Search

The 4-α-glucanotransferase (4-α-GTase or amylomaltase) is an essential enzyme in maltodextrin metabolism. Generally, most bacterial 4-α-GTase is classified into glycoside hydrolase (GH) family 77. However, hyperthermophiles have unique 4-α-GTases belonging to GH family 57. These enzymes are the main amylolytic protein in hyperthermophiles, but their mode of action in maltooligosaccharide utilization is poorly understood. In the present study, we investigated the catalytic properties of 4-α-GTase from the hyperthermophile Pyrococcus sp. ST04 (PSGT) in the presence of maltooligosaccharides of various lengths. Unlike 4-α-GTases in GH family 77, GH family 57 PSGT produced maltotriose in the early stage of reaction and preferred maltose and maltotriose over glucose as the acceptor. The kinetic analysis showed that maltotriose had the lowest KM value, which increased amylose degradation activity by 18.3-fold. Structural models of PSGT based on molecular dynamic simulation revealed two aromatic amino acids interacting with the substrate at the +2 and +3 binding sites, and the mutational study demonstrated they play a critical role in maltotriose binding. These results clarify the mode of action in carbohydrate utilization and explain acceptor binding mechanism of GH57 family 4-α-GTases in hyperthermophilic archaea [ABSTRACT FROM AUTHOR]

Although copper is in many cases an essential micronutrient for cellular life, higher concentrations are toxic. Therefore, all living cells have developed strategies to maintain copper homeostasis. In this manuscript, we have analyzed the transcriptome-wide response of Pyrococcus furiosus to increased copper concentrations and described the essential role of the putative copper-sensing metalloregulator CopR in the detoxification process. To this end, we employed biochemical and biophysical methods to characterize the role of CopR. Additionally, a copR knockout strain revealed an amplified sensitivity in comparison to the parental strain towards increased copper levels, which designates an essential role of CopR for copper homeostasis. To learn more about the CopR-regulated gene network, we performed differential gene expression and ChIP-seq analysis under normal and 20 μM copper-shock conditions. By integrating the transcriptome and genome-wide binding data, we found that CopR binds to the upstream regions of many copper-induced genes. Negative-stain transmission electron microscopy and 2D class averaging revealed an octameric assembly formed from a tetramer of dimers for CopR, similar to published crystal structures from the Lrp family. In conclusion, we propose a model for CopR-regulated transcription and highlight the regulatory network that enables Pyrococcus to respond to increased copper concentrations.

Synthesis and assembly of ribosomal components are fundamental cellular processes and generally well-conserved within the main groups of organisms. Yet, provocative variations to the general schemes exist. We have discovered an unusual processing pathway of pre-rRNA in extreme thermophilic archaea exemplified by Pyrococcus furiosus. The large subunit (LSU) rRNA is produced as a circularly permuted form through circularization followed by excision of Helix 98. As a consequence, the terminal domain VII that comprise the binding site for the signal recognition particle is appended to the 5´ end of the LSU rRNA that instead terminates in Domain VI carrying the Sarcin-Ricin Loop, the primary interaction site with the translational GTPases. To our knowledge, this is the first example of a true post-transcriptional circular permutation of a main functional molecule and the first example of rRNA fragmentation in archaea.

Although copper is in many cases an essential micronutrient for cellular life, higher concentrations are toxic. Therefore, all living cells have developed strategies to maintain copper homeostasis. In this manuscript, we have analyzed the transcriptome-wide response of Pyrococcus furiosus to increased copper concentrations and described the essential role of the putative copper-sensing metalloregulator CopR in the detoxification process. To this end, we employed biochemical and biophysical methods to characterize the role of CopR. Additionally, a copR knockout strain revealed an amplified sensitivity in comparison to the parental strain towards increased copper levels, which designates an essential role of CopR for copper homeostasis. To learn more about the CopR-regulated gene network, we performed differential gene expression and ChIP-seq analysis under normal and 20 μM copper-shock conditions. By integrating the transcriptome and genome-wide binding data, we found that CopR binds to the upstream regions of many copper-induced genes. Negative-stain transmission electron microscopy and 2D class averaging revealed an octameric assembly formed from a tetramer of dimers for CopR, similar to published crystal structures from the Lrp family. In conclusion, we propose a model for CopR-regulated transcription and highlight the regulatory network that enables Pyrococcus to respond to increased copper concentrations. [ABSTRACT FROM AUTHOR]

Organic and inorganic cyanides are widely distributed in nature, yet not much is known about the ability of microorganisms to use these compounds as a source of nitrogen and/or carbon at high temperatures (>80 °C). Here we studied the capacity of organic and inorganic cyanides to support growth of an hyperthermophilic Pyrococcus strain isolated from Deception Island, Antarctica. This microorganism was capable of growing with aromatic nitriles, aliphatic nitriles, heterocyclic nitriles, amino aromatic nitriles and inorganic cyanides as nitrogen and/or carbon source. This is the first report of an hyperthermophilic microorganism able to incorporate these compounds in its nitrogen and carbon metabolism. Based on enzymatic activity and genomic information, it is possibly that cells of this Pyrococcus strain growing with nitriles or cyanide, might use the carboxylic acid and/or the ammonia generated through the nitrilase enzymatic activity, as a carbon and/or nitrogen source respectively. This work expands the temperature range at which microorganisms can use organic and inorganic cyanides to growth, having important implications to understand microbial metabolisms that can support life on Earth and the possibility to support life elsewhere., (© 2024. The Author(s), under exclusive licence to Springer Nature Japan KK, part of Springer Nature.)

