Hyperthermophilic Archaea Research Articles

DUTP pyrophosphatases from hyperthermophilic eubacterium and archaeon: Structural and functional examinations on the suitability for PCR application.

Deoxyuridine triphosphate pyrophosphatase (DUT) suppresses incorporation of uracil into genomic DNA during replication. Thermostable DUTs from hyperthermophilic archaea such as Thermococcus pacificus enhance PCR amplification by preventing misincorporation of dUTP generated by spontaneous deamination of dCTP. However, it is necessary to elucidate whether DUTs do not cause dNTP imbalances during PCR by unwanted side activity. Moreover, it has been unknown what structural features define the thermostability of those DUTs. Here, DUT from a hyperthermophilic eubacterium, Aquifex aeolicus (Aa-DUT), was characterized together with those from T. pacificus (Tp-DUT). Aa-DUT was as thermostable as Tp-DUT up to at least 95°C. The crystal structures of the two thermostable enzymes were determined, which revealed that the structures of Aa-DUT and Tp-DUT resembled those of monofunctional and bifunctional DUTs, respectively. Generally, bifunctional DUTs harbor the dCTP deaminase activity in addition to the DUT activity. However, not only Aa-DUT but also Tp-DUT showed poor activity towards dCTP, indicating both enzymes are monofunctional. We further examined eight types of parameters related to thermostability of protein structure and found that the thermostability of Aa-DUT and Tp-DUT might be accomplished by increased numbers of ion pairs on the protein surface. Finally, we verified that Aa-DUT promoted PCR amplification with Pfu DNA polymerase to the same extent as Tp-DUT. Collectively, we conclude that both DUTs from hyperthermophiles maintain the enzymatic activity at high temperatures without consuming dCTP due to the lack of the deaminate activity.

An Insight into the Mechanism of DNA Cleavage by DNA Endonuclease from the Hyperthermophilic Archaeon Pyrococcus furiosus.

Hyperthermophilic archaea such as Pyrococcus furiosus survive under very aggressive environmental conditions by occupying niches inaccessible to representatives of other domains of life. The ability to survive such severe living conditions must be ensured by extraordinarily efficient mechanisms of DNA processing, including repair. Therefore, in this study, we compared kinetics of conformational changes of DNA Endonuclease Q from P. furiosus during its interaction with various DNA substrates containing an analog of an apurinic/apyrimidinic site (F-site), hypoxanthine, uracil, 5,6-dihydrouracil, the α-anomer of adenosine, or 1,N6-ethenoadenosine. Our examination of DNA cleavage activity and fluorescence time courses characterizing conformational changes of the dye-labeled DNA substrates during the interaction with EndoQ revealed that the enzyme induces multiple conformational changes of DNA in the course of binding. Moreover, the obtained data suggested that the formation of the enzyme-substrate complex can proceed through dissimilar kinetic pathways, resulting in different types of DNA conformational changes, which probably allow the enzyme to perform its biological function at an extreme temperature.

Exploiting Archaeal/Thermostable Enzymes in Synthetic Chemistry: Back to the Future?

Billions of years of evolution have led to the selection of (hyper)thermophiles capable of flourishing at elevated temperatures. The corresponding native (hyper)thermophilic enzymes retain their tertiary structures at near‐boiling water temperatures. And yet, while hyper/thermophilic enzymes offer special opportunities in biocatalysis and in hybrid bio/chemocatalytic approaches to modern synthesis in both academia and industry, these enzymes remain underexplored in biocatalysis. Among the strategic advantages that can be leveraged in running biocatalytic transformations at higher temperatures are included more favorable kinetics, removal of volatile byproducts to drive reactions forward, improved substrate solubility and product separation, and accelerated stereodynamics for dynamic kinetic resolutions. These topics are discussed and illustrated with contemporary examples of note, in sections organized by stratagem. Finally, the reader is alerted in particular to archaeal enzymes that have proven useful in non‐natural synthetic chemistry ventures, and at the same time is referred to a rich area of archaea whose genomes have been sequenced but whose enzymatic activities of interest have not yet been mined. Though hyperthermophilic archaea are among the most ancient of organisms, their enzymes may hold the key to many future innovations in biocatalytic chemistry – perhaps we really do need to go ‘back to the future.’

