Genetic examination and mass balance analysis of pyruvate/amino acid oxidation pathways in the hyperthermophilic archaeon Thermococcus kodakarensis.
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ABSTRACT:
SUBMITTER: Nohara K
PROVIDER: S-EPMC4248822 | biostudies-literature | 2014 Nov
REPOSITORIES: biostudies-literature
Publications
Genetic examination and mass balance analysis of pyruvate/amino acid oxidation pathways in the hyperthermophilic archaeon Thermococcus kodakarensis.
Nohara Kenta K Orita Izumi I Nakamura Satoshi S Imanaka Tadayuki T Fukui Toshiaki T
Journal of bacteriology 20140825 22
The present study investigated the simultaneous oxidation of pyruvate and amino acids during H2-evolving growth of the hyperthermophilic archaeon Thermococcus kodakarensis. The comparison of mass balance between a cytosolic hydrogenase (HYH)-deficient strain (the ΔhyhBGSL strain) and the parent strain indicated that NADPH generated via H2 uptake by HYH was consumed by reductive amination of 2-oxoglutarate catalyzed by glutamate dehydrogenase. Further examinations were done to elucidate functions ...[more]
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Project description:Amino acid catabolism in Thermococcales is presumed to proceed via three steps: oxidative deamination of amino acids by glutamate dehydrogenase (GDH) or aminotransferases, oxidative decarboxylation by 2-oxoacid:ferredoxin oxidoreductases (KOR), and hydrolysis of acyl-coenzyme A (CoA) by ADP-forming acyl-CoA synthetases (ACS). Here, we performed a genetic examination of enzymes involved in Glu catabolism in Thermococcus kodakarensis. Examination of amino acid dehydrogenase activities in cell extracts of T. kodakarensis KUW1 (?pyrF ?trpE) revealed high NADP-dependent GDH activity, along with lower levels of NAD-dependent activity. NADP-dependent activities toward Gln/Ala/Val/Cys and an NAD-dependent threonine dehydrogenase activity were also detected. In KGDH1, a gene disruption strain of T. kodakarensis GDH (Tk-GDH), only threonine dehydrogenase activity was detected, indicating that all other activities were dependent on Tk-GDH. KGDH1 could not grow in a medium in which growth was dependent on amino acid catabolism, implying that Tk-GDH is the only enzyme that can discharge the electrons (to NADP(+)/NAD(+)) released from amino acids in their oxidation to 2-oxoacids. In a medium containing excess pyruvate, KGDH1 displayed normal growth, but higher degrees of amino acid catabolism were observed compared to those for KUW1, suggesting that Tk-GDH functions to suppress amino acid oxidation and plays an anabolic role under this condition. We further constructed disruption strains of 2-oxoglutarate:ferredoxin oxidoreductase and succinyl-CoA synthetase. The two strains displayed growth defects in both media compared to KUW1. Succinate generation was not observed in these strains, indicating that the two enzymes are solely responsible for Glu catabolism among the multiple KOR and ACS enzymes in T. kodakarensis.
| S-EPMC3624576 | biostudies-other
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:The hyperthermophilic archaeon Thermococcus kodakarensis utilizes amino acids as a carbon and energy source. Multiple aminotransferases, along with glutamate dehydrogenase, are presumed to be involved in the catabolic conversion of amino acids. T. kodakarensis harbors seven Class I aminotransferase homologs on its genome. Here we examined the biochemical properties and physiological roles of two Class I aminotransferases. The TK0548 protein was produced in Escherichia coli and the TK2268 protein in T. kodakarensis. Purified TK0548 protein preferred Phe, Trp, Tyr, and His, and to a lower extent, Leu, Met and Glu. The TK2268 protein preferred Glu and Asp, with lower activities toward Cys, Leu, Ala, Met and Tyr. Both proteins recognized 2-oxoglutarate as the amino acceptor. The TK0548 protein exhibited the highest k cat/K m value toward Phe, followed by Trp, Tyr, and His. The TK2268 protein exhibited highest k cat/K m values for Glu and Asp. The TK0548 and TK2268 genes were individually disrupted, and both disruption strains displayed a retardation in growth on a minimal amino acid medium, suggesting their involvement in amino acid metabolism. Activities in the cell-free extracts of the disruption strains and the host strain were examined. The results suggested that the TK0548 protein contributes to the conversion of Trp, Tyr and His, and the TK2268 protein to that of Asp and His. Although other aminotransferases seem to contribute to the transamination of Phe, Trp, Tyr, Asp, and Glu, our results suggest that the TK0548 protein is responsible for the majority of aminotransferase activity toward His in T. kodakarensis. The genetic examination carried out in this study provides insight into the contributions of the two aminotransferases toward specific amino acids in vivo, an aspect which had not been thoroughly considered thus far.
