metabolites and 218 differentially expressed genes (DEGs). Both summer day metabolomes and metatranscriptomes suggested active mitochondria-driven energy production, with fungal DEGs involved in fatty acids biosynthesis and succinate assimilation, and three differentially abundant acylcarnitines that support fatty acid transport into the mitochondrion for oxidative phosphorylation. In contrast, bacteria displayed DEGs for cell division arrest and components of reactive oxygen species scavenging systems. Under the winter night condition, both bacteria and yeast exhibited a similar prosperous state with DEGs encoding translation, protein repair and turnover, as well as cell cycle related functions. Thus, eukaryotes and prokaryotes may engage in distinct strategies to survive in clouds, depending on environmental conditions. This study consolidates our understanding of microbial roles and interactions in cloud water, paving the way for deeper insights into the chemistry of atmospheric systems.

Metabolically active microorganisms are increasingly acknowledged as actors of cloud chemical reactivity able to use organic compounds present in clouds (e.g. organic acids, aldhedydes) for their metabolism (Vaïtilingom et al., 2013). Uncharacterized biological activity may play a major role especially during the night, while during daytime the abiotic degradation of organic compounds would be driven and dominated by hydroxyl radical (•OH) chemistry (Vaïtilingom et al., 2011). To better understand and predict the impact of biological activity on atmospheric chemical reactivity, the metabolic pathways of the whole cloud microbiome and their modulations by environmental conditions (temperature, light, oxidants) must now be assessed.The METACLOUD project addresses metabolic acclimatation of cloud microorganisms under two contrasted situations simulating a summer day (17°C, with solar light and presence of hydrogen peroxide) and a winter night (at 5°C, in the dark and without hydrogen peroxide). A focus is made on formaldehyde assimilations as this compound is a key intermediate both in cloud radical chemistry and in many C1 biological pathway, using fluxomics (LC-HRMS and IC-HRMS) on 13C-formaldehyde supplemented samples. Experiments were conducted in specially designed photobioreactors, either on (1) freshly sampled cloud water from the research station at the top of the puy de Dôme station (1465m asl, PUY, France) including naturally present microorganisms, or (2) an artificial consortium assembled from microbial strains isolated from cloud water sampled at PUY and resuspended in an artificial medium mimicking the composition of marine cloud water (major inorganic and organic compounds).Metatranscriptomes and metabolomes indicate metabolic acclimations of the cloud microbiome to model summer/winter conditions, especially linked with fatty acid regulation and central metabolism (e.g. citrate cycle). First results with 13C-formaldehyde showed carbon incorporation from this molecule into several classes of metabolites (e.g. nucleotides, amino acids, central metabolites), illustrating the complex biological fate of this compound in the environment. The data will be used to implement biological activity on cloud chemistry models. Vaïtilingom M. et al. (2011) Atmospheric chemistry of carboxylic acids: microbial implication versus photochemistry. Atmos. Chem. Phys. 11, 8721-8733. doi: 10.5194/acp-11-8721-2011.Vaïtilingom M. et al. (2013) Potential impact of microbial activity on the oxidant capacity and organic carbon budget in clouds. Proc. Nat. Acad. Sci. USA 110, 559-564. doi: 10.1073/pnas.1205743110.