Removal of phosphoglycolate in hyperthermophilic archaea

Many organisms that utilize the Calvin-Benson-Bassham (CBB) cycle for autotrophic growth harbor metabolic pathways to remove and/or salvage 2-phosphoglycolate, the product of the oxygenase activity of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco). It has been presumed that the occurrence of 2-phosphoglycolate salvage is linked to the CBB cycle, and in particular, the C2 pathway to the CBB cycle and oxygenic photosynthesis. Here, we examined 2-phosphoglycolate salvage in the hyperthermophilic archaeon Thermococcus kodakarensis, an obligate anaerobe that harbors a Rubisco that functions in the pentose bisphosphate pathway. T. kodakarensis harbors enzymes that have the potential to convert 2-phosphoglycolate to glycine and serine, and their genes were identified by biochemical and/or genetic analyses. 2-phosphoglycolate phosphatase activity increased 1.6-fold when cells were grown under microaerobic conditions compared to anaerobic conditions. Among two candidates, TK1734 encoded a phosphatase specific for 2-phosphoglycolate, and the enzyme was responsible for 80% of the 2-phosphoglycolate phosphatase activity in T. kodakarensis cells. The TK1734 disruption strain displayed growth impairment under microaerobic conditions, which was relieved upon addition of sodium sulfide. In addition, glycolate was detected in the medium when T. kodakarensis was grown under microaerobic conditions. The results suggest that T. kodakarensis removes 2-phosphoglycolate via a phosphatase reaction followed by secretion of glycolate to the medium. As the Rubisco in T. kodakarensis functions in the pentose bisphosphate pathway and not in the CBB cycle, mechanisms to remove 2-phosphoglycolate in this archaeon emerged independent of the CBB cycle.

Pleomorphic viruses establish stable relationship with marine hyperthermophilic archaea.

Non-lytic viruses with enveloped pleomorphic virions (family Pleolipoviridae) are ubiquitous in hypersaline environments across the globe and are associated with nearly all major lineages of halophilic archaea. However, their existence in other ecosystems remains largely unknown. Here, we show that evolutionarily-related viruses also infect hyperthermophilic archaea thriving in deep-sea hydrothermal vents. Archaeoglobus veneficus pleomorphic virus 1 (AvPV1), the first virus described for any member of the class Archaeoglobi, encodes a morphogenetic module typical of pleolipoviruses, including the characteristic VP4-like membrane fusion protein. We show that AvPV1 is a non-lytic virus chronically produced in liquid cultures without substantially affecting the growth dynamics of its host with a stable virus-to-host ratio of ~1. Mining of genomic and metagenomic databases revealed broad distribution of AvPV1-like viruses in geographically remote hydrothermal vents. Comparative genomics, coupled with phylogenetic analysis of VP4-like fusogens revealed deep divergence of pleomorphic viruses infecting halophilic, methanogenic, and hyperthermophilic archaea, signifying niche separation and coevolution of the corresponding virus-host pairs. Hence, we propose a new virus family, "Thalassapleoviridae," for classification of the marine hyperthermophilic virus AvPV1 and its relatives. Collectively, our results provide insights into the diversity and evolution of pleomorphic viruses beyond hypersaline environments.

Uncovering the temporal dynamics and regulatory networks of thermal stress response in a hyperthermophile using transcriptomics and proteomics.

Extreme environments provide unique challenges for life, and the study of extremophiles can shed light on the mechanisms of adaptation to such conditions. Pyrococcus furiosus, a hyperthermophilic archaeon, is a model organism for studying thermal stress response mechanisms. In this study, we used an integrated analysis of RNA-sequencing and mass spectrometry data to investigate the transcriptomic and proteomic responses of P. furiosus to heat and cold shock stress and recovery. Our results reveal the rapid and dynamic changes in gene and protein expression patterns associated with these stress responses, as well as the coordinated regulation of different gene sets in response to different stressors. These findings provide valuable insights into the molecular adaptations that facilitate life in extreme environments and advance our understanding of stress response mechanisms in hyperthermophilic archaea.

Dissecting the Arginine and Lysine Biosynthetic Pathways and Their Relationship in Haloarchaeon Natrinema gari J7-2 via Endogenous CRISPR-Cas System-Based Genome Editing.

