Showing papers on "Methanosarcina barkeri published in 2013"

Guided cobalamin biosynthesis supports Dehalococcoides mccartyi reductive dechlorination activity

Abstract: Dehalococcoides mccartyi strains are corrinoid-auxotrophic Bacteria and axenic cultures that require vitamin B12 (CN-Cbl) to conserve energy via organohalide respiration. Cultures of D. mccartyi strains BAV1, GT and FL2 grown with limiting amounts of 1 µg l(-1) CN-Cbl quickly depleted CN-Cbl, and reductive dechlorination of polychlorinated ethenes was incomplete leading to vinyl chloride (VC) accumulation. In contrast, the same cultures amended with 25 µg l(-1) CN-Cbl exhibited up to 2.3-fold higher dechlorination rates, 2.8-9.1-fold increased growth yields, and completely consumed growth-supporting chlorinated ethenes. To explore whether known cobamide-producing microbes supply Dehalococcoides with the required corrinoid cofactor, co-culture experiments were performed with the methanogen Methanosarcina barkeri strain Fusaro and two acetogens, Sporomusa ovata and Sporomusa sp. strain KB-1, as Dehalococcoides partner populations. During growth with H2/CO2, M. barkeri axenic cultures produced 4.2 ± 0.1 µg l(-1) extracellular cobamide (factor III), whereas the Sporomusa cultures produced phenolyl- and p-cresolyl-cobamides. Neither factor III nor the phenolic cobamides supported Dehalococcoides reductive dechlorination activity suggesting that M. barkeri and the Sporomusa sp. cannot fulfil Dehalococcoides' nutritional requirements. Dehalococcoides dechlorination activity and growth occurred in M. barkeri and Sporomusa sp. co-cultures amended with 10 µM 5',6'-dimethylbenzimidazole (DMB), indicating that a cobalamin is a preferred corrinoid cofactor of strains BAV1, GT and FL2 when grown with chlorinated ethenes as electron acceptors. Even though the methanogen and acetogen populations tested did not produce cobalamin, the addition of DMB enabled guided biosynthesis and generated a cobalamin that supported Dehalococcoides' activity and growth. Guided cobalamin biosynthesis may offer opportunities to sustain and enhance Dehalococcoides activity in contaminated subsurface environments.

Genomically and biochemically accurate metabolic reconstruction of Methanosarcina barkeri Fusaro, iMG746

Abstract: Methanosarcina barkeri is an Archaeon that produces methane anaerobically as the primary byproduct of its metabolism. M. barkeri can utilize several substrates for ATP and biomass production including methanol, acetate, methyl amines, and a combination of hydrogen and carbon dioxide. In 2006, a metabolic reconstruction of M. barkeri, iAF692, was generated based on a draft genome annotation. The iAF692 reconstruction enabled the first genome-Scale simulations for Archaea. Since the publication of the first metabolic reconstruction of M. barkeri, additional genomic, biochemical, and phenotypic data have clarified several metabolic pathways. We have used this newly available data to improve the M. barkeri metabolic reconstruction. Modeling simulations using the updated model, iMG746, have led to increased accuracy in predicting gene knockout phenotypes and simulations of batch growth behavior. We used the model to examine knockout lethality data and make predictions about metabolic regulation under different growth conditions. Thus, the updated metabolic reconstruction of M. barkeri metabolism is a useful tool for predicting cellular behavior, studying the methanogenic lifestyle, guiding experimental studies, and making predictions relevant to metabolic engineering applications.

A Functional Approach To Uncover the Low-Temperature Adaptation Strategies of the Archaeon Methanosarcina barkeri

Abstract: Low-temperature anaerobic digestion (LTAD) technology is underpinned by a diverse microbial community. The methanogenic archaea represent a key functional group in these consortia, undertaking CO2 reduction as well as acetate and methylated C1 metabolism with subsequent biogas (40 to 60% CH4 and 30 to 50% CO2) formation. However, the cold adaptation strategies, which allow methanogens to function efficiently in LTAD, remain unclear. Here, a pure-culture proteomic approach was employed to study the functional characteristics of Methanosarcina barkeri (optimum growth temperature, 37°C), which has been detected in LTAD bioreactors. Two experimental approaches were undertaken. The first approach aimed to characterize a low-temperature shock response (LTSR) of M. barkeri DSMZ 800T grown at 37°C with a temperature drop to 15°C, while the second experimental approach aimed to examine the low-temperature adaptation strategies (LTAS) of the same strain when it was grown at 15°C. The latter experiment employed cell viability and growth measurements (optical density at 600 nm [OD600]), which directly compared M. barkeri cells grown at 15°C with those grown at 37°C. During the LTSR experiment, a total of 127 proteins were detected in 37°C and 15°C samples, with 20 proteins differentially expressed with respect to temperature, while in the LTAS experiment 39% of proteins identified were differentially expressed between phases of growth. Functional categories included methanogenesis, cellular information processing, and chaperones. By applying a polyphasic approach (proteomics and growth studies), insights into the low-temperature adaptation capacity of this mesophilically characterized methanogen were obtained which suggest that the metabolically diverse Methanosarcinaceae could be functionally relevant for LTAD systems.

Inhibitory effects of saturated fatty acids on methane production by methanogenic Archaea

Abstract: The present study investigated the inhibitory effects of saturated fatty acids on methanogenesis in Archaea, and whether or not competitive inactivation of the methanogens’ coenzyme M (HS-CoM) is involved in the inhibition. Strains tested in batch cultures were Methanosarcina barkeri, Methanosarcina mazei, Methanococcus voltae, all incubated at 37°C, and Methanothermobacter thermoautotrophicus, incubated at 65°C. The fatty acids C10, C12, C14 and C18 were supplemented at 1 mg . ml–1 in cultivation medium. The methanogens were susceptible to C10 and C12, and less so to C14. Only M. thermoautotrophicus was affected by C18. In M. mazei cultures, excessive HS-CoM did not prevent the action of C14 which might suggest that competitive inhibition of HS-CoM is not the reason for the SFA-induced effect on methanogenesis. The results indicate that, as a prerequisite to inhibit methanogenesis in Archaea, medium- and long-chain saturated fatty acids have to be at least partially molten.

Structure and Reaction Mechanism of Pyrrolysine Synthase (PylD).

Abstract: Pyrrolysine (Pyl, 4), the recently discovered 22nd genetically encoded amino acid, has almost instantaneously become a hotspot of protein biochemistry, although its natural occurrence appears to be limited to just three proteins that are involved in the breakdown of methylamines in a small subgroup of archaea and bacteria. The novel amino acid is incorporated by a cognate pair of a tRNA and its synthetase (specified as pylT and pylS, respectively) through recognition of an amber stop codon (UAG). Biosynthesis of 4 is accomplished from two molecules of lysine (1) by sequential action of PylB, PylC, and PylD (Scheme 1). Specifically, the iron–sulfur S-adenosylmethionine protein PylB catalyzes the conversion of 1 to (3R)-3methyl-d-ornithine (3MO, 2), which is subsequently hooked up ATP-dependently to the e amino group of a second lysine molecule by PylC resulting in 3. Dehydrogenation at the C5 position of the methylornithine moiety of the isopeptide and subsequent ring closure catalyzed by PylD completes the biosynthesis of the unusual amino acid pyrrolysine. The present report on PylD describes the structural investigation and implementation on the reaction mechanism of the enzymes required for the biosynthesis of pyrrolysine and may open novel opportunities for the harnessing of the system for biotechnology purposes. We expressed the pylD gene of Methanosarcina barkeri Fusaro in an Escherichia coli strain. The recombinant protein was purified by metal-affinity chromatography and showed in vitro catalytic activity with Km = 3.6 mm 0.5 mm (Figure S1 in the Supporting Information) and kcat = 0.76 s 1 0.04 s 1 using the surrogate l-lysine-N-d-ornithine (LysN-d-Orn, 3a) as substrate. PylD was crystallized together with 3a and/ or a pyridine nucleotide cofactor (NADH or NAD). Crystals diffracted to a maximum resolution of 1.8 and starting phases were obtained by a combination of single-wavelength anomalous diffraction (SAD) methods using a selenomethionine derivative and twofold noncrystallographic symmetry (NCS) averaging. Real-space electron density map averaging was performed with MAIN in combination with CCP4 routines. Model building was carried out with MAIN and refinement was completed with REFMAC5 (see Table S1). After structural elucidation of the selenomethionine-labeled PylD holoenzyme (PylD:holo (peak), PDB ID: 4JK3), we crystallized and determined the structure of native PylD in the presence of NAD (PylD:holo, PDB ID: 4J43) to 2.2 resolution (Rfree = 20.1%, Table S1). Its molecular architecture is shown schematically in Figure 1a: the C-terminal segment (residues 139–259) resembles a Rossmann motif of five parallel b strands (S6–S10) with 21345 topology, which is Nand C-terminally flanked by helices H5 and H10, respectively (secondary-structure nomenclature: see Figure S3), yielding in the DALI search a highest Z-score of 15.6 for the Rhodospirillum rubrum Transhydrogenase Domain I (PDB ID: 1L7D). The N-terminal segment (residues 1–138) comprises a b sheet of five strands (S1–S5) whose overall orientation is orthogonal to that of the Rossmann motif of the C-terminal part. Interestingly, the C-terminal helix (H10) of PylD is wedged between the two b sheets supporting the correct fold and orientation of the N-terminal half of the dehydrogenase. In contrast to the Rossmann fold, the DALI search for proteins resembling the topology of the N-terminal segment resulted in only some similarities with the tRNA binding domain of certain tRNA synthetases (Zscore< 8). The nicotinamide adenine dinucleotide coenzyme NAD is bound to PylD in an extended conformation, inside a groove at the C-terminal pole of the Rossmann b sheet; both furanose rings have C2’-endo conformation. A typical VXGXGXXGXXXA motif (residues 146–157) is part of the coenzyme binding site and the backbone elements are predominantly involved in a network of hydrogen bonds with Scheme 1. Biosynthesis of pyrrolysine. Note: PylB generates only 3MO (2); PylC and PylD also catalyze the reactions of 2a and 3a, respectively.