Showing papers on "Methanosarcina barkeri published in 2014"

Direct Interspecies Electron Transfer between Geobacter metallireducens and Methanosarcina barkeri

Abstract: Direct interspecies electron transfer (DIET) is potentially an effective form of syntrophy in methanogenic communities, but little is known about the diversity of methanogens capable of DIET. The ability of Methanosarcina barkeri to participate in DIET was evaluated in coculture with Geobacter metallireducens. Cocultures formed aggregates that shared electrons via DIET during the stoichiometric conversion of ethanol to methane. Cocultures could not be initiated with a pilin-deficient G. metallireducens strain, suggesting that long-range electron transfer along pili was important for DIET. Amendments of granular activated carbon permitted the pilin-deficient G. metallireducens isolates to share electrons with M. barkeri, demonstrating that this conductive material could substitute for pili in promoting DIET. When M. barkeri was grown in coculture with the H2-producing Pelobacter carbinolicus, incapable of DIET, M. barkeri utilized H2 as an electron donor but metabolized little of the acetate that P. carbinolicus produced. This suggested that H2, but not electrons derived from DIET, inhibited acetate metabolism. P. carbinolicus-M. barkeri cocultures did not aggregate, demonstrating that, unlike DIET, close physical contact was not necessary for interspecies H2 transfer. M. barkeri is the second methanogen found to accept electrons via DIET and the first methanogen known to be capable of using either H2 or electrons derived from DIET for CO2 reduction. Furthermore, M. barkeri is genetically tractable, making it a model organism for elucidating mechanisms by which methanogens make biological electrical connections with other cells.

The Alternative Route to Heme in the Methanogenic Archaeon Methanosarcina barkeri

Abstract: In living organisms heme is formed from the common precursor uroporphyrinogen III by either one of two substantially different pathways. In contrast to eukaryotes and most bacteria which employ the so-called “classical” heme biosynthesis pathway, the archaea use an alternative route. In this pathway, heme is formed from uroporphyrinogen III via the intermediates precorrin-2, sirohydrochlorin, siroheme, 12,18-didecarboxysiroheme, and iron-coproporphyrin III. In this study the heme biosynthesis proteins AhbAB, AhbC, and AhbD from Methanosarcina barkeri were functionally characterized. Using an in vivo enzyme activity assay it was shown that AhbA and AhbB (Mbar_A1459 and Mbar_A1460) together catalyze the conversion of siroheme into 12,18-didecarboxysiroheme. The two proteins form a heterodimeric complex which might be subject to feedback regulation by the pathway end-product heme. Further, AhbC (Mbar_A1793) was shown to catalyze the formation of iron-coproporphyrin III in vivo. Finally, recombinant AhbD (Mbar_A1458) was produced in E. coli and purified indicating that this protein most likely contains two [4Fe-4S] clusters. Using an in vitro enzyme activity assay it was demonstrated that AhbD catalyzes the conversion of iron-coproporphyrin III into heme.

Methanosarcina spelaei sp. nov., a methanogenic archaeon isolated from a floating biofilm of a subsurface sulphurous lake.

Abstract: A novel methanogenic archaeon, strain MC-15T, was isolated from a floating biofilm on a sulphurous subsurface lake in Movile Cave (Mangalia, Romania). Cells were non-motile sarcina-like cocci with a diameter of 2–4 µm, occurring in aggregates. The strain was able to grow autotrophically on H2/CO2. Additionally, acetate, methanol, monomethylamine, dimethylamine and trimethylamine were utilized, but not formate or dimethyl sulfide. Trypticase peptone and yeast extract were not required for growth. Optimal growth was observed at 33 °C, pH 6.5 and a salt concentration of 0.05 M NaCl. The predominant membrane lipids of MC-15T were archaeol and hydroxyarchaeol phosphatidylethanolamine, phosphatidylglycerol, and phosphatidylinositol as well as hydroxyarchaeol phosphatidylserine and archaeol glycosaminyl phosphatidylinositol. The closely related species, Methanosarcina vacuolata and Methanosarcina horonobensis, had a similar composition of major membrane lipids to strain MC-15T. The 16S rRNA gene sequence of strain MC-15T was similar to those of Methanosarcina vacuolata DSM 1232T (sequence similarity 99.3 %), Methanosarcina horonobensis HB-1T (98.8 %), Methanosarcina barkeri DSM 800T (98.7 %) and Methanosarcina siciliae T4/MT (98.4 %). DNA–DNA hybridization revealed 43.3 % relatedness between strain MC-15T and Methanosarcina vacuolata DSM 1232T. The G+C content of the genomic DNA was 39.0 mol%. Based on physiological, phenotypic and genotypic differences, strain MC-15T represents a novel species of the genus Methanosarcina , for which the name Methanosarcina spelaei sp. nov. is proposed. The type strain is MC-15T ( = DSM 26047T = JCM 18469T).