The evolutionary relationship between arginine and lysine biosynthetic pathways has been well established in bacteria and hyperthermophilic archaea but remains largely unknown in haloarchaea. Here, the endogenous CRISPR-Cas system was harnessed to edit arginine and lysine biosynthesis-related genes in the haloarchaeon Natrinema gari J7-2. The ΔargW, ΔargX, ΔargB, and ΔargD mutant strains display an arginine auxotrophic phenotype, while the ΔdapB mutant shows a lysine auxotrophic phenotype, suggesting that strain J7-2 utilizes the ArgW-mediated pathway and the diaminopimelate (DAP) pathway to synthesize arginine and lysine, respectively. Unlike the ArgD in Escherichia coli acting as a bifunctional aminotransferase in both the arginine biosynthesis pathway and the DAP pathway, the ArgD in strain J7-2 participates only in arginine biosynthesis. Meanwhile, in strain J7-2, the function of argB cannot be compensated for by its evolutionary counterpart ask in the DAP pathway. Moreover, strain J7-2 cannot utilize α-aminoadipate (AAA) to synthesize lysine via the ArgW-mediated pathway, in contrast to hyperthermophilic archaea that employ a bifunctional LysW-mediated pathway to synthesize arginine (or ornithine) and lysine from glutamate and AAA, respectively. Additionally, the replacement of a 5-amino-acid signature motif responsible for substrate specificity of strain J7-2 ArgX with that of its hyperthermophilic archaeal homologs cannot endow the ΔdapB mutant with the ability to biosynthesize lysine from AAA. The in vitro analysis shows that strain J7-2 ArgX acts on glutamate rather than AAA. These results suggest that the arginine and lysine biosynthetic pathways of strain J7-2 are highly specialized during evolution. IMPORTANCE Due to their roles in amino acid metabolism and close evolutionary relationship, arginine and lysine biosynthetic pathways represent interesting models for probing functional specialization of metabolic routes. The current knowledge with respect to arginine and lysine biosynthesis is limited for haloarchaea compared to that for bacteria and hyperthermophilic archaea. Our results demonstrate that the haloarchaeon Natrinema gari J7-2 employs the ArgW-mediated pathway and the DAP pathway for arginine and lysine biosynthesis, respectively, and the two pathways are functionally independent of each other; meanwhile, ArgX is a key determinant of substrate specificity of the ArgW-mediated pathway in strain J7-2. This study provides new clues about haloarchaeal amino acid metabolism and confirms the convenience and efficiency of endogenous CRISPR-Cas system-based genome editing in haloarchaea.

Extracellular cytochrome nanowires appear to be ubiquitous in prokaryotes

Electrically conductive appendages from the anaerobic bacterium Geobacter sulfurreducens, recently identified as extracellular cytochrome nanowires (ECNs), have received wide attention due to numerous potential applications. However, whether other organisms employ similar ECNs for electron transfer remains unknown. Here, using cryoelectron microscopy, we describe the atomic structures of two ECNs from two major orders of hyperthermophilic archaea present in deep-sea hydrothermal vents and terrestrial hot springs. Homologs of Archaeoglobus veneficus ECN are widespread among mesophilic methane-oxidizing Methanoperedenaceae, alkane-degrading Syntrophoarchaeales archaea, and in the recently described megaplasmids called Borgs. The ECN protein subunits lack similarities in their folds; however, they share a common heme arrangement, suggesting an evolutionarily optimized heme packing for efficient electron transfer. The detection of ECNs in archaea suggests that filaments containing closely stacked hemes may be a common and widespread mechanism for long-range electron transfer in both prokaryotic domains of life.

Production of carbon-containing pyrite spherules induced by hyperthermophilic Thermococcales: a biosignature?

Thermococcales, a major order of hyperthermophilic archaea inhabiting iron- and sulfur-rich anaerobic parts of hydrothermal deep-sea vents, are known to induce the formation of iron phosphates, greigite (Fe3S4) and abundant quantities of pyrite (FeS2), including pyrite spherules. In the present study, we report the characterization of the sulfide and phosphate minerals produced in the presence of Thermococcales using X-ray diffraction, synchrotron-based X ray absorption spectroscopy and scanning and transmission electron microscopies. Mixed valence Fe(II)-Fe(III) phosphates are interpreted as resulting from the activity of Thermococcales controlling phosphorus-iron-sulfur dynamics. The pyrite spherules (absent in abiotic control) consist of an assemblage of ultra-small nanocrystals of a few ten nanometers in size, showing coherently diffracting domain sizes of few nanometers. The production of these spherules occurs via a sulfur redox swing from S0 to S-2 and then to S-1, involving a comproportionation of (-II) and (0) oxidation states of sulfur, as supported by S-XANES data. Importantly, these pyrite spherules sequester biogenic organic compounds in small but detectable quantities, possibly making them good biosignatures to be searched for in extreme environments.

