Complete Genome Sequence of the Radioresistant Bacterium Deinococcus aetherius ST0316, Isolated from Air Dust Collected in the Lower Stratosphere above Japan.

Deinococcus aetherius ST0316 is a radioresistant bacterium that possess proficient DNA repair capacity. Here, we report the complete genome sequence of D. aetherius, which was obtained by hybrid assembly using short- and long-read sequencing. This sequence will be important information for elucidating the unique DNA repair mechanism of Deinococcus bacteria.

Amino Acid Specificity of Ancestral Aminoacyl-tRNA Synthetase Prior to the Last Universal Common Ancestor Commonote commonote.

Extant organisms commonly use 20 amino acids in protein synthesis. In the translation system, aminoacyl-tRNA synthetase (ARS) selectively binds an amino acid and transfers it to the cognate tRNA. It is postulated that the amino acid repertoire of ARS expanded during the development of the translation system. In this study we generated composite phylogenetic trees for seven ARSs (SerRS, ProRS, ThrRS, GlyRS-1, HisRS, AspRS, and LysRS) which are thought to have diverged by gene duplication followed by mutation, before the evolution of the last universal common ancestor. The composite phylogenetic tree shows that the AspRS/LysRS branch diverged from the other five ARSs at the deepest node, with the GlyRS/HisRS branch and the other three ARSs (ThrRS, ProRS and SerRS) diverging at the second deepest node. ThrRS diverged next, and finally ProRS and SerRS diverged from each other. Based on the phylogenetic tree, sequences of the ancestral ARSs prior to the evolution of the last universal common ancestor were predicted. The amino acid specificity of each ancestral ARS was then postulated by comparison with amino acid recognition sites of ARSs of extant organisms. Our predictions demonstrate that ancestral ARSs had substantial specificity and that the number of amino acid types amino-acylated by proteinaceous ARSs was limited before the appearance of a fuller range of proteinaceous ARS species. From an assumption that 10 amino acid species are required for folding and function, proteinaceous ARS possibly evolved in a translation system composed of preexisting ribozyme ARSs, before the evolution of the last universal common ancestor.

Space Exposure of Amino Acids and Their Precursors during the Tanpopo Mission.

Amino acids have been detected in extraterrestrial bodies such as carbonaceous chondrites (CCs), which suggests that extraterrestrial organics could be the source of the first life on Earth, and interplanetary dust particles (IDPs) or micrometeorites (MMs) are promising carriers of extraterrestrial organic carbon. Some amino acids found in CCs are amino acid precursors, but these have not been well characterized. The Tanpopo mission was conducted in Earth orbit from 2015 to 2019, and the stability of glycine (Gly), hydantoin (Hyd), isovaline (Ival), 5-ethyl-5-methylhydantoin (EMHyd), and complex organics formed by proton irradiation from CO, NH3, and H2O (CAW) in space were analyzed by high-performance liquid chromatography and/or gas chromatography/mass spectrometry. The target substances showed a logarithmic decomposition over 1-3 years upon space exposure. Recoveries of Gly and CAW were higher than those of Hyd, Ival, and EMHyd. Ground simulation experiments showed different results: Hyd was more stable than Gly. Solar ultraviolet light was fatal to all organics, and they required protection when carried by IDPs/MMs. Thus, complex amino acid precursors (such as CAW) were possibly more robust than simple precursors during transportation to primitive Earth. The Tanpopo 2 mission is currently being conducted to expose organics to more probable space conditions.

Mutation Analysis of the rpoB Gene in the Radiation-Resistant Bacterium Deinococcus radiodurans R1 Exposed to Space during the Tanpopo Experiment at the International Space Station.