| S-EPMC9986279 | biostudies-literature
Project description:Background: Prokaryotes have relatively small genomes, densely-packed and apparently dominated by protein-encoding sequences. However, data now generated by high throughput RNA sequencing (RNA-seq) reveal surprisingly more-complex transcriptomes with many previously unrecognized and unanticipated non-coding small and antisense transcripts. To date, such studies have investigated primarily Bacteria. Here, we report the transcripts present in Thermococcus kodakarensis, a model hyperthermophilic Archaeon, synthesized under different growth and metabolic conditions. Results: cDNA libraries, generated from RNA preparations isolated from cells growing in media with sulfur or pyruvate, with sulfur to stationary phase, and growing with pyruvate but with sulfur added 20 min before RNA isolation, have been deep-sequenced. The results identify >2,700 sites of transcription initiation, establish a genome-wide map of transcripts, and consensus sequences for transcription initiation and post-transcription regulatory elements in T. kodakarensis. Primary transcription start sites (TSS) are identified upstream of 1,254 annotated genes, including ~78 % of those predicted by promoter locations, and an additional 644 primary TSS and their promoters have been identified within genes. Most of the mRNAs have a 5'-untranslated region (5'-UTR) between 10 and 50 nt long (median length = 16 nt), ~20 % have 5'-UTRs from 50 to 300 nt long, ~14 % are leaderless with 5'-UTRs ≤8 nt, and ~50% contain a consensus ribosome binding sequence. The results also identify TSS for 1,018 antisense transcripts, most with sequences complementary to either the 5'- or 3'-region of a sense mRNA. The data confirm the presence of transcripts from all three CRISPR loci, the RNase P and 7S RNAs, all tRNAs and rRNAs and 69 snoRNAs predicted to be encoded in the T. kodakarensis genome. Two transcripts, putatively identified as riboswitches, were present in RNA preparations isolated from growing but not from stationary phase cells. The procedure used is designed to identify TSS but, assuming that the number of cDNA reads correlates with transcript abundance, the data obtained also provide a semi-quantitative overview of global operon expression. They document substantial differences in gene expression under different physiological conditions and are consistent with previous observations of substrate-dependent specific gene expression. Many previously unrecognized and 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. Conclusion: We have identified >2,700 TSS that include almost all of the primary sites of transcription initiation upstream of annotated genes, and also 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 results reveal the presence of many non-coding RNAs and predict extensive RNA-based regulation in T. kodakarensis.
2014-08-18 | GSE56262 | GEO
Project description:The euryarchaeon Thermococcus kodakarensis is a well-characterized anaerobic hyperthermophilic heterotroph and due to the availability of genetic engineering systems it has become one of the model organisms for studying Archaea. Despite this prominent role among the Euryarchaeota, no data about the ploidy level of this species is available. While polyploidy has been shown to exist in various Euryarchaeota, especially Halobacteria, the chromosome copy number of species belonging to one of the major orders within that phylum, i.e., the Thermococcales (including Thermococcus spp. and Pyrococcus spp.), has never been determined. This prompted us to investigate the chromosome copy number of T. kodakarensis. In this study, we demonstrate that T. kodakarensis is polyploid with a chromosome copy number that varies between 7 and 19 copies, depending on the growth phase. An apparent correlation between the presence of histones and polyploidy in Archaea is observed.
