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Hydrogenotrophic methanogenesis occurs in multiple environments, ranging from the intestinal tracts of animals to anaerobic sediments and hot springs. Energy conservation in hydrogenotrophic methanogens was long a mystery; only within the last decade was it reported that net energy conservation for growth depends on electron bifurcation. In this work, we focus on Methanococcus maripaludis, a well-studied hydrogenotrophic marine methanogen. To better understand hydrogenotrophic methanogenesis and compare it with methylotrophic methanogenesis that utilizes oxidative phosphorylation rather than electron bifurcation, we have built iMR539, a genome scale metabolic reconstruction that accounts for 539 of the 1,722 protein-coding genes of M. maripaludis strain S2. Our reconstructed metabolic network uses recent literature to not only represent the central electron bifurcation reaction but also incorporate vital biosynthesis and assimilation pathways, including unique cofactor and coenzyme syntheses. We show that our model accurately predicts experimental growth and gene knockout data, with 93% accuracy and a Matthews correlation coefficient of 0.78. Furthermore, we use our metabolic network reconstruction to probe the implications of electron bifurcation by showing its essentiality, as well as investigating the infeasibility of aceticlastic methanogenesis in the network. Additionally, we demonstrate a method of applying thermodynamic constraints to a metabolic model to quickly estimate overall free-energy changes between what comes in and out of the cell. Finally, we describe a novel reconstruction-specific computational toolbox we created to improve usability. Together, our results provide a computational network for exploring hydrogenotrophic methanogenesis and confirm the importance of electron bifurcation in this process., Importance: Understanding and applying hydrogenotrophic methanogenesis is a promising avenue for developing new bioenergy technologies around methane gas. Although a significant portion of biological methane is generated through this environmentally ubiquitous pathway, existing methanogen models portray the more traditional energy conservation mechanisms that are found in other methanogens. We have constructed a genome scale metabolic network of Methanococcus maripaludis that explicitly accounts for all major reactions involved in hydrogenotrophic methanogenesis. Our reconstruction demonstrates the importance of electron bifurcation in central metabolism, providing both a window into hydrogenotrophic methanogenesis and a hypothesis-generating platform to fuel metabolic engineering efforts., (Copyright © 2016 Richards et al.)

Flavin-based electron bifurcation has recently been characterized as an essential energy conservation mechanism that is utilized by hydrogenotrophic methanogenic Archaea to generate low-potential electrons in an ATP-independent manner. Electron bifurcation likely takes place at the flavin associated with the α subunit of heterodisulfide reductase (HdrA). In Methanococcus maripaludis the electrons for this reaction come from either formate or H2 via formate dehydrogenase (Fdh) or Hdr-associated hydrogenase (Vhu). However, how these enzymes bind to HdrA to deliver electrons is unknown. Here, we present evidence that the δ subunit of hydrogenase (VhuD) is central to the interaction of both enzymes with HdrA. When M. maripaludis is grown under conditions where both Fdh and Vhu are expressed, these enzymes compete for binding to VhuD, which in turn binds to HdrA. Under these conditions, both enzymes are fully functional and are bound to VhuD in substoichiometric quantities. We also show that Fdh copurifies specifically with VhuD in the absence of other hydrogenase subunits. Surprisingly, in the absence of Vhu, growth on hydrogen still occurs; we show that this involves F420-reducing hydrogenase. The data presented here represent an initial characterization of specific protein interactions centered on Hdr in a hydrogenotrophic methanogen that utilizes multiple electron donors for growth.

Methanogens catalyze the critical methane-producing step (called methanogenesis) in the anaerobic decomposition of organic matter. Here, we present the first predictive model of global gene regulation of methanogenesis in a hydrogenotrophic methanogen, Methanococcus maripaludis. We generated a comprehensive list of genes (protein-coding and noncoding) for M. maripaludis through integrated analysis of the transcriptome structure and a newly constructed Peptide Atlas. The environment and gene-regulatory influence network (EGRIN) model of the strain was constructed from a compendium of transcriptome data that was collected over 58 different steady-state and time-course experiments that were performed in chemostats or batch cultures under a spectrum of environmental perturbations that modulated methanogenesis. Analyses of the EGRIN model have revealed novel components of methanogenesis that included at least three additional protein-coding genes of previously unknown function as well as one noncoding RNA. We discovered that at least five regulatory mechanisms act in a combinatorial scheme to intercoordinate key steps of methanogenesis with different processes such as motility, ATP biosynthesis, and carbon assimilation. Through a combination of genetic and environmental perturbation experiments we have validated the EGRIN-predicted role of two novel transcription factors in the regulation of phosphate-dependent repression of formate dehydrogenase-a key enzyme in the methanogenesis pathway. The EGRIN model demonstrates regulatory affiliations within methanogenesis as well as between methanogenesis and other cellular functions.

