Structures of the sulfite detoxifying F420-dependent enzyme from Methanococcales.

Methanogenic archaea are main actors in the carbon cycle but are sensitive to reactive sulfite. Some methanogens use a sulfite detoxification system that combines an F420H2-oxidase with a sulfite reductase, both of which are proposed precursors of modern enzymes. Here, we present snapshots of this coupled system, named coenzyme F420-dependent sulfite reductase (Group I Fsr), obtained from two marine methanogens. Fsr organizes as a homotetramer, harboring an intertwined six-[4Fe-4S] cluster relay characterized by spectroscopy. The wire, spanning 5.4 nm, electronically connects the flavin to the siroheme center. Despite a structural architecture similar to dissimilatory sulfite reductases, Fsr shows a siroheme coordination and a reaction mechanism identical to assimilatory sulfite reductases. Accordingly, the reaction of Fsr is unidirectional, reducing sulfite or nitrite with F420H2. Our results provide structural insights into this unique fusion, in which a primitive sulfite reductase turns a poison into an elementary block of life.

The Oxoglutarate Binding Site and Regulatory Mechanism Are Conserved in Ammonium Transporter Inhibitors GlnKs from Methanococcales.

Protein inhibition is a natural regulatory process to control cellular metabolic fluxes. PII-family signal-transducing effectors are in this matter key regulators of the nitrogen metabolism. Their interaction with their various targets is governed by the cellular nitrogen level and the energy charge. Structural studies on GlnK, a PII-family inhibitor of the ammonium transporters (Amt), showed that the T-loops responsible for channel obstruction are displaced upon the binding of 2-oxoglutarate, magnesium and ATP in a conserved cleft. However, GlnK from Methanocaldococcus jannaschii was shown to bind 2-oxoglutarate on the tip of its T-loop, causing a moderate disruption to GlnK-Amt interaction, raising the question if methanogenic archaea use a singular adaptive strategy. Here we show that membrane fractions of Methanothermococcus thermolithotrophicus released GlnKs only in the presence of Mg-ATP and 2-oxoglutarate. This observation led us to structurally characterize the two GlnK isoforms apo or in complex with ligands. Together, our results show that the 2-oxoglutarate binding interface is conserved in GlnKs from Methanococcales, including Methanocaldococcus jannaschii, emphasizing the importance of a free carboxy-terminal group to facilitate ligand binding and to provoke the shift of the T-loop positions.

Shifts in methanogenic archaea communities and methane dynamics along a subtropical estuarine land use gradient.

In coastal aquatic ecosystems, prokaryotic communities play an important role in regulating the cycling of nutrients and greenhouse gases. In the coastal zone, estuaries are complex and delicately balanced systems containing a multitude of specific ecological niches for resident microbes. Anthropogenic influences (i.e. urban, industrial and agricultural land uses) along the estuarine continuum can invoke physical and biochemical changes that impact these niches. In this study, we investigate the relative abundance of methanogenic archaea and other prokaryotic communities, distributed along a land use gradient in the subtropical Burnett River Estuary, situated within the Great Barrier Reef catchment, Australia. Microbiological assemblages were compared to physicochemical, nutrient and greenhouse gas distributions in both pore and surface water. Pore water samples from within the most urbanised site showed a high relative abundance of methanogenic Euryarchaeota (7.8% of all detected prokaryotes), which coincided with elevated methane concentrations in the water column, ranging from 0.51 to 0.68 µM at the urban and sewage treatment plant (STP) sites, respectively. These sites also featured elevated dissolved organic carbon (DOC) concentrations (0.66 to 1.16 mM), potentially fuelling methanogenesis. At the upstream freshwater site, both methane and DOC concentrations were considerably higher (2.68 µM and 1.8 mM respectively) than at the estuarine sites (0.02 to 0.66 µM and 0.39 to 1.16 mM respectively) and corresponded to the highest relative abundance of methanotrophic bacteria. The proportion of sulfate reducing bacteria in the prokaryotic community was elevated within the urban and STP sites (relative abundances of 8.0%- 10.5%), consistent with electron acceptors with higher redox potentials (e.g. O2, NO3-) being scarce. Overall, this study showed that ecological niches in anthropogenically altered environments appear to give an advantage to specialized prokaryotes invoking a potential change in the thermodynamic landscape of the ecosystem and in turn facilitating the generation of methane-a potent greenhouse gas.

Temporal and spatial impact of Spartina alterniflora invasion on methanogens community in Chongming Island, China.

