Intracellular pyruvate flux in the methane-producing archaeon Methanococcus maripaludis

During growth of the methanogenic archaeon Methanococcus maripaludis on alanine as the sole nitrogen source under H(2)/CO(2), alanine was incorporated into amino acids derived from pyruvate including leucine, isoleucine, and valine. Thus, growth with alanine was an efficient means of labeling intracellular pools of pyruvate in this lithotroph. Cells were grown with 18% [U-(13)C]alanine, and the distribution of the isotope in the branched-chain amino acids was determined by (13)C-NMR. Carbons derived from pyruvate contained 14.5% (13)C, indicating that most of the cellular pyruvate was obtained from alanine. In contrast, carbons derived from acetyl-CoA contained only 3-5% (13)C, indicating that only small amounts of acetyl-CoA were formed from pyruvate. Thus, autotrophic acetyl-CoA biosynthesis continued even in the presence of an organic carbon source. Moreover, the labeling of acetyl-CoA was lower than would be predicted if pyruvate was a C-1 donor for acetyl-CoA biosynthesis. Carbon derived from the C-1 of acetyl-CoA contained less (13)C than carbon derived from the C-2 of acetyl-CoA, and this difference was attributed to the acetyl-CoA:CO(2) exchange activity of acetyl-CoA synthase. No enrichment was detected for the C-1 of valine, which was derived from the C-1 of pyruvate. This result was attributed to the pyruvate:CO(2) exchange activity of pyruvate oxidoreductase and may have important implications for isotope tracer studies utilizing pyruvate. Lastly, these results demonstrate that the breakdown of pyruvate by methanococci is very limited even under conditions where it is the sole nitrogen and major carbon source.     

Hydrogenase-independent uptake and metabolism of electrons by the archaeon Methanococcus maripaludis

Direct, shuttle-free uptake of extracellular, cathode-derived electrons has been postulated as a novel mechanism of electron metabolism in some prokaryotes that may also be involved in syntrophic electron transport between two microorganisms. Experimental proof for direct uptake of cathodic electrons has been mostly indirect and has been based on the absence of detectable concentrations of molecular hydrogen. However, hydrogen can be formed as a transient intermediate abiotically at low cathodic potentials (<−414 mV) under conditions of electromethanogenesis. Here we provide genetic evidence for hydrogen-independent uptake of extracellular electrons. Methane formation from cathodic electrons was observed in a wild-type strain of the methanogenic archaeon Methanococcus maripaludis as well as in a hydrogenase-deletion mutant lacking all catabolic hydrogenases, indicating the presence of a hydrogenase-independent mechanism of electron catabolism. In addition, we discovered a new route for hydrogen or formate production from cathodic electrons: Upon chemical inhibition of methanogenesis with 2-bromo-ethane sulfonate, hydrogen or formate accumulated in the bioelectrochemical cells instead of methane. These results have implications for our understanding on the diversity of microbial electron uptake and metabolism.     

Impact of translational selection on codon usage bias in the archaeon Methanococcus maripaludis

Patterns of codon usage have been extensively studied among Bacteria and Eukaryotes, but there has been little investigation of species from the third domain of life, the Archaea. Here, we examine the nature of codon usage bias in a methanogenic archaeon, Methanococcus maripaludis. Genome-wide patterns of codon usage are dominated by a strong A + T bias, presumably largely reflecting mutation patterns. Nevertheless, there is variation among genes in the use of a subset of putatively translationally optimal codons, which is strongly correlated with gene expression level. In comparison with Bacteria such as Escherichia coli, the strength of selected codon usage bias in highly expressed genes in M. maripaludis seems surprisingly high given its moderate growth rate. However, the pattern of selected codon usage differs between M. maripaludis and E. coli: in the archaeon, strongly selected codon usage bias is largely restricted to twofold degenerate amino acids (AAs). Weaker bias among the codons for fourfold degenerate AAs is consistent with the small number of tRNA genes in the M. maripaludis genome.     

The DNA polymerase gene from the methanogenic archaeon Methanococcus voltae.

Previous studies have identified intervening sequences that encode homing endonucleases within the genes encoding several archaeal DNA polymerases. We report the sequence of the gene encoding the DNA polymerase of Methanococcus voltae and describe evidence that it lacks analogous intervening sequences.     

