Methanogenic Archaeon Methanococcus Maripaludis Research Articles

REP-X: An Evolution-guided Strategy for the Rational Design of Cysteine-less Protein Variants

Site-specific labeling of proteins is often a prerequisite for biophysical and biochemical characterization. Chemical modification of a unique cysteine residue is among the most facile methods for site-specific labeling of proteins. However, many proteins have multiple reactive cysteines, which must be mutated to other residues to enable labeling of unique positions. This trial-and-error process often results in cysteine-free proteins with reduced activity or stability. Herein we describe a general methodology to rationally engineer cysteine-less proteins. Briefly, natural variation across orthologues is exploited to identify suitable cysteine replacements compatible with protein activity and stability. As a proof-of-concept, we recount the successful engineering of a cysteine-less mutant of the group II chaperonin from methanogenic archaeon Methanococcus maripaludis. A webapp, REP-X (Replacement at Endogenous Positions from eXtant sequences), which enables users to design their own cysteine-less protein variants, will make this rational approach widely available.

Engineering the Autotroph Methanococcus maripaludis for Geraniol Production.

The rapid autotrophic growth of the methanogenic archaeon Methanococcus maripaludis on H2 and CO2 makes it an attractive microbial chassis to inexpensively produce biochemicals. To explore this potential, a synthetic gene encoding geraniol synthase (GES) derived from Ocimum basilicum was cloned into a M. maripaludis expression vector under selection for puromycin resistance. Recombinant expression of GES in M. maripaludis during autotrophic growth on H2/CO2 or formate yielded geraniol at 2.8 and 4.0 mg g(-1) of dry weight, respectively. The yield of geraniol decreased 2-3-fold when organic carbon sources were added to stimulate heterotrophic growth. In the absence of puromycin, geraniol production during autotrophic growth on formate increased to 4.6 mg g(-1) of dry weight. A conceptual model centered on the autotrophic acetyl coenzyme A biosynthetic pathway identified strategies to divert more autotrophic carbon flux to geraniol production.

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.

Biosynthesis of 4-Thiouridine in tRNA in the Methanogenic Archaeon Methanococcus maripaludis

4-Thiouridine (s(4)U) is a conserved modified nucleotide at position 8 of bacterial and archaeal tRNAs and plays a role in protecting cells from near-UV killing. Escherichia coli employs the following two enzymes for its synthesis: the cysteine desulfurase IscS, which forms a Cys persulfide enzyme adduct from free Cys; and ThiI, which adenylates U8 and transfers sulfur from IscS to form s(4)U. The C-terminal rhodanese-like domain (RLD) of ThiI is responsible for the sulfurtransferase activity. The mechanism of s(4)U biosynthesis in archaea is not known as many archaea lack cysteine desulfurase and an RLD of the putative ThiI. Using the methanogenic archaeon Methanococcus maripaludis, we show that deletion of ThiI (MMP1354) abolished the biosynthesis of s(4)U but not of thiamine. MMP1354 complements an Escherichia coli ΔthiI mutant for s(4)U formation, indicating that MMP1354 is sufficient for sulfur incorporation into s(4)U. In the absence of an RLD, MMP1354 uses Cys(265) and Cys(268) located in the PP-loop pyrophosphatase domain to generate persulfide and disulfide intermediates for sulfur transfer. In vitro assays suggest that S(2-) is a physiologically relevant sulfur donor for s(4)U formation catalyzed by MMP1354 (K(m) for Na(2)S is ∼1 mm). Thus, methanogenic archaea developed a strategy for sulfur incorporation into s(4)U that differs from bacteria; this may be an adaptation to life in sulfide-rich environments.

Methanococcus maripaludis: an archaeon with multiple functional MCM proteins?

There are a large number of proteins involved in the control of eukaryotic DNA replication, which act together to ensure DNA is replicated only once every cell cycle. Key proteins involved in the initiation and elongation phases of DNA replication include the MCM (minchromosome maintenance) proteins, MCM2-MCM7, a family of six related proteins believed to act as the replicative helicase. Genome sequencing has revealed that the archaea possess a simplified set of eukaryotic replication homologues. The complexity of the DNA replication machinery in eukaryotes has led to a number of archaeal species being adapted as model organisms for the study of the DNA replication process. Most archaea sequenced to date possess a single MCM homologue that forms a hexameric complex. Recombinant MCMs from several archaea have been used in the biochemical characterization of the protein, revealing that the MCM complex has ATPase, DNA-binding and -unwinding activities. Unusually, the genome of the methanogenic archaeon Methanococcus maripaludis contains four MCM homologues, all of which contain the conserved motifs required for function. The availability of a wide range of genetic tools for the manipulation of M. maripaludis and the relative ease of growth of this organism in the laboratory makes it a good potential model for studying the role of multiple MCMs in DNA replication.