To investigate microbial viability and DNA damage, dried cell pellets of the radiation-resistant bacterium Deinococcus radiodurans were exposed to various space environmental conditions at the Exposure Facility of the International Space Station (ISS) as part of the Tanpopo mission. Mutation analysis was done by sequencing the rpoB gene encoding RNA polymerase ß-subunit of the rifampicin-resistant mutants. Samples included bacteria exposed to the space environment with and without exposure to UV radiation as well as control samples held in the ISS cabin and at ground. The mutation sites of the rpoB gene obtained from the space-exposed and ISS/ground control samples were similar to the rpoB mutation sites previously reported in D. radiodurans. Most mutations were found at or near the rifampicin binding site in the RNA polymerase ß-subunit. Mutation sites found in UV-exposed samples were mostly shared with non-exposed and ISS/ground control samples. These results suggest that most mutations found in our experiments were induced during procedures that were applied across all treatments: preparation, transfer from our laboratory to the ISS, return from the ISS, and storage before analysis. Some mutations may be enhanced by specific factors in the space experiments, but the mutations were also found in the spontaneous and control samples. Our experiment suggests that the dried cells of the microorganism D. radiodurans can travel without space-specific deterioration that may induce excess mutations relative to travel at Earth's surface. However, upon arrival at a recipient location, they must still be able to survive and repair the general damage induced during travel.

Scientific Targets of Tanpopo: Astrobiology Exposure and Micrometeoroid Capture Experiments at the Japanese Experiment Module Exposed Facility of the International Space Station.

The Tanpopo experiment was the first Japanese astrobiology mission on board the Japanese Experiment Module Exposed Facility on the International Space Station (ISS). The experiments were designed to address two important astrobiological topics, panspermia and the chemical evolution process toward the generation of life. These experiments also tested low-density aerogel and monitored the microdebris environment around low Earth orbit. The following six subthemes were identified to address these goals: (1) Capture of microbes in space: Estimation of the upper limit of microbe density in low Earth orbit; (2) Exposure of microbes in space: Estimation of the survival time course of microbes in the space environment; (3) Capture of cosmic dust on the ISS and analysis of organics: Detection of the possible presence of organic compounds in cosmic dust; (4) Alteration of organic compounds in space environments: Evaluation of decomposition time courses of organic compounds in space; (5) Space verification of the Tanpopo hyper-low-density aerogel: Durability and particle-capturing capability of aerogel; (6) Monitoring of the number of space debris: Time-dependent change in space debris environment. Subthemes 1 and 2 address the panspermia hypothesis, whereas 3 and 4 address the chemical evolution. The last two subthemes contribute to space technology development. Some of the results have been published previously or are included in this issue. This article summarizes the current status of the Tanpopo experiments.

Evolution of Superoxide Dismutases and Catalases in Cyanobacteria: Occurrence of the Antioxidant Enzyme Genes before the Rise of Atmospheric Oxygen.

Knowledge on the evolution of antioxidant systems in cyanobacteria is crucial for elucidating the cause and consequence of the rise of atmospheric oxygen in the Earth's history. In this study, to elucidate the origin and evolution of cyanobacterial antioxidant enzymes, we analyzed the occurrence of genes encoding four types of superoxide dismutases and three types of catalases in 85 complete cyanobacterial genomes, followed by phylogenetic analyses. We found that Fe superoxide dismutase (FeSOD), Mn superoxide dismutase (MnSOD), and Mn catalase (MnCat) are widely distributed among modern cyanobacteria, whereas CuZn superoxide dismutase (CuZnSOD), bifunctional catalase (KatG), and monofunctional catalase (KatE) are less common. Ni superoxide dismutase (NiSOD) is distributed among marine Prochlorococcus and Synechococcus species. Phylogenetic analyses suggested that bacterial MnSOD evolved from cambialistic Fe/MnSOD before the diversification of major bacterial lineages. The analyses suggested that FeSOD evolved from MnSOD before the origin of cyanobacteria. MnCat also evolved in the early stages of bacterial evolution, predating the emergence of cyanobacteria. KatG, KatE, and NiSOD appeared 2.3-2.5 billion years ago. Thus, almost all cyanobacterial antioxidant enzymes emerged before or during the rise of atmospheric oxygen. The loss and appearance of these enzymes in marine cyanobacteria may be also related to the change in the metal concentration induced by the increased oxygen concentration in the ocean.

DNA Damage and Survival Time Course of Deinococcal Cell Pellets During 3 Years of Exposure to Outer Space.