Synthesis and assembly of ribosomal components are fundamental cellular processes and generally well-conserved within the main groups of organisms. Yet, provocative variations to the general schemes exist. We have discovered an unusual processing pathway of pre-rRNA in extreme thermophilic archaea exemplified by Pyrococcus furiosus. The large subunit (LSU) rRNA is produced as a circularly permuted form through circularization followed by excision of Helix 98. As a consequence, the terminal domain VII that comprise the binding site for the signal recognition particle is appended to the 5´ end of the LSU rRNA that instead terminates in Domain VI carrying the Sarcin-Ricin Loop, the primary interaction site with the translational GTPases. To our knowledge, this is the first example of a true post-transcriptional circular permutation of a main functional molecule and the first example of rRNA fragmentation in archaea. [ABSTRACT FROM AUTHOR]

The key difference in the modified Embden–Meyerhof glycolytic pathway in hyperthermophilic Archaea, such as Pyrococcus furiosus, occurs at the conversion from glyceraldehyde-3-phosphate (GAP) to 3-phosphoglycerate (3-PG) where the typical intermediate 1,3-bisphosphoglycerate (1,3-BPG) is not present. The absence of the ATP-yielding step catalyzed by phosphoglycerate kinase (PGK) alters energy yield, redox energetics, and kinetics of carbohydrate metabolism. Either of the two enzymes, ferredoxin-dependent glyceraldehyde-3-phosphate ferredoxin oxidoreductase (GAPOR) or NADP+-dependent non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase (GAPN), responsible for this "bypass" reaction, could be deleted individually without impacting viability, albeit with differences in native fermentation product profiles. Furthermore, P. furiosus was viable in the gluconeogenic direction (growth on pyruvate or peptides plus elemental sulfur) in a ΔgapnΔgapor strain. Ethanol was utilized as a proxy for potential heterologous products (e.g., isopropanol, butanol, fatty acids) that require reducing equivalents (e.g., NAD(P)H, reduced ferredoxin) generated from glycolysis. Insertion of a single gene encoding the thermostable NADPH-dependent primary alcohol dehydrogenase (adhA) (Tte_0696) from Caldanaerobacter subterraneus, resulted in a strain producing ethanol via the previously established aldehyde oxidoreductase (AOR) pathway. This strain demonstrated a high ratio of ethanol over acetate (> 8:1) at 80 °C and enabled ethanol production up to 85 °C, the highest temperature for bio-ethanol production reported to date. [ABSTRACT FROM AUTHOR]

Pyrococcus furiosus DSM 3638 is a model organism for hyperthermophilic archaea with an optimal growth temperature near 100°C. The genome was sequenced about 18 years ago. However, some publications suggest that in contrast to other Pyrococcus species, the genome of P. furiosus DSM 3638 is prone to genomic rearrangements. Therefore, we re-sequenced the genome using third generation sequencing techniques. The new de novo assembled genome is 1,889,914 bp in size and exhibits high sequence identity to the published sequence. However, two major deviations were detected: (1) The genome is 18,342 bp smaller than the NCBI reference genome due to a recently described deletion. (2) The region between PF0349 and PF0388 is inverted most likely due an assembly problem for the original sequence. In addition, numerous minor variations, ranging from single nucleotide exchanges, deletions or insertions were identified. The total number of insertion sequence (IS) elements is also reduced from 30 to 24 in the new sequence. Re-sequencing of a 2-year-old “lab culture” using Nanopore sequencing confirmed the overall stability of the P. furiosus DSM 3638 genome even under normal lab conditions without taking any special care. To improve genome annotation, the updated DNA sequence was combined with an RNA sequencing approach. Here, RNAs from eight different growth conditions were pooled to increase the number of detected transcripts. Furthermore, a differential RNA-Seq approach was employed for the identification of transcription start sites (TSSs). In total, 2515 TSSs were detected and classified into 834 primary (pTSS), 797 antisense (aTSS), 739 internal and 145 secondary TSSs. Our analysis of the upstream regions revealed a well conserved archaeal promoter structure. Interrogation of the distances between pTSSs and aTSSs revealed a significant number of antisense transcripts, which are a result of bidirectional transcription from the same TATA box. This mechanism of antisense transcript production could be further confirmed by in vitro transcription experiments. We assume that bidirectional transcription gives rise to non-functional antisense RNAs and that this is a widespread phenomenon in archaea due to the architecture of the TATA element and the symmetric structure of the TATA-binding protein.

In practical applications, the gelatinisation temperature of starch is high. Most current glycogen branching enzymes (GBEs, EC 2.4.1.18) exhibit optimum activity at moderate or low temperatures and quickly lose their activity at higher temperatures, limiting the application of GBEs in starch modification. Therefore, we used the PROSS strategy combined with PDBePISA analysis of the dimer interface to further improve the heat resistance of hyperthermophilic bacteria Pyrococcus horikoshii OT3 GBE. The results showed that the melting temperature of mutant T508K increased by 3.1 °C compared to wild-type (WT), and the optimum reaction temperature increased by 10 °C for all mutants except V140I. WT almost completely lost its activity after incubation at 95 °C for 60 h, while all of the combined mutants maintained >40 % of their residual activity. Further, the content of the α-1,6 glycosidic bond of corn starch modified by H415W and V140I/H415W was approximately 2.68-fold and 1.92-fold higher than that of unmodified corn starch and corn starch modified by WT, respectively. Additionally, the glucan chains of DP < 13 were significantly increased in mutant modified corn starch. This method has potential for improving the thermal stability of GBE, which can be applied in starch branching in the food industry., Competing Interests: Declaration of competing interest There are no conflicts of interest to declare., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Pyrococcus furiosus DSM 3638 is a model organism for hyperthermophilic archaea with an optimal growth temperature near 100°C. The genome was sequenced about 18 years ago. However, some publications suggest that in contrast to other Pyrococcus species, the genome of P. furiosus DSM 3638 is prone to genomic rearrangements. Therefore, we re-sequenced the genome using third generation sequencing techniques. The new de novo assembled genome is 1,889,914 bp in size and exhibits high sequence identity to the published sequence. However, two major deviations were detected: (1) The genome is 18,342 bp smaller than the NCBI reference genome due to a recently described deletion. (2) The region between PF0349 and PF0388 is inverted most likely due an assembly problem for the original sequence. In addition, numerous minor variations, ranging from single nucleotide exchanges, deletions or insertions were identified. The total number of insertion sequence (IS) elements is also reduced from 30 to 24 in the new sequence. Re-sequencing of a 2-year-old "lab culture" using Nanopore sequencing confirmed the overall stability of the P. furiosus DSM 3638 genome even under normal lab conditions without taking any special care. To improve genome annotation, the updated DNA sequence was combined with an RNA sequencing approach. Here, RNAs from eight different growth conditions were pooled to increase the number of detected transcripts. Furthermore, a differential RNA-Seq approach was employed for the identification of transcription start sites (TSSs). In total, 2515 TSSs were detected and classified into 834 primary (pTSS), 797 antisense (aTSS), 739 internal and 145 secondary TSSs. Our analysis of the upstream regions revealed a well conserved archaeal promoter structure. Interrogation of the distances between pTSSs and aTSSs revealed a significant number of antisense transcripts, which are a result of bidirectional transcription from the same TATA box. This mechanism of antisense transcript production could be further confirmed by in vitro transcription experiments. We assume that bidirectional transcription gives rise to non-functional antisense RNAs and that this is a widespread phenomenon in archaea due to the architecture of the TATA element and the symmetric structure of the TATA-binding protein. [ABSTRACT FROM AUTHOR]

