Increased biohydrogen production by deletion of uncharacterized DNA-binding protein discovered in hyperthermophilic archaeon Thermococcus onnurineus NA1
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ABSTRACT:
ORGANISM(S): Thermococcus onnurineus NA1
PROVIDER: GSE164395 | GEO | 2023/09/11
REPOSITORIES: GEO
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Project description:Increased biohydrogen production by deletion of uncharacterized DNA-binding protein discovered in hyperthermophilic archaeon Thermococcus onnurineus NA1
| PRJNA690534 | ENA
Project description:Hyperthermophilic archeaon, Thermococcus onnurineus NA1 has known as a strict anaerobe. To date, a few of studies have been reported that strict anaerobe can grow using oxygen. However, the research of the growth enhancement of strict anaerobic archaeon belonging to the order of Thermococcales using the oxygen, in which has never been reported so far. In this study, we showed that the growth of T. onnurineus NA1 strain increased under various oxygen concentrations and we observed that oxygen was decreased in the headspace during the growth of cell. Genome-wide transcriptomic analysis was carried out to evaluate alterations in gene expression induced by O2 and to explain the physiological effects of oxidative stress on the growth of T. onnurineus NA1.
2021-03-10 | GSE114281 | GEO
Project description:Expression analysis of Thermococcus onnurineus NA1 KCTC10859 and an Frh-deficient mutant, Frh overexpression mutant and TON_0282 deletion mutant in a T. onnurineus NA1 strain.
| PRJNA348351 | ENA
Project description:Investigation of whole genome gene expression level changes in an Frh-deficient mutant, Frh overexpression mutant and TON_0282 deletion mutant, compared to the wild-type strain. In this study, as a first step to assess the role of the frhAGB-encoding hydrogenases and TON_0282 gene, we constructed three mutants by deleting each gene in a T. onnurineus NA1.
2018-02-19 | GSE88718 | GEO
Project description:A strong promoter increases transcription of the genes of interest and enhances the production of various valuable substances. For a hyperthermophilic archaeon Thermococcus onnurineus NA1, which can produce H2 by carbon monoxide oxidation, we searched for a novel endogenous strong promoter by transcriptome analysis using high-throughput RNA sequencing. Based on the relative transcript abundance, we selected one promoter to encode a hypothetical gene, of which homologs were found only in several Thermococcales strains. This promoter, PTN0510, was introduced into the front of CO-responsible hydrogenase gene cluster encoding a carbon monoxide dehydrogenase (CODH), a hydrogenase and a Na+/H+ antiporter. In the resulting mutant strain, KS0510, transcription and translation level of the gene cluster increased by 4- to 14-folds and 1.5- to 1.9-folds, respectively, in comparison with those of wild-type strain. Additionally, H2 production rate of KS0510 mutant was 4.8-fold higher than that of wild-type strain. The PTN0510 was identified to be much stronger than the well-known two strong promoters, gdh and slp promoters from Thermococcus strains, through RT-qPCR and western blotting analyses and kinetics of H2 production. In this study, we demonstrated that the RNA-seq approach is a good strategy to mine a novel strong promoter of use to a Thermococcus strain when developed as a biotechnologically promising strain to produce valuable metabolites through a metabolic engineering. RNA expreesion profile of T. onnurineus NA1 genes in CO-containing media, in duplicate, using Illumina HiSeq 2500
2015-04-28 | E-GEOD-64036 | biostudies-arrayexpress
Project description:BACKGROUND:Thermococcus litoralis is a heterotrophic facultative sulfur dependent hyperthermophilic Archaeon, which was isolated from a shallow submarine thermal spring. It has been successfully used in a two-stage fermentation system, where various keratinaceous wastes of animal origin were converted to biohydrogen. In this system T. litoralis performed better than its close relative, P. furiosus. Therefore, new alternative enzymes involved in peptide and hydrogen metabolism were assumed in T. litoralis. RESULTS:An about 10.5 kb long genomic region was isolated and sequenced from Thermococcus litoralis. In silico analysis revealed that the region contained a putative operon consisting of eight genes: the fdhAB genes coding for a formate dehydrogenase and the mhyCDEFGH genes encoding a [NiFe] hydrogenase belonging to the group of the H2-evolving, energy-conserving, membrane-bound hydrogenases. Reverse transcription linked quantitative Real-Time PCR and Western blotting experiments showed that the expression of the fdh-mhy operon was up-regulated during fermentative growth on peptides and down-regulated in cells cultivated in the presence of sulfur. Immunoblotting and protein separation experiments performed on cell fractions indicated that the formate dehydrogenase part of the complex is associated to the membrane-bound [NiFe] hydrogenase. CONCLUSION:The formate dehydrogenase together with the membrane-bound [NiFe] hydrogenase formed a formate hydrogenlyase (formate dehydrogenase coupled hydrogenase, FDH-MHY) complex. The expression data suggested that its physiological role is linked to the removal of formate likely generated during anaerobic peptide fermentation.
| S-EPMC2432063 | biostudies-literature
Project description:A strong promoter increases transcription of the genes of interest and enhances the production of various valuable substances. For a hyperthermophilic archaeon Thermococcus onnurineus NA1, which can produce H2 by carbon monoxide oxidation, we searched for a novel endogenous strong promoter by transcriptome analysis using high-throughput RNA sequencing. Based on the relative transcript abundance, we selected one promoter to encode a hypothetical gene, of which homologs were found only in several Thermococcales strains. This promoter, PTN0510, was introduced into the front of CO-responsible hydrogenase gene cluster encoding a carbon monoxide dehydrogenase (CODH), a hydrogenase and a Na+/H+ antiporter. In the resulting mutant strain, KS0510, transcription and translation level of the gene cluster increased by 4- to 14-folds and 1.5- to 1.9-folds, respectively, in comparison with those of wild-type strain. Additionally, H2 production rate of KS0510 mutant was 4.8-fold higher than that of wild-type strain. The PTN0510 was identified to be much stronger than the well-known two strong promoters, gdh and slp promoters from Thermococcus strains, through RT-qPCR and western blotting analyses and kinetics of H2 production. In this study, we demonstrated that the RNA-seq approach is a good strategy to mine a novel strong promoter of use to a Thermococcus strain when developed as a biotechnologically promising strain to produce valuable metabolites through a metabolic engineering.