A genome-scale metabolic model of Methanococcus maripaludis S2 for CO2 capture and conversion to methane

Abstract: Methane is a major energy source for heating and electricity. Its production by methanogenic bacteria is widely known in nature. M. maripaludis S2 is a fully sequenced hydrogenotrophic methanogen and an excellent laboratory strain with robust genetic tools. However, a quantitative systems biology model to complement these tools is absent in the literature. To understand and enhance its methanogenesis from CO2, this work presents the first constraint-based genome-scale metabolic model (iMM518). It comprises 570 reactions, 556 distinct metabolites, and 518 genes along with gene-protein-reaction (GPR) associations, and covers 30% of open reading frames (ORFs). The model was validated using biomass growth data and experimental phenotypic studies from the literature. Its comparison with the in silico models of Methanosarcina barkeri, Methanosarcina acetivorans, and Sulfolobus solfataricus P2 shows M. maripaludis S2 to be a better organism for producing methane. Using the model, genes essential for growth were identified, and the efficacies of alternative carbon, hydrogen and nitrogen sources were studied. The model can predict the effects of reengineering M. maripaludis S2 to guide or expedite experimental efforts.

Optimized Plasmid Systems for the Incorporation of Multiple Different Unnatural Amino Acids by Evolved Orthogonal Ribosomes

Abstract: Incorporation of multiple different unnatural amino acids into the same polypeptide remains a significant challenge. Orthogonal ribosomes, which are evolvable as they direct the translation of a single dedicated orthogonal mRNA, can provide an avenue to produce such polypeptides routinely. Recent advances in engineering orthogonal ribosomes have created a prototype system to enable genetically encoded introduction of two different functional groups, albeit with limited efficiency. Here, we systematically investigated the limiting factors of this system by using assays to measure the levels and activities of individual components; we identified Methanosarcina barkeri PylRS as a limiting factor for protein yield. Balancing the expression levels of individual components significantly improved growth rate and protein yield. This optimization of the system is likely to increase the scope of evolved orthogonal ribosome-mediated incorporation of multiple different unnatural amino acids.

The structure, function and properties of sirohaem decarboxylase – an enzyme with structural homology to a transcription factor family that is part of the alternative haem biosynthesis pathway

Abstract: Some bacteria and archaea synthesize haem by an alternative pathway, which involves the sequestration of sirohaem as a metabolic intermediate rather than as a prosthetic group. Along this pathway the two acetic acid side-chains attached to C12 and C18 are decarboxylated by sirohaem decarboxylase, a heterodimeric enzyme composed of AhbA and AhbB, to give didecarboxysirohaem. Further modifications catalysed by two related radical SAM enzymes, AhbC and AhbD, transform didecarboxysirohaem into Fe-coproporphyrin III and haem respectively. The characterization of sirohaem decarboxylase is reported in molecular detail. Recombinant versions of Desulfovibrio desulfuricans, Desulfovibrio vulgaris and Methanosarcina barkeri AhbA/B have been produced and their physical properties compared. The D. vulgaris and M. barkeri enzyme complexes both copurify with haem, whose redox state influences the activity of the latter. The kinetic parameters of the D. desulfuricans enzyme have been determined, the enzyme crystallized and its structure has been elucidated. The topology of the enzyme reveals that it shares a structural similarity to the AsnC/Lrp family of transcription factors. The active site is formed in the cavity between the two subunits and a AhbA/B-product complex with didecarboxysirohaem has been obtained. A mechanism for the decarboxylation of the kinetically stable carboxyl groups is proposed.

The alternative haem biosynthesis pathway : structure, function and properties of sirohaem decarboxylase

