Assimilatory sulfate reduction in the marine methanogen Methanothermococcus thermolithotrophicus.

Methanothermococcus thermolithotrophicus is the only known methanogen that grows on sulfate as its sole sulfur source, uniquely uniting methanogenesis and sulfate reduction. Here we use physiological, biochemical and structural analyses to provide a snapshot of the complete sulfate reduction pathway of this methanogenic archaeon. We find that later steps in this pathway are catalysed by atypical enzymes. PAPS (3'-phosphoadenosine 5'-phosphosulfate) released by APS kinase is converted into sulfite and 3'-phosphoadenosine 5'-phosphate (PAP) by a PAPS reductase that is similar to the APS reductases of dissimilatory sulfate reduction. A non-canonical PAP phosphatase then hydrolyses PAP. Finally, the F420-dependent sulfite reductase converts sulfite to sulfide for cellular assimilation. While metagenomic and metatranscriptomic studies suggest that the sulfate reduction pathway is present in several methanogens, the sulfate assimilation pathway in M. thermolithotrophicus is distinct. We propose that this pathway was 'mix-and-matched' through the acquisition of assimilatory and dissimilatory enzymes from other microorganisms and then repurposed to fill a unique metabolic role.

Comparative Transcriptomics Sheds Light on Remodeling of Gene Expression during Diazotrophy in the Thermophilic Methanogen Methanothermococcus thermolithotrophicus.

Some marine thermophilic methanogens are able to perform energy-consuming nitrogen fixation despite deriving only little energy from hydrogenotrophic methanogenesis. We studied this process in Methanothermococcus thermolithotrophicus DSM 2095, a methanogenic archaeon of the order Methanococcales that contributes to the nitrogen pool in some marine environments. We successfully grew this archaeon under diazotrophic conditions in both batch and fermenter cultures, reaching the highest cell density reported so far. Diazotrophic growth depended strictly on molybdenum and, in contrast to other diazotrophs, was not inhibited by tungstate or vanadium. This suggests an elaborate control of metal uptake and a specific metal recognition system for the insertion into the nitrogenase cofactor. Differential transcriptomics of M. thermolithotrophicus grown under diazotrophic conditions with ammonium-fed cultures as controls revealed upregulation of the nitrogenase machinery, including chaperones, regulators, and molybdate importers, as well as simultaneous upregulation of an ammonium transporter and a putative pathway for nitrate and nitrite utilization. The organism thus employs multiple synergistic strategies for uptake of nitrogen nutrients during the early exponential growth phase without altering transcription levels for genes involved in methanogenesis. As a counterpart, genes coding for transcription and translation processes were downregulated, highlighting the maintenance of an intricate metabolic balance to deal with energy constraints and nutrient limitations imposed by diazotrophy. This switch in the metabolic balance included unexpected processes, such as upregulation of the CRISPR-Cas system, probably caused by drastic changes in transcription levels of putative mobile and virus-like elements. IMPORTANCE The thermophilic anaerobic archaeon M. thermolithotrophicus is a particularly suitable model organism to study the coupling of methanogenesis to diazotrophy. Likewise, its capability of simultaneously reducing N2 and CO2 into NH3 and CH4 with H2 makes it a viable target for biofuel production. We optimized M. thermolithotrophicus cultivation, resulting in considerably higher cell yields and enabling the successful establishment of N2-fixing bioreactors. Improved understanding of the N2 fixation process would provide novel insights into metabolic adaptations that allow this energy-limited extremophile to thrive under diazotrophy, for instance, by investigating its physiology and uncharacterized nitrogenase. We demonstrated that diazotrophic growth of M. thermolithotrophicus is exclusively dependent on molybdenum, and complementary transcriptomics corroborated the expression of the molybdenum nitrogenase system. Further analyses of differentially expressed genes during diazotrophy across three cultivation time points revealed insights into the response to nitrogen limitation and the coordination of core metabolic processes.

Identification of a novel N-linked glycan on the archaellins and S-layer protein of the thermophilic methanogen, Methanothermococcus thermolithotrophicus.