Thermostable DNA ligases from hyperthermophiles in biotechnology.

DNA ligase is an important enzyme ubiquitous in all three kingdoms of life that can ligate DNA strands, thus playing essential roles in DNA replication, repair and recombination in vivo. In vitro, DNA ligase is also used in biotechnological applications requiring in DNA manipulation, including molecular cloning, mutation detection, DNA assembly, DNA sequencing, and other aspects. Thermophilic and thermostable enzymes from hyperthermophiles that thrive in the high-temperature (above 80°C) environments have provided an important pool of useful enzymes as biotechnological reagents. Similar to other organisms, each hyperthermophile harbors at least one DNA ligase. In this review, we summarize recent progress on structural and biochemical properties of thermostable DNA ligases from hyperthermophiles, focusing on similarities and differences between DNA ligases from hyperthermophilic bacteria and archaea, and between these thermostable DNA ligases and non-thermostable homologs. Additionally, altered thermostable DNA ligases are discussed. Possessing improved fidelity or thermostability compared to the wild-type enzymes, they could be potential DNA ligases for biotechnology in the future. Importantly, we also describe current applications of thermostable DNA ligases from hyperthermophiles in biotechnology.

The Impact of Single-Stranded DNA-Binding Protein SSB and Putative SSB-Interacting Proteins on Genome Integrity in the Thermophilic Crenarchaeon Sulfolobus acidocaldarius

The study of DNA repair in hyperthermophiles has the potential to elucidate the mechanisms of genome integrity maintenance systems under extreme conditions. Previous biochemical studies have suggested that the single-stranded DNA-binding protein (SSB) from the hyperthermophilic crenarchaeon Sulfolobus is involved in the maintenance of genome integrity, namely, in mutation avoidance, homologous recombination (HR), and the repair of helix-distorting DNA lesions. However, no genetic study has been reported that elucidates whether SSB actually maintains genome integrity in Sulfolobus in vivo. Here, we characterized mutant phenotypes of the ssb-deleted strain Δssb in the thermophilic crenarchaeon S. acidocaldarius. Notably, an increase (29-fold) in mutation rate and a defect in HR frequency was observed in Δssb, indicating that SSB was involved in mutation avoidance and HR in vivo. We characterized the sensitivities of Δssb, in parallel with putative SSB-interacting protein-encoding gene-deleted strains, to DNA-damaging agents. The results showed that not only Δssb but also Δalhr1 and ΔSaci_0790 were markedly sensitive to a wide variety of helix-distorting DNA-damaging agents, indicating that SSB, a novel helicase SacaLhr1, and a hypothetical protein Saci_0790, were involved in the repair of helix-distorting DNA lesions. This study expands our knowledge of the impact of SSB on genome integrity and identifies novel and key proteins for genome integrity in hyperthermophilic archaea in vivo.

Archaeal DNA-import apparatus is homologous to bacterial conjugation machinery

Conjugation is a major mechanism of horizontal gene transfer promoting the spread of antibiotic resistance among human pathogens. It involves establishing a junction between a donor and a recipient cell via an extracellular appendage known as the mating pilus. In bacteria, the conjugation machinery is encoded by plasmids or transposons and typically mediates the transfer of cognate mobile genetic elements. Much less is known about conjugation in archaea. Here, we determine atomic structures by cryo-electron microscopy of three conjugative pili, two from hyperthermophilic archaea (Aeropyrum pernix and Pyrobaculum calidifontis) and one encoded by the Ti plasmid of the bacterium Agrobacterium tumefaciens, and show that the archaeal pili are homologous to bacterial mating pili. However, the archaeal conjugation machinery, known as Ced, has been ‘domesticated’, that is, the genes for the conjugation machinery are encoded on the chromosome rather than on mobile genetic elements, and mediates the transfer of cellular DNA.

