Non-essential MCM-related proteins mediate a response to DNA damage in the archaeon Methanococcus maripaludis.
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ABSTRACT:
SUBMITTER: Walters AD
PROVIDER: S-EPMC5737143 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:The methanogenic archaea Methanocaldococcus jannaschii and Methanothermobacter thermautotrophicus contain a dual-specificity prolyl-tRNA synthetase (ProCysRS) that accurately forms both prolyl-tRNA (Pro-tRNA) and cysteinyl-tRNA (Cys-tRNA) suitable for in vivo translation. This intriguing enzyme may even perform its dual role in organisms that possess a canonical single-specificity cysteinyl-tRNA synthetase (CysRS), raising the question as to whether this latter aminoacyl-tRNA synthetase is indeed required for cell viability. To test the postulate that all synthetase genes are essential, we disrupted the cysS gene (encoding CysRS) of Methanococcus maripaludis. The knockout strain was viable under normal growth conditions. Biochemical analysis showed that the pure M. maripaludis ProCysRS was capable of forming Cys-tRNA, implying that the dual-specificity enzyme compensates in vivo for the loss of CysRS. The canonical CysRS has a higher affinity for cysteine than ProCysRS, a reason why M. maripaludis may have acquired cysS by a late lateral gene transfer. These data challenge the notion that all twenty aminoacyl-tRNA synthetases are essential for the viability of a cell.
| S-EPMC64675 | biostudies-literature
Project description: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.
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Project description: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.
| S-EPMC103557 | biostudies-literature
Project description:Most microorganisms in nature spend the majority of time in a state of slow or zero growth and slow metabolism under limited energy or nutrient flux rather than growing at maximum rates. Yet, most of our knowledge has been derived from studies on fast-growing bacteria. Here, we systematically characterized the physiology of the methanogenic archaeon Methanococcus maripaludis during slow growth. M. maripaludis was grown in continuous culture under energy (formate)-limiting conditions at different dilution rates ranging from 0.09 to 0.002 h-1, the latter corresponding to 1% of its maximum growth rate under laboratory conditions (0.23 h-1). While the specific rate of methanogenesis correlated with growth rate as expected, the fraction of cellular energy used for maintenance increased and the maintenance energy per biomass decreased at slower growth. Notably, proteome allocation between catabolic and anabolic pathways was invariant with growth rate. Unexpectedly, cells maintained their maximum methanogenesis capacity over a wide range of growth rates, except for the lowest rates tested. Cell size, cellular DNA, RNA, and protein content as well as ribosome numbers also were largely invariant with growth rate. A reduced protein synthesis rate during slow growth was achieved by a reduction in ribosome activity rather than via the number of cellular ribosomes. Our data revealed a resource allocation strategy of a methanogenic archaeon during energy limitation that is fundamentally different from commonly studied versatile chemoheterotrophic bacteria such as E. coli.
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Project description:Methyl coenzyme M reductase (MCR) is a complex enzyme that catalyzes the final step in biological methanogenesis. To better understand its assembly, the recombinant MCR from the thermophile Methanothermococcus okinawensis (rMCRok) was expressed in the mesophile Methanococcus maripaludis The rMCRok was posttranslationally modified correctly and contained McrD and the unique nickel tetrapyrrole coenzyme F430 Subunits of the native M. maripaludis (MCRmar) were largely absent, suggesting that the recombinant enzyme was formed by an assembly of cotranscribed subunits. Strong support for this hypothesis was obtained by expressing a chimeric operon comprising the His-tagged mcrA from M. maripaludis and the mcrBDCG from M. okinawensis in M. maripaludis The His-tagged purified rMCR then contained the M. maripaludis McrA and the M. okinawensis McrBDG. The present study prompted us to form a working model for MCR assembly, which can be further tested by the heterologous expression system established here.IMPORTANCE Approximately 1.6% of the net primary production of plants, algae, and cyanobacteria are processed by biological methane production in anoxic environments. This accounts for about 74% of the total global methane production, up to 25% of which is consumed by anaerobic oxidation of methane (AOM). Methyl coenzyme M reductase (MCR) is the key enzyme in both methanogenesis and AOM. MCR is assembled as a dimer of two heterotrimers, where posttranslational modifications and F430 cofactors are embedded in the active sites. However, this complex assembly process remains unknown. Here, we established a heterologous expression system for MCR to learn how MCR is assembled.