Abstract Background Chloromethane (CH3Cl) is the most abundant halogenated organic compound in the atmosphere and substantially responsible for the destruction of the stratospheric ozone layer. Since anthropogenic CH3Cl sources have become negligible with the application of the Montreal Protocol (1987), natural sources, such as vegetation and soils, have increased proportionally in the global budget. CH3Cl-degrading methylotrophs occurring in soils might be an important and overlooked sink. Results and conclusions The objective of our study was to link the biotic CH3Cl sink with the identity of active microorganisms and their biochemical pathways for CH3Cl degradation in a deciduous forest soil. When tested in laboratory microcosms, biological CH3Cl consumption occurred in leaf litter, senescent leaves, and organic and mineral soil horizons. Highest consumption rates, around 2 mmol CH3Cl g−1 dry weight h−1, were measured in organic soil and senescent leaves, suggesting that top soil layers are active (micro-)biological CH3Cl degradation compartments of forest ecosystems. The DNA of these [13C]-CH3Cl-degrading microbial communities was labelled using stable isotope probing (SIP), and the corresponding taxa and their metabolic pathways studied using high-throughput metagenomics sequencing analysis. [13C]-labelled Metagenome-Assembled Genome closely related to the family Beijerinckiaceae may represent a new methylotroph family of Alphaproteobacteria, which is found in metagenome databases of forest soils samples worldwide. Gene markers of the only known pathway for aerobic CH3Cl degradation, via the methyltransferase system encoded by the CH3Cl utilisation genes (cmu), were undetected in the DNA-SIP metagenome data, suggesting that biological CH3Cl sink in this deciduous forest soil operates by a cmu-independent metabolism.

Dichloromethane (DCM, methylene chloride) is a toxic halogenated volatile organic compound massively used for industrial applications, and consequently often detected in the environment as a major pollutant. DCM biotransformation suggests a sustainable decontamination strategy of polluted sites. Among methylotrophic bacteria able to use DCM as a sole source of carbon and energy for growth, Methylorubrum extorquens DM4 is a longstanding reference strain. Here, the primary 5′-ends of transcripts were obtained using a differential RNA-seq (dRNA-seq) approach to provide the first transcription start site (TSS) genome-wide landscape of a methylotroph using DCM or methanol. In total, 7231 putative TSSs were annotated and classified with respect to their localization to coding sequences (CDSs). TSSs on the opposite strand of CDS (antisense TSS) account for 31% of all identified TSSs. One-third of the detected TSSs were located at a distance to the start codon inferior to 250 nt (average of 84 nt) with 7% of leaderless mRNA. Taken together, the global TSS map for bacterial growth using DCM or methanol will facilitate future studies in which transcriptional regulation is crucial, and efficient DCM removal at polluted sites is limited by regulatory processes.

The complete genome of Sphingomonas aerolata PDD-32b-11, a bacterium isolated from cloud water, was sequenced. It features four circular replicons, a chromosome of 3.99 Mbp, and three plasmids. Two putative rhodopsin-encoding genes were detected which might act as proton pumps to harvest light energy.

The genome of the basidiomycete yeast Dioszegia hungarica strain PDD-24b-2 isolated from cloud water at the summit of puy de Dome (France) was sequenced using a hybrid PacBio and Illumina sequencing strategy. The obtained assembled genome of 20.98 Mb and a GC content of 57% is structured in 16 large-scale contigs ranging from 90 kb to 5.56 Mb, and another 27.2 kb contig representing the complete circular mitochondrial genome. In total, 8,234 proteins were predicted from the genome sequence. The mitochondrial genome shows 16.2% cgu codon usage for arginine but has no canonical cognate tRNA to translate this codon. Detected transposable element (TE)-related sequences account for about 0.63% of the assembled genome. A dataset of 2,068 hand-picked public environmental metagenomes, representing over 20 Tbp of raw reads, was probed for D. hungarica related ITS sequences, and revealed worldwide distribution of this species, particularly in aerial habitats. Growth experiments suggested a psychrophilic phenotype and the ability to disperse by producing ballistospores. The high-quality assembled genome obtained for this D. hungarica strain will help investigate the behavior and ecological functions of this species in the environment.

Background: Chloromethane (CH3Cl) is the most abundant chlorinated volatile organic compound in the atmosphere and contributes to stratospheric ozone depletion. CH3Cl has mainly natural sources such as emissions from vegetation. In particular, ferns have been recognized as strong emitters. Mitigation of CH3Cl to the atmosphere by methylotrophic bacteria, a global sink for this compound, is likely underestimated and remains poorly characterized. Results and Conclusions: We investigated chloromethane-degrading taxa associated with intact and living tree fern plants of the species Cyathea australis by stable isotope probing (SIP) with 13C-labelled CH3Cl combined with metagenomic DNA sequencing. Metagenome assembled genomes (MAGs) related to Methylobacterium and Friedmanniella were identified as being involved in the degradation of CH3Cl in the phyllosphere, i.e., the aerial parts of the tree fern, while a MAG related to Sorangium was linked to CH3Cl degradation in the fern rhizosphere. The only known metabolic pathway for CH3Cl degradation, via a methyltransferase system including the gene cmuA, was not detected in metagenomes or MAGs identified by SIP. Hence, a yet uncharacterised methylotrophic cmuA-independent pathway likely drives CH3Cl degradation in the investigated tree ferns.

Chloromethane (CH3 Cl) is the most abundant halogenated volatile organic compound in the atmosphere and contributes to stratospheric ozone depletion. CH3 Cl has mainly natural sources such as emissions from vegetation. In particular, ferns have been recognized as strong emitters. Mitigation of CH3 Cl to the atmosphere by methylotrophic bacteria, a global sink for this compound, is likely underestimated and remains poorly characterized. We identified and characterized CH3 Cl-degrading bacteria associated with intact and living tree fern plants of the species Cyathea australis by stable isotope probing (SIP) with 13 C-labelled CH3 Cl combined with metagenomics. Metagenome-assembled genomes (MAGs) related to Methylobacterium and Friedmanniella were identified as being involved in the degradation of CH3 Cl in the phyllosphere, i.e., the aerial parts of the tree fern, while a MAG related to Sorangium was linked to CH3 Cl degradation in the fern rhizosphere. The only known metabolic pathway for CH3 Cl degradation, via a methyltransferase system including the gene cmuA, was not detected in metagenomes or MAGs identified by SIP. Hence, a yet uncharacterized methylotrophic cmuA-independent pathway may drive CH3 Cl degradation in the investigated tree ferns.

Several bacteria are able to degrade the major industrial solvent dichloromethane (DCM) by using the conserved dehalogenase DcmA, the only system for DCM degradation characterised at the sequence level so far. Using differential proteomics, we rapidly identified key determinants of DCM degradation for Hyphomicrobium sp. MC8b, an unsequenced facultative methylotrophic DCM-degrading strain. For this, we designed a pan-proteomics database comprising the annotated genome sequences of 13 distinct Hyphomicrobium strains. Compared to growth with methanol, growth with DCM induces drastic changes in the proteome of strain MC8b. Dichloromethane dehalogenase DcmA was detected by differential pan-proteomics, but only with poor sequence coverage, suggesting atypical characteristics of the DCM dehalogenation system in this strain. More peptides were assigned to DcmA by error-tolerant search, warranting subsequent sequencing of the genome of strain MC8b, which revealed a highly divergent set of dcm genes in this strain. This suggests that the dcm enzymatic system is less strongly conserved than previously believed, and that substantial molecular evolution of dcm genes has occurred beyond their horizontal transfer in the bacterial domain. Our study showed the power of pan-proteomics for quick characterization of new strains belonging to branches of the Tree of Life that are densely genome-sequenced.

Organohalides are organic molecules formed biotically and abiotically, both naturally and through industrial production. They are usually toxic and represent a health risk for living organisms, including humans. Bacteria capable of degrading organohalides for growth express dehalogenase genes encoding enzymes that cleave carbon-halogen bonds. Such bacteria are of potential high interest for bioremediation of contaminated sites. Dehalogenase genes are often part of gene clusters that may include regulators, accessory genes and genes for transporters and other enzymes of organohalide degradation pathways. Organohalides and their degradation products affect the activity of regulatory factors, and extensive genome-wide modulation of gene expression helps dehalogenating bacteria to cope with stresses associated with dehalogenation, such as intracellular increase of halides, dehalogenase-dependent acid production, organohalide toxicity, and misrouting and bottlenecks in metabolic fluxes. This review focuses on transcriptional regulation of gene clusters for dehalogenation in bacteria, as studied in laboratory experiments and in situ. The diversity in gene content, organization, and regulation of such gene clusters is highlighted for representative organohalide-degrading bacteria. Selected examples illustrate a key, overlooked role of regulatory processes, often strain-specific, for efficient dehalogenation and productive growth in presence of organohalides.

Chloromethane is a halogenated volatile organic compound, produced in large quantities by terrestrial vegetation. After its release to the troposphere and transport to the stratosphere, its photolysis contributes to the degradation of stratospheric ozone. A better knowledge of chloromethane sources (production) and sinks (degradation) is a prerequisite to estimate its atmospheric budget in the context of global warming. The degradation of chloromethane by methylotrophic communities in terrestrial environments is a major underestimated chloromethane sink. Methylotrophs isolated from soils, marine environments and more recently from the phyllosphere have been grown under laboratory conditions using chloromethane as the sole carbon source. In addition to anaerobes that degrade chloromethane, the majority of cultivated strains were isolated in aerobiosis for their ability to use chloromethane as sole carbon and energy source. Among those, the Proteobacterium Methylobacterium (recently reclassified as Methylorubrum) harbours the only characterized ‘chloromethane utilization’ (cmu) pathway, so far. This pathway may not be representative of chloromethane-utilizing populations in the environment as cmu genes are rare in metagenomes. Recently, combined ‘omics’ biological approaches with chloromethane carbon and hydrogen stable isotope fractionation measurements in microcosms, indicated that microorganisms in soils and the phyllosphere (plant aerial parts) represent major sinks of chloromethane in contrast to more recently recognized microbe-inhabited environments, such as clouds. Cultivated chloromethane-degraders lacking cmu genes display a singular isotope fractionation signature of chloromethane. Moreover, CH3Cl labelling of active methylotrophic communities by stable isotope probing in soils identify taxa that differ from those known for chloromethane degradation. These observations suggest that new biomarkers for detecting active microbial chloromethane-utilizers in the environment are needed to assess the contribution of microorganisms to the global chloromethane cycle. Bringel et al. 150 |

Chloromethane is a halogenated volatile organic compound, produced in large quantities by terrestrial vegetation. After its release to the troposphere and transport to the stratosphere, its photolysis contributes to the degradation of stratospheric ozone. A better knowledge of chloromethane sources (production) and sinks (degradation) is a prerequisite to estimate its atmospheric budget in the context of global warming. The degradation of chloromethane by methylotrophic communities in terrestrial environments is a major underestimated chloromethane sink. Methylotrophs isolated from soils, marine environments and more recently from the phyllosphere have been grown under laboratory conditions using chloromethane as the sole carbon source. In addition to anaerobes that degrade chloromethane, the majority of cultivated strains were isolated in aerobiosis for their ability to use chloromethane as sole carbon and energy source. Among those, the Proteobacterium Methylobacterium (recently reclassified as Methylorubrum) harbours the only characterisized 'chloromethane utilization' (cmu) pathway, so far. This pathway is not representative of chloromethane-utilizing populations in the environment as cmu genes are rare in metagenomes. Recently, combined 'omics' biological approaches with chloromethane carbon and hydrogen stable isotope fractionation measurements in microcosms, indicated that microorganisms in soils and the phyllosphere (plant aerial parts) represent major sinks of chloromethane in contrast to more recently recognized microbe-inhabited environments, such as clouds. Cultivated chloromethane-degraders lacking the cmu genes display a singular isotope fractionation signature of chloromethane. Moreover, 13CH3Cl labelling of active methylotrophic communities by stable isotope probing in soils identify taxa that differ from the taxa known for chloromethane degradation. These observations suggest that new biomarkers for detecting active microbial chloromethane-utilizers in the environment are needed to assess the contribution of microorganisms to the global chloromethane cycle.

Flowers are essential but vulnerable plant organs, exposed to pollinators and florivores; however, flower chemical defenses are rarely investigated. We show here that two clustered terpene synthase and cytochrome P450 encoding genes (TPS11 and CYP706A3) on chromosome 5 of Arabidopsis (Arabidopsis thaliana) are tightly coexpressed in floral tissues, upon anthesis and during floral bud development. TPS11 was previously reported to generate a blend of sesquiterpenes. By heterologous coexpression of TPS11 and CYP706A3 in yeast (Saccharomyces cerevisiae) and Nicotiana benthamiana, we demonstrate that CYP706A3 is active on TPS11 products and also further oxidizes its own primary oxidation products. Analysis of headspace and soluble metabolites in cyp706a3 and 35S:CYP706A3 mutants indicate that CYP706A3-mediated metabolism largely suppresses sesquiterpene and most monoterpene emissions from opening flowers, and generates terpene oxides that are retained in floral tissues. In flower buds, the combined expression of TPS11 and CYP706A3 also suppresses volatile emissions and generates soluble sesquiterpene oxides. Florivory assays with the Brassicaceae specialist Plutella xylostella demonstrate that insect larvae avoid feeding on buds expressing CYP706A3 and accumulating terpene oxides. Composition of the floral microbiome appears also to be modulated by CYP706A3 expression. TPS11 and CYP706A3 simultaneously evolved within Brassicaceae and form the most versatile functional gene cluster described in higher plants so far.

Chloromethane is a halogenated volatile organic compound, produced in large quantities by terrestrial vegetation. After its release to the troposphere and transport to the stratosphere, its photolysis contributes to the degradation of stratospheric ozone. A better knowledge of chloromethane sources (production) and sinks (degradation) is a prerequisite to estimate its atmospheric budget in the context of global warming. The degradation of chloromethane by methylotrophic communities in terrestrial environments is a major underestimated chloromethane sink. Methylotrophs isolated from soils, marine environments and more recently from the phyllosphere have been grown under laboratory conditions using chloromethane as the sole carbon source. In addition to anaerobes that degrade chloromethane, the majority of cultivated strains were isolated in aerobiosis for their ability to use chloromethane as sole carbon and energy source. Among those, the Proteobacterium Methylobacterium (recently reclassified as Methylorubrum) harbours the only characterized ‘chloromethane utilization’ (cmu) pathway, so far. This pathway may not be representative of chloromethane-utilizing populations in the environment as cmu genes are rare in metagenomes. Recently, combined ‘omics’ biological approaches with chloromethane carbon and hydrogen stable isotope fractionation measurements in microcosms, indicated that microorganisms in soils and the phyllosphere (plant aerial parts) represent major sinks of chloromethane in contrast to more recently recognized microbe-inhabited environments, such as clouds. Cultivated chloromethane-degraders lacking cmu genes display a singular isotope fractionation signature of chloromethane. Moreover, CH3Cl labelling of active methylotrophic communities by stable isotope probing in soils identify taxa that differ from those known for chloromethane degradation. These observations suggest that new biomarkers for detecting active microbial chloromethane-utilizers in the environment are needed to assess the contribution of microorganisms to the global chloromethane cycle. Curr. Issues Mol. Biol. (2019) Vol. 33 caister.com/cimb Bringel et al. 150 |

Ecological, signaling, metabolic, and chemical processes in plant-microorganism systems and in plant-derived material may link the use of chlorinated pesticides in the environment with plant chloromethane emission. This neglected factor should be taken into account to assess global planetary budgets of chloromethane and impacts on atmospheric ozone depletion.

Chloromethane (CHCl, methyl chloride) is the most abundant volatile halocarbon in the atmosphere and involved in stratospheric ozone depletion. The global CHCl budget, and especially the CHCl sink from microbial degradation in soil, still involves large uncertainties. These may potentially be resolved by a combination of stable isotope analysis and bacterial diversity studies. We determined the stable isotope fractionation of CHCl hydrogen and carbon and investigated bacterial diversity during CHCl degradation in three soils with different properties (forest, grassland, and agricultural soils) and at different temperatures and headspace mixing ratios of CHCl. The extent of chloromethane degradation decreased in the order forest > grassland > agricultural soil. Rates ranged from 0.7 to 2.5 μg g dry wt. d for forest soil, from 0.1 to 0.9 μg g dry wt. d for grassland soil, and from 0.1 to 0.4 μg g dry wt. d for agricultural soil and increased with increasing temperature and CHCl supplementation. The measured mean stable hydrogen enrichment factor of CHCl of -50 ± 13‰ was unaffected by temperature, mixing ratio, or soil type. In contrast, the stable carbon enrichment factor depended on CHCl degradation rates and ranged from -38 to -11‰. Bacterial community composition correlated with soil properties was independent from CHCl degradation or isotope enrichment. Nevertheless, increased abundance after CHCl incubation was observed in 21 bacterial operational taxonomical units (OTUs at the 97% 16S RNA sequence identity level). This suggests that some of these bacterial taxa, although not previously associated with CHCl degradation, may play a role in the microbial CHCl sink in soil.