The H2 -dependent methylene-tetrahydromethanopterin dehydrogenase (Hmd), also known as the [Fe]-hydrogenase, is found only in methanogens without cytochromes. In contrast to the binuclear metal centers of the [NiFe]- and [FeFe]-hydrogenases, the [Fe]-hydrogenase contains only a single Fe atom, which is coordinated by a novel guanylylpyridinol cofactor in the active site. The biosynthesis of the cofactor is not well understood and the responsible genes are unknown. However, seven genes (hmd co-occurring genes, hcg) encoding proteins of unknown function are always associated with the hmd gene. In the model methanogen Methanococcus maripaludis, we used a genetic background in which a deletion of hmd had a distinct growth phenotype, and made null-mutations in each hcg gene as well as in a gene encoding the Hmd paralog HmdII, which is hypothesized to function as a scaffold for cofactor synthesis. Deletions in all seven hcg genes resulted in the same growth phenotype as a deletion in hmd, suggesting they are required for Hmd function. In all cases, genetic complementation of the mutation restored the wild-type phenotype. A deletion in hmdII had no effect., (© 2013 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.)

Hydrogenotrophic methanogenic Archaea are defined by an H2 requirement for growth. Despite this requirement, many hydrogenotrophs are also capable of growth with formate as an electron donor for methanogenesis. While certain responses of these organisms to hydrogen availability have been characterized, responses to formate starvation have not been reported. Here we report that during continuous culture of Methanococcus maripaludis under defined nutrient conditions, growth yields relative to methane production decreased markedly with either H2 excess or formate excess. Analysis of the growth yields of several mutants suggests that this phenomenon occurs independently of the storage of intracellular carbon or a transcriptional response to methanogenesis. Using microarray analysis, we found that the expression of genes encoding coenzyme F420-dependent steps of methanogenesis, including one of two formate dehydrogenases, increased with H2 starvation but with formate occurred at high levels regardless of limitation or excess. One gene, encoding H2-dependent methylene-tetrahydromethanopterin dehydrogenase, decreased in expression with either H2 limitation or formate limitation. Expression of genes for the second formate dehydrogenase, molybdenum-dependent formylmethanofuran dehydrogenase, and molybdenum transport increased specifically with formate limitation. Of the two formate dehydrogenases, only the first could support growth on formate in batch culture where formate was in excess.

Unlabelled: Hydrogenotrophic methanogenic Archaea require reduced ferredoxin as an anaplerotic source of electrons for methanogenesis. H(2) oxidation by the hydrogenase Eha provides these electrons, consistent with an H(2) requirement for growth. Here we report the identification of alternative pathways of ferredoxin reduction in Methanococcus maripaludis that operate independently of Eha to stimulate methanogenesis. A suppressor mutation that increased expression of the glycolytic enzyme glyceraldehyde-3-phosphate:ferredoxin oxidoreductase resulted in a strain capable of H(2)-independent ferredoxin reduction and growth with formate as the sole electron donor. In this background, it was possible to eliminate all seven hydrogenases of M. maripaludis. Alternatively, carbon monoxide oxidation by carbon monoxide dehydrogenase could also generate reduced ferredoxin that feeds into methanogenesis. In either case, the reduced ferredoxin generated was inefficient at stimulating methanogenesis, resulting in a slow growth phenotype. As methanogenesis is limited by the availability of reduced ferredoxin under these conditions, other electron donors, such as reduced coenzyme F(420), should be abundant. Indeed, when F(420)-reducing hydrogenase was reintroduced into the hydrogenase-free mutant, the equilibrium of H(2) production via an F(420)-dependent formate:H(2) lyase activity shifted markedly toward H(2) compared to the wild type., Importance: Hydrogenotrophic methanogens are thought to require H(2) as a substrate for growth and methanogenesis. Here we show alternative pathways in methanogenic metabolism that alleviate this H(2) requirement and demonstrate, for the first time, a hydrogenotrophic methanogen that is capable of growth in the complete absence of H(2). The demonstration of alternative pathways in methanogenic metabolism suggests that this important group of organisms is metabolically more versatile than previously thought.