Biohydrogen Production from Food Waste Using Glucose-Adapted Hyperthermophilic Archaeon

PurposeGlucose is one of the most important carbon and energy source for heterotrophic growth in all living organisms. However, glucose has been reported as a poor substrate to support the growth of hyperthermophilic archaea belonging to the order Thermococcales. To enhance glucose-assisted growth of Thermococcus onnurineus NA1, adaptive evolution process was applied. In an effort for industrial applications, glucose-adapted cells were further tested for H2 producing potential using food processing waste as a promising zero-value substrate containing polysaccharides composed of glucose.MethodsAdaptive evolution of T. onnurineus NA1 was performed by transferring cells to fresh medium containing glucose until cell growth increased. Genome sequencing was conducted to identify genetic changes in adapted cells. H2 production in the parent strain and glucose-adapted cells was analyzed using either glucose or potato peel waste as substrate.ResultsThe glucose-adapted cells, WG-100T, had 10.8-fold and 14.7-fold increases in cell density and glucose consumption, respectively, compared to the parent strain. Genome sequencing of WG-100T revealed a total of 17 genomic changes in genes, including those encoding transcription factors and several proteins involved in various transport systems. WG-100T produced H2 using potato peel waste through simultaneous saccharification and fermentation.ConclusionThis study showed that the performance of the Thermococcales strain was improved by adaptive evolution, resulting in faster use of glucose. In addition, it was shown that the use of a hyperthermophile made it possible to produce biohydrogen without pretreatment of food processing waste for saccharification.Graphical

Domesticated conjugation machinery promotes DNA exchange in hyperthermophilic archaea.

Conjugation, a major mechanism of horizontal gene transfer promoting the spread of antibiotic resistance among human pathogens, involves establishing a junction between a donor and a recipient cell. The mating pilus plays a prominent role in this process. In bacteria, the conjugation machinery is encoded by plasmids or transposons and typically mediates the transfer of cognate mobile genetic elements. Much less has been known about conjugation in archaea, with no homologs of bacterial mating pilins having been identified in sequence searches. Here, using cryo-electron microscopy, we determined high-resolution structures of three conjugative pili, two from hyperthermophilic archaea, Aeropyrum pernix and Pyrobaculum calidifontis, and one encoded by the Ti plasmid of the bacterium Agrobacterium tumefaciens. We show that the archaeal pili are homologous to bacterial mating pili and display intricate pilin-lipid interaction networks. Unlike in bacteria, the conjugation machinery in hyperthermophilic archaea has been domesticated and mediates the transfer of cellular DNA. We suggest that the constitutive expression of the pili may be due to the chronic damage of DNA at high temperatures that is constantly being repaired using homologous recombination.

First person – Sherman Foo

ABSTRACT First Person is a series of interviews with the first authors of a selection of papers published in Journal of Cell Science, helping researchers promote themselves alongside their papers. Sherman Foo is first author on ‘ Diacylglycerol at the inner nuclear membrane fuels nuclear envelope expansion in closed mitosis’, published in JCS. Sherman conducted the research described in this article while a PhD student in the labs of Snezhana Oliferenko (The Francis Crick Institute, London, UK, and Randall Centre for Cell and Molecular Biophysics, King's College London, UK) and Markus R. Wenk (Singapore Lipidomics Incubator, National University of Singapore). He is now a postdoc in the lab of Buzz Baum at Medical Research Council Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge, UK, where he is currently studying cellular organization of hyperthermophilic archaea, with a particular focus on the surface layer and cell cycle of Sulfolobus acidocaldarius.

Biochemical characterization and mechanistic insight of the family IV uracil DNA glycosylase from Sulfolobus islandicus REY15A

Uracil DNA glycosylase (UDG) can remove uracil from DNA, thus playing an essential role in maintaining genomic stability. Family IV UDG members are mostly widespread in hyperthermophilic Archaea and bacteria. In this work, we characterized the family IV UDG from the hyperthermophilic crenarchaeon Sulfolobus islandicus REY15A (Sis-UDGIV) biochemically, and dissected the roles of nine conserved residues in uracil excision by mutational analyses. Biochemical data demonstrate that Sis-UDGIV displays maximum efficiency for uracil excision at 50 °C ~ 70 °C and at pH 7.0–9.0. Additionally, the enzyme has displays a weak activity without a divalent metal ion, but maximum activity with Mg2+. Our mutational analyses show that residues E48 and F55 in Sis-UDGIV are essential for uracil removal, and residues E48, F55, R87, R92 and K146 are responsible for binding DNA. Importantly, we systemically revealed the roles of four conserved cysteine residues C14, C17, C86 and C102 in Sis-UDGIV that are required for being ligands of FeS cluster in maintaining the overall protein conformation and stability by circular dichroism analyses. Overall, our work has provided insights into biochemical function and DNA-binding specificity of archaeal family IV UDGs.