Motility in archaea is facilitated by a unique structure termed the archaellum. N-Glycosylation of the major structural proteins (archaellins) is important for their subsequent incorporation into the archaellum filament. The identity of some of these N-glycans has been determined, but archaea exhibit extensive variation in their glycans, meaning that further investigations can shed light not only on the specific details of archaellin structure and function, but also on archaeal glycobiology in general. Here we describe the structural characterization of the N-linked glycan modifications on the archaellins and S-layer protein of Methanothermococcus thermolithotrophicus, a methanogen that grows optimally at 65 °C. SDS-PAGE and MS analysis revealed that the sheared archaella are composed principally of two of the four predicted archaellins, FlaB1 and FlaB3, which are modified with a branched, heptameric glycan at all N-linked sequons except for the site closest to the N termini of both proteins. NMR analysis of the purified glycan determined the structure to be α-d-glycero-d-manno-Hep3OMe6OMe-(1-3)-[α-GalNAcA3OMe-(1-2)-]-ß-Man-(1-4)-[ß-GalA3OMe4OAc6CMe-(1-4)-α-GalA-(1-2)-]-α-GalAN-(1-3)-ß-GalNAc-Asn. A detailed investigation by hydrophilic interaction liquid ion chromatography-MS discovered the presence of several, less abundant glycan variants, related to but distinct from the main heptameric glycan. In addition, we confirmed that the S-layer protein is modified with the same heptameric glycan, suggesting a common N-glycosylation pathway. The M. thermolithotrophicus archaellin N-linked glycan is larger and more complex than those previously identified on the archaellins of related mesophilic methanogens, Methanococcus voltae and Methanococcus maripaludis This could indicate that the nature of the glycan modification may have a role to play in maintaining stability at elevated temperatures.

Methanofervidicoccus abyssi gen. nov., sp. nov., a hydrogenotrophic methanogen, isolated from a hydrothermal vent chimney in the Mid-Cayman Spreading Center, the Caribbean Sea.

A novel hydrogenotrophic methanogen, strain HHBT, was isolated from a deep-sea hydrothermal vent chimney sample collected from Beebe Vent Field at the Mid-Cayman Spreading Center, Caribbean Sea. The cells were non-motile regular to irregular cocci possessing several flagella. The novel isolate grew at 60-80 °C, pH 5.0-7.4 and with 1-4 % of NaCl (w/v). The isolate utilized H2/CO2 as the only substrates for growth and methane production. The results of phylogenetic analyses of both 16S rRNA and mcrA gene sequences and comparative genome analysis indicated that HHBT represented a member of the order Methanococcales, and was closely related to the members of the genera Methanothermococcus and Methanotorris. The most closely related species were Methanothermococcus okinawensis IH1T and Methanotorris igneus Kol 5T in comparison of 16S rRNA gene sequences (each with 93 % identity), and Methanotorris formicicus Mc-S-70T in the case of deduced amino acid sequence similarity of mcrA genes (92 % similarity). The ANI and AAI values between HHBT and the members of the genera Methanothermococcus and Methanotorris were 69-72 % and 66-70 %, respectively. Although many of the morphological and physiological characteristics were quite similar between HHBT and the species of the genera Methanothermococcus and Methanotorris, they were distinguishable by the differences in susceptibility to antibiotics, formate utilization, growth temperature and NaCl ranges. On the basis of these phenotypic, phylogenetic and genomic properties, we propose that strain HHBT represents a novel species, of a novel genus, Methanofervidicoccus abyssi gen. nov., sp. nov. The type strain is HHBT (=JCM 32161T=DSM 105918T).

Crystal structure of archaeal HMG-CoA reductase: insights into structural changes of the C-terminal helix of the class-I enzyme.

3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) catalyses the last step in mevalonate biosynthesis. HMGR is the target of statin inhibitors that regulate cholesterol concentration in human blood. Here, we report the properties and structures of HMGR from an archaeon Methanothermococcus thermolithotrophicus (mHMGR). The structures of the apoenzyme and the NADPH complex are highly similar to those of human HMGR. A notable exception is C-terminal helix (Lα10-11) that is straight in both mHMGR structures. This helix is kinked and closes the active site in the human enzyme ternary complex, pointing to a substrate-induced structural rearrangement of C-terminal in class-I HMGRs during the catalytic cycle.

Biological methane production under putative Enceladus-like conditions.

The detection of silica-rich dust particles, as an indication for ongoing hydrothermal activity, and the presence of water and organic molecules in the plume of Enceladus, have made Saturn's icy moon a hot spot in the search for potential extraterrestrial life. Methanogenic archaea are among the organisms that could potentially thrive under the predicted conditions on Enceladus, considering that both molecular hydrogen (H2) and methane (CH4) have been detected in the plume. Here we show that a methanogenic archaeon, Methanothermococcus okinawensis, can produce CH4 under physicochemical conditions extrapolated for Enceladus. Up to 72% carbon dioxide to CH4 conversion is reached at 50 bar in the presence of potential inhibitors. Furthermore, kinetic and thermodynamic computations of low-temperature serpentinization indicate that there may be sufficient H2 gas production to serve as a substrate for CH4 production on Enceladus. We conclude that some of the CH4 detected in the plume of Enceladus might, in principle, be produced by methanogens.

