Thermococcus kodakarensis has two functional PCNA homologs but only one is required for viability

Proliferating cell nuclear antigen (PCNA) monomers assemble to form a ring-shaped clamp complex that encircles duplex DNA. PCNA binding to other proteins tethers them to the DNA providing contacts and interactions for many other enzymes essential for DNA metabolic processes. Most eukarya and euryarchaea have only one PCNA homolog but Thermococcus kodakarensis uniquely has two, designated PCNA1 and PCNA2, encoded by TK0535 and TK0582, respectively. Here, we establish that both PCNA1 and PCNA2 form homotrimers that stimulate DNA synthesis by archaeal DNA polymerases B and D and ATP hydrolysis by the replication factor C complex. In exponentially growing cells, PCNA1 is abundant and present at an ~100-fold higher concentration than PCNA2 monomers. Deletion of TK0582 (PCNA2) had no detectable effects on viability or growth whereas repeated attempts to construct a T. kodakarensis strain with TK0535 (PCNA1) deleted were unsuccessful. The implications of these observations for PCNA1 function and the origin of the two PCNA-encoding genes in T. kodakarensis are discussed.     

Thermococcus kodakarensis Genetics: TK1827-Encoded β-Glycosidase,New Positive-Selection Protocol,and Targeted and Repetitive Deletion Technology

Inactivation of TK1761, the reporter gene established for Thermococcus kodakarensis, revealed the presence of a second β-glycosidase that we have identified as the product of TK1827. This enzyme (pTK1827) has been purified and shown to hydrolyze glucopyranoside but not mannopyranoside, have optimal activity at 95°C and from pH 8 to 9.5, and have a functional half-life of ∼7 min at 100°C. To generate a strain with both TK1761 and TK1827 deleted, a new selection/counterselection protocol has been developed, and the levels of β-glycosidase activity in T. kodakarensis strains with TK1761 and/or TK1827 deleted and with these genes expressed from heterologous promoters are described. Genetic tools and strains have been developed that extend the use of this selection/counterselection procedure to delete any nonessential gene from the T. kodakarensis chromosome. Using this technology, TK0149 was deleted to obtain an agmatine auxotroph that grows on nutrient-rich medium only when agmatine is added. Transformants can therefore be selected rapidly, and replicating plasmids can be maintained in this strain growing in rich medium by complementation of the TK0149 deletion.Members of the Thermococcales, hyperthermophilic Euryarchaea, grow readily under laboratory conditions and are the focus of many basic and applied research projects (2, 8). Their investigation has, however, been limited by the lack of genetics, and it was a seminal advance therefore when Thermococcus kodakarensis (formerly Thermococcus kodakaraensis [1]) was shown to be naturally competent and to recombine added DNA into its genome (21, 23). T. kodakarensis is attractive as an experimental system as a fermentative heterotroph that grows rapidly on a variety of different substrates (1), optimally at 85°C, including substrates from which it generates substantial levels of hydrogen (11). The T. kodakarensis genome sequence and genome microarray assays are established (7, 12, 18) and, since the discovery of transformation (21), inactivation and manipulation of chromosomal genes has revealed novel biochemical pathways, facilitated in vivo evaluations of the archaeal gene expression machinery, and simplified enzyme purifications (3, 5, 6, 9, 10, 12, 15-20, 22-24, 28). For complementation assays and to facilitate heterologous gene expression in T. kodakarensis, shuttle plasmids have been constructed that replicate and confer selectable phenotypes in both T. kodakarensis and Escherichia coli (19), and TK1761 expression has been established as a reporter system that can be used to identify and quantify regulatory elements in T. kodakarensis (18, 20).During the development of the TK1761 reporter system, a T. kodakarensis strain, designated TS416, was constructed with a nonsense mutation in TK1761 (18). This mutation had no discernible effects on growth, confirming that TK1761 was not an essential gene, but lysates of T. kodakarensis TS416 retained a low level β-glycosidase activity. T. kodakarensis apparently, therefore, had a second β-glycosidase, but since this activity was very low and remained constant with changes in TK1761 expression, its presence did not compromise the use of TK1761 expression as a reporter system in the laboratory media used. The existence of a second β-glycosidase did, nevertheless, raise a potential concern for TK1761 assays in cells grown under different conditions in which expression of the gene encoding this second β-glycosidase might not be constant. To address this, we purified and characterized the second β-glycosidase and, to eliminate the concern for the reporter assay, we constructed a T. kodakarensis strain with both TK1761 and the gene (TK1827) that encodes the second β-glycosidase deleted.To construct the double-deletion strain, we developed a new selection/counterselection protocol and have extended this into a procedure that can be used to delete any nonessential gene from the T. kodakarensis genome. A two-gene cassette has been constructed that can be integrated into the T. kodakarensis chromosome at any desired locus by homologous recombination of flanking genes. Expression of the cassette provides a positive selection for transformants and confers sensitivity to 6-methyl purine (6MP^s). Mutants then isolated that are spontaneously resistant to 6-methyl purine (6MP^r) have both the cassette and the adjacent target gene(s) precisely deleted.Sato et al. (21, 23) established the T. kodakarensis transformation protocol by selecting transformants of tryptophan (trpE) and uracil (pyrF) auxotrophs that grew on minimal medium without tryptophan or uracil. Overexpression of the hydroxy-methylglutaryl-coenzyme A reductase encoded by PF1848, cloned from Pyrococcus furiosus, was later found to confer to resistance to simvastatin (15) and mevinolin (19), allowing the selection of T. kodakarensis transformants on rich media that contain either antibiotic. Mutants spontaneously resistant to these antibiotics do, however, occur at experimentally significant frequencies, and these are very expensive reagents for routine use and prohibitively expensive for incorporation into large preparative cultures. With this in mind, to develop an alternative selection that might be used in nutrient-rich media, we used the 6MP cassette system to delete TK0149. As predicted (6), the T. kodakarensis ΔTK0149 strain generated was an agmatine auxotroph that only grows in nutrient-rich media when agmatine is added. When transformed with DNA expressing TK0149, transformants of this strain can be selected directly on standard nutrient-rich media, and complementation of the ΔTK0149 mutation can be used to maintain the presence of an expression plasmid in T. kodakarensis cells grown in large-volume, rich-medium cultures for enzyme purification.     