Genetic and mass spectrometry analyses of the unusual type IV-like pili of the archaeon Methanococcus maripaludis

The structure of pili from the archaeon Methanococcus maripaludis is unlike that of any bacterial pili. However, genetic analysis of the genes involved in the formation of these pili has been lacking until this study. Pili were isolated from a nonflagellated (ΔflaK) mutant and shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to consist primarily of subunits with an apparent molecular mass of 17 kDa. In-frame deletions were created in three genes, MMP0233, MMP0236, and MMP0237, which encode proteins with bacterial type IV pilin-like signal peptides previously identified by in silico methodology as likely candidates for pilus structural proteins. Deletion of MMP0236 or MMP0237 resulted in mutant cells completely devoid of pili on the cell surface, while deletion of the third pilin-like gene, MMP0233, resulted in cells greatly reduced in the number of pili on the surface. Complementation with the deleted gene in each case returned the cells to a piliated state. Surprisingly, mass spectrometry analysis of purified pili identified the major structural pilin as another type IV pilin-like protein, MMP1685, whose gene is located outside the first pilus locus. This protein was found to be glycosylated with an N-linked branched pentasaccharide glycan. Deletion and complementation analysis confirmed that MMP1685 is required for piliation.     

Isolation of acetate auxotrophs of the methane-producing archaeon Methanococcus maripaludis by random insertional mutagenesis.

To learn more about autotrophic growth of methanococci, we isolated nine conditional mutants of Methanococcus maripaludis after transformation of the wild type with a random library in pMEB.2, a suicide plasmid bearing the puromycin-resistance cassette pac. These mutants grew poorly in mineral medium and required acetate or complex organic supplements such as yeast extract for normal growth. One mutant, JJ104, was a leaky acetate auxotroph. A plasmid, pWDK104, was recovered from this mutant by electroporation of a plasmid preparation into Escherichia coli. Transformation of wild-type M. maripaludis with pWDK104 produced JJ104-1, a mutant with the same phenotype as JJ104, thus establishing that insertion of pWDK104 into the genome was responsible for the phenotype. pWDK104 contained portions of the methanococcal genes encoding an ABC transporter closely related to MJ1367-MJ1368 of M. jannaschii. Because high levels of molybdate, tungstate, and selenite restored growth to wild-type levels, this transporter may be specific for these oxyanions. A second acetate auxotroph, JJ117, had an absolute growth requirement for either acetate or cobalamin, and wild-type growth was observed only in the presence of both. Cobinamide, 5', 6'-dimethylbenzimidazole, and 2-aminopropanol did not replace cobalamin. This phenotype was correlated with tandem insertions in the genome but not single insertions and appeared to have resulted from an indirect effect on cobamide metabolism. Plasmids rescued from other mutants contained portions of ORFs denoted in M. jannaschii as endoglucanase (MJ0555), transketolase (MJ0681), thiamine biosynthetic protein thiI (MJ0931), and several hypothetical proteins (MJ1031, MJ0835, and MJ0835.1).     

Characterization of pURB500 from the archaeon Methanococcus maripaludis and construction of a shuttle vector.

The complete sequence of the 8,285-bp plasmid pURB500 from Methanococcus maripaludis C5 was determined. Sequence analysis identified 18 open reading frames as well as two regions of potential iterons and complex secondary structures. The shuttle vector, pDLT44, for M. maripaludis JJ was constructed from the entire pURB500 plasmid and pMEB.2, an Escherichia coli vector containing a methanococcal puromycin-resistance marker (P. Gernhardt, O. Possot, M. Foglino, L. Sibold, and A. Klein, Mol. Gen. Genet. 221:273-279, 1990). By using polyethylene glycol transformation, M. maripaludis JJ was transformed at a frequency of 3.3 x 10(7) transformants per microg of pDLT44. The shuttle vector was stable in E. coli under ampicillin selection but was maintained at a lower copy number than pMEB.2. Based on the inability of various restriction fragments of pURB500 to support maintenance in M. maripaludis JJ, multiple regions of pURB500 were required. pDLT44 did not replicate in Methanococcus voltae.     

Regulation of formate dehydrogenase activity in Methanococcus thermolithotrophicus.