Electron Bifurcation Makes the Puzzle Pieces Fall Energetically into Place in Methanogenic Energy Conservation.

Elaborate arrays of iron-sulfur clusters link active sites via a flavin that bifurcates electrons through two energetically independent paths. The structure of the heterodisulfide reductase provides insight into how methanogens conserve energy through coupling hydrogen oxidation to coordinated exergonic heterodisulfide and endergonic ferredoxin reduction in an overall thermodynamically favorable reaction.

Methanogenic heterodisulfide reductase (HdrABC-MvhAGD) uses two noncubane [4Fe-4S] clusters for reduction.

In methanogenic archaea, the carbon dioxide (CO2) fixation and methane-forming steps are linked through the heterodisulfide reductase (HdrABC)-[NiFe]-hydrogenase (MvhAGD) complex that uses flavin-based electron bifurcation to reduce ferredoxin and the heterodisulfide of coenzymes M and B. Here, we present the structure of the native heterododecameric HdrABC-MvhAGD complex at 2.15-angstrom resolution. HdrB contains two noncubane [4Fe-4S] clusters composed of fused [3Fe-4S]-[2Fe-2S] units sharing 1 iron (Fe) and 1 sulfur (S), which were coordinated at the CCG motifs. Soaking experiments showed that the heterodisulfide is clamped between the two noncubane [4Fe-4S] clusters and homolytically cleaved, forming coenzyme M and B bound to each iron. Coenzymes are consecutively released upon one-by-one electron transfer. The HdrABC-MvhAGD atomic model serves as a structural template for numerous HdrABC homologs involved in diverse microbial metabolic pathways.

The methanogenic CO2 reducing-and-fixing enzyme is bifunctional and contains 46 [4Fe-4S] clusters.

Biological methane formation starts with a challenging adenosine triphosphate (ATP)-independent carbon dioxide (CO2) fixation process. We explored this enzymatic process by solving the x-ray crystal structure of formyl-methanofuran dehydrogenase, determined here as Fwd(ABCDFG)2 and Fwd(ABCDFG)4 complexes, from Methanothermobacter wolfeii The latter 800-kilodalton apparatus consists of four peripheral catalytic sections and an electron-supplying core with 46 electronically coupled [4Fe-4S] clusters. Catalysis is separately performed by subunits FwdBD (FwdB and FwdD), which are related to tungsten-containing formate dehydrogenase, and subunit FwdA, a binuclear metal center carrying amidohydrolase. CO2 is first reduced to formate in FwdBD, which then diffuses through a 43-angstrom-long tunnel to FwdA, where it condenses with methanofuran to formyl-methanofuran. The arrangement of [4Fe-4S] clusters functions as an electron relay but potentially also couples the four tungstopterin active sites over 206 angstroms.

Profiling of Indigenous Microbial Community Dynamics and Metabolic Activity During Enrichment in Molasses-Supplemented Crude Oil-Brine Mixtures for Improved Understanding of Microbial Enhanced Oil Recovery.

Anaerobic incubations using crude oil and brine from a North Sea reservoir were conducted to gain increased understanding of indigenous microbial community development, metabolite production, and the effects on the oil-brine system after addition of a complex carbon source, molasses, with or without nitrate to boost microbial growth. Growth of the indigenous microbes was stimulated by addition of molasses. Pyrosequencing showed that specifically Anaerobaculum, Petrotoga, and Methanothermococcus were enriched. Addition of nitrate favored the growth of Petrotoga over Anaerobaculum. The microbial growth caused changes in the crude oil-brine system: formation of oil emulsions, and reduction of interfacial tension (IFT). Reduction in IFT was associated with microbes being present at the oil-brine interphase. These findings suggest that stimulation of indigenous microbial growth by addition of molasses has potential as microbial enhanced oil recovery (MEOR) strategy in North Sea oil reservoirs.

Characterization of the supporting role of SecE in protein translocation.

SecYEG functions as a membrane channel for protein export. SecY constitutes the protein-conducting pore, which is enwrapped by SecE in a V-shaped manner. In its minimal form SecE consists of a single transmembrane segment that is connected to a surface-exposed amphipathic α-helix via a flexible hinge. These two domains are the major sites of interaction between SecE and SecY. Specific cleavage of SecE at the hinge region, which destroys the interaction between the two SecE domains, reduced translocation. When SecE and SecY were disulfide bonded at the two sites of interaction, protein translocation was not affected. This suggests that the SecY and SecE interactions are static, while the hinge region provides flexibility to allow the SecY pore to open.