| S-EPMC4502288 | biostudies-literature
Project description:Lipoic acid is a sulfur-containing cofactor and a component of the glycine cleavage system (GCS) involved in C1 compound metabolism and the 2-oxoacid dehydrogenases that catalyze the oxidative decarboxylation of 2-oxoacids. Lipoic acid is found in all domains of life and is generally synthesized as a lipoyl group on the H-protein of the GCS or the E2 subunit of 2-oxoacid dehydrogenases. Lipoyl synthase catalyzes the insertion of two sulfur atoms to the C-6 and C-8 carbon atoms of the octanoyl moiety on the octanoyl-H-protein or octanoyl-E2 subunit. Although the hyperthermophilic archaeon Thermococcus kodakarensis seemed able to synthesize lipoic acid, a classical lipoyl synthase (LipA) gene homolog cannot be found on the genome. In this study, we aimed to identify the lipoyl synthase in this organism. Genome information analysis suggested that the TK2109 and TK2248 genes, which had been annotated as biotin synthase (BioB), are both involved in lipoic acid metabolism. Based on the chemical reaction catalyzed by BioB, we predicted that the genes encode proteins that catalyze the lipoyl synthase reaction. Genetic analysis of TK2109 and TK2248 provided evidence that these genes are involved in lipoic acid biosynthesis. The purified TK2109 and TK2248 recombinant proteins exhibited lipoyl synthase activity toward a chemically synthesized octanoyl-octapeptide. These in vivo and in vitro analyses indicated that the TK2109 and TK2248 genes encode a structurally novel lipoyl synthase. TK2109 and TK2248 homologs are widely distributed among the archaeal genomes, suggesting that in addition to the LipA homologs, the two proteins represent a new group of lipoyl synthases in archaea.IMPORTANCE Lipoic acid is an essential cofactor for GCS and 2-oxoacid dehydrogenases, and α-lipoic acid has been utilized as a medicine and attracted attention as a supplement due to its antioxidant activity. The biosynthesis pathways of lipoic acid have been established in Bacteria and Eucarya but not in Archaea Although some archaeal species, including Sulfolobus, possess a classical lipoyl synthase (LipA) gene homolog, many archaeal species, including T. kodakarensis, do not. In addition, the biosynthesis mechanism of the octanoyl moiety, a precursor for lipoyl group biosynthesis, is also unknown for many archaea. As the enzyme identified in T. kodakarensis most likely represents a new group of lipoyl synthases in Archaea, the results obtained in this study provide an important step in understanding how lipoic acid is synthesized in this domain and how the two structurally distinct lipoyl synthases evolved in nature.