Insights into Antarctic microbiomes: diversity patterns for terrestrial and marine habitats.

Microorganisms in Antarctica are recognized for having crucial roles in ecosystems functioning and biogeochemical cycles. To explore the diversity and composition of microbial communities through different terrestrial and marine Antarctic habitats, we analyze 16S rRNA sequence datasets from fumarole and marine sediments, soil, snow and seawater environments. We obtained measures of alpha- and beta-diversities, as well as we have identified the core microbiome and the indicator microbial taxa of a particular habitat. Our results showed a unique microbial community structure according to each habitat, including specific taxa composing each microbiome. Marine sediments harbored the highest microbial diversity among the analyzed habitats. In the fumarole sediments, the core microbiome was composed mainly of thermophiles and hyperthermophilic Archaea, while in the majority of soil samples Archaea was absent. In the seawater samples, the core microbiome was mainly composed by cultured and uncultured orders usually identified on Antarctic pelagic ecosystems. Snow samples exhibited common taxa previously described for habitats of the Antarctic Peninsula, which suggests long-distance dispersal processes occurring from the Peninsula to the Continent. This study contributes as a baseline for further efforts on evaluating the microbial responses to environmental conditions and future changes.

Acceptor dependent catalytic properties of GH57 4-α-glucanotransferase from Pyrococcus sp. ST04.

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.

Direct adenylation from 5'-OH-terminated oligonucleotides by a fusion enzyme containing Pfu RNA ligase and T4 polynucleotide kinase.

5′-Adenylated oligonucleotides (AppOligos) are widely used for single-stranded DNA/RNA ligation in next-generation sequencing (NGS) applications such as microRNA (miRNA) profiling. The ligation between an AppOligo adapter and target molecules (such as miRNA) no longer requires ATP, thereby minimizing potential self-ligations and simplifying library preparation procedures. AppOligos can be produced by chemical synthesis or enzymatic modification. However, adenylation via chemical synthesis is inefficient and expensive, while enzymatic modification requires pre-phosphorylated substrate and additional purification. Here we cloned and characterized the Pfu RNA ligase encoded by the PF0353 gene in the hyperthermophilic archaea Pyrococcus furiosus. We further engineered fusion enzymes containing both Pfu RNA ligase and T4 polynucleotide kinase. One fusion enzyme, 8H-AP, was thermostable and can directly catalyze 5′-OH-terminated DNA substrates to adenylated products. The newly discovered Pfu RNA ligase and the engineered fusion enzyme may be useful tools for applications using AppOligos.

Genomic attributes of thermophilic and hyperthermophilic bacteria and archaea.

Thermophiles and hyperthermophiles are immensely useful in understanding the evolution of life, besides their utility in environmental and industrial biotechnology. Advancements in sequencing technologies have revolutionized the field of microbial genomics. The massive generation of data enhances the sequencing coverage multi-fold and allows to analyse the entire genomic features of microbes efficiently and accurately. The mandate of a pure isolate can also be bypassed where whole metagenome-assembled genomes and single cell-based sequencing have fulfilled the majority of the criteria to decode various attributes of microbial genomes. A boom has, therefore, been seen in analysing the extremophilic bacteria and archaea using sequence-based approaches. Due to extensive sequence analysis, it becomes easier to understand the gene flow and their evolution among the members of bacteria and archaea. For instance, sequencing unveiled that Thermotoga maritima shares around 24% of genes of archaeal origin. Comparative and functional genomics provide an analytical view to understanding the microbial diversity of thermophilic bacteria and archaea, their interactions with other microbes, their adaptations, gene flow, and evolution over time. In this review, the genomic features of thermophilic bacteria and archaea are dealt with comprehensively.