Project description:Our goal is to convert methane efficiently into liquid fuels that may be more readily transported. Since aerobic oxidation of methane is less efficient, we focused on anaerobic processes to capture methane, which are accomplished by anaerobic methanotrophic archaea (ANME) in consortia. However, no pure culture capable of oxidizing and growing on methane anaerobically has been isolated. In this study, Methanosarcina acetivorans, an archaeal methanogen, was metabolically engineered to take up methane, rather than to generate it. To capture methane, we cloned the DNA coding for the enzyme methyl-coenzyme M reductase (Mcr) from an unculturable archaeal organism from a Black Sea mat into M. acetivorans to effectively run methanogenesis in reverse. The engineered strain produces primarily acetate, and our results demonstrate that pure cultures can grow anaerobically on methane.
Project description:Transcriptional profiling of methanotrophic bacteria (pmoA gene) in methane oxidation biocover soil by depth Three-different depth condition in methane oxidation biocover soil: top, middle and botton layer soil: genomic DNA extract. Three replicate per array.
Project description:Natural and anthropogenic wetlands are main sources of the atmospheric greenhouse gas methane. Methane emissions from wetlands are mitigated by methanotrophic microorganisms and by processes at the oxic-anoxic interface, such as sulfur cycling, that reduce the activity of methanogens. In this study, we obtained a pure culture (strain HY1) of a versatile wetland methanotroph that oxidizes various organic and inorganic compounds. This strain represents (i) the first isolate that can aerobically oxidize both methane and reduced sulfur compounds and (ii) a new alphapoteobacterial species, named Candidatus Methylovirgula thiovorans. Genomic and proteomic analyses showed that soluble methane monooxygenase and XoxF-type alcohol dehydrogenases are the only enzymes for methane and methanol oxidation, respectively. Unexpectedly, strain HY1 harbors various pathways for respiratory sulfur oxidation and oxidized reduced sulfur compounds to sulfate using the Sox-rDsr pathway (without SoxCD) and the S4I system. It employed the Calvin-Benson-Bassham cycle for CO2 fixation during chemolithoautotrophic growth on the reduced sulfur compounds. Methane and thiosulfate were independently and simultaneously oxidized by strain HY1 for growth. Proteomic and microrespiratory analyses showed that the metabolic pathways for methane and thiosulfate oxidation were induced in the presence of their substrates. The discovery of this versatile methanotroph demonstrates that methanotrophy and thiotrophy is compatible in a single bacterium and adds a new aspect to interactions of methane and sulfur cycles in oxic-anoxic interface environments.
Project description:We established simple synthetic microbial communities in a microcosm model system to determine the mechanisms that underlay cross-feeding in microbial methane-consuming communities. Co-occurring strains from Lake Washington sediment were used that are involved in methane consumption, a methanotroph and two non-methanotrophic methylotrophs.
Project description:Chemosynthetic symbioses between bacteria and invertebrates occur worldwide in a wide range of marine habitats. Although they have been intensively investigated, molecular physiological studies of chemoautotrophic bacteria colonizing the surface of animals (ectosymbioses) are scarce. Stilbonematinae nematodes are the only known invertebrates capable of cultivating monocultures of thiotrophic Gammaproteobacteria on their surface. Crucially, as these nematodes migrate through the redox zone of marine sediments, the ectosymbionts directly experience drastic variations in oxygen concentration. Here, by applying an array of omics, Raman microspectroscopy and stable isotope labeling-based techniques, we investigated the effect of varying concentrations of dissolved oxygen on physiology and metabolism of Candidatus Thiosymbion oneisti, the longitudinally dividing ectosymbiont of Laxus oneistus. We show that, unexpectedly, sulfur oxidation genes were upregulated in anoxic relative to oxic conditions, and that carbon fixation genes and incorporation of 13C-labeled bicarbonate were not. Instead, several genes involved in carbon fixation in addition to genes responsible for assimilating organic carbon compounds and polyhydroxyalkanoate (PHA) biosynthesis, as well as nitrogen fixation and urea utilization genes were upregulated in oxic versus anoxic conditions. Furthermore, in the presence of oxygen, stress-related genes were upregulated together with vitamin and cofactor biosynthesis genes likely necessary to withstand its deleterious effects. Based on this first global physiological study of an uncultured, chemosynthetic ectosymbiont, we propose that, in anoxic pore water, it proliferates by utilizing nitrate to oxidize reduced sulfur compounds, whereas, when exposed to oxygen, it exploits aerobic respiration to facilitate energetically costly assimilation of carbon and nitrogen to survive oxidative stress. Both anaerobic sulfur oxidation and its decoupling from carbon fixation represent unprecedented adaptations among chemosynthetic symbionts. We postulate that Ca. T. oneisti originated from an obligate anaerobic, denitrifying sulfur-oxidizer, which, while transitioning from the free-living to the symbiotic lifestyle, evolved mechanisms to survive the oxidative stress inherent to a life attached to an animal.