| S-EPMC7657641 | biostudies-literature
Project description:Members of Thermococcales harbor a number of genes encoding putative aminotransferase class III enzymes. Here, we characterized the TK1211 protein from the hyperthermophilic archaeon Thermococcus kodakarensis The TK1211 gene was expressed in T. kodakarensis under the control of the strong, constitutive promoter of the cell surface glycoprotein gene TK0895 (P csg ). The purified protein did not display aminotransferase activity but exhibited racemase activity. An examination of most amino acids indicated that the enzyme was a racemase with relatively high activity toward Leu and Met. Kinetic analysis indicated that Leu was the most preferred substrate. A TK1211 gene disruption strain (ΔTK1211) was constructed and grown on minimal medium supplemented with l- or d-Leu or l- or d-Met. The wild-type T. kodakarensis is not able to synthesize Leu and displays Leu auxotrophy, providing a direct means to examine the Leu racemase activity of the TK1211 protein in vivo When we replaced l-Leu with d-Leu in the medium, the host strain with an intact TK1211 gene displayed an extended lag phase but displayed cell yield similar to that observed in medium with l-Leu. In contrast, the ΔTK1211 strain displayed growth in medium with l-Leu but could not grow with d-Leu. The results indicate that TK1211 encodes a Leu racemase that is active in T. kodakarensis cells and that no other protein exhibits this activity, at least to an extent that can support growth. Growth experiments with l- or d-Met also confirmed the Met racemase activity of the TK1211 protein in T. kodakarensisIMPORTANCE Phylogenetic analysis of aminotransferase class III proteins from all domains of life reveals numerous groups of protein sequences. One of these groups includes a large number of sequences from Thermococcales species and can be divided into four subgroups. Representatives of three of these subgroups have been characterized in detail. This study reveals that a representative from the remaining uncharacterized subgroup is an amino acid racemase with preference toward Leu and Met. Taken together with results of previous studies on enzymes from Pyrococcus horikoshii and Thermococcus kodakarensis, members of the four subgroups now can be presumed to function as a broad-substrate-specificity amino acid racemase (subgroup 1), alanine/serine racemase (subgroup 2), ornithine ω-aminotransferase (subgroup 3), or Leu/Met racemase (subgroup 4).
| S-EPMC8088516 | biostudies-literature
Project description:TrpY from Methanothermobacter thermautotrophicus is a regulator that inhibits transcription of the Trp biosynthesis (trp) operon. Here, we show that the TrpY homolog in Thermococcus kodakarensis is not involved in such regulation. There are 87 genes on the T. kodakarensis genome predicted to encode transcriptional regulators (TRs). By screening for TRs that specifically bind to the promoter of the trp operon of T. kodakarensis, we identified TK0271. The gene resides in the aro operon, responsible for the biosynthesis of chorismate, a precursor for Trp, Tyr, and Phe. TK0271 was expressed in Escherichia coli, and the protein, here designated Tar ( Thermococcalesaromatic amino acid regulator), was purified. Tar specifically bound to the trp promoter with a dissociation constant (Kd ) value of approximately 5 nM. Tar also bound to the promoters of the Tyr/Phe biosynthesis (tyr-phe) and aro operons. The protein recognized a palindromic sequence (TGGACA-N8-TGTCCA) conserved in these promoters. In vitro transcription assays indicated that Tar activates transcription from all three promoters. We cultivated T. kodakarensis in amino acid-based medium and found that transcript levels of the trp, tyr-phe, and aro operons increased in the absence of Trp, Tyr, or Phe. We further constructed a TK0271 gene disruption strain (ΔTK0271). Growth of ΔTK0271 was similar to that of the host strain in medium including Trp, Tyr, and Phe but was significantly impaired in the absence of any one of these amino acids. The results suggest that Tar is responsible for the transcriptional activation of aromatic amino acid biosynthesis genes in T. kodakarensisIMPORTANCE The mechanisms of transcriptional regulation in archaea are still poorly understood. In this study, we identified a transcriptional regulator in the hyperthermophilic archaeon Thermococcus kodakarensis that activates the transcription of three operons involved in the biosynthesis of aromatic amino acids. The study represents one of only a few that identifies a regulator in Archaea that activates transcription. The results also imply that transcriptional regulation of genes with the same function is carried out by diverse mechanisms in the archaea, depending on the lineage.