Methane production by methanogens in wetland is recognized as a significant contributor to global warming. Spartina alterniflora (S. alterniflora), which is an invasion plant in China's wetland, was reported to have enormous effects on methane production. But studies on shifts in the methanogen community in response to S. alterniflora invasion at temporal and spatial scales in the initial invasion years are rare. Sediments derived from the invasive species S. alterniflora and the native species Phragmites australis (P. australis) in pairwise sites and an invasion chronosequence patch (4 years) were analyzed to investigate the abundance and community structure of methanogens using quantitative real-time PCR (qPCR) and Denaturing gradient gel electrophoresis (DGGE) cloning of the methyl-coenzyme M reductase A (mcrA) gene. For the pairwise sites, the abundance of methanogens in S. alterniflora soils was lower than that of P. australis soils. For the chronosequence patch, the abundance and diversity of methanogens was highest in the soil subjected to two years invasion, in which we detected some rare groups including Methanocellales and Methanococcales. These results indicated a priming effect at the initial invasion stages of S. alterniflora for microorganisms in the soil, which was also supported by the diverse root exudates. The shifts of methanogen communities after S. alterniflora invasion were due to changes in pH, salinity and sulfate. The results indicate that root exudates from S. alterniflora have a priming effect on methanogens in the initial years after invasion, and the predominate methylotrophic groups (Methanosarcinales) may adapt to the availability of diverse substrates and reflects the potential for high methane production after invasion by S. alterniflora.

Characterization of the Cation Binding Sites in the NCKX2 Na+/Ca2+-K+ Exchanger.

NCKX1-5 are proteins involved in K+-dependent Na+/Ca2+ exchange in various signal tissues. Here we present a homology model of NCKX2 based on the crystal structure of the NCX_Mj transporter found in Methanoccocus jannaschii. Molecular dynamics simulations were performed on the resultant wild-type NCKX2 model and two mutants (D548N and D575N) loaded with either four Na+ ions or one Ca2+ ion and one K+ ion, in line with the experimentally observed transport stoichiometry. The selectivity of the active site in wild-type NCKX2 for Na+, K+, and Li+ and the electrostatic interactions of the positive Na+ ions in the negatively charged active site of wild-type NCKX2 and the two mutants were evaluated from free energy perturbation calculations. For validation of the homology model, our computational results were compared to available experimental data obtained from numerous prior functional studies. The NCKX2 homology model is in good agreement with the discussed experimental data and provides valuable insights into the structure of the active site, which is lined with acidic and polar residues. The binding of the potassium and calcium ions is accomplished via Asp 575 and 548, respectively. Mutation of these residues to Asn alters the functionality of NCKX2 because of the elimination of the favorable carboxylate-cation interactions. The knowledge obtained from the NCKX2 model can be transferred to other isoforms of the NCKX family: newly discovered pathological mutations in NCKX4 and NCKX5 affect residues that are involved in ion binding and/or transport according to our homology model.

Abundance and potential metabolic activity of methanogens in well-aerated forest and grassland soils of an alpine region.

Although methanogens were recently discovered to occur in aerated soils, alpine regions have not been extensively studied for their presence so far. Here, the abundance of archaea and the methanogenic guilds Methanosarcinales, Methanococcales, Methanobacteriales, Methanomicrobiales and Methanocella spp. was studied at 16 coniferous forest and 14 grassland sites located at the montane and subalpine belts of the Northern Limestone Alps (calcareous) and the Austrian Central Alps (siliceous) using quantitative real-time PCR. Abundance of archaea, methanogens and the methanogenic potentials were significantly higher in grasslands than in forests. Furthermore, methanogenic potentials of calcareous soils were higher due to pH. Methanococcales, Methanomicrobiales and Methanocella spp. were detected in all collected samples, which indicates that they are autochthonous, while Methanobacteriales were absent from 4 out of 16 forest soils. Methanosarcinales were absent from 10 out of 16 forest soils and 2 out of 14 grassland soils. Nevertheless, together with Methanococcales they represented the majority of the 16S rRNA gene copies quantified from the grassland soils. Contrarily, forest soils were clearly dominated by Methanococcales. Our results indicate a higher diversity of methanogens in well-aerated soils than previously believed and that pH mainly influences their abundances and activities.

Identification of structurally diverse methanofuran coenzymes in methanococcales that are both N-formylated and N-acetylated.

Methanofuran (MF) is a coenzyme necessary for the first step of methanogenesis from CO2. The well-characterized MF core structure is 4-[N-(γ-l-glutamyl-γ-l-glutamyl)-p-(ß-aminoethyl)phenoxymethyl]-2-(aminomethyl)furan (APMF-γ-Glu2). Three different MF structures that differ on the basis of the composition of their side chains have been determined previously. Here, we use liquid chromatography coupled with high-resolution mass spectrometry and a variety of biochemical methods to deduce the unique structures of MFs present in four different methanogens in the order Methanococcales. This is the first detailed characterization of the MF occurring in methanogens of this order. MF in each of these organisms contains the expected APMF-γ-Glu2; however, the composition of the side chain is different from that of the previously described MF structures. In Methanocaldococcus jannaschii, additional γ-linked glutamates that range from 7 to 12 residues are present. The MF coenzymes in Methanococcus maripaludis, Methanococcus vannielii, and Methanothermococcus okinawensis also have additional glutamate residues but interestingly also contain a completely different chemical moiety in the middle of the side chain that we have identified as N-(3-carboxy-2- or 3-hydroxy-1-oxopropyl)-l-aspartic acid. This addition results in the terminal γ-linked glutamates being incorporated in the opposite orientation. In addition to these nonacylated MF coenzymes, we also identified the corresponding N-formyl-MF and, surprisingly, N-acetyl-MF derivatives. N-Acetyl-MF has never been observed or implied to be functioning in nature and may represent a new route for acetate formation in methanogens.