Methanococcus thermolithotrophicus can use either H2 or formate as the electron donor for methanogenesis from CO2. Resuspended-cell experiments revealed that the ability to use H2 as the source of electrons for methanogenesis was constitutive; cells grown on formate or H2-CO2 were equally capable of H2-CO2 methanogenesis. The ability to metabolize formate at high rates was observed only in cells previously grown on formate. Two such strains were distinguished: strain F and strain HF. Strain F was repeatedly grown exclusively on formate for over 3 years; this strain showed a constitutive capacity to metabolize formate to methane, even after subsequent repeated transfers to medium containing only H2-CO2. Strain HF could only metabolize formate to methane when grown in the presence of formate with no H2 present; this strain was recently derived from another strain (H) that had been exclusively grown on H2-CO2 and which upon initial transfer to formate medium could only metabolize formate to methane at a very slow rate. Initial adaptation of strain H to growth on formate was preceded by a long lag. The specific activities of hydrogenase and formate dehydrogenase in cell extracts derived from these different strains confirmed these findings. Similar levels of hydrogenase were observed in all strains, independent of the presence of H2 in the growth medium medium. High levels of formate dehydrogenase were also constitutive in strain F. Only low formate dehydrogenase activities were observed in strain H. High levels of formate dehydrogenase were observed in strain HF only when these cells were grown with formate in the absence of H2. In all strains the two- to threefold fluctuations of both hydrogenase and formate dehydrogenase cell-free activities were observed during growth, with peak activities reached in the middle of the exponential phase.     

Disruption of the operon encoding Ehb hydrogenase limits anabolic CO2 assimilation in the archaeon Methanococcus maripaludis

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.     

Occurrence of selenocysteine in the selenium-dependent formate dehydrogenase of Methanococcus vannielii.

The selenium-dependent formate dehydrogenase of Methanococcus vannielii was isolated from bacteria grown in the presence of [${}^{75}\text{Se}$]selenite. Purification under strictly anaerobic conditions resulted in the simultaneous enrichment of formate dehydrogenase activity, ${}^{75}\text{Se}$, and a brown chromophore that absorbs maximally at 380 nm. Acid hydrolysis of the enzyme after reduction with borohydride and alkylation with iodoacetamide, released a radioactive selenoamino acid derivative that was identified as [${}^{75}\text{Se}$]carboxymethyl-selenocysteine. This is the third selenoenzyme shown to contain selenocysteine.     