Abstract: Haem, a cyclic tetrapyrrole, is found in organisms from all three domains of life. Haem is a prosthetic group for many proteins involved in essential biological processes such as respiration and oxygen transport. Synthesis of haem in eukaryotes and most bacteria follows a well defined route with highly conserved intermediates. However, an alternative haem biosynthesis pathway in Archaea and some bacteria was recently elucidated. This newly discovered pathway utilises sirohaem as a metabolic intermediate rather than a prosthetic group. The alternative haem biosynthesis pathway is catalysed by the Ahb enzymes A, B, C and D. Initial decarboxylation of sirohaem occurs at the two acetic acid side-chains attached to carbons C12 and C18 to give didecarboxysirohaem, a process catalysed by AhbA and AhbB. Subsequently, the radical SAM enzyme AhbC converts didecarboxysirohaem to Fe-coproporphyrin III. Finally, AhbD catalyses the conversion of Fe-coproporphyrin III into haem in another SAM dependent reaction. This study focused on the AhbA and AhbB proteins from three sources, Desulfovibrio desulfuricans, Desulfovibrio vulgaris and Methanosarcina barkeri. Purifications of individual recombinantly produced proteins revealed that both AhbA and AhbB are highly unstable. However, low concentrations of D. desulfuricans and D. vulgaris AhbA and AhbB proteins were isolated and were discovered to have decarboxylase activity. Simultaneous overproduction of AhbA and AhbB proteins facilitated the co-purification of stable heteromeric AhbA/B complexes from all three organisms. However, despite their sequence similarities, distinctly different properties were observed between the homologues including differences in oliogmeric state and haem/product binding capabilities. The D. desulfuricans enzyme has been crystallised and its structure has been elucidated in both apo- and product-bound forms. Mutagenesis of the D. desulfuricans complex has provided further information about active site residues and a mechanism of action has been proposed. Finally, novel functional chimeric complexes were produced using the Desulfovibrio proteins.

Interspecies Electron Transfer in environmental and engineered syntrophic systems

Abstract: Syntrophic Interspecies Electron Transfer (IET) has been observed in anoxic environments and mainly requires intermediates such as hydrogen or formate as electron carriers. However direct IET (DIET) is also possible and requires that electrons are transferred via membrane-bound proteins or conductive appendages. Engineering of multiple artificial syntrophies was attempted to study these concepts. Growth of cultures proved very difficult due to long establishment times and slow adaptation. Three artificial syntrophies were confirmed by growth curves, chemical analysis, and microscopic analysis. These included a tri-culture of Syntrophobacter wolinii, Methanobrevibacter smithii and Methanosaeta concilii, degrading propionate, bidirectional co cultures with Shewanella oneidensis and Methanosarcina barkeri first growing on lactate and secondly on acetate with hydrous ferric oxide (HFO) or a carbon anode. Bidirectional syntrophies indicated that syntrophic associations can extend beyond those systems observed and isolated to date. This work also focused on the possibilities of stimulating and enhancing DIET in environmental cultures. A number of hypotheses were tested, including the presence of proton carrying buffers and increase in salinity. Since direct electron transport requires cation/proton transport, the presence of buffers such as phosphate should enhance rates where DIET dominates. This was tested by varying initial conductivity (1.5mS/cm, X10, X30) and total phosphate: chloride ratios (1:0, 1:1, 2:1, 1:2 and 0:1), in a square factorial analysis, during propionic acid oxidation and ethanol oxidation, with crushed anaerobic granules as inoculum. Microbial communities were analysed through 16S rRNA gene pyrosequencing and fluorescent in situ hybridisation (FISH). With propionic acid oxidation, key microbial shifts with increasing salinity were from Syntrophomonas sp to Syntrophobacter sp and Candidatus Cloacamonas sp within bacteria and Methanobacterium sp to Methanolinea sp. Results suggested an indirect IET (IIET) system. With ethanol, there was a higher percentage of Geobacter sp, capable of DIET, and Methanosaeta sp, especially at 1:1 and X10 conductivity where the best rate was recorded. Low methane yield was observed at X30 conductivity, likely due to the reduction of ethanol to higher organic acids. In both assays, responses were better at 1:1 ratio, with the best kinetic values recorded at X10 conductivity. Acclimatisation with propionic acid at high conductivities (X30), over 6 months was also tested to reduce the impact of salt shock. New granules were acclimatised at 1:0, 1:1 and 0:1 ratio. Activity was low at ratio 1:0 but improved at 1:1. Complete oxidation with KCl only was attributed to emerging Comamonas sp as presumptive oxidiser.n

A new DIET for Methanosarcina barkeri: direct interspecies electron transfer in a genetically tractable methanogen.

Abstract: Project Goals: The long-term goal of our project, which is entitled “Systems Level Analysis of the Function and Adaptive Responses of Methanogenic Consortia”, is to develop genome-scale metabolic models of microbial communities that play an important role in the global carbon cycle that can be coupled with the appropriate physical-chemical models to predict how the microbial communities will respond to environmental perturbations, such as climate change. The short-term objective of the current research is to elucidate the mechanisms for direct interspecies electron transfer (DIET), the diversity of methanogens that are capable of DIET, and the prevalence of DIET in soils and sediments that make important contributions to atmospheric methane.