The hypothesis called "panspermia" proposes an interplanetary transfer of life. Experiments have exposed extremophilic organisms to outer space to test microbe survivability and the panspermia hypothesis. Microbes inside shielding material with sufficient thickness to protect them from UV-irradiation can survive in space. This process has been called "lithopanspermia," meaning rocky panspermia. We previously proposed sub-millimeter cell pellets (aggregates) could survive in the harsh space environment based on an on-ground laboratory experiment. To test our hypothesis, we placed dried cell pellets of the radioresistant bacteria Deinococcus spp. in aluminum plate wells in exposure panels attached to the outside of the International Space Station (ISS). We exposed microbial cell pellets with different thickness to space environments. The results indicated the importance of the aggregated form of cells for surviving in harsh space environment. We also analyzed the samples exposed to space from 1 to 3 years. The experimental design enabled us to get and extrapolate the survival time course to predict the survival time of Deinococcus radiodurans. Dried deinococcal cell pellets of 500 µm thickness were alive after 3 years of space exposure and repaired DNA damage at cultivation. Thus, cell pellets 1 mm in diameter have sufficient protection from UV and are estimated to endure the space environment for 2-8 years, extrapolating the survival curve and considering the illumination efficiency of the space experiment. Comparison of the survival of different DNA repair-deficient mutants suggested that cell aggregates exposed in space for 3 years suffered DNA damage, which is most efficiently repaired by the uvrA gene and uvdE gene products, which are responsible for nucleotide excision repair and UV-damage excision repair. Collectively, these results support the possibility of microbial cell aggregates (pellets) as an ark for interplanetary transfer of microbes within several years.

Mitogenome analysis of dwarf pufferfish (Carinotetraodon travancoricus) endemic to southwest India and its implications in the phylogeny of Tetraodontidae.

The Tetraodontidae (pufferfishes), is primarily a family of marine and estuarine fishes with a limited number of freshwater species. Freshwater invasions can be observed in South America, Southeast Asia and central Africa. In the present study, we have analysed the complete mitogenome of freshwater pufferfish, Carinotetraodon travancoricus (dwarf pufferfish or Malabar pufferfish) endemic to southwest India. The genome is 16487 bp in length and consist of 13 protein-coding genes, 22 transfer RNA genes, two ribosomal RNA genes and one control region like all the other vertebrate mitogenomes. The protein-coding genes ranged from 165 bp (ATP synthase subunit 8) to 1812 bp (NADH dehydrogenase subunit 5) and comprised of 11310 bp in total, constituting 68.5% of the complete mitogenome. Some overlaps have been observed in protein-coding genes by a total of 7 bp. The AT skew (0.032166) and GC skew (-0.29746) of the mitogenome indicated that heavy strand consists equal amount of A and T, but the overall base composition was mainly C skewed. The noncoding D-loop region comprised 869 bp. The conserved motifs ATGTA and its complement TACAT associated with thermostable hairpin structure formation were identified in the control region. The phylogenetic analysis depicted a sister group relationship of C. travancoricus with euryhaline species Dichotomyctere nigroviridis and D. ocellatus with 100% bootstrap value rather than with the other freshwater members of Carinotetraodon species from Southeast Asia. The data from this study will be useful for proper identification, genetic differentiation, management and conservation of the dwarf Indian pufferfish.

Establishment of mesophilic-like catalytic properties in a thermophilic enzyme without affecting its thermal stability.

Thermophilic enzymes are generally more thermally stable but are less active at moderate temperatures than are their mesophilic counterparts. Thermophilic enzymes with improved low-temperature activity that retain their high stability would serve as useful tools for industrial processes especially when robust biocatalysts are required. Here we show an effective way to explore amino acid substitutions that enhance the low-temperature catalytic activity of a thermophilic enzyme, based on a pairwise sequence comparison of thermophilic/mesophilic enzymes. One or a combination of amino acid(s) in 3-isopropylmalate dehydrogenase from the extreme thermophile Thermus thermophilus was/were substituted by a residue(s) found in the Escherichia coli enzyme at the same position(s). The best mutant, which contained three amino acid substitutions, showed a 17-fold higher specific activity at 25 °C compared to the original wild-type enzyme while retaining high thermal stability. The kinetic and thermodynamic parameters of the mutant showed similar patterns along the reaction coordinate to those of the mesophilic enzyme. We also analyzed the residues at the substitution sites from a structural and phylogenetic point of view.

Draft Genome Sequence of the Radioresistant Bacterium Deinococcus aerius TR0125, Isolated from the High Atmosphere above Japan.

Deinococcus aerius strain TR0125 is a bacterium isolated from the high atmosphere above Japan that shows strong resistance to desiccation, UV-C, and gamma radiation. Here, we report the draft genome sequence of D. aerius (4.5 Mb), which may provide useful genetic information supporting its biochemical features.