The discovery of hyperthermophiles has dramatically changed our understanding of the habitats in which life can thrive. However, the extreme high temperatures in which these organisms live have severely restricted the development of genetic tools. The archaeon Pyrococcus yayanosii A1 is a strictly anaerobic and piezophilic hyperthermophile that is an ideal model for studies of extreme environmental adaptation. In the present study, we identified a high hydrostatic pressure (HHP)-inducible promoter (Phhp) that controls target gene expression under HHP. We developed an HHP-inducible toxin-antitoxin cassette (HHP-TAC) containing a counterselectable marker in which a putative toxin-encoding gene virulence-associated protein C (PF0776 (VapC)), controlled by the HHP-inducible promoter, was used in conjunction with the antitoxin-encoding gene PF0775 (VapB), which was fused to a constitutive promoter (PhmtB), and a positive marker with the 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase-encoding gene from P. furiosus controlled by the constitutive 34 promoter Pgdh. The HHP-TAC was constructed to realize markerless gene disruption directly in P. yayanosii A1 in rich medium. The pop-out recombination step was performed using an HHP-inducible method. As proof, the PYCH_13690 gene, which encodes a 4-α-glucanotransferase, was successfully deleted from the strain P. yayanosii A1. The results showed that the capacity for starch hydrolysis in the Δ1369- 39 mutant decreased dramatically compared with that in the wild-type strain. The inducible toxin-antitoxin (TA) system developed in this study greatly increases the genetic tools available for use in hyperthermophiles. Importance Genetic manipulations in hyperthermophiles have been studied for over 20 years. However, the extremely high temperatures under which these organisms grow have limited the development of genetic tools. In this study, an HHP-inducible promoter was used to control the expression of a toxin. Compared with sugar-inducible and cold-shock-inducible promoters, the HHP-inducible promoter rarely has negative effects on the overall physiology and central metabolism of microorganisms, especially piezophilic hyperthermophiles. Previous studies have used auxotrophic strains as hosts, which may interfere with studies of adaptation and metabolism. Using an inducible TA system as a counterselectable marker enables the generation of a markerless gene disruption strain without the use of auxotrophic mutants and counterselection with 5-fluoroorotic acid. TA systems are widely distributed in bacteria and archaea and can be used to overcome the limitations of high growth temperatures and dramatically extend the selectivity of genetic tools in hyperthermophiles. [ABSTRACT FROM AUTHOR]

PolD is an archaeal replicative DNA polymerase (DNAP) made of a proofreading exonuclease subunit (DP1) and a larger polymerase catalytic subunit (DP2). Recently, we reported the individual crystal structures of the DP1 and DP2 catalytic cores, thereby revealing that PolD is an atypical DNAP that has all functional properties of a replicative DNAP but with the catalytic core of an RNA polymerase (RNAP). We now report the DNA-bound cryo–electron microscopy (cryo-EM) structure of the heterodimeric DP1–DP2 PolD complex from Pyrococcus abyssi, revealing a unique DNA-binding site. Comparison of PolD and RNAPs extends their structural similarities and brings to light the minimal catalytic core shared by all cellular transcriptases. Finally, elucidating the structure of the PolD DP1–DP2 interface, which is conserved in all eukaryotic replicative DNAPs, clarifies their evolutionary relationships with PolD and sheds light on the domain acquisition and exchange mechanism that occurred during the evolution of the eukaryotic replisome. [ABSTRACT FROM AUTHOR]

Elucidating the lipidome of Archaea is essential to understand their tolerance to extreme environmental conditions. Previous characterizations of the lipid composition of Pyrococcus species, a model genus of hyperthermophilic archaea belonging to the Thermococcales order, led to conflicting results, which hindered the comprehension of their membrane structure and the putative adaptive role of their lipids. In an effort to clarify the lipid composition data of the Pyrococcus genus, we thoroughly investigated the distribution of both the core lipids (CL) and intact polar lipids (IPL) of the model Pyrococcus furiosus and, for the first time, of Pyrococcus yayanosii, the sole obligate piezophilic hyperthermophilic archaeon known to date. We showed a low diversity of IPL in the lipid extract of P. furiosus, which nonetheless allowed the first report of phosphatidyl inositol-based glycerol mono- and trialkyl glycerol tetraethers. With up to 13 different CL structures identified, the acid methanolysis of Pyrococcus furiosus revealed an unprecedented CL diversity and showed strong discrepancies with the IPL compositions reported here and in previous studies. By contrast, P. yayanosii displayed fewer CL structures but a much wider variety of polar heads. Our results showed severe inconsistencies between IPL and CL relative abundances. Such differences highlight the diversity and complexity of the Pyrococcus plasma membrane composition and demonstrate that a large part of its lipids remains uncharacterized. Reassessing the lipid composition of model archaea should lead to a better understanding of the structural diversity of their lipidome and of their physiological and adaptive functions.