Methanococcus maripaludis grown syntrophically with Desulfovibrio vulgaris was compared with M. maripaludis monocultures grown under hydrogen limitation using transcriptional, proteomic and metabolite analyses. These measurements indicate a decrease in transcript abundance for energy-consuming biosynthetic functions in syntrophically grown M. maripaludis, with an increase in transcript abundance for genes involved in the energy-generating central pathway for methanogenesis. Compared with growth in monoculture under hydrogen limitation, the response of paralogous genes, such as those coding for hydrogenases, often diverged, with transcripts of one variant increasing in relative abundance, whereas the other was little changed or significantly decreased in abundance. A common theme was an apparent increase in transcripts for functions using H(2) directly as reductant, versus those using the reduced deazaflavin (coenzyme F(420)). The greater importance of direct reduction by H(2) was supported by improved syntrophic growth of a deletion mutant in an F(420)-dependent dehydrogenase of M. maripaludis. These data suggest that paralogous genes enable the methanogen to adapt to changing substrate availability, sustaining it under environmental conditions that are often near the thermodynamic threshold for growth. Additionally, the discovery of interspecies alanine transfer adds another metabolic dimension to this environmentally relevant mutualism.

Plants and microorganisms reduce environmental inorganic nitrogen to ammonium, which then enters various metabolic pathways solely via conversion of 2-oxoglutarate (2OG) to glutamate and glutamine. Cellular 2OG concentrations increase during nitrogen starvation. We recently identified a family of 2OG-sensing proteins--the nitrogen regulatory protein NrpR--that bind DNA and repress transcription of nitrogen assimilation genes. We used X-ray crystallography to determine the structure of NrpR regulatory domain. We identified the NrpR 2OG-binding cleft and show that residues predicted to interact directly with 2OG are conserved among diverse classes of 2OG-binding proteins. We show that high levels of 2OG inhibit NrpRs ability to bind DNA. Electron microscopy analyses document that NrpR adopts different quaternary structures in its inhibited 2OG-bound state compared with its active apo state. Our results indicate that upon 2OG release, NrpR repositions its DNA-binding domains correctly for optimal interaction with DNA thereby enabling gene repression., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Escherichia coli transforms the methanogenic archaeon Methanococcus maripaludis at frequencies ranging from 0.2 x 10(-6) to 2 x 10(-6) per recipient cell. Transformation requires cell-to-cell contact, oriT, and tra functions, is insensitive to DNase I, and otherwise displays hallmarks of conjugation.

In methanogenic Archaea, the final step of methanogenesis generates methane and a heterodisulfide of coenzyme M and coenzyme B (CoM-S-S-CoB). Reduction of this heterodisulfide by heterodisulfide reductase to regenerate HS-CoM and HS-CoB is an exergonic process. Thauer et al. [Thauer, et al. 2008 Nat Rev Microbiol 6:579-591] recently suggested that in hydrogenotrophic methanogens the energy of heterodisulfide reduction powers the most endergonic reaction in the pathway, catalyzed by the formylmethanofuran dehydrogenase, via flavin-based electron bifurcation. Here we present evidence that these two steps in methanogenesis are physically linked. We identify a protein complex from the hydrogenotrophic methanogen, Methanococcus maripaludis, that contains heterodisulfide reductase, formylmethanofuran dehydrogenase, F(420)-nonreducing hydrogenase, and formate dehydrogenase. In addition to establishing a physical basis for the electron-bifurcation model of energy conservation, the composition of the complex also suggests that either H(2) or formate (two alternative electron donors for methanogenesis) can donate electrons to the heterodisulfide-H(2) via F(420)-nonreducing hydrogenase or formate via formate dehydrogenase. Electron flow from formate to the heterodisulfide rather than the use of H(2) as an intermediate represents a previously unknown path of electron flow in methanogenesis. We further tested whether this path occurs by constructing a mutant lacking F(420)-nonreducing hydrogenase. The mutant displayed growth equal to wild-type with formate but markedly slower growth with hydrogen. The results support the model of electron bifurcation and suggest that formate, like H(2), is closely integrated into the methanogenic pathway.

The euryarchaeal transcriptional repressor NrpR regulates a variety of nitrogen assimilation genes by 2-oxoglutarate-reversible binding to conserved palindromic operators. The number and positioning of these operators varies among promoter regions of regulated genes, suggesting NrpR can bind in different patterns. Particularly intriguing is the contrast between the nif and glnK(1) promoter regions of Methanococcus maripaludis, where two operators are present but with different configurations. Here we study NrpR binding and regulation at the glnK(1) promoter, where the two operator sequences overlap and occur on opposite faces of the double helix. We find that both operators function in binding, with a dimer of NrpR binding simultaneously to each overlapping operator. We show in vivo that the first operator plays a primary role in regulation and the second operator plays an enhancing role. This is the first demonstration of overlapping operators functioning in Archaea.