Project description:Crude oil is the one of the most important natural assets of humankind, yet it is a major environmental pollutant, in particular, in marine environments. One of the largest crude oil polluted areas in the word is the semi-enclosed Mediterranean Sea, where the metabolic potential of indigenous populations towards the chronic pollution at a large scale is yet to be defined, particularly in anaerobic and micro-anaerobic marine sites. Here, we provided a novel insight into the active microbial metabolism in sediments from three environments along the coastline of Italy. Microbial proteomes exhibited prevalence in anaerobic metabolism, not related to the biodegradation directly, suggesting the strong limitation by oxygen induced by the carbon overload. They also point at previously unrecognized metabolic coupling between methane and methanol utilizers as well as sulfur reducers in marine petroleum polluted sediments.
2015-07-24 | PXD001490 | Pride
Project description:Aerobic and anaerobic methane oxidation in a seasonally anoxic basin, Mariager Fjord, Denmark
Project description:Our goal is to convert methane efficiently into liquid fuels that may be more readily transported. Since aerobic oxidation of methane is less efficient, we focused on anaerobic processes to capture methane, which are accomplished by anaerobic methanotrophic archaea (ANME) in consortia. However, no pure culture capable of oxidizing and growing on methane anaerobically has been isolated. In this study, Methanosarcina acetivorans, an archaeal methanogen, was metabolically engineered to take up methane, rather than to generate it. To capture methane, we cloned the DNA coding for the enzyme methyl-coenzyme M reductase (Mcr) from an unculturable archaeal organism from a Black Sea mat into M. acetivorans to effectively run methanogenesis in reverse. The engineered strain produces primarily acetate, and our results demonstrate that pure cultures can grow anaerobically on methane. Differential gene analysis of two growth conditions (three biological replicates each) was performed: (i) M. acetivorans/pES1-MATmcr3 grown on methane and (ii) M. acetivorans/pES1-MATmcr3 grown on methanol. All starter cultures (200 mL) were grown on methanol for 5 days, and harvested by centrifugation. Cell pellets were washed three times with HS medium, and resuspended using 5 mL HS medium, 2 µg/mL puromycin, and 0.1 mM FeCl3. For condition (i), methane was filled into the headspace of the cultures. For condition (ii), 150 mM methanol was added. All cultures were incubated at 37C for 5 days, followed by rapid centrifugation in the presence of 50 µL RNAlater solution (Ambion, Austin, TX) per mL of culture. Total RNA was isolated using RNeasy Mini kit (Qiagen, Valencia, CA) were then digested with terminator 5â-phosphate-dependent exonuclease (Epicentre, Madison, WI) to partially remove ribosomal RNA. Digested RNA were cleaned up using AgenCourt RNAClean XP beads (AgenCourt Bioscience, Beverly, MA) and used for cDNA library construction using the TruSeq Stranded mRNA Library kit (Illumina). Pooled and barcoded cDNA library was then sequenced on a HiSeq sequencing platform (Illumina). Obtained reads were mapped to the reference genome of M. acetivorans (Genbank accession NC_003552.1) using STAR. The mapped reads were assembled using Cufflink v2.2.1 to identify potential novel transcripts. Assembled, unannotated novel transcripts for all the strains were combined with the list of known genes. Differential expression of genes and potential novel transcripts were determined using Cuffdiff at a significance cutoff at q < 0.07 with a false discovery rate of 0.05. Expression levels of gene transcripts are expressed as fragments per kilobase of transcript per million mapped fragments (FPKM), and expression changes are determined by the ratio of FPKM of culture replicates grown on methane to FPKM of culture replicates grown on methanol.
Project description:Aerobic methanotrophic bacteria use methane as their sole source of carbon and energy and serve as a major sink for the potent greenhouse gas methane in freshwater ecosystems. Despite this important environmental role, little is known about the molecular details of how these organisms interact in the environment. Many bacterial species use quorum sensing systems to regulate gene expression in a density-dependent manner. We have identified a quorum sensing system in the genome of Methylobacter tundripaludum, a dominant methane-oxidizer in methane enrichments of sediment from Lake Washington (Seattle, WA, USA). We determined that M. tundripaludum primarily produces N-3-hydroxydecanoyl-L-homoserine lactone (3-OH-C10-HSL) and that production is governed by a positive feedback loop. We then further characterized this system by determining which genes are regulated by quorum sensing in this methane-oxidizer using RNA-seq, and discovered this system regulates the expression of a novel nonribosomal peptide synthetase biosynthetic gene cluster. These results identify and characterize a mode of cellular communication in an aerobic methane-oxidizing bacterium.