| S-EPMC5847650 | biostudies-literature
Project description:Methanococcus maripaludis is a strictly anaerobic, methane-producing archaeon. Aromatic amino acids (AroAAs) are biosynthesized in this autotroph either by the de novo pathway, with chorismate as an intermediate, or by the incorporation of exogenous aryl acids via indolepyruvate oxidoreductase (IOR). In order to evaluate the roles of these pathways, the gene that encodes the third step in the de novo pathway, 3-dehydroquinate dehydratase (DHQ), was deleted. This mutant required all three AroAAs for growth, and no DHQ activity was detectible in cell extracts, compared to 6.0 +/- 0.2 mU mg(-1) in the wild-type extract. The growth requirement for the AroAAs could be fulfilled by the corresponding aryl acids phenylacetate, indoleacetate, and p-hydroxyphenylacetate. The specific incorporation of phenylacetate into phenylalanine by the IOR pathway was demonstrated in vivo by labeling with [1-(13)C]phenylacetate. M. maripaludis has two IOR homologs. A deletion mutant for one of these homologs contained 76, 74, and 42% lower activity for phenylpyruvate, p-hydoxyphenylpyruvate, and indolepyruvate oxidation, respectively, than the wild type. Growth of this mutant in minimal medium was inhibited by the aryl acids, but the AroAAs partially restored growth. Genetic complementation of the IOR mutant also restored much of the wild-type phenotype. Thus, aryl acids appear to regulate the expression or activity of the de novo pathway. The aryl acids did not significantly inhibit the activity of the biosynthetic enzymes chorismate mutase, prephenate dehydratase, and prephenate dehydrogenase in cell extracts, so the inhibition of growth was probably not due to an effect on these enzymes.
| S-EPMC451642 | biostudies-literature
Project description: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.
| S-EPMC4817615 | biostudies-literature
Project description:A comprehensive whole-genome analysis of gene function by transposon mutagenesis and deep sequencing methodology has been implemented successfully in a representative of the Archaea domain. Libraries of transposon mutants were generated for the hydrogenotrophic, methanogenic archaeon Methanococcus maripaludis S2 using a derivative of the Tn5 transposon. About 89,000 unique insertions were mapped to the genome, which allowed for the classification of 526 genes or about 30% of the genome as possibly essential or strongly advantageous for growth in rich medium. Many of these genes were homologous to eukaryotic genes that encode fundamental processes in replication, transcription, and translation, providing direct evidence for their importance in Archaea. Some genes classified as possibly essential were unique to the archaeal or methanococcal lineages, such as that encoding DNA polymerase PolD. In contrast, the archaeal homolog to the gene encoding DNA polymerase B was not essential for growth, a conclusion confirmed by construction of an independent deletion mutation. Thus PolD, and not PolB, likely plays a fundamental role in DNA replication in methanococci. Similarly, 121 hypothetical ORFs were classified as possibly essential and likely play fundamental roles in methanococcal information processing or metabolism that are not established outside this group of prokaryotes.
| S-EPMC3607031 | biostudies-literature
Project description: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.
| S-EPMC3030896 | biostudies-literature
Project description:In Archaea selenocysteine (Sec) is synthesized in three steps. First seryl-tRNA synthetase acylates tRNA(Sec) with serine to generate Ser-tRNA(Sec). Then phosphoseryl-tRNA(Sec) kinase (PSTK) forms Sep-tRNA(Sec) , which is converted to Sec-tRNA(Sec) by Sep-tRNA:Sec-tRNA synthase (SepSecS) in the presence of selenophosphate produced by selenophosphate synthetase (SelD). A complete in vivo analysis of the archaeal Sec biosynthesis pathway is still unavailable, and the existence of a redundant pathway or of a rescue mechanism based on the conversion of Sep-tRNA(Sec) to Cys-tRNA(Sec) during selenium starvation, cannot be excluded. Here we present a mutational analysis of Sec biosynthesis in Methanococcus maripaludis strain Mm900. Sec formation is abolished upon individually deleting the genes encoding SelD, PSTK or SepSecS; the resulting mutant strains could no longer grow on formate while growth with H(2) + CO(2) remained unaffected. However, deletion of the PSTK and SepSecS genes was not possible unless the selenium-free [NiFe]-hydrogenases Frc and Vhc were expressed. This required the prior deletion of either the gene encoding SelD or that of HrsM, a LysR-type regulator suppressing transcription of the frc and vhc operons in the presence of selenium. These results show that M. maripaludis Mm900 is facultatively selenium-dependent with a single pathway of Sec-tRNA(Sec) formation.
| S-EPMC3124581 | biostudies-other