Background: Methanogenic Archaea play key metabolic roles in anaerobic ecosystems, where they use H2 and other substrates to produce methane. Methanococcus maripaludis is a model for studies of the global response to nutrient limitations., Results: We used high-coverage quantitative proteomics to determine the response of M. maripaludis to growth-limiting levels of H2, nitrogen, and phosphate. Six to ten percent of the proteome changed significantly with each nutrient limitation. H2 limitation increased the abundance of a wide variety of proteins involved in methanogenesis. However, one protein involved in methanogenesis decreased: a low-affinity [Fe] hydrogenase, which may dominate over a higher-affinity mechanism when H2 is abundant. Nitrogen limitation increased known nitrogen assimilation proteins. In addition, the increased abundance of molybdate transport proteins suggested they function for nitrogen fixation. An apparent regulon governed by the euryarchaeal nitrogen regulator NrpR is discussed. Phosphate limitation increased the abundance of three different sets of proteins, suggesting that all three function in phosphate transport., Conclusion: The global proteomic response of M. maripaludis to each nutrient limitation suggests a wider response than previously appreciated. The results give new insight into the function of several proteins, as well as providing information that should contribute to the formulation of a regulatory network model.

Methanococcus maripaludis, an H(2)- and formate-utilizing methanogen, produced H(2) at high rates from formate. The rates and kinetics of H(2) production depended upon the growth conditions, and H(2) availability during growth was a major factor. Specific activities of resting cells grown with formate or H(2) were 0.4 to 1.4 U mg(-1) (dry weight). H(2) production in formate-grown cells followed Michaelis-Menten kinetics, and the concentration of formate required for half-maximal activity (K(f)) was 3.6 mM. In contrast, in H(2)-grown cells this process followed sigmoidal kinetics, and the K(f) was 9 mM. A key enzyme for formate-dependent H(2) production was formate dehydrogenase, Fdh. H(2) production and growth were severely reduced in a mutant containing a deletion of the gene encoding the Fdh1 isozyme, indicating that it was the primary Fdh. In contrast, a mutant containing a deletion of the gene encoding the Fdh2 isozyme possessed near-wild-type activities, indicating that this isozyme did not play a major role. H(2) production by a mutant containing a deletion of the coenzyme F(420)-reducing hydrogenase Fru was also severely reduced, suggesting that the major pathway of H(2) production comprised Fdh1 and Fru. Because a Deltafru-Deltafrc mutant retained 10% of the wild-type activity, an additional pathway is present. Mutants possessing deletions of the gene encoding the F(420)-dependent methylene-H(4)MTP dehydrogenase (Mtd) or the H(2)-forming methylene-H(4)MTP dehydrogenase (Hmd) also possessed reduced activity, which suggested that this second pathway was comprised of Fdh1-Mtd-Hmd. In contrast to H(2) production, the cellular rates of methanogenesis were unaffected in these mutants, which suggested that the observed H(2) production was not a direct intermediate of methanogenesis. In conclusion, high rates of formate-dependent H(2) production demonstrated the potential of M. maripaludis for the microbial production of H(2) from formate.

Reduced coenzyme F420 (F420H2) is an essential intermediate in methanogenesis from CO2. During methanogenesis from H2 and CO2, F420H2 is provided by the action of F420-reducing hydrogenases. However, an alternative pathway has been proposed, where H2-dependent methylenetetrahydromethanopterin dehydrogenase (Hmd) and F420H2-dependent methylenetetrahydromethanopterin dehydrogenase (Mtd) together reduce F420 with H2. Here we report the construction of mutants of Methanococcus maripaludis that are defective in each putative pathway. Their analysis demonstrates that either pathway supports growth on H2 and CO2. Furthermore, we show that during growth on formate instead of H2, where F420H2 is a direct product of formate oxidation, H2 production occurs. H2 presumably arises from the oxidation of F420H2, and the analysis of the mutants during growth on formate suggests that this too can occur by either pathway. We designate the alternative pathway for the interconversion of H2 and F420H2 the Hmd-Mtd cycle.

Continuous culture, transcriptome arrays, and measurements of cellular amino acid pools and tRNA charging levels were used to determine the response of Methanococcus maripaludis to leucine limitation. For comparison, the responses to phosphate and H2 limitations were measured as well. In addition, the effect of growth rate was determined. Leucine limitation resulted in a broad response. tRNA(Leu) charging decreased, but only small increases in mRNA were seen for amino acid biosynthesis genes. However, the cellular levels of free isoleucine and valine showed significant increases, indicating a coordinate regulation of branched-chain amino acids at a post-mRNA level. Leucine limitation also resulted in increased mRNA abundance for ribosomal protein genes, increased rRNA abundance, and decreased mRNA abundance for genes of methanogenesis. In contrast, phosphate limitation induced a specific response, a marked increase in mRNA levels for a phosphate transporter. Some mRNA levels responded to more than one factor; for example, transcripts for flagellum synthesis genes decreased under conditions of leucine limitation and increased under H2 limitation. Increased growth rate resulted in increased mRNA levels for ribosomal protein genes, increased rRNA abundance, and increased mRNA for a gene encoding an S-layer protein.