Conformational fluctuations of UreG, an intrinsically disordered enzyme.

UreG proteins are small GTP binding (G) proteins that catalyze the hydrolysis of GTP necessary for the maturation of urease, a virulence factor in bacterial pathogenesis. UreG proteins are the first documented cases of intrinsically disordered enzymes. The comprehension of the dynamics of folding-unfolding events occurring in this protein could shed light on the enzymatic mechanism of UreG. Here, we used the recently developed replica exchange with solute tempering (REST2) computational methodology to explore the conformational space of UreG from Helicobacter pylori (HpUreG) and to identify its structural fluctuations. The same simulation and analysis protocol has been applied to HypB from Methanocaldococcus jannaschii (MjHypB), which is closely related to UreG in both sequence and function, even though it is not intrinsically disordered. A comparison of the two systems reveals that both HpUreG and MjHypB feature a substantial rigidity of the protein regions involved in catalysis, justifying its residual catalytic activity. On the other hand, HpUreG tends to unfold more than MjHypB in portions involved in protein-protein interactions with metallochaperones necessary for the formation of multiprotein complexes known to be involved in urease activation.

Crystallization and preliminary X-ray diffraction analysis of the Nif3-family protein MJ0927 from Methanocaldococcus jannaschii.

MJ0927 is a member of the Nif3 family and is widely distributed across living organisms. Although several crystal structures of Nif3 proteins have been reported, structural information on archaeal Nif3 is still limited. To understand the structural differences between bacterial and archaeal Nif3 proteins, MJ0927 from Methanocaldococcus jannaschii was purified and crystallized using the sitting-drop vapour-diffusion method. The crystals diffracted to a resolution of 2.47 Šand belonged to the orthorhombic space group C222, with unit-cell parameters a = 81.21, b = 172.94, c = 147.42 Å. Determination of this structure may provide insights into the function of MJ0927.

Avatar pre-tRNAs help elucidate the properties of tRNA-splicing endonucleases that produce tRNA from permuted genes.

Unusual tRNA genes, found in some algae, have their mature terminal 3' portion in front of their 5' portion in the genome. The transcripts from such genes must be cleaved by a pre-tRNA endonuclease to form a functional tRNA. We present a mechanism for the generation of "corrected" tRNAs from such a "permuted" pre-tRNA configuration. We used two avatar (av) or model pre-tRNAs and two splicing endonucleases with distinct mechanisms of recognition of the pre-tRNA. The splicing results are compatible with an evolutionary route in which permuted genes result from a duplication event followed by DNA rearrangement. The model pre-tRNAs permit description of the features that a transcript, derived from a rearranged duplicated gene, must have to give rise to functional tRNA. The two tRNA endonucleases are a eukaryal enzyme that normally acts in a mature domain-dependent mode and an archaeal enzyme that acts in a mature domain-independent mode. Both av pre-tRNAs are able to fold into two conformations: 1 and 2. We find that only conformation 2 can yield a corrected functional tRNA. This result is consistent with contemporary algae representing snapshots of different evolutionary stages, with duplicated genes preceding recombinatorial events generating a permutated gene. In a scenario elucidated by the use of the av pre-tRNAs, algal permuted tRNA genes could have further lost one of two mature domains, eliminating steric problems for the algal tRNA endonuclease, which remains a typical eukaryal enzyme capable of correcting the permuted transcript to a functional tRNA.

Linking energy production and protein synthesis in hydrogenotrophic methanogens.

Hydrogenotrophic methanogens possessing the hydrogen-dependent dehydrogenase Hmd also encode paralogs of this protein whose function is poorly understood. Here we present biochemical evidence that the two inactive Hmd paralogs of Methanocaldococcus jannaschii, HmdII and HmdIII, form binary and ternary complexes with several components of the protein translation apparatus. HmdII and HmdIII, but not the active dehydrogenase Hmd, bind with micromolar binding affinities to a number of tRNAs and form ternary complexes with tRNA(Pro) and prolyl-tRNA synthetase (ProRS). Fluorescence spectroscopy experiments also suggest that binding of HmdII and ProRS involves distinct binding determinants on the tRNA. These biochemical data suggest the possibility of a regulatory link between energy production and protein translation pathways that may allow a rapid cellular response to altered environmental conditions.

A new class of adenylate kinase in methanogens is related to uridylate kinase.

The protein derived from the Methanocaldococcus jannaschii MJ0458 gene is annotated as a δ-1-pyrroline 5-carboxylate synthetase and is predicted to be related to aspartokinase and uridylate kinase. Analysis of the predicted protein sequence indicated that it is a unique kinase with few similarities to either uridylate or adenylate kinase. Here, we report that the MJ0458 gene product is a second type of archaeal adenylate kinase, AdkB. This enzyme is different from the established archaeal-specific adenylate kinase in both sequence and predicted tertiary structure.