Hydroxylation of a conserved tRNA modification establishes non-universal genetic code in echinoderm mitochondria.

The genetic code is not frozen but still evolving, which can result in the acquisition of 'dialectal' codons that deviate from the universal genetic code. RNA modifications in the anticodon region of tRNAs play a critical role in establishing such non-universal genetic codes. In echinoderm mitochondria, the AAA codon specifies asparagine instead of lysine. By analyzing mitochondrial (mt-) tRNALys isolated from the sea urchin (Mesocentrotus nudus), we discovered a novel modified nucleoside, hydroxy-N6-threonylcarbamoyladenosine (ht6A), 3' adjacent to the anticodon (position 37). Biochemical analysis revealed that ht6A37 has the ability to prevent mt-tRNALys from misreading AAA as lysine, thereby indicating that hydroxylation of N6-threonylcarbamoyladenosine (t6A) contributes to the establishment of the non-universal genetic code in echinoderm mitochondria.

Reconstructed ancestral enzymes suggest long-term cooling of Earth's photic zone since the Archean.

Paleotemperatures inferred from the isotopic compositions (δ18O and δ30Si) of marine cherts suggest that Earth's oceans cooled from 70 ± 15 °C in the Archean to the present ∼15 °C. This interpretation, however, has been subject to question due to uncertainties regarding oceanic isotopic compositions, diagenetic or metamorphic resetting of the isotopic record, and depositional environments. Analyses of the thermostability of reconstructed ancestral enzymes provide an independent method by which to assess the temperature history inferred from the isotopic evidence. Although previous studies have demonstrated extreme thermostability in reconstructed archaeal and bacterial proteins compatible with a hot early Earth, taxa investigated may have inhabited local thermal environments that differed significantly from average surface conditions. We here present thermostability measurements of reconstructed ancestral enzymatically active nucleoside diphosphate kinases (NDKs) derived from light-requiring prokaryotic and eukaryotic phototrophs having widely separated fossil-based divergence ages. The ancestral environmental temperatures thereby determined for these photic-zone organisms--shown in modern taxa to correlate strongly with NDK thermostability--are inferred to reflect ancient surface-environment paleotemperatures. Our results suggest that Earth's surface temperature decreased over geological time from ∼65-80 °C in the Archean, a finding consistent both with previous isotope-based and protein reconstruction-based interpretations. Interdisciplinary studies such as those reported here integrating genomic, geologic, and paleontologic data hold promise for providing new insight into the coevolution of life and environment over Earth history.

Diversification of mitochondrial genome of Daphnia galeata (Cladocera, Crustacea): Comparison with phylogenetic consideration of the complete sequences of clones isolated from five lakes in Japan.

To characterize genetic diversity and gene flow among Daphnia galeata populations, the complete nucleotide (nt) sequences of the mitochondrial (mt) DNAs of D. galeata clones isolated from five lakes in Japan (Lakes Shirakaba, Suwa, Kizaki, Kasumigaura, and Biwa) were determined. Comparison of non-synonymous (amino acid altering) substitution rates with synonymous substitution rates of D. galeata mt protein-coding genes demonstrated that ATPase8 and COI genes were the most and least susceptible, respectively, to the evolutional forces selecting the aa substitutions. Several non-synonymous substitutions were found in ATPase8 and ATPase6 even in the comparison that no synonymous substitution was found. Comparison of the total number of nt variations among the mt DNAs suggested the phylogenetic relationship ((((Shirakaba/Suwa, Kizaki), Kasumigaura), Biwa), D. pulex). Maximum-likelihood analysis using the total nt sequences of mt protein-coding genes confirmed this relationship with bootstrap values higher than 98%. All the mtDNAs of the analyzed Japanese D. galeata clones contained a control region of essentially the same structure that is distinct from those of the previously reported European Daphnia species of the D. longispina complex. The two control regions of different structures spread among mtDNAs of the Japanese and European Daphnia species, respectively, probably after the divergence of the Japanese D. galeata under different selection pressures associated with their habitats.

Updated mitochondrial phylogeny of Pteriomorph and Heterodont Bivalvia, including deep-sea chemosymbiotic Bathymodiolus mussels, vesicomyid clams and the thyasirid clam Conchocele cf. bisecta.

The mitochondrial genomