Identification of Proteins and Genes Expressed by Methylophaga thiooxydans During Growth on Dimethylsulfide and Their Presence in Other Members of the Genus

Dimethylsulfide is a volatile organic sulfur compound that provides the largest input of biogenic sulfur from the oceans to the atmosphere, and thence back to land, constituting an important link in the global sulfur cycle. Microorganisms degrading DMS affect fluxes of DMS in the environment, but the underlying metabolic pathways are still poorly understood. Methylophaga thiooxydans is a marine methylotrophic bacterium capable of growth on DMS as sole source of carbon and energy. Using proteomics and transcriptomics we identified genes expressed during growth on dimethylsulfide and methanol to refine our knowledge of the metabolic pathways that are involved in DMS and methanol degradation in this strain. Amongst the most highly expressed genes on DMS were the two methanethiol oxidases driving the oxidation of this reactive and toxic intermediate of DMS metabolism. Growth on DMS also increased expression of the enzymes of the tetrahydrofolate linked pathway of formaldehyde oxidation, in addition to the tetrahydromethanopterin linked pathway. Key enzymes of the inorganic sulfur oxidation pathway included flavocytochrome c sulfide dehydrogenase, sulfide quinone oxidoreductase, and persulfide dioxygenases. A sulP permease was also expressed during growth on DMS. Proteomics and transcriptomics also identified a number of highly expressed proteins and gene products whose function is currently not understood. As the identity of some enzymes of organic and inorganic sulfur metabolism previously detected in Methylophaga has not been characterized at the genetic level yet, highly expressed uncharacterized genes provide new targets for further biochemical and genetic analysis. A pan-genome analysis of six available Methylophaga genomes showed that only two of the six investigated strains, M. thiooxydans and M. sulfidovorans have the gene encoding methanethiol oxidase, suggesting that growth on methylated sulfur compounds of M. aminisulfidivorans is likely to involve different enzymes and metabolic intermediates. Hence, the pathways of DMS-utilization and subsequent C1 and sulfur oxidation are not conserved across Methylophaga isolates that degrade methylated sulfur compounds

Microorganisms associated with Sporobolus anglicus, an invasive dimethylsulfoniopropionate producing salt marsh plant, are an unrecognized sink for dimethylsulfide

Background: Saltmarshes are hotspots of organosulfur compound cycling due to production of dimethylsulfoniopropionate (DMSP) by benthic microorganisms, macroalgae, and saltmarsh vegetation. Degradation of DMSP is a source of dimethylsulfide (DMS), an important precursor for formation of secondary organic aerosol. Microorganisms degrading DMS play a role in controlling the amount of DMS available for emission into the atmosphere. Previous work has implicated sediment microbial populations as a major sink for DMS. Here, we show that Sporobolus anglicus (previously known as Spartina anglica), a widely distributed saltmarsh plant, is colonized by DMS-degrading microorganisms. Methods: Dimethylsulfide degradation potential was assessed by gas chromatography and 13C-DMS stable isotope probing, microbial community diversity and functional genetic potential in phyllosphere and rhizosphere samples was assessed by high-throughput sequencing of 16S rRNA gene amplicons, cloning and sequencing of methanethiol oxidase genes, and by metagenomic analysis of phyllosphere microbial communities. Results: The DMS degradation potential of microbial communities recovered from phyllosphere and rhizosphere samples was similar. Active DMS-degraders were identified by 13C-DMS stable isotope probing and included populations related to Methylophaga and other Piscirickettsiaceae in rhizosphere samples. DMS-degraders in the phyllosphere included Xanthomonadaceae and Halothiobacillaceae. The diversity in sediment samples of the methanethiol oxidase (mtoX) gene, a marker for metabolism of methanethiol during DMS and DMSP degradation, was similar to previously detected saltmarsh mtoX, including those of Methylophaga and Methylococcaeae. Phyllosphere mtoX genes were distinct from sediment mtoX and did not include close relatives of cultivated bacteria. Microbial diversity in the phyllosphere of S. anglicus was distinct compared to those of model plants such as rice, soybean, clover and Arabidopsis and showed a dominance of Gammaproteobacteria rather than Alphaproteobacteria. Conclusion: The potential for microbial DMS degradation in the phyllosphere and rhizosphere of Sporobolus anglicus suggest that DMS cycling in saltmarshes is more complex than previously recognised and calls for a more detailed assessment of how aboveground activities affect fluxes of DMS

Comparative genomics analyses indicate differential methylated amine utilization trait within members of the genus Gemmobacter

Methylated amines are ubiquitous in the environment and play a role in regulating the earth's climate via a set of complex biological and chemical reactions. Microbial degradation of these compounds is thought to be a major sink. Recently we isolated a facultative methylotroph, Gemmobacter sp. LW‐1, an isolate from the unique environment Movile Cave, Romania, which is capable of methylated amine utilization as a carbon source. Here, using a comparative genomics approach, we investigate how widespread methylated amine utilization is within members of the bacterial genus Gemmobacter. Seven genomes of different Gemmobacter species isolated from diverse environments, such as activated sludge, fresh water, sulphuric cave waters (Movile Cave) and the marine environment were available from the public repositories and used for the analysis. Our results indicate that methylamine utilization is a distinctive feature of selected members of the genus Gemmobacter, namely G. aquatilis, G. lutimaris, G. sp. HYN0069, G. caeni and G. sp. LW‐1 have the genetic potential while others (G. megaterium and G. nectariphilus) have not

Identification of proteins and genes expressed by Methylophaga thiooxydans during growth on dimethylsulfide and their presence in other members of the genus

Dimethylsulfide is a volatile organic sulfur compound that provides the largest input of biogenic sulfur from the oceans to the atmosphere, and thence back to land, constituting an important link in the global sulfur cycle. Microorganisms degrading DMS affect fluxes of DMS in the environment, but the underlying metabolic pathways are still poorly understood. Methylophaga thiooxydans is a marine methylotrophic bacterium capable of growth on DMS as sole source of carbon and energy. Using proteomics and transcriptomics we identified genes expressed during growth on dimethylsulfide and methanol to refine our knowledge of the metabolic pathways that are involved in DMS and methanol degradation in this strain. Amongst the most highly expressed genes on DMS were the two methanethiol oxidases driving the oxidation of this reactive and toxic intermediate of DMS metabolism. Growth on DMS also increased expression of the enzymes of the tetrahydrofolate linked pathway of formaldehyde oxidation, in addition to the tetrahydromethanopterin linked pathway. Key enzymes of the inorganic sulfur oxidation pathway included flavocytochrome c sulfide dehydrogenase, sulfide quinone oxidoreductase, and persulfide dioxygenases. A sulP permease was also expressed during growth on DMS. Proteomics and transcriptomics also identified a number of highly expressed proteins and gene products whose function is currently not understood. As the identity of some enzymes of organic and inorganic sulfur metabolism previously detected in Methylophaga has not been characterised at the genetic level yet, highly expressed uncharacterised genes provide new targets for further biochemical and genetic analysis. A pan-genome analysis of six available Methylophaga genomes showed that only two of the six investigated strains, M. thiooxydans and M. sulfidovorans have the gene encoding methanethiol oxidase, suggesting that growth on methylated sulfur compounds of M. aminisulfidivorans is likely to involve different enzymes and metabolic intermediates. Hence, the pathways of DMS-utilization and subsequent C1 and sulfur oxidation are not conserved across Methylophaga isolates that degrade methylated sulfur compounds

Environmental genomics and proteomics of plant-associated microbial dimethylsulfide degradation in a coastal salt marsh

The methylated sulfur compound dimethylsulfide (DMS) plays a major role in the biogeochemical sulfur cycle and atmospheric chemistry. Bacteria are a main sink for DMS in the global sulfur cycle and can utilise DMS as a sole carbon and energy source. This study investigated the diversity and activity of bacteria capable of DMS degradation and associated with the salt marsh plant Spartina anglica known to be a producer of the DMS precursor dimethylsulfoniopropionate (DMSP). Initially, it was shown that S. anglica is rich in DMSP throughout the entire seasonal cycle in the Stiffkey salt marsh providing a likely hotspot for DMSP- and DMS-degrading bacteria. DMS uptake experiments demonstrated that DMS degradation takes place in the phyllosphere and rhizosphere of S. anglica and denaturing gradient gel electrophoresis (DGGE) and high-throughput amplicon sequencing of the 16S rRNA revealed the dominance of bacteria related to α - and γ- Proteobacteria, as well as Flavobacteria in the phyllosphere of S. anglica, whereas the rhizosphere was mainly colonised by members of the classes γ-, δ-, α-, and ε-Proteobacteria and Bacteroidia. The diversity of DMS-degrading bacteria associated with S. anglica was first assessed by enrichment culture. DGGE analysis and high-throughput sequencing diversity of DMS enriched samples using the 16S rRNA gene as a marker suggested the dominance of Piscirickettsiaceae, Methylophaga and Methylophaga-like bacteria in DMS-enrichments of phyllosphere and rhizosphere samples of S. anglica. A functional gene marker analyses was carried out using the gene encoding methanethiol oxidase (mtoX), a key enzyme in DMS degradation and the gene encoding a DMSP lyase (dddP) to determine the diversity of bacteria degrading DMS and DMSP, respectively, in the phyllosphere and rhizosphere of S. anglica. The analysis for mtoX showed a great diversity of this gene in phyllosphere and rhizosphere of S. anglica and that major clades of mtoX clustered together with Sedimenticola, Methylohalobius, Methylophaga and other mtoX clones previously detected in surface sediments of the same salt marsh. The results for the functional marker gene analysis for the dddP gene suggested the dominance of Ruegeria-like species and Roseobacter-like bacteria but also of unidentified Ddd+ bacteria in the phyllosphere and rhizosphere of S. anglica. In order to identify the active DMS degraders in the phyllosphere and rhizosphere of S. anglica stable-isotope probing (SIP) combined with DGGE and highthroughput sequencing of the 16S rRNA gene was carried out. The SIP experiments revealed the dominance of Piscirickettsiaceae, Methylophaga and Methylophaga-like microorganisms in the rhizosphere of S. anglica. However, the DMS-degrading microbial community in the phyllosphere seemed more diverse than in the rhizosphere and microorganisms like Halothiobacillus, Xanthomonadaceae, Rhodanobacter but also Piscirickettsiaceae seemed to be involved. A comparative proteomic and transcriptomic experiment of Methylophaga thiooxydans, a microorganism found in phyllosphere and rhizosphere of S. anglica, revealed the general pathways involved in methanol but especially DMS degradation. During DMS cycling the protein and the protein-encoding gene for the methanethiol oxidase (MtoX/mtoX) was highly expressed. A metaproteogenomic experiment provided an insight into the taxonomy and functional diversity of the microbial community associated with the Spartina anglica phyllosphere. Analysis of the metagenome provided evidence that the microbial community associated with S. anglica is dominated by γ-Proteobacteria such as Halomonadales, Alteromonadales, Oceanospirillales, and Thiotrichales and the alphaproteobacterial order Rhodobacterales and showed therefore a major difference to the bacterial community composition in the phyllosphere of for instance A. thaliana, clover, soybean and rice. The detection of DMSP lyase encoding genes and genes encoding proteins for DMS degradation confirmed the genetic potential for the observed DMSP and DMS degradation activity previously measured in the phyllosphere of S. anglica. The metaproteomic experiment allowed a first insight into the proteins expressed in the phyllosphere of S. anglica which also suggested that mainly γ-Proteobacteria and α-Proteobacteria are dominant populations occurring in this habitat. New insights were gained into the activity and diversity of DMS-degrading microbial communities associated with a salt marsh plant that represents a significant component of salt marsh plant communities world wide. Not only was the taxonomic and functional diversity of DMS-degrading microorganisms associated with S. anglica greater then previously realised, the observation of considerable potential of above-ground plant-associated DMS degradation in the phyllosphere demonstrates a previously unrealised sink in the DMS cycle in coastal ecosystems, which is clearly more complex than previously appreciated