Starting up microbial enhanced oil recovery.

This chapter gives the reader a practical introduction into microbial enhanced oil recovery (MEOR) including the microbial production of natural gas from oil. Decision makers who consider the use of one of these technologies are provided with the required scientific background as well as with practical advice for upgrading an existing laboratory in order to conduct microbiological experiments. We believe that the conversion of residual oil into natural gas (methane) and the in situ production of biosurfactants are the most promising approaches for MEOR and therefore focus on these topics. Moreover, we give an introduction to the microbiology of oilfields and demonstrate that in situ microorganisms as well as injected cultures can help displace unrecoverable oil in place (OIP). After an initial research phase, the enhanced oil recovery (EOR) manager must decide whether MEOR would be economical. MEOR generally improves oil production but the increment may not justify the investment. Therefore, we provide a brief economical assessment at the end of this chapter. We describe the necessary state-of-the-art scientific equipment to guide EOR managers towards an appropriate MEOR strategy. Because it is inevitable to characterize the microbial community of an oilfield that should be treated using MEOR techniques, we describe three complementary start-up approaches. These are: (i) culturing methods, (ii) the characterization of microbial communities and possible bio-geochemical pathways by using molecular biology methods, and (iii) interfacial tension measurements. In conclusion, we hope that this chapter will facilitate a decision on whether to launch MEOR activities. We also provide an update on relevant literature for experienced MEOR researchers and oilfield operators. Microbiologists will learn about basic principles of interface physics needed to study the impact of microorganisms living on oil droplets. Last but not least, students and technicians trying to understand processes in oilfields and the techniques to examine them will, we hope, find a valuable source of information in this review.

Anaerobic coculture of microalgae with Thermosipho globiformans and Methanocaldococcus jannaschii at 68°C enhances generation of n-alkane-rich biofuels after pyrolysis.

We tested different alga-bacterium-archaeon consortia to investigate the production of oil-like mixtures, expecting that n-alkane-rich biofuels might be synthesized after pyrolysis. Thermosipho globiformans and Methanocaldococcus jannaschii were cocultured at 68°C with microalgae for 9 days under two anaerobic conditions, followed by pyrolysis at 300°C for 4 days. Arthrospira platensis (Cyanobacteria), Dunaliella tertiolecta (Chlorophyta), Emiliania huxleyi (Haptophyta), and Euglena gracilis (Euglenophyta) served as microalgal raw materials. D. tertiolecta, E. huxleyi, and E. gracilis cocultured with the bacterium and archaeon inhibited their growth and CH(4) production. E. huxleyi had the strongest inhibitory effect. Biofuel generation was enhanced by reducing impurities containing alkanenitriles during pyrolysis. The composition and amounts of n-alkanes produced by pyrolysis were closely related to the lipid contents and composition of the microalgae. Pyrolysis of A. platensis and D. tertiolecta containing mainly phospholipids and glycolipids generated short-carbon-chain n-alkanes (n-tridecane to n-nonadecane) and considerable amounts of isoprenoids. E. gracilis also produced mainly short n-alkanes. In contrast, E. huxleyi containing long-chain (31 and 33 carbon atoms) alkenes and very long-chain (37 to 39 carbon atoms) alkenones, in addition to phospholipids and glycolipids, generated a high yield of n-alkanes of various lengths (n-tridecane to n-pentatriacontane). The gas chromatography-mass spectrometry (GC-MS) profiles of these n-alkanes were similar to those of native petroleum crude oils despite containing a considerable amount of n-hentriacontane. The ratio of phytane to n-octadecane was also similar to that of native crude oils.

Structural insights into the mechanisms of Mg2+ uptake, transport, and gating by CorA.

Despite the importance of Mg(2+) for numerous cellular activities, the mechanisms underlying its import and homeostasis are poorly understood. The CorA family is ubiquitous and is primarily responsible for Mg(2+) transport. However, the key questions-such as, the ion selectivity, the transport pathway, and the gating mechanism-have remained unanswered for this protein family. We present a 3.2 Å resolution structure of the archaeal CorA from Methanocaldococcus jannaschii, which is a unique complete structure of a CorA protein and reveals the organization of the selectivity filter, which is composed of the signature motif of this family. The structure reveals that polar residues facing the channel coordinate a partially hydrated Mg(2+) during the transport. Based on these findings, we propose a unique gating mechanism involving a helical turn upon the binding of Mg(2+) to the regulatory intracellular binding sites, and thus converting a polar ion passage into a narrow hydrophobic pore. Because the amino acids involved in the uptake, transport, and gating are all conserved within the entire CorA family, we believe this mechanism is general for the whole family including the eukaryotic homologs.