| S-EPMC6737238 | biostudies-literature
Project description:Thermococcus kodakarensis is a hyperthermophilic archaeon that harbors a complete set of genes for chitin degradation to fructose 6-phosphate. However, wild-type T. kodakarensis KOD1 does not display growth on chitin. In this study, we developed a T. kodakarensis strain that can grow on chitin via genetic and adaptive engineering. First, a chitinase overproduction strain (KC01) was constructed by replacing the chitinase gene promoter with a strong promoter from the cell surface glycoprotein gene, resulting in increased degradation of swollen chitin and accumulation of N-,N'-diacetylchitobiose in the medium. To enhance N-,N'-diacetylchitobiose assimilation in KC01, genes encoding diacetylchitobiose deacetylase, exo-β-d-glucosaminidase, and glucosamine-6-phosphate deaminase were also overexpressed to obtain strain KC04. To strengthen the glycolytic flux of KC04, the gene encoding Tgr (transcriptional repressor of glycolytic genes) was disrupted to obtain strain KC04Δt. In both KC04 and KC04Δt strains, degradation of swollen chitin was further enhanced. In the culture broth of these strains, the accumulation of glucosamine was observed. KC04Δt was repeatedly inoculated in a swollen-chitin-containing medium for 13 cultures. This adaptive engineering strategy resulted in the isolation of a strain (KC04ΔtM1) that showed almost complete degradation of 0.4% (wt/vol) swollen chitin after 90 h. The strain produced high levels of acetate and ammonium in the culture medium, and, moreover, molecular hydrogen was generated. This strongly suggests that strain KC04ΔtM1 has acquired the ability to convert chitin to fructose 6-phosphate via deacetylation and deamination and further convert fructose 6-phosphate to acetate via glycolysis coupled to hydrogen generation.IMPORTANCE Chitin is a linear homopolymer of β-1,4-linked N-acetylglucosamine and is the second most abundant biomass next to cellulose. Compared to the wealth of research focused on the microbial degradation and conversion of cellulose, studies addressing microbial chitin utilization are still limited. In this study, using the hyperthermophilic archaeon Thermococcus kodakarensis as a host, we have constructed a strain that displays chitin-dependent hydrogen generation. The apparent hydrogen yield per unit of sugar consumed was slightly higher with swollen chitin than with starch. As gene manipulation in T. kodakarensis is relatively simple, the strain constructed in this study can also be used as a parent strain for the development and expansion of chitin-dependent biorefinery, in addition to its capacity to produce hydrogen.
| S-EPMC5514669 | biostudies-literature
Project description:Long-chain and/or branched-chain polyamines are unique polycations found in thermophiles. Cytoplasmic polyamines were analyzed for cells cultivated at various growth temperatures in the hyperthermophilic archaeon Thermococcus kodakarensis. Spermidine [34] and N4-aminopropylspermine [3(3)43] were identified as major polyamines at 60°C, and the amounts of N4-aminopropylspermine [3(3)43] increased as the growth temperature rose. To identify genes involved in polyamine biosynthesis, a gene disruption study was performed. The open reading frames (ORFs) TK0240, TK0474, and TK0882, annotated as agmatine ureohydrolase genes, were disrupted. Only the TK0882 gene disruptant showed a growth defect at 85°C and 93°C, and the growth was partially retrieved by the addition of spermidine. In the TK0882 gene disruptant, agmatine and N1-aminopropylagmatine accumulated in the cytoplasm. Recombinant TK0882 was purified to homogeneity, and its ureohydrolase characteristics were examined. It possessed a 43-fold-higher kcat/Km value for N1-aminopropylagmatine than for agmatine, suggesting that TK0882 functions mainly as N1-aminopropylagmatine ureohydrolase to produce spermidine. TK0147, annotated as spermidine/spermine synthase, was also studied. The TK0147 gene disruptant showed a remarkable growth defect at 85°C and 93°C. Moreover, large amounts of agmatine but smaller amounts of putrescine accumulated in the disruptant. Purified recombinant TK0147 possessed a 78-fold-higher kcat/Km value for agmatine than for putrescine, suggesting that TK0147 functions primarily as an aminopropyl transferase to produce N1-aminopropylagmatine. In T. kodakarensis, spermidine is produced mainly from agmatine via N1-aminopropylagmatine. Furthermore, spermine and N4-aminopropylspermine were detected in the TK0147 disruptant, indicating that TK0147 does not function to produce spermine and long-chain polyamines.
| S-EPMC2944531 | biostudies-literature