Reduction of substrate by nitrogenase requires direct electron transfer from the Fe protein to the MoFe protein. Inhibition of nitrogenase activity in Methanococcus maripaludis occurs when the regulatory protein NifI(1,2) binds the MoFe protein. This inhibition is relieved by 2-oxoglutarate. Here we present evidence that NifI(1,2) binding prevents association of the two nitrogenase components. Increasing amounts of Fe protein competed with NifI(1,2), decreasing its inhibitory effect. NifI(1,2) prevented the co-purification of MoFe protein with a mutant form of the Fe protein that forms a stable complex with the MoFe protein, and NifI(1,2) was unable to bind to an AlF4--stabilized Fe protein:MoFe protein complex. NifI(1,2) inhibited ATP- and MoFe protein-dependent oxidation of the Fe protein, and 2OG relieved this inhibition. These results support a model where NifI(1,2) competes with the Fe protein for binding to MoFe protein and prevents electron transfer.

NrpR is an euryarchaeal transcriptional repressor of nitrogen assimilation genes. Previous studies with Methanococcus maripaludis demonstrated that NrpR binds to palindromic operator sequences, blocking transcription initiation. The metabolite 2-oxoglutarate, an indicator of cellular nitrogen deficiency, induces transcription by lowering the affinity of NrpR for operator DNA. In this report we build on existing genetic tools for M. maripaludis to develop a screen for change-of-function mutations in a transcriptional regulator and demonstrate the use of an X-Gal (5-bromo-4-chloro-3-indolyl-beta-d-galactopyranoside) screen for strict anaerobes. We use the approach to address the primary structural requirements for the response of NrpR to 2-oxoglutarate. nrpR genes from the mesophilic M. maripaludis and the hyperthermophilic Methanopyrus kandleri were targeted for mutagenesis. M. maripaludis nrpR encodes a protein with two homologous NrpR domains while the M. kandleri nrpR homolog encodes a single NrpR domain. Random point mutagenesis and alanine replacement mutagenesis identified two amino acid residues of M. kandleri NrpR involved in induction of gene expression under nitrogen-deficient conditions and thus in the response to 2-oxoglutarate. Mutagenesis of the corresponding regions in either domain of M. maripaludis NrpR resulted in a similar effect, demonstrating a conserved structure-function relationship between the two repressors. The results indicate that in M. maripaludis, both NrpR domains participate in the 2-oxoglutarate response. The approach used here has wide adaptability to other regulatory systems in methanogenic Archaea and other strict anaerobes.

NrpR is a transcriptional repressor of nitrogen assimilation genes that was recently discovered and characterized in the methanogenic archaeon Methanococcus maripaludis. NrpR homologues are widely distributed in Euryarchaeota and present in a few bacterial species. They exist in three different domain configurations: a single ORF encoding one NrpR domain following an N-terminal helix-turn-helix (HTH); a single ORF encoding two NrpR domains fused in tandem following an N-terminal HTH; and two separate ORFs, one with a single domain following an N-terminal HTH and one with a single domain without a HTH. Phylogenetic analysis indicated that the NrpR family forms five distinct groups: the single domain HTH type, the two domains of the double domain HTH type and the two separately encoded domains. To determine the function of diverse NrpR homologues, representative genes in were expressed an Methanococcus maripaludis nrpR deletion mutant. Homologues from species that possess a single gene restored regulated repression, regardless of domain structure. In the case of Methanosarcina acetivorans that contains two genes, both were required. The results show that distantly related NrpR homologues that are present in widely dispersed phyla regulate the expression of nitrogen assimilation genes in a similar fashion.

The use of molecular hydrogen as electron donor for energy generation is a defining characteristic of the hydrogenotrophic methanogens, an ancient group that dominates the phylum Euryarchaeota. We present here a global study of changes in mRNA abundance in response to hydrogen availability for a hydrogenotrophic methanogen. Cells of Methanococcus maripaludis were grown by using continuous culture to deconvolute the effects of hydrogen limitation and growth rate, and microarray analyses were conducted. Hydrogen limitation markedly increased mRNA levels for genes encoding enzymes of the methanogenic pathway that reduce or oxidize the electron-carrying deazaflavin, coenzyme F(420). F(420)-dependent redox functions in energy-generating metabolism are characteristic of the methanogenic Archaea, and the results show that their regulation is distinct from other redox processes in the cell. Rapid growth increased mRNA levels of the gene for an unusual hydrogenase, the hydrogen-dependent methylenetetrahydromethanopterin dehydrogenase.