Characterization of Energy-Conserving Hydrogenase B in Methanococcus maripaludis

The Methanococcus maripaludis energy-conserving hydrogenase B (Ehb) generates low potential electrons required for autotrophic CO₂ assimilation. To analyze the importance of individual subunits in Ehb structure and function, markerless in-frame deletions were constructed in a number of M. maripaludis ehb genes. These genes encode the large and small hydrogenase subunits (ehbN and ehbM, respectively), a polyferredoxin and ferredoxin (ehbK and ehbL, respectively), and an ion translocator (ehbF). In addition, a gene replacement mutation was constructed for a gene encoding a putative membrane-spanning subunit (ehbO). When grown in minimal medium plus acetate (McA), all ehb mutants had severe growth deficiencies except the ΔehbO::pac strain. The membrane-spanning ion translocator (ΔehbF) and the large hydrogenase subunit (ΔehbN) deletion strains displayed the severest growth defects. Deletion of the ehbN gene was of particular interest because this gene was not contiguous to the ehb operon. In-gel activity assays and Western blots confirmed that EhbN was part of the membrane-bound Ehb hydrogenase complex. The ΔehbN strain was also sensitive to growth inhibition by aryl acids, indicating that Ehb was coupled to the indolepyruvate oxidoreductase (Ior), further supporting the hypothesis that Ehb provides low potential reductants for the anabolic oxidoreductases in M. maripaludis.Hydrogenotrophic methanococci specialize in utilizing H₂ as an electron donor, and these organisms possess six different Ni-Fe hydrogenases. These enzymes include two F_420--reducing hydrogenases, two non-F₄₂₀-reducing hydrogenases, and two membrane-bound hydrogenases (Eha and Ehb [5]). The F₄₂₀-reducing hydrogenases reduce coenzyme F₄₂₀, which subsequently reduces methenyltetrahydromethanopterin and methylenetetrahydromethanopterin, intermediates in the pathway of methanogenesis. In Methanococcus voltae, the F₄₂₀-reducing hydrogenase is also reported to reduce the 2-mercaptoethanesulfonate:7-mercaptoheptanoylthreonine phosphate heterodisulfide formed in the final step of methanogenesis (2). In contrast, Methanothermobacter marburgensis utilizes the non-F₄₂₀-reducing hydrogenase to reduce the heterodisulfide (22, 25).The two membrane-bound hydrogenases couple the chemiosmotic energy of ion gradients to H₂ oxidation and ferredoxin reduction. In the aceticlastic methanogen Methanosarcina barkeri, the homologous enzyme is called energy conserving hydrogenase or Ech and performs a variety of physiological functions, including the generation of a proton motive force during CO oxidation and concomitant proton reduction in aceticlastic methanogenesis and the generation of low potential electron donors for CO₂ reduction to formylmethanofuran in the first step of methanogenesis and the reductive carboxylation of acetyl coenzyme A (acetyl-CoA) to pyruvate in carbon assimilation (11, 12). In the hydrogenotrophic methanogens, it is predicted that the two energy-conserving hydrogenases (Eha and Ehb) have distinct roles (26). The Ehb appears to reduce low potential electron carriers utilized in autotrophic CO₂ fixation (16). Anabolic enzymes likely to be coupled to Ehb in this manner include (i) the carbon monoxide dehydrogenase/acetyl-CoA synthase (CODH/ACS) and the pyruvate oxidoreductase (Por), which catalyze the first two steps of carbon assimilation; (ii) the α-ketoglutarate oxidoreductase (Kor), which catalyzes the final step in the incomplete reductive tricarboxylic acid cycle; and (iii) the indolepyruvate oxidoreductase (Ior) and the 2-oxoisovalerate oxidoreductase (Vor), which are involved in amino acid biosynthesis from aryl and branched-chain acids, respectively. Support for these conclusions comes in large part from the phenotype of an M. maripaludis ehb gene replacement mutant S40, which was only capable of limited growth in the absence of acetate and amino acids (16). Furthermore, expression of CODH/ACS, Por, and Vor were significantly upregulated in the mutant, providing further evidence for a role of Ehb in these processes (16). In contrast, there is no direct evidence for the role of Eha. By analogy with the Methanosarcina Ech, it could be involved in generating reducing equivalents for the reduction of CO₂ to formylmethanofuran. Alternatively, hydrogenotrophic methanogens may have an alternative method of CO₂ reduction (27), and Eha could have another function entirely.In spite of some functional similarities between the Ech of the aceticlastic methanogens and Eha or Ehb of hydrogenotrophs, the structures of their operons are very different (Fig. ​(Fig.1).1). Based upon sequence comparisons, all of these membrane-bound hydrogenases possess conserved large and small hydrogenase subunits, a 2[4Fe-4S] ferredoxin, and an integral membrane ion translocator (3, 8, 26). Otherwise, the structures are very different. The purified Ech from Methanosarcina barkeri contains six polypeptides encoded by the six genes of the ech operon (8, 11). The Eha and Ehb hydrogenases have never been purified. The eha and ehb operons from the hydrogenotrophic methanogen Methanothermobacter thermautotrophicus comprise 20 and 17 genes, respectively (23, 26). Most of these genes are predicted to encode transmembrane proteins, although there are also several polyferredoxins and hydrophilic proteins (26). Many of these genes are not homologous to the M. barkeri ech genes. The Methanococcus maripaludis genome contains homologs to the M. thermautotrophicus eha and ehb genes, although only nine of the ehb genes are contiguous on the genome (Fig. ​(Fig.1).1). In the present study, the Ehb from the hydrogenotrophic methanogen Methanococcus maripaludis was analyzed. M. maripaludis is a model organism that can be easily genetically modified. Furthermore, its genome has been sequenced, and many of its biochemical pathways have been characterized.Open in a separate windowFIG. 1.Genetic map of Methanosarcina barkeri ech (A), Methanothermobacter marburgensis ehb (MTH1235-1251) (B), and Methanococcus maripaludis ehb (MMP1631-1629) (C) operons. Genes encoding integral membrane proteins found only in Ehb are indicated in blue, integral membrane proteins conserved in both Ech and Ehb are blue with diagonal stripes, hydrogenase small subunits are yellow, hydrogenase large subunits are red, 4Fe-4S motif-containing proteins are brown, and other hydrophilic proteins present in Ehb but absent from Ech are gray. Notably, M. maripaludis contains homologs to all of the M. marburgensis ehb genes, but many are unlinked to the major gene cluster and not shown. Based upon references 5, 8, 11, and 26.