Transcription factor B recruiting factor 1 (TFB-RF1; PF1088) is a transcription regulator which activates transcription on archaeal promoters containing weak TFB recognition elements (BRE) by recruiting TFB to the promoter. The mechanism of activation is described in detail, but nothing is known about the biological function of this protein in Pyrococcus furiosus. The protein is located in an operon structure together with the hypothetical gene pf1089 and western blot as well as end-point RT-PCR experiments revealed an extremely low expression rate of both proteins. Furthermore, conditions to induce the expression of the operon are not known. By introducing an additional copy of tfb-RF1 using a Pyrococcus shuttle vector we could circumvent the lacking expression of both proteins under standard growth conditions as indicated by western blot as well as end-point RT-PCR experiments. A ChIP-seq experiment revealed an additional binding site of TFB-RF1 in the upstream region of the pf1011/1012 operon, beside the expected target of the pf1089/tfb-RF1 region. This operon codes for a putative ABC transporter which is most-related to a multidrug export system and in vitro analysis using gel shift assays, DNase I footprinting and in vitro transcription confirmed the activator function of TFB-RF1 on the corresponding promoter. These findings are also in agreement with in vivo data, as RT-qPCR experiments also indicate transcriptional activation of both operons. Taken together, the overexpression strategy of tfb-RF1 enabled the identification of an additional operon of the TFB-RF1 regulon which indicates a transport-related function and provides a promising starting position to decipher the physiological function of the TFB-RF1 gene regulatory network in P. furiosus.

Acetate and acetyl-CoA play fundamental roles in all of biology, including anaerobic prokaryotes from the domains Bacteria and Archaea, which compose an estimated quarter of all living protoplasm in Earth’s biosphere. Anaerobes from the domain Archaea contribute to the global carbon cycle by metabolizing acetate as a growth substrate or product. They are components of anaerobic microbial food chains converting complex organic matter to methane, and many fix CO2 into cell material via synthesis of acetyl-CoA. They are found in a diversity of ecological habitats ranging from the digestive tracts of insects to deep-sea hydrothermal vents, and synthesize a plethora of novel enzymes with biotechnological potential. Ecological investigations suggest that still more acetate-metabolizing species with novel properties await discovery.

Chitinase D (designated as Pc-ChiD) was found in a hyperthermophilic archaeon, Pyrococcus chitonophagus (previously described as Thermococcus chitonophagus), that was isolated from media containing only chitin as carbon source. Pc-ChiD displays chitinase activity and is thermostable at temperatures up to 95°C, suggesting its potential for industrial use. Pc-ChiD has a secretion signal peptide and two chitin-binding domains (ChBDs) in the N-terminal domain. However, the Cterminal domain shares no sequence similarity with previously identified saccharide-degrading enzymes and does not contain the DXDXE motif conserved in the glycoside hydrolase (GH) 18 family chitinases. To elucidate its overall structure and reaction mechanism, we determined the first crystal structures of Pc-ChiD, both in the ligand-free form and in complexes with substrates. Structure analyses revealed that the C-terminal domain of Pc-ChiD, Pc-ChiD(ΔBD), consists of a third putative substrate-binding domain, which cannot be predicted from the amino acid sequence, and a catalytic domain structurally similar to that found in not the GH18 family but the GH23 family. Based on the similarity with GH23 family chitinase, the catalytic residues of Pc-ChiD were predicted and confirmed by mutagenesis analyses. Moreover, the specific C-terminal 100 residues of Pc-ChiD are important to fix the putative substrate-binding domain next to the catalytic domain, contributing to the structure stability as well as the long chitin chain binding. Our findings reveal the structure of a unique archaeal chitinase that is distinct from previously known members of the GH23 family [ABSTRACT FROM AUTHOR]

Toxin-antitoxin (TA) system is bacterial or archaeal genetic module consisting of toxin and antitoxin gene that be organized as a bicistronic operon. TA system could elicit programmed cell death, which is supposed to play important roles for the survival of prokaryotic population under various physiological stress conditions. The phage abortive infection system (AbiE family) belongs to bacterial type IV TA system. However, no archaeal AbiE family TA system has been reported so far. In this study, a putative AbiE TA system (PygAT), which is located in a genomic island PYG1 in the chromosome of Pyrococcus yayanosii CH1, was identified and characterized. In Escherichia coli, overexpression of the toxin gene pygT inhibited its growth while the toxic effect can be suppressed by introducing the antitoxin gene pygA in the same cell. PygAT also enhances the stability of shuttle plasmids with archaeal plasmid replication protein Rep75 in E. coli. In P. yayanosii, disruption of antitoxin gene pygA cause a significantly growth delayed under high hydrostatic pressure (HHP). The antitoxin protein PygA can specifically bind to the PygAT promoter region and regulate the transcription of pygT gene in vivo. These results show that PygAT is a functional TA system in P. yayanosii, and also may play a role in the adaptation to HHP environment. [ABSTRACT FROM AUTHOR]

Transcription factor B recruiting factor 1 (TFB-RF1; PF1088) is a transcription regulator which activates transcription on archaeal promoters containing weak TFB recognition elements (BRE) by recruiting TFB to the promoter. The mechanism of activation is described in detail, but nothing is known about the biological function of this protein in Pyrococcus furiosus. The protein is located in an operon structure together with the hypothetical gene pf1089 and western blot as well as end-point RT-PCR experiments revealed an extremely low expression rate of both proteins. Furthermore, conditions to induce the expression of the operon are not known. By introducing an additional copy of tfb-RF1 using a Pyrococcus shuttle vector we could circumvent the lacking expression of both proteins under standard growth conditions as indicated by western blot as well as end-point RT-PCR experiments. A ChIP-seq experiment revealed an additional binding site of TFB-RF1 in the upstream region of the pf1011/1012 operon, beside the expected target of the pf1089/tfb-RF1 region. This operon codes for a putative ABC transporter which is most-related to a multidrug export system and in vitro analysis using gel shift assays, DNase I footprinting and in vitro transcription confirmed the activator function of TFB-RF1 on the corresponding promoter. These findings are also in agreement with in vivo data, as RT-qPCR experiments also indicate transcriptional activation of both operons. Taken together, the overexpression strategy of tfb-RF1 enabled the identification of an additional operon of the TFB-RF1 regulon which indicates a transport-related function and provides a promising starting position to decipher the physiological function of the TFB-RF1 gene regulatory network in P. furiosus. [ABSTRACT FROM AUTHOR]