The rate of production of methane in many environments depends upon mutualistic interactions between sulfate-reducing bacteria and methanogens. To enhance our understanding of these relationships, we took advantage of the fully sequenced genomes of Desulfovibrio vulgaris and Methanococcus maripaludis to produce and analyze the first multispecies stoichiometric metabolic model. Model results were compared to data on growth of the co-culture on lactate in the absence of sulfate. The model accurately predicted several ecologically relevant characteristics, including the flux of metabolites and the ratio of D. vulgaris to M. maripaludis cells during growth. In addition, the model and our data suggested that it was possible to eliminate formate as an interspecies electron shuttle, but hydrogen transfer was essential for syntrophic growth. Our work demonstrated that reconstructed metabolic networks and stoichiometric models can serve not only to predict metabolic fluxes and growth phenotypes of single organisms, but also to capture growth parameters and community composition of simple bacterial communities.

Posttranslational regulation of nitrogenase, or switch-off, in the methanogenic archaeon Methanococcus maripaludis requires both nifI(1) and nifI(2), which encode members of the PII family of nitrogen-regulatory proteins. Previous work demonstrated that nitrogenase activity in cell extracts was inhibited in the presence of NifI(1) and NifI(2), and that 2-oxoglutarate (2OG), a potential signal of nitrogen limitation, relieved this inhibition. To further explore the role of the NifI proteins in switch-off, we found proteins that interact with NifI(1) and NifI(2) and determined whether 2OG affected these interactions. Anaerobic purification of His-tagged NifI(2) resulted in copurification of NifI(1) and the dinitrogenase subunits NifD and NifK, and 2OG or a deletion mutation affecting the T-loop of NifI(2) prevented copurification of dinitrogenase but did not affect copurification of NifI(1). Similar results were obtained with His-tagged NifI(1). Gel-filtration chromatography demonstrated an interaction between purified NifI(1,2) and dinitrogenase that was inhibited by 2OG. The NifI proteins themselves formed a complex of approximately 85 kDa, which appeared to further oligomerize in the presence of 2OG. NifI(1,2) inhibited activity of purified nitrogenase when present in a 1:1 molar ratio to dinitrogenase, and 2OG fully relieved this inhibition. These results suggest a model for switch-off of nitrogenase activity, where direct interaction of a NifI(1,2) complex with dinitrogenase causes inhibition, which is relieved by 2OG. The presence of nifI(1) and nifI(2) in the nif operons of all nitrogen-fixing Archaea and some anaerobic Bacteria suggests that this mode of nitrogenase regulation may operate in a wide variety of diazotrophs.

For the archaeon Methanococcus maripaludis, a fully sequenced and annotated model species of hydrogenotrophic methanogen, we report validation of quantitative protein level expression ratios on a proteome-wide basis. Using an approach based on quantitative multidimensional capillary HPLC and quadrupole ion trap mass spectrometry, coverage of gene expression approached that currently achievable with transcription microarrays. Comprehensive mass spectrometry-based proteomics and spotted cDNA arrays were used to compare global protein and mRNA levels in a wild-type (S2) and mutant strain (S40) of M. maripaludis. Using linear regression with 652 expression ratios generated by both the proteomic and microarray methods, a product moment correlation coefficient of 0.24 was observed. The correlation improved to 0.61 if only genes showing significant expression changes were included. A novel two-stage method of outlier detection was used for the protein measurements when Dixon's Q-test by itself failed to give satisfactory results. The log(2) transformations of the number of peptides or isotopic peptide pairs associated with each ORF, divided by the predicted molecular weight, were found to have moderately positive correlations with two bioinformatic predictors of gene expression based on codon bias. We detected peptides derived from 939 proteins or 55% of the genome coding capacity. Of these, 60 were overexpressed, and 34 were underexpressed in the mutant. Of the 1722 ORFs encoded in the genome, 1597 or 93% were probed by cDNA arrays. Of these, 50 were more highly expressed, and 45 showed lower expression levels in the mutant relative to the wild type. 15 ORFs were shown to be overexpressed by both methods, and two ORFs were shown to be overexpressed by proteomics and underexpressed by microarray.

Methanococcus maripaludis is a mesophilic archaeon that reduces CO2 to methane with H2 or formate as an energy source. It contains two membrane-bound energy-conserving hydrogenases, Eha and Ehb. To determine the role of Ehb, a deletion in the ehb operon was constructed to yield the mutant, strain S40. Growth of S40 was severely impaired in minimal medium. Both acetate and yeast extract were necessary to restore growth to nearly wild-type levels, suggesting that Ehb was involved in multiple steps in carbon assimilation. However, no differences in the total hydrogenase specific activities were found between the wild type and mutant in either cell extracts or membrane-purified fractions. Methanogenesis by resting cells with pyruvate as the electron donor was also reduced by 30% in S40, suggesting a defect in pyruvate oxidation. CO dehydrogenase/acetyl coenzyme A (CoA) synthase and pyruvate oxidoreductase had higher specific activities in the mutant, and genes encoding these enzymes, as well as AMP-forming acetyl-CoA synthetase, were expressed at increased levels. These observations support a role for Ehb in anabolic CO2 assimilation in methanococci.