Protein abundance ratios for global studies of prokaryotes

The use of multidimensional capillary HPLC combined with MS/MS has allowed high qualitative and quantitative proteome coverage of prokaryotic organisms. The determination of protein abundance change between two or more conditions has matured to the point that false discovery rates can be very low and for smaller proteomes coverage is sufficiently high to explicitly consider false negative error. Selected aspects of using these methods for global protein abundance assessments are reviewed. These include instrumental issues that influence the reliability of abundance ratios; a comparison of sources of nonlinearity, errors, and data compression in proteomics and spotted cDNA arrays; strengths and weaknesses of spectral counting versus stable isotope metabolic labeling; and a survey of microbiological applications of global abundance analysis at the protein level. Proteomic results for two organisms that have been studied extensively using these methods are reviewed in greater detail. Spectral counting and metabolic labeling data are compared and the utility of proteomics for global gene regulation studies are discussed for the methanogenic Archaeon Methanococcus maripaludis. The oral pathogen Porphyromonas gingivalis is discussed as an example of an organism where a large percentage of the proteome differs in relative abundance between the intracellular and extracellular phenotype.

Diverse homologues of the archaeal repressor NrpR function similarly in nitrogen regulation

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.

2‐Oxoglutarate and the PII homologues NifI1 and NifI2 regulate nitrogenase activity in cell extracts of Methanococcus maripaludis

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.

Continuous culture of Methanococcus maripaludis under defined nutrient conditions

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.

A novel repressor of nif and glnA expression in the methanogenic archaeon Methanococcus maripaludis.

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.

Regulatory response of Methanococcus maripaludis to alanine, an intermediate nitrogen source.

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.

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.

Function and regulation of glnA in the methanogenic archaeon Methanococcus maripaludis.

The glnA gene in the domains Bacteria and Archaea encodes glutamine synthetase, a universally distributed enzyme that functions in ammonia assimilation and glutamine synthesis. We investigated the regulation and function of glnA in the methanogenic archaeon Methanococcus maripaludis. The deduced amino acid sequence of the gene demonstrated its membership in class GSI-alpha of glutamine synthetases. The gene appeared to be expressed as a monocistronic operon. glnA mRNA levels and specific activities of glutamine synthetase were regulated similarly by nitrogen. Three transcription start sites were identified, corresponding to two overlapping nitrogen-regulated promoters and one weaker constitutive promoter. An inverted repeat immediately upstream of the regulated transcription start sites mediated repression under noninducing conditions. Thus, mutations that altered the sequence of the inverted repeat resulted in derepression. The inverted repeat had sequence similarity with a repeat that we previously identified as the nif operator of M. maripaludis, suggesting a common mechanism of nitrogen regulation. Efforts to produce a glnA null mutant failed, suggesting that glnA is an essential gene in M. maripaludis.

The nif gene operon of the methanogenic archaeon Methanococcus maripaludis.

Nitrogen fixation occurs in two domains, Archaea and Bacteria. We have characterized a nif (nitrogen fixation) gene cluster in the methanogenic archaeon Methanococcus maripaludis. Sequence analysis revealed eight genes, six with sequence similarity to known nif genes and two with sequence similarity to glnB. The gene order, nifH, ORF105 (similar to glnB), ORF121 (similar to glnB), nifD, nifK, nifE, nifN, and nifX, was the same as that found in part in other diazotrophic methanogens and except for the presence of the glnB-like genes, also resembled the order found in many members of the Bacteria. Using transposon insertion mutagenesis, we determined that an 8-kb region required for nitrogen fixation corresponded to the nif gene cluster. Northern analysis revealed the presence of either a single 7.6-kb nif mRNA transcript or 10 smaller mRNA species containing portions of the large transcript. Polar effects of transposon insertions demonstrated that all of these mRNAs arose from a single promoter region, where transcription initiated 80 bp 5' to nifH. Distinctive features of the nif gene cluster include the presence of the six primary nif genes in a single operon, the placement of the two glnB-like genes within the cluster, the apparent physical separation of the cluster from any other nif genes that might be in the genome, the fragmentation pattern of the mRNA, and the regulation of expression by a repression mechanism described previously. Our study and others with methanogenic archaea reporting multiple mRNAs arising from gene clusters with only a single putative promoter sequence suggest that mRNA processing following transcription may be a common occurrence in methanogens.

Transcriptional regulation in Archaea: in vivo demonstration of a repressor binding site in a methanogen.

The status of the Archaea as one of the three primary Domains emphasizes the importance of understanding their molecular fundamentals. Basic transcription in the Archaea resembles eucaryal transcription. However, little is known about transcriptional regulation. We have taken an in vivo approach, using genetics to address transcriptional regulation in the methanogenic Archaeon Methanococcus maripaludis. We identified a repressor binding site that regulates nif (nitrogen fixation) gene expression. The repressor binding site was palindromic (an inverted repeat) and was located just after the transcription start site of nifH. Mutations that changed the sequence of the palindrome resulted in marked decreases in repression by ammonia, even when the palindromic nature of the site was retained. The same mutations greatly decreased binding to the site by components of cell extract. These results provide the first partial description of a transcriptional regulatory mechanism in the methanogenic Archaea. This work also illustrates the utility of genetic approaches in Methanococcus that have not been widely used in the methanogens: directed mutagenesis and reporter gene fusions with lacZ.