Data_Sheet_1_Microorganisms associated with Sporobolus anglicus, an invasive dimethylsulfoniopropionate producing salt marsh plant, are an unrecognized sink for dimethylsulfide.zip

BackgroundSaltmarshes are hotspots of organosulfur compound cycling due to production of dimethylsulfoniopropionate (DMSP) by benthic microorganisms, macroalgae, and saltmarsh vegetation. Degradation of DMSP is a source of dimethylsulfide (DMS), an important precursor for formation of secondary organic aerosol. Microorganisms degrading DMS play a role in controlling the amount of DMS available for emission into the atmosphere. Previous work has implicated sediment microbial populations as a major sink for DMS. Here, we show that Sporobolus anglicus (previously known as Spartina anglica), a widely distributed saltmarsh plant, is colonized by DMS-degrading microorganisms.MethodsDimethylsulfide degradation potential was assessed by gas chromatography and 13C-DMS stable isotope probing, microbial community diversity and functional genetic potential in phyllosphere and rhizosphere samples was assessed by high-throughput sequencing of 16S rRNA gene amplicons, cloning and sequencing of methanethiol oxidase genes, and by metagenomic analysis of phyllosphere microbial communities.ResultsThe DMS degradation potential of microbial communities recovered from phyllosphere and rhizosphere samples was similar. Active DMS-degraders were identified by 13C-DMS stable isotope probing and included populations related to Methylophaga and other Piscirickettsiaceae in rhizosphere samples. DMS-degraders in the phyllosphere included Xanthomonadaceae and Halothiobacillaceae. The diversity in sediment samples of the methanethiol oxidase (mtoX) gene, a marker for metabolism of methanethiol during DMS and DMSP degradation, was similar to previously detected saltmarsh mtoX, including those of Methylophaga and Methylococcaeae. Phyllosphere mtoX genes were distinct from sediment mtoX and did not include close relatives of cultivated bacteria. Microbial diversity in the phyllosphere of S. anglicus was distinct compared to those of model plants such as rice, soybean, clover and Arabidopsis and showed a dominance of Gammaproteobacteria rather than Alphaproteobacteria.ConclusionThe potential for microbial DMS degradation in the phyllosphere and rhizosphere of Sporobolus anglicus suggest that DMS cycling in saltmarshes is more complex than previously recognised and calls for a more detailed assessment of how aboveground activities affect fluxes of DMS.</p

A putatively new family of alphaproteobacterial chloromethane degraders from a deciduous forest soil revealed by stable isotope probing and metagenomics

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

Methanol consumption drives the bacterial chloromethane sink in a forest 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 [C-13]-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 C-13-labeled upon [C-13]-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

Microbial sink of an abundant halogenated hydrocarbon in soil and at the soil-plant-atmosphere interphase - Microorganisms as sinks of atmospheric chloromethane

International audienc

Methylotrophs and methylotroph populations for chloromethane degradation

International audienceChloromethane 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 (pro-duction) 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 phyl-losphere (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 frac-tionation signature of chloromethane. Moreover, 13 CH 3 Cl 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 chlo-romethane-utilizers in the environment are needed to assess the contribution of microorganisms to the global chloromethane cycle