Summary Post-translational regulation of nitrogen fixation, or switch-off, in the methanogenic archaeon Methanococcus maripaludis does not involve detectable covalent modification of the dinitrogenase reductase as in some bacteria, and the genes encoding the PII homologues NifI(1) and NifI(2) are both required, indicating a novel mechanism. To further understand the mechanism of switch-off, we assayed nitrogenase activity in cell extracts from wild-type and nifI mutant strains in the absence or presence of potential signals of nitrogen status. Activity in extracts from a DeltanifI(1)nifI(2) strain was sixfold higher than in extracts from wild-type cells. Addition of 2-oxoglutarate to wild-type extracts enhanced activity up to fivefold, a level similar to that observed in DeltanifI(1)nifI(2) extracts. 2-Oxoglutarate did not affect activity in DeltanifI(1)nifI(2) or single nifI mutant extracts. Furthermore, extracts from genetically complimented nifI mutants regained wild-type characteristics, indicating an in vitro correlation with in vivo effects. Extraction and quantification of 2-oxoglutarate indicated concentrations 10-fold higher in nitrogen-fixing cells than in switched-off and ammonium-grown cells. We propose a model for switch-off where the NifI proteins have an inhibitory effect on nitrogenase activity that is counteracted by high levels of 2-oxoglutarate, which acts as a signal of nitrogen limitation.

The methanogenic archaean Methanococcus maripaludis can use ammonia, alanine, or dinitrogen as a nitrogen source for growth. The euryarchaeal nitrogen repressor NrpR controls the expression of the nif (nitrogen fixation) operon, resulting in full repression with ammonia, intermediate repression with alanine, and derepression with dinitrogen. NrpR binds to two tandem operators in the nif promoter region, nifOR(1) and nifOR(2). Here we have undertaken both in vivo and in vitro approaches to study the way in which NrpR, nifOR(1), nifOR(2), and the effector 2-oxoglutarate (2OG) combine to regulate nif expression, leading to a comprehensive understanding of this archaeal regulatory system. We show that NrpR binds as a dimer to nifOR(1) and cooperatively as two dimers to both operators. Cooperative binding occurs only with both operators present. nifOR(1) has stronger binding and by itself can mediate the repression of nif transcription during growth on ammonia, unlike the weakly binding nifOR(2). However, nifOR(2) in combination with nifOR(1) is critical for intermediate repression during growth on alanine. Accordingly, NrpR binds to both operators together with higher affinity than to nifOR(1) alone. NrpR responds directly to 2OG, which weakens its binding to the operators. Hence, 2OG is an intracellular indicator of nitrogen deficiency and acts as an inducer of nif transcription via NrpR. This model is upheld by the recent finding (J. A. Dodsworth and J. A. Leigh, submitted for publication) in our laboratory that 2OG levels in M. maripaludis vary with growth on different nitrogen sources.

Among the archaea, Methanococcus maripaludis has the unusual ability to use L- or D-alanine as a nitrogen source. To understand how this occurs, we tested the roles of three adjacent genes encoding homologs of alanine dehydrogenase, alanine racemase, and alanine permease. To produce mutations in these genes, we devised a method for markerless mutagenesis that builds on previously established genetic tools for M. maripaludis. The technique uses a negative selection strategy that takes advantage of the ability of the M. maripaludis hpt gene encoding hypoxanthine phosphoribosyltransferase to confer sensitivity to the base analog 8-azahypoxanthine. In addition, we developed a negative selection method to stably incorporate constructs into the genome at the site of the upt gene encoding uracil phosphoribosyltransferase. Mutants with in-frame deletion mutations in the genes for alanine dehydrogenase and alanine permease lost the ability to grow on either isomer of alanine, while a mutant with an in-frame deletion mutation in the gene for alanine racemase lost only the ability to grow on D-alanine. The wild-type gene for alanine dehydrogenase, incorporated into the upt site, complemented the alanine dehydrogenase mutation. Hence, the permease is required for the transport of either isomer, the dehydrogenase is specific for the L isomer, and the racemase converts the D isomer to the L isomer. Phylogenetic analysis indicated that all three genes had been acquired by lateral gene transfer from the low-moles-percent G+C gram-positive bacteria.

To study global regulation in the methanogenic archaeon Methanococcus maripaludis, we devised a system for steady-state growth in chemostats. New Brunswick Bioflo 110 bioreactors were equipped with controlled delivery of hydrogen, nitrogen, carbon dioxide, hydrogen sulfide, and anaerobic medium. We determined conditions and media compositions for growth with three different limiting nutrients, hydrogen, phosphate, and leucine. To investigate leucine limitation we constructed and characterized a mutant in the leuA gene for 2-isopropylmalate synthase, demonstrating for the first time the function of this gene in the Archaea. Steady state specific growth rates in these studies ranged from 0.042 to 0.24 h(-1). Plots of culture density vs. growth rate for each condition showed the behavior predicted by growth modeling. The results show that growth behavior is normal and reproducible and validate the use of the chemostat system for metabolic and global regulation studies in M. maripaludis.

Methanococcus maripaludis is a mesophilic species of Archaea capable of producing methane from two substrates: hydrogen plus carbon dioxide and formate. To study the latter, we identified the formate dehydrogenase genes of M. maripaludis and found that the genome contains two gene clusters important for formate utilization. Phylogenetic analysis suggested that the two formate dehydrogenase gene sets arose from duplication events within the methanococcal lineage. The first gene cluster encodes homologs of formate dehydrogenase alpha (FdhA) and beta (FdhB) subunits and a putative formate transporter (FdhC) as well as a carbonic anhydrase analog. The second gene cluster encodes only FdhA and FdhB homologs. Mutants lacking either fdhA gene exhibited a partial growth defect on formate, whereas a double mutant was completely unable to grow on formate as a sole methanogenic substrate. Investigation of fdh gene expression revealed that transcription of both gene clusters is controlled by the presence of H(2) and not by the presence of formate.

Nitrogen assimilation in the methanogenic archaeon Methanococcus maripaludis is regulated by transcriptional repression involving a palindromic 'nitrogen operator' repressor binding sequence. Here we report the isolation of the nitrogen repressor, NrpR, from M. maripaludis using DNA affinity purification. Deletion of the nrpR gene resulted in loss of nitrogen operator binding activity in cell extracts and loss of repression of nif (nitrogen-fixation) and glnA (glutamine synthetase) gene expression in vivo. Genetic complementation of the nrpR mutation restored all functions. NrpR contained a putative N-terminal winged helix-turn-helix motif followed by two mutually homologous domains of unknown function. Comparison of the migration of NrpR in gel-filtration chromatography with its subunit molecular weight (60 kDa) suggested that NrpR was a tetramer. Several lines of evidence suggested that the level of NrpR itself is not regulated, and the binding affinity of NrpR to the nitrogen operator is controlled by an unknown mechanism. Homologues of NrpR were found only in certain species in the kingdom Euryarchaeota. Full length homologues were found in Methanocaldococcus jannaschii and Methanothermobacter thermoautotrophicus, and homologues lacking one or more of the three polypeptide domains were found in Archaeoglobus fulgidus, Methanopyrus kandleri, Methanosarcina acetivorans, and Methanosarcina mazei. NrpR represents a new family of regulators unique to the Euryarchaeota.

In the methanogenic archaeon Methanococcus maripaludis, growth with ammonia results in conditions of nitrogen excess. Complete repression of nitrogen fixation (nif) gene transcription occurs, and glutamine synthetase (glnA) gene transcription falls to a basal constitutive level. In addition, ammonia completely switches off nitrogenase enzyme activity. In contrast, growth with dinitrogen as the sole nitrogen source results in nitrogen starvation, full expression of nif and glnA, and high activity of nitrogenase. Here we report that a third nitrogen source, alanine, results in an intermediate regulatory response. Growth with alanine resulted in intermediate transcription of nif and glnA, and addition of alanine to a nitrogen-fixing (diazotrophic) culture caused partial switch-off of nitrogenase. This uniformity of response occurred despite differences in regulatory mechanisms. Nitrogenase switch-off requires the nitrogen sensor homologs NifI(1) and NifI(2), while transcriptional regulation of nif and glnA relies on a different, unknown sensor mechanism. In addition, although nif and glnA transcription are governed by a common repressor, the numbers and arrangements of repressor binding sites differ. Thus, the nif promoter region contains two operators situated downstream of the transcription start site, while the glnA promoter region contains only one operator just upstream of two closely spaced transcription start sites. In a previous study of nif expression using ammonia, we were able to detect a role only for the first nif operator in repression. Here we show that nif repression by alanine requires the second operator as well. In contrast, in the case of glnA the single operator was sufficient for repression by ammonia or alanine. These results suggest a uniform cellular response to nitrogen that is mediated by a different mechanism in each case.