The natural trans-acting ribozyme RNase P RNA (RPR) is composed of two domains in which the catalytic (C-) domain mediates cleavage of various substrates. The C-domain alone, after removal of the second specificity (S-) domain, catalyzes this reaction as well, albeit with reduced efficiency. Here we provide experimental evidence indicating that efficient cleavage mediated by the Escherichia coli C-domain (Eco CP RPR) with and without the C5 protein likely depends on an interaction referred to as the "P6-mimic". Moreover, the P18 helix connects the C- and S-domains between its loop and the P8 helix in the S-domain (the P8/ P18-interaction). In contrast to the "P6-mimic", the presence of P18 does not contribute to the catalytic performance by the C-domain lacking the S-domain in cleavage of an all ribo model hairpin loop substrate while deletion or disruption of the P8/ P18-interaction in full-size RPR lowers the catalytic efficiency in cleavage of the same model hairpin loop substrate in keeping with previously reported data using precursor tRNAs. Consistent with that P18 is not required for cleavage mediated by the C-domain we show that the archaeal Pyrococcus furiosus RPR C-domain, which lacks the P18 helix, is catalytically active in trans without the S-domain and any protein. Our data also suggest that the S-domain has a larger impact on catalysis for E. coli RPR compared to P. furiosus RPR. Finally, we provide data indicating that the absence of the S-domain and P18, or the P8/ P18-interaction in full-length RPR influences the charge distribution near the cleavage site in the RPR-substrate complex to a small but reproducible extent. [ABSTRACT FROM AUTHOR]

Compound K (C-K) is a crucial pharmaceutical and cosmetic component because of disease prevention and skin anti-aging effects. For industrial application of this active compound, the protopanaxadiol (PPD)-type ginsenosides should be transformed to C-K. β-Glycosidase from Sulfolobus solfataricus has been reported as an efficient C-K-producing enzyme, using glycosylated PPD-type ginsenosides as substrates. β-Glycosidase from S. solfataricus can hydrolyze β--glucopyranoside in ginsenosides Rc, C-Mc1, and C-Mc, but not α--arabinofuranoside in these ginsenosides. To determine candidate residues involved in α--arabinofuranosidase activity, compound Mc (C-Mc) was docking to β-glycosidase from S. solfataricus in homology model and sequence was aligned with β-glycosidase from Pyrococcus furiosus that has α--arabinofuranosidase activity. A L213A variant β-glycosidase with increased α--arabinofuranosidase activity was selected by substitution of other amino acids for candidate residues. The increased α--arabinofuranosidase activity of the L213A variant was confirmed through the determination of substrate specificity, change in binding energy, transformation pathway, and C-K production from ginsenosides Rc and C-Mc. The L213A variant β-glycosidase catalyzed the conversion of Rc to Rd by hydrolyzing α--arabinofuranoside linked to Rc, whereas the wild-type β-glycosidase did not. The variant enzyme converted ginsenosides Rc and C-Mc into C-K with molar conversions of 97%, which were 1.5- and 2-fold higher, respectively, than those of the wild-type enzyme. Therefore, protein engineering is a useful tool for enhancing the hydrolytic activity on specific glycoside linked to ginsenosides. [ABSTRACT FROM AUTHOR]

The wyosine hypermodification found exclusively at G37 of tRNA Phe in eukaryotes and archaea is a very complicated process involving multiple steps and enzymes, and the derivatives are essential for the maintenance of the reading frame during translation. In the archaea Pyrococcus abyssi , two key enzymes from the Trm5 family, named PaTrm5a and PaTrm5b respectively, start the process by forming N1-methylated guanosine (m 1 G37). In addition, PaTrm5a catalyzes the further methylation of C7 on 4-demethylwyosine (imG-14) to produce isowyosine (imG2) at the same position. The structural basis of the distinct methylation capacities and possible conformational changes during catalysis displayed by the Trm5 enzymes are poorly studied. Here we report the 3.3 Å crystal structure of the mono-functional PaTrm5b, which shares 32% sequence identity with PaTrm5a. Interestingly, structural superposition reveals that the PaTrm5b protein exhibits an extended conformation similar to that of tRNA-bound Trm5b from Methanococcus jannaschii (MjTrm5b), but quite different from the open conformation of apo-PaTrm5a or well folded apo-MjTrm5b reported previously. Truncation of the N-terminal D1 domain leads to reduced tRNA binding as well as the methyltransfer activity of PaTrm5b. The differential positioning of the D1 domains from three reported Trm5 structures were rationalized, which could be attributable to the dissimilar inter-domain interactions and crystal packing patterns. This study expands our understanding on the methylation mechanism of the Trm5 enzymes and wyosine hypermodification. [ABSTRACT FROM AUTHOR]

Nitriles are important chemical building blocks for the synthesis of intermediates in fine chemical and pharmaceutical industries. Here, we report a new highly thermostable nitrilase from an Antarctic Pyrococcus sp. MC-FB, a hyperthermophilic archaeon. A gene that encoded a nitrilase was identified and subsequently cloned and overexpressed in Escherichia coli. The recombinant nitrilase, named NitMC-FB, is active as a homodimer (60 kDa) with an optimal temperature and pH of 90 °C and 7.0, respectively. NitMC-FB hydrolyzes preferentially aromatic nitriles, being the first aromatic nitrilase from an archaeon described so far. The K and V parameters were determined to be 13.9 mM and 3.7 μmol/min*mg, respectively, with 2-cyanopyridine as the substrate. Additionally, the recombinant nitrilase is highly thermostable with a half-life of 8 h at 90 °C. [ABSTRACT FROM AUTHOR]

Background: Alpha amylase hydrolyzes α-bonds of polysaccharides such as starch and produces maltooligosaccharides. Its starch saccharification applications make it an essential enzyme in the textile, food and brewing industries. Commercially available α-amylase is mostly produced from Bacillus or Aspergillus. A hyper-thermostable and Ca 2++ independent α-amylase from Pyrococcus furiosus (PFA) expressed in E.coli forms insoluble inclusion bodies and thus is not feasible for industrial applications. Results: We expressed PFA in Nicotiana tabacum and found that plant-produced PFA forms functional aggregates with an accumulation level up to 3.4 g/kg FW (fresh weight) in field conditions. The aggregates are functional without requiring refolding and therefore have potential to be applied as homogenized plant tissue without extraction or purification. PFA can also be extracted from plant tissue upon dissolution in a mild reducing buffer containing SDS. Like the enzyme produced in P. furiosus and in E. coli, plant produced PFA preserves hyper-thermophilicity and hyper-thermostability and has a long shelf life when stored in lyophilized leaf tissue. With tobacco's large biomass and high yield, hyper-thermostable α-amylase was produced at a scale of 42 kg per hectare. Conclusions: Tobacco may be a suitable bioreactor for industrial production of active hyperthermostable alpha amylase. [ABSTRACT FROM AUTHOR]

Archaea-specific D-family DNA polymerase (PolD) forms a dimeric heterodimer consisting of two large polymerase subunits and two small exonuclease subunits. According to the protein-protein interactions identified among the domains of large and small subunits of PolD, a symmetrical model for the domain topology of the PolD holoenzyme is proposed. The experimental evidence supports various aspects of the model. The conserved amphipathic nature of the N-terminal putative α-helix of the large subunit plays a key role in the homodimeric assembly and the self-cyclization of the large subunit and is deeply involved in the archaeal PolD stability and activity. We also discuss the evolutional transformation from archaeal D-family to eukaryotic B-family polymerase on the basis of the structural information.

Deep-sea hydrothermal vent environments are characterized by high hydrostatic pressure and sharp temperature and chemical gradients. Horizontal gene transfer is thought to play an important role in the microbial adaptation to such an extreme environment. In this study, a 21.4-kb DNA fragment was identified as a genomic island, designated PYG1, in the genomic sequence of the piezophilic hyperthermophile Pyrococcus yayanosii. According to the sequence alignment and functional annotation, the genes in PYG1 could tentatively be divided into five modules, with functions related to mobility, DNA repair, metabolic processes and the toxin-antitoxin system. Integrase can mediate the site-specific integration and excision of PYG1 in the chromosome of P. yayanosii A1. Gene replacement of PYG1 with a SimR cassette was successful. The growth of the mutant strain ∆PYG1 was compared with its parent strain P. yayanosii A2 under various stress conditions, including different pH, salinity, temperature and hydrostatic pressure. The ∆PYG1 mutant strain showed reduced growth when grown at 100 °C, while the biomass of ∆PYG1 increased significantly when cultured at 80 MPa. Differential expression of the genes in module Ⅲ of PYG1 was observed under different temperature and pressure conditions. This study demonstrates the first example of an archaeal integrative genomic island that could affect the adaptation of the hyperthermophilic piezophile P. yayanosii to high temperature and high hydrostatic pressure.

Glycoside hydrolase family 12 endocellulase (GH family12) plays a key role in the degradation of β-glucan and cellulose. Hyperthermostable GH family 12 endocellulase from the archaeon Pyrococcus furiosus (EGPf) catalyzes the hydrolysis of β(1 → 4) glucosidic linkages in cellulose and β-glucan containing β(1 → 3),(1 → 4) mixed-linkages. Therefore, in the combination with the hyperthermophilic β-glucosidase from P. furiosus (BGLPf), non-crystalline cellulose and β-glucan can be degraded to glucose completely by EGPf at high temperature. X-ray crystallography and protein engineering were used to reveal how the β(1 → 4) and β(1 → 3) linkages in β-glucan substrates are recognized by the enzyme. Structural and functional analyses clarified that the active site of EGPf consists of six subsites: the reducing end subsites (+1 and + 2) recognize both β(1 → 4) and β(1 → 3) linkages of various substrates in a productive binding mode, and recognition is controlled by Trp121 and Gln208 located at subsite +2. It was also revealed that the deep cleft in subsite −4 can accommodate the torsion angles of substrates consisting of β(1 → 3),(1 → 4) mixed-linkages due to the changing tilt of the Trp62 side chain. From the structural similarity, it is proposed that the substrate specificity of family 12 endocellulases towards β(1 → 3),(1 → 4) mixed-linkage substrates are controlled by the subsites (+1, +2, and −4). Furthermore, the function of family 12 endocellulase could be improved by protein engineering method using the information of the analysis. [Display omitted] • Endocellulase (GH family12) can cleave both the β(1 → 4) linkages of non-crystalline cellulose and the β(1 → 4) linkages of β(1 → 3),(1 → 4) mixed-linkages in β-glucan. • The substrate recognition mechanism for the saccharification of biomass containing β(1 → 3) linkages was revealed. • The reducing end subsites (+1 and + 2) of the enzyme recognize both β(1 → 4) and β(1 → 3) linkages of various substrates. • The deep cleft in subsite −4 can accommodate the torsion angles of substrates consisting of β(1 → 3),(1 → 4) mixed-linkages. [ABSTRACT FROM AUTHOR]

Deep-sea hydrothermal vent environments are characterized by high hydrostatic pressure and sharp temperature and chemical gradients. Horizontal gene transfer is thought to play an important role in the microbial adaptation to such an extreme environment. In this study, a 21.4-kb DNA fragment was identified as a genomic island, designated PYG1, in the genomic sequence of the piezophilic hyperthermophile Pyrococcus yayanosii. According to the sequence alignment and functional annotation, the genes in PYG1 could tentatively be divided into five modules, with functions related to mobility, DNA repair, metabolic processes and the toxin-antitoxin system. Integrase can mediate the site-specific integration and excision of PYG1 in the chromosome of P. yayanosii A1. Gene replacement of PYG1 with a SimR cassette was successful. The growth of the mutant strain ΔPYG1 was compared with its parent strain P. yayanosii A2 under various stress conditions, including different pH, salinity, temperature, and hydrostatic pressure. The 1PYG1 mutant strain showed reduced growth when grown at 100°C, while the biomass of ΔPYG1 increased significantly when cultured at 80 MPa. Differential expression of the genes in module III of PYG1 was observed under different temperature and pressure conditions. This study demonstrates the first example of an archaeal integrative genomic island that could affect the adaptation of the hyperthermophilic piezophile P. yayanosii to high temperature and high hydrostatic pressure. [ABSTRACT FROM AUTHOR]

In many hyperthermophilic archaea the DNA binding protein TrmBL2 or one of its homologues is abundantly expressed. TrmBL2 is thought to play a significant role in modulating the chromatin architecture in combination with the archaeal histone proteins and Alba. However, its precise physiological role is poorly understood. It has been previously shown that upon binding TrmBL2 covers double-stranded DNA, which leads to the formation of a thick and fibrous filament. Here we investigated the filament formation process as well as the stabilization of DNA by TrmBL2 from Pyroccocus furiosus in detail. We used magnetic tweezers that allow to monitor changes of the DNA mechanical properties upon TrmBL2 binding on the single-molecule level. Extended filaments formed in a cooperative manner and were considerably stiffer than bare double-stranded DNA. Unlike Alba, TrmBL2 did not form DNA cross-bridges. The protein was found to bind double- and single-stranded DNA with similar affinities. In mechanical disruption experiments of DNA hairpins this led to stabilization of both, the double- (before disruption) and the single-stranded (after disruption) DNA forms. Combined, these findings suggest that the biological function of TrmBL2 is not limited to modulating genome architecture and acting as a global repressor but that the protein acts additionally as a stabilizer of DNA secondary structure. [ABSTRACT FROM AUTHOR]

Here we analyze the first complete genome sequence of Pyrococcus chitonophagus. The archaeon was previously suggested to belong to the Thermococcus rather than the Pyrococcus genus. Whole genome phylogeny as well as whole proteome comparisons using all available complete genomes in Thermococcales clearly showed that the species belongs to the Pyrococcus genus. P. chitonophagus was originally isolated from a hydrothermal vent site and it has been described to effectively degrade chitin debris, and therefore is considered to play a major role in the sea water ecology and metabolic activity of microbial consortia within hot sea water ecosystems. Indeed, an obvious feature of the P. chitonophagus genome is that it carries proteins showing complementary activities for chitin degradation, i.e. endo- and exo-chitinase, diacetylchitobiose deacetylase and exo-β- d glucosaminidase activities. This finding supports the hypothesis that compared to other Thermococcales species P. chitonophagus is adapted to chitin degradation. [ABSTRACT FROM AUTHOR]

The potential of the hyperthermophilic β-glycosidase from Pyrococcus woesei (DSM 3773) for the synthesis of glycosides under microwave irradiation (MWI) at low temperatures was investigated. Transgalactosylation reactions with β-N-acetyl-d-glucosamine as acceptor substrate (GlcNAc-linker-tBoc) under thermal heating (TH, 85 °C) and under MWI at 100 and 300 W resulted in the formation of (Galβ(1,4)GlcNAc-linker-tBoc) as the main product in all reactions. Most importantly, MWI at temperatures far below the temperature optimum of the hyperthermophilic glycosidase led to higher product yields with only minor amounts of side products β(1,6-linked disaccharide and trisaccharides). At high acceptor concentrations (50 mM), transgalactosylation reactions under MWI at 300 W gave similar product yields when compared to TH at 85 °C. In summary, we demonstrate that MWI is useful as a novel experimental set-up for the synthesis of defined galacto-oligosaccharides. In conclusion, glycosylation reactions under MWI at low temperatures have the potential as a general strategy for regioselective glycosylation reactions of hyperthermophilic glycosidases using heat-labile acceptor or donor substrates. [ABSTRACT FROM AUTHOR]

DNA polymerases are versatile tools used in numerous important molecular biological core technologies like the ubiquitous polymerase chain reaction (PCR), cDNA cloning, genome sequencing and nucleic acid based diagnostics. Taking into account the multiple DNA amplification techniques in use, different DNA polymerases must be optimized for each type of application. One of the current tendencies is to reengineer or to discover new DNA polymerases with increased performance and broadened substrate spectra. At present, there is a great demand for such enzymes in applications, e.g., forensics or paleogenomics. Current major limitations hinge on the inability of conventional PCR enzymes, such as Taq, to amplify degraded or low amounts of template DNA. Besides, a wide range of PCR inhibitors can also impede reactions of nucleic acid amplification. Here we looked at the PCR performances of the proof-reading D-type DNA polymerase from P. abyssi, Pab-polD. Fragments, 3 kilobases in length, were specifically PCR-amplified in its optimized reaction buffer. Pab-polD showed not only a greater resistance to high denaturation temperatures than Taq during cycling, but also a superior tolerance to the presence of potential inhibitors. Proficient proof-reading Pab-polD enzyme could also extend a primer containing up to two mismatches at the 3’ primer termini. Overall, we found valuable biochemical properties in Pab-polD compared to the conventional Taq, which makes the enzyme ideally suited for cutting-edge PCR-applications.

Base deamination is a typical form of DNA damage, and it must be repaired quickly to maintain the genome integrity of living organisms. Endonuclease Q (EndoQ), recently found in the hyperthermophilic archaea, is an enzyme that cleaves the phosphodiester bond 5′ from the damaged nucleotide in the DNA strand, and may primarily function to start the repair process for the damaged bases. Endonuclease V (EndoV) also hydrolyzes the second phosphodiester bond 3′ from the damaged nucleotide, although the hyperthermophilic archaeal EndoV is a strictly hypoxanthine-specific endonuclease. To understand the relationships of the EndoQ and EndoV functions in hyperthermophilic archaea, we analyzed their interactions in hypoxanthine repair. EndoQ and EndoV do not directly interact with each other in either the presence or absence of DNA. However, EndoQ and EndoV individually worked on deoxyinosine (dI)-containing DNA at each cleavage site. EndoQ has higher affinity to dI-containing DNA than EndoV, and cells produce higher amounts of EndoQ, as compared to EndoV. These data support the proposal that EndoQ primarily functions for, at least, dI-containing DNA. [ABSTRACT FROM AUTHOR]

Background: R.PabI is an exceptional restriction enzyme that functions as a DNA glycosylase. The enzyme excises an unmethylated base from its recognition sequence to generate apurinic/apyrimidinic (AP) sites, and also displays AP lyase activity, cleaving the DNA backbone at the AP site to generate the 3'-phospho alpha, beta-unsaturated aldehyde end in addition to the 5'-phosphate end. The resulting ends are difficult to religate with DNA ligase. The enzyme was originally isolated in Pyrococcus, a hyperthermophilic archaeon, and additional homologs subsequently identified in the epsilon class of the Gram-negative bacterial phylum Proteobacteria, such as Helicobacter pylori. Results: Systematic analysis of R.PabI homologs and their neighboring genes in sequenced genomes revealed co-occurrence of R.PabI with M.PabI homolog methyltransferase genes. R.PabI and M.PabI homolog genes are occasionally found at corresponding (orthologous) loci in different species, such as Helicobacter pylori, Helicobacter acinonychis and Helicobacter cetorum, indicating long-term maintenance of the gene pair. One R.PabI and M.PabI homolog gene pair is observed immediately after the GMP synthase gene in both Campylobacter and Helicobacter, representing orthologs beyond genera. The mobility of the PabI family of restriction-modification (RM) system between genomes is evident upon comparison of genomes of sibling strains/species. Analysis of R.PabI and M.PabI homologs in H. pylori revealed an insertion of integrative and conjugative elements (ICE), and replacement with a gene of unknown function that may specify a membrane-associated toxin (hrgC). In view of the similarity of HrgC with toxins in type I toxin-antitoxin systems, we addressed the biological significance of this substitution. Our data indicate that replacement with hrgC occurred in the common ancestor of hspAmerind and hspEAsia. Subsequently, H. pylori with and without hrgC were intermixed at this locus, leading to complex distribution of hrgC in East Asia and the Americas. In Malaysia, hrgC was horizontally transferred from hspEAsia to hpAsia2 strains. Conclusions: The PabI family of RM system behaves as a mobile, selfish genetic element, similar to the other families of Type II RM systems. Our analysis additionally revealed some cases of long-term inheritance. The distribution of the hrgC gene replacing the PabI family in the subpopulations of H. pylori, hspAmerind, hspEAsia and hpAsia2, corresponds to the two human migration events, one from East Asia to Americas and the other from China to Malaysia. [ABSTRACT FROM AUTHOR]

Acetate and acetyl-CoA play fundamental roles in all of biology, including anaerobic prokaryotes from the domains Bacteria and Archaea, which compose an estimated quarter of all living protoplasm in Earth's biosphere. Anaerobes from the domain Archaea contribute to the global carbon cycle by metabolizing acetate as a growth substrate or product. They are components of anaerobic microbial food chains converting complex organic matter to methane, and many fix CO2 into cell material via synthesis of acetyl-CoA. They are found in a diversity of ecological habitats ranging from the digestive tracts of insects to deep-sea hydrothermal vents, and synthesize a plethora of novel enzymes with biotechnological potential. Ecological investigations suggest that still more acetate-metabolizing species with novel properties await discovery. [ABSTRACT FROM AUTHOR]

The crystal structure of the aminopeptidase APDkam589 from the thermophilic crenarchaeon Desulfurococcus kamchatkensis was determined at a resolution of 3.0 Å. In the crystal, the monomer of APDkam589 and its symmetry-related monomers are densely packed to form a 12-subunit complex. Single-particle electron-microscopy analysis confirms that APDkam589 is present as a compact dodecamer in solution. The APDkam589 molecule is built similarly to the molecules of the PhTET peptidases, which have the highest sequence identity to APDkam589 among known structures and were isolated from the more thermostable archaeon Pyrococcus horikoshii. A comparison of the interactions of the subunits in APDkam589 with those in PhTET1, PhTET2 and PhTET3 reveals that APDkam589 has a much lower total number of salt bridges, which correlates with the lower thermostability of APDkam589. The monomer of APDkam589 has six Trp residues, five of which are on the external surface of the dodecamer. A superposition of the structure of APDkam589 with those having a high sequence similarity to APDkam589 reveals that, although the positions of Trp45, Trp252 and Trp358 are not conserved in the sequences, the spatial locations of the Trp residues in these models are similar. [ABSTRACT FROM AUTHOR]

The cytoplasmic [NiFe]-hydrogenase I (SHI) of the hyperthermophile Pyrococcus furiosus evolves hydrogen gas (H 2 ) from NADPH. It has been previously used for biohydrogen production from sugars using a mixture of enzymes in an in vitro cell-free synthetic pathway. The theoretical yield (12 H 2 /glucose) is three times greater than microbial fermentation (4 H 2 /glucose), making the in vitro approach very promising for large scale biohydrogen production. Further development of this process at an industrial scale is limited by the availability of the H 2 -producing SHI. To overcome the obstacles of the complex biosynthetic and maturation pathway for the [NiFe] site of SHI, the four gene operon encoding the enzyme was overexpressed in P. furiosus and included a polyhistidine affinity tag. The one-step purification resulted in a 50-fold increase in yield compared to the four-step purification procedure for the native enzyme. A trimeric form was also identified that lacked the [NiFe]-catalytic subunit but catalyzed NADPH oxidation with a specific activity similar to that of the tetrameric form. The presence of an active trimeric intermediate confirms the proposed maturation pathway where, in the terminal step, the NiFe-containing catalytic subunit assembles with NADPH-oxidizing trimeric form to give the active holoenzyme. [ABSTRACT FROM AUTHOR]

The hyperthermophile Pyrococcus yayanosii CH1 is the only high-pressure-requiring microorganism obtained thus far within the archaea domain or among all non-psychrophiles in any domain. In this study, we developed a genetic manipulation system for P. yayanosii after first isolating a facultatively piezophilic derivative strain, designated P. yayanosii A1. The 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase gene was overexpressed in strain P. yayanosii A1 and was demonstrated to confer host cell resistance against simvastatin. Furthermore, using simvastatin as a selection marker, the endogenous pyrF of P. yayanosii A1 was disrupted through homologous recombination, thus generating the additional host strain P. yayanosii A2 (Δ pyrF). A markerless gene disruption vector was constructed by incorporating a pyrF- sim cassette that enables the combined use of simvastatin resistance for positive selection and 5-FOA for counter selection. The utility of this versatile disruption system was demonstrated by deleting the carbon-nitrogen hydrolase of P. yayanosii strain A1. These results demonstrate that a variety of genetic tools are now in place to study unknown gene function and the molecular mechanisms of piezophilic adaptation in P. yayanosii. [ABSTRACT FROM AUTHOR]