Halogenated volatile organic compounds (VOCs) emitted by terrestrial ecosystems, such as chloromethane (CH3Cl), have pronounced effects on troposphere and stratosphere chemistry and climate. The magnitude of the global CH3Cl sink is uncertain since it involves a largely uncharacterized microbial sink. CH3Cl represents a growth substrate for some specialized methylotrophs, while methanol (CH3OH), formed in much larger amounts in terrestrial environments, may be more widely used by such microorganisms. Direct measurements of CH3Cl degradation rates in two field campaigns and in microcosms allowed the identification of top soil horizons (i.e., organic plus mineral A horizon) as the major biotic sink in a deciduous forest. Metabolically active members of Alphaproteobacteria and Actinobacteria were identified by taxonomic and functional gene biomarkers following stable isotope labeling (SIP) of microcosms with CH3Cl and CH3OH, added alone or together as the [13C]-isotopologue. Well-studied reference CH3Cl degraders, such as Methylobacterium extorquens CM4, were not involved in the sink activity of the studied soil. Nonetheless, only sequences of the cmuA chloromethane dehalogenase gene highly similar to those of known strains were detected, suggesting the relevance of horizontal gene transfer for CH3Cl degradation in forest soil. Further, CH3Cl consumption rate increased in the presence of CH3OH. Members of Alphaproteobacteria and Actinobacteria were also 13C-labeled upon [13C]-CH3OH amendment. These findings suggest that key bacterial CH3Cl degraders in forest soil benefit from CH3OH as an alternative substrate. For soil CH3Cl-utilizing methylotrophs, utilization of several one-carbon compounds may represent a competitive advantage over heterotrophs that cannot utilize one-carbon compounds.

Chloromethane (CH3Cl) is the most abundant halogenated trace gas in the atmosphere. It plays an important role in natural stratospheric ozone destruction. Current estimates of the global CH3Cl budget are approximate. The strength of the CH3Cl global sink by microbial degradation in soils and plants is under discussion. Some plants, particularly ferns, have been identified as substantial emitters of CH3Cl. Their ability to degrade CH3Cl remains uncertain. In this study, we investigated the potential of leaves from 3 abundant ferns (Osmunda regalis, Cyathea cooperi, Dryopteris filix-mas) to produce and degrade CH3Cl by measuring their production and consumption rates and their stable carbon and hydrogen isotope signatures. Investigated ferns are able to degrade CH3Cl at rates from 2.1 to 17 and 0.3 to 0.9μggdw−1day−1 for C. cooperi and D. filix-mas respectively, depending on CH3Cl supplementation and temperature. The stable carbon isotope enrichment factor of remaining CH3Cl was −39±13‰, whereas negligible isotope fractionation was observed for hydrogen (−8±19‰). In contrast, O. regalis did not consume CH3Cl, but produced it at rates ranging from 0.6 to 128μggdw−1day−1, with stable isotope values of −97±8‰ for carbon and −202±10‰ for hydrogen, respectively. Even though the 3 ferns showed clearly different formation and consumption patterns, their leaf-associated bacterial diversity was not notably different. Moreover, we did not detect genes associated with the only known chloromethane utilization pathway “cmu” in the microbial phyllosphere of the investigated ferns. Our study suggests that still unknown CH3Cl biodegradation processes on plants play an important role in global cycling of atmospheric CH3Cl.