2015-04-28 | GSE64036 | GEO
Project description:Prokaryotes have relatively small genomes, densely-packed with protein-encoding sequences. RNA sequencing has, however, revealed surprisingly complex transcriptomes and here we report the transcripts present in the model hyperthermophilic Archaeon, Thermococcus kodakarensis, under different physiological conditions.Sequencing cDNA libraries, generated from RNA isolated from cells under different growth and metabolic conditions has identified >2,700 sites of transcription initiation, established a genome-wide map of transcripts, and consensus sequences for transcription initiation and post-transcription regulatory elements. The primary transcription start sites (TSS) upstream of 1,254 annotated genes, plus 644 primary TSS and their promoters within genes, are identified. Most mRNAs have a 5'-untranslated region (5'-UTR) 10 to 50 nt long (median = 16 nt), but ~20% have 5'-UTRs from 50 to 300 nt long and ~14% are leaderless. Approximately 50% of mRNAs contain a consensus ribosome binding sequence. The results identify TSS for 1,018 antisense transcripts, most with sequences complementary to either the 5'- or 3'-region of a sense mRNA, and confirm the presence of transcripts from all three CRISPR loci, the RNase P and 7S RNAs, all tRNAs and rRNAs and 69 predicted snoRNAs. Two putative riboswitch RNAs were present in growing but not in stationary phase cells. The procedure used is designed to identify TSS but, assuming that the number of cDNA reads correlates with transcript abundance, the results also provide a semi-quantitative documentation of the differences in T. kodakarensis genome expression under different growth conditions and confirm previous observations of substrate-dependent specific gene expression. Many previously unanticipated small RNAs have been identified, some with relative low GC contents (? 50%) and sequences that do not fold readily into base-paired secondary structures, contrary to the classical expectations for non-coding RNAs in a hyperthermophile.The results identify >2,700 TSS, including almost all of the primary sites of transcription initiation upstream of annotated genes, plus many secondary sites, sites within genes and sites resulting in antisense transcripts. The T.?kodakarensis genome is small (~2.1 Mbp) and tightly packed with protein-encoding genes, but the transcriptomes established also contain many non-coding RNAs and predict extensive RNA-based regulation in this model Archaeon.
| S-EPMC4247193 | biostudies-literature
Project description:In this study, we developed a gene disruption system for Thermococcus barophilus using simvastatin for positive selection and 5-fluoroorotic acid (5-FOA) for negative selection or counterselection to obtain markerless deletion mutants using single- and double-crossover events. Disruption plasmids carrying flanking regions of each targeted gene were constructed and introduced by transformation into wild-type T. barophilus MP cells. Initially, a pyrF deletion mutant was obtained as a starting point for the construction of further markerless mutants. A deletion of the hisB gene was also constructed in the UBOCC-3256 (?pyrF) background, generating a strain (UBOCC-3260) that was auxotrophic for histidine. A functional pyrF or hisB allele from T. barophilus was inserted into the chromosome of UBOCC-3256 (?pyrF) or UBOCC-3260 (?pyrF ?hisB), allowing homologous complementation of these mutants. The piezophilic genetic tools developed in this study provide a way to construct strains with multiple genetic backgrounds that will allow further genetic studies for hyperthermophilic piezophilic archaea.
| S-EPMC3993132 | biostudies-literature
Project description:Extracellular vesicles (EVs) produced by a sulfur-reducing, hyperthermophilic archaeon, "Thermococcus onnurineus" NA1(T), were purified and characterized. A maximum of four EV bands, showing buoyant densities between 1.1899 and 1.2828 g cm(-3), were observed after CsCl ultracentrifugation. The two major EV bands, B (buoyant density at 25°C [ρ(25)] = 1.2434 g cm(-3)) and C (ρ(25) = 1.2648 g cm(-3)), were separately purified and counted using a qNano particle analyzer. These EVs, showing different buoyant densities, were identically spherical in shape, and their sizes varied from 80 to 210 nm in diameter, with 120- and 190-nm sizes predominant. The average size of DNA packaged into EVs was about 14 kb. The DNA of the EVs in band C was sequenced and assembled. Mapping of the T. onnurineus NA1(T) EV (ToEV) DNA sequences onto the reference genome of the parent archaeon revealed that most genes of T. onnurineus NA1(T) were packaged into EVs, except for an ∼9.4-kb region from TON_0536 to TON_0544. The absence of this specific region of the genome in the EVs was confirmed from band B of the same culture and from bands B and C purified from a different batch culture. The presence of the 3'-terminal sequence and the absence of the 5'-terminal sequence of TON_0536 were repeatedly confirmed. On the basis of these results, we hypothesize that the unpackaged part of the T. onnurineus NA1(T) genome might be related to the process that delivers DNA into ToEVs and/or the mechanism generating the ToEVs themselves.
| S-EPMC4551206 | biostudies-literature