Mutational analysis of Sep-tRNA:Cys-tRNA synthase reveals critical residues for tRNA-dependent cysteine formation.

In methanogenic archaea, Sep-tRNA:Cys-tRNA synthase (SepCysS) converts Sep-tRNA(Cys) to Cys-tRNA(Cys). The mechanism of tRNA-dependent cysteine formation remains unclear due to the lack of functional studies. In this work, we mutated 19 conserved residues in Methanocaldococcus jannaschii SepCysS, and employed an in vivo system to determine the activity of the resulting variants. Our results show that three active-site cysteines (Cys39, Cys42 and Cys247) are essential for SepCysS activity. In addition, combined with structural modeling, our mutational and functional analyses also reveal multiple residues that are important for the binding of PLP, Sep and tRNA. Our work thus represents the first systematic functional analysis of conserved residues in archaeal SepCysSs, providing insights into the catalytic and substrate binding mechanisms of this poorly characterized enzyme.

1H, 13C, 15N assignment and secondary structure determination of glutamine amido transferase subunit of gaunosine monophosphate synthetase from Methanocaldococcus jannaschii.

Sequence specific resonance assignments have been obtained for (1)H, (13)C and (15)N nuclei of the 21 kDa (188 residues long) glutamine amido transferase subunit of guanosine monophosphate synthetase from Methanocaldococcus jannaschii. From an analysis of (1)H and (13)C(α), (13)C(ß) secondary chemical shifts, (3) JH(N)H(α) scalar coupling constants and sequential, short and medium range (1)H-(1)H NOEs, it was deduced that the glutamine amido transferase subunit has eleven strands and five helices as the major secondary structural elements in its tertiary structure.

Structure and mutational analysis of the archaeal GTP:AdoCbi-P guanylyltransferase (CobY) from Methanocaldococcus jannaschii: insights into GTP binding and dimerization.

In archaea and bacteria, the late steps in adenosylcobalamin (AdoCbl) biosynthesis are collectively known as the nucleotide loop assembly (NLA) pathway. In the archaeal and bacterial NLA pathways, two different guanylyltransferases catalyze the activation of the corrinoid. Structural and functional studies of the bifunctional bacterial guanylyltransferase that catalyze both ATP-dependent corrinoid phosphorylation and GTP-dependent guanylylation are available, but similar studies of the monofunctional archaeal enzyme that catalyzes only GTP-dependent guanylylation are not. Herein, the three-dimensional crystal structure of the guanylyltransferase (CobY) enzyme from the archaeon Methanocaldococcus jannaschii (MjCobY) in complex with GTP is reported. The model identifies the location of the active site. An extensive mutational analysis was performed, and the functionality of the variant proteins was assessed in vivo and in vitro. Substitutions of residues Gly8, Gly153, or Asn177 resulted in ≥94% loss of catalytic activity; thus, variant proteins failed to support AdoCbl synthesis in vivo. Results from isothermal titration calorimetry experiments showed that MjCobY(G153D) had 10-fold higher affinity for GTP than MjCobY(WT) but failed to bind the corrinoid substrate. Results from Western blot analyses suggested that the above-mentioned substitutions render the protein unstable and prone to degradation; possible explanations for the observed instability of the variants are discussed within the framework of the three-dimensional crystal structure of MjCobY(G153D) in complex with GTP. The fold of MjCobY is strikingly similar to that of the N-terminal domain of Mycobacterium tuberculosis GlmU (MtbGlmU), a bifunctional acetyltransferase/uridyltransferase that catalyzes the formation of uridine diphosphate-N-acetylglucosamine (UDP-GlcNAc).

Growth of methanogens under defined hydrogen conditions.

Hydrogen (H(2)) is a primary electron donor for methanogenesis and its availability can have profound effects on gene expression and the physiology of energy conservation. The rigorous evaluation of the effects of hydrogen conditions requires the comparison of cultures that are grown under hydrogen limitation and hydrogen excess. The growth of methanogens under defined hydrogen conditions is complicated by the dynamics of hydrogen dissolution and its utilization by the cells. In batch culture, gassing and agitation conditions must be carefully calibrated, and even then variations in growth rate and cell density are hard to avoid. Using chemostats, continuous cultures can be achieved whose nutritional states are known, while growth rate and cell density are invariant. Cultures whose growth is limited by hydrogen can be compared to cultures whose growth is limited by some other nutrient and are therefore under hydrogen excess.