Genetic Examination and Mass Balance Analysis of Pyruvate/Amino Acid Oxidation Pathways in the Hyperthermophilic Archaeon Thermococcus kodakarensis

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 of three enzymes potentially involved in pyruvate oxidation: pyruvate formate-lyase (PFL), pyruvate:ferredoxin oxidoreductase (POR), and 2-oxoisovalerate:ferredoxin oxidoreductase (VOR) under the HYH-deficient background in T. kodakarensis. No significant change was observed by deletion of pflDA, suggesting that PFL had no critical role in pyruvate oxidation. The growth properties and mass balances of ΔporDAB and ΔvorDAB strains indicated that POR and VOR specifically functioned in oxidation of pyruvate and branched-chain amino acids, respectively, and the lack of POR or VOR was compensated for by promoting the oxidation of another substrate driven by the remaining oxidoreductase. The H₂ yields from the consumed pyruvate and amino acids were increased from 31% by the parent strain to 67% and 82% by the deletion of hyhBGSL and double deletion of hyhBGSL and vorDAB, respectively. Significant discrepancies in the mass balances were observed in excess formation of acetate and NH₃, suggesting the presence of unknown metabolisms in T. kodakarensis grown in the rich medium containing pyruvate.     

Characterization of Two Members among the Five ADP-Forming Acyl Coenzyme A (Acyl-CoA) Synthetases Reveals the Presence of a 2-(Imidazol-4-yl)Acetyl-CoA Synthetase in Thermococcus kodakarensis

The genome of Thermococcus kodakarensis, along with those of most Thermococcus and Pyrococcus species, harbors five paralogous genes encoding putative α subunits of nucleoside diphosphate (NDP)-forming acyl coenzyme A (acyl-CoA) synthetases. The substrate specificities of the protein products for three of these paralogs have been clarified through studies on the individual enzymes from Pyrococcus furiosus and T. kodakarensis. Here we have examined the biochemical properties of the remaining two acyl-CoA synthetase proteins from T. kodakarensis. The TK0944 and TK2127 genes encoding the two α subunits were each coexpressed with the β subunit-encoding TK0943 gene. In both cases, soluble proteins with an α₂β₂ structure were obtained and their activities toward various acids in the ADP-forming reaction were examined. The purified TK0944/TK0943 protein (ACS III_Tk) accommodated a broad range of acids that corresponded to those generated in the oxidative metabolism of Ala, Val, Leu, Ile, Met, Phe, and Cys. In contrast, the TK2127/TK0943 protein exhibited relevant levels of activity only toward 2-(imidazol-4-yl)acetate, a metabolite of His degradation, and was thus designated 2-(imidazol-4-yl)acetyl-CoA synthetase (ICS_Tk), a novel enzyme. Kinetic analyses were performed on both proteins with their respective substrates. In T. kodakarensis, we found that the addition of histidine to the medium led to increases in intracellular ADP-forming 2-(imidazol-4-yl)acetyl-CoA synthetase activity, and 2-(imidazol-4-yl)acetate was detected in the culture medium, suggesting that ICS_Tk participates in histidine catabolism. The results presented here, together with those of previous studies, have clarified the substrate specificities of all five known NDP-forming acyl-CoA synthetase proteins in the Thermococcales.     

An Archaeal Glutamate Decarboxylase Homolog Functions as an Aspartate Decarboxylase and Is Involved in β-Alanine and Coenzyme A Biosynthesis

β-Alanine is a precursor for coenzyme A (CoA) biosynthesis and is a substrate for the bacterial/eukaryotic pantothenate synthetase and archaeal phosphopantothenate synthetase. β-Alanine is synthesized through various enzymes/pathways in bacteria and eukaryotes, including the direct decarboxylation of Asp by aspartate 1-decarboxylase (ADC), the degradation of pyrimidine, or the oxidation of polyamines. However, in most archaea, homologs of these enzymes are not present; thus, the mechanisms of β-alanine biosynthesis remain unclear. Here, we performed a biochemical and genetic study on a glutamate decarboxylase (GAD) homolog encoded by TK1814 from the hyperthermophilic archaeon Thermococcus kodakarensis. GADs are distributed in all three domains of life, generally catalyzing the decarboxylation of Glu to γ-aminobutyrate (GABA). The recombinant TK1814 protein displayed not only GAD activity but also ADC activity using pyridoxal 5′-phosphate as a cofactor. Kinetic studies revealed that the TK1814 protein prefers Asp as its substrate rather than Glu, with nearly a 20-fold difference in catalytic efficiency. Gene disruption of TK1814 resulted in a strain that could not grow in standard medium. Addition of β-alanine, 4′-phosphopantothenate, or CoA complemented the growth defect, whereas GABA could not. Our results provide genetic evidence that TK1814 functions as an ADC in T. kodakarensis, providing the β-alanine necessary for CoA biosynthesis. The results also suggest that the GAD activity of TK1814 is not necessary for growth, at least under the conditions applied in this study. TK1814 homologs are distributed in a wide range of archaea and may be responsible for β-alanine biosynthesis in these organisms.     

The crystal structure of a novel phosphopantothenate synthetase from the hyperthermophilic archaea, Thermococcus onnurineus NA1

Pantothenate is the essential precursor of coenzyme A (CoA), a fundamental cofactor in all aspects of metabolism. In bacteria and eukaryotes, pantothenate synthetase (PS) catalyzes the last step in the pantothenate biosynthetic pathway, and pantothenate kinase (PanK) phosphorylates pantothenate for its entry into the CoA biosynthetic pathway. However, genes encoding PS and PanK have not been identified in archaeal genomes. Recently, a comparative genomic analysis and the identification and characterization of two novel archaea-specific enzymes show that archaeal pantoate kinase (PoK) and phosphopantothenate synthetase (PPS) represent counterparts to the PS/PanK pathway in bacteria and eukaryotes. The TON1374 protein from Thermococcus onnurineus NA1 is a PPS, that shares 54% sequence identity with the first reported archaeal PPS candidate, MM2281, from Methanosarcina mazei and 91% sequence identity with TK1686, the PPS from Thermococcus kodakarensis. Here, we report the apo and ATP-complex structures of TON1374 and discuss the substrate-binding mode and reaction mechanism.     

MJ1647, an open reading frame in the genome of the hyperthermophile Methanococcus jannaschii, encodes a very thermostable archaeal histone with a C-terminal extension

All archaeal histones studied to date have similar lengths, 66 to 69 amino acid residues that form three α-helices separated by two β-strand loop regions which together constitute a histone fold. In contrast, the eukaryal nucleosome core histones are larger, 102 to 135 residues in length, with N-terminal and C-terminal extensions flanking the histone fold that participate in gene regulation and higher-order chromatin assembly. In the Methanococcus jannaschii genome, MJ1647 was annotated as an open reading frame predicted to encode an archaeal histone with an approximately 27-amino-acid C-terminal extension, and we here document the DNA binding and assembly properties and thermodynamic stability parameters of the recombinant product of MJ1647 synthesized in Escherichia coli with (rMJ1647) and without (rMJ1647Δ) the C-terminal extension. The presence of the C-terminal extension did not prevent homodimer formation or inhibit DNA binding, but the complexes formed by rMJ1647, presumably archaeal nucleosomes containing a (rMJ1647)₄ tetramer, were apparently less stable than those formed by (rMJ1647Δ)₄. The presence of the C-terminal extension increased the thermostability of rMJ1647 when compared with rMJ1647Δ in 0.2 M KCl at pH 4 but not in the absence of KCl at pH 1. Based on thermal unfolding transitions, rMJ1647 and rHAfB generated by expression of AF0337 cloned from the genome of the related hyperthermophile Archaeoglobus fulgidus in E. coli were found to have higher thermodynamic stabilities than all previously studied archaeal histones. Received: September 2, 1999 / Accepted: October 18, 1999     

DNA Repair Pathway Selection Caused by Defects in TEL1, SAE2, and De Novo Telomere Addition Generates Specific Chromosomal Rearrangement Signatures

Whole genome sequencing of cancer genomes has revealed a diversity of recurrent gross chromosomal rearrangements (GCRs) that are likely signatures of specific defects in DNA damage response pathways. However, inferring the underlying defects has been difficult due to insufficient information relating defects in DNA metabolism to GCR signatures. By analyzing over 95 mutant strains of Saccharomyces cerevisiae, we found that the frequency of GCRs that deleted an internal CAN1/URA3 cassette on chrV L while retaining a chrV L telomeric hph marker was significantly higher in tel1Δ, sae2Δ, rad53Δ sml1Δ, and mrc1Δ tof1Δ mutants. The hph-retaining GCRs isolated from tel1Δ mutants contained either an interstitial deletion dependent on non-homologous end-joining or an inverted duplication that appeared to be initiated from a double strand break (DSB) on chrV L followed by hairpin formation, copying of chrV L from the DSB toward the centromere, and homologous recombination to capture the hph-containing end of chrV L. In contrast, hph-containing GCRs from other mutants were primarily interstitial deletions (mrc1Δ tof1Δ) or inverted duplications (sae2Δ and rad53Δ sml1Δ). Mutants with impaired de novo telomere addition had increased frequencies of hph-containing GCRs, whereas mutants with increased de novo telomere addition had decreased frequencies of hph-containing GCRs. Both types of hph-retaining GCRs occurred in wild-type strains, suggesting that the increased frequencies of hph retention were due to the relative efficiencies of competing DNA repair pathways. Interestingly, the inverted duplications observed here resemble common GCRs in metastatic pancreatic cancer.     

An alternative beads‐on‐a‐string chromatin architecture in Thermococcus kodakarensis

We have applied chromatin sequencing technology to the euryarchaeon Thermococcus kodakarensis, which is known to possess histone‐like proteins. We detect positioned chromatin particles of variable sizes associated with lengths of DNA differing as multiples of 30 bp (ranging from 30 bp to >450 bp) consistent with formation from dynamic polymers of the archaeal histone dimer. T. kodakarensis chromatin particles have distinctive underlying DNA sequence suggesting a genomic particle‐positioning code and are excluded from gene‐regulatory DNA suggesting a functional organization. Beads‐on‐a‐string chromatin is therefore conserved between eukaryotes and archaea but can derive from deployment of histone‐fold proteins in a variety of multimeric forms.     

Characterization and gene deletion analysis of four homologues of group 3 pyridine nucleotide disulfide oxidoreductases from Thermococcus kodakarensis

Enzymatic characterization of the four group 3 pyridine nucleotide disulfide oxidoreductase (PNDOR) homologues TK1299, TK0304, TK0828, and TK1481 from Thermococcus kodakarensis was performed, with a focus on their CoA-dependent NAD(P)H: elemental sulfur (S⁰) oxidoreductase (NSR) and NAD(P)H oxidase (NOX) activities. TK1299 exhibited NSR activity with a preference for NADPH and showed strict CoA-dependency similar to that of the Pyrococcus furiosus homologue PF1186. During the assays, the non-enzymatic formation of H₂S from S⁰ and free CoA–SH was observed, and the addition of enzyme and NADPH enhanced H₂S evolution. A catalytic cycle of TK1299 was proposed suggesting that CoA–SH acted to solubilize S⁰ by forming CoA persulfides, followed by reduction of an enzyme–S–S–CoA intermediate produced after both enzymatic and non-enzymatic evolution of H₂S from the CoA persulfide, with NADPH as an electron donor. TK1481 showed NSR activity independently of CoA–SH, implying a direct reaction with S⁰. TK1299, TK1481, and TK0304 exhibited high NOX activity, and the NADH-dependent activities were inhibited by the addition of free CoA–SH. Multiple disruptions of the four group 3 PNDOR homologues in T. kodakarensis demonstrated that none of these homologues were essential for S⁰-dependent growth. Many disruptants grew better than the parent strain, but a few multiple disruptants showed decreased growth properties after aerobic inoculation into a pyruvate-containing medium without S⁰, suggesting the complicated participation of these group 3 PNDORs in sensitivity/resistance to dissolved oxygen when S⁰ was absent.     

Deletion of alternative pathways for reductant recycling in Thermococcus kodakarensis increases hydrogen production

Hydrogen (H₂) production by Thermococcus kodakarensis compares very favourably with the levels reported for the most productive algal, fungal and bacterial systems. T. kodakarensis can also consume H₂ and is predicted to use several alternative pathways to recycle reduced cofactors, some of which may compete with H₂ production for reductant disposal. To explore the reductant flux and possible competition for H₂ production in vivo, T. kodakarensis TS517 was mutated to precisely delete each of the alternative pathways of reductant disposal, H₂ production and consumption. The results obtained establish that H₂ is generated predominantly by the membrane‐bound hydrogenase complex (Mbh), confirm the essential role of the SurR (TK1086p) regulator in vivo, delineate the roles of sulfur (S°) regulon proteins and demonstrate that preventing H₂ consumption results in a substantial net increase in H₂ production. Constitutive expression of TK1086 (surR) from a replicative plasmid restored the ability of T. kodakarensis TS1101 (ΔTK1086) to grow in the absence of S° and stimulated H₂ production, revealing a second mechanism to increase H₂ production. Transformation of T. kodakarensis TS1101 with plasmids that express SurR variants constructed to direct the constitutive synthesis of the Mbh complex and prevent expression of the S° regulon was only possible in the absence of S° and, under these conditions, the transformants exhibited wild‐type growth and H₂ production. With S° present, they grew slower but synthesized more H₂ per unit biomass than T. kodakarensis TS517.     

Conserved eukaryotic histone-fold residues substituted into an archaeal histone increase DNA affinity but reduce complex flexibility

Although the archaeal and eukaryotic nucleosome core histones evolved from a common ancestor, conserved lysine residues are present at DNA-binding locations in all four eukaryotic histones that are not present in the archaeal histones. Introduction of lysine residues at the corresponding locations into an archaeal histone, HMfB, generated a variant with increased affinity for DNA that formed more compact complexes with DNA. However, these complexes no longer facilitated the circularization of short DNA molecules and had lost the flexibility to wrap DNA alternatively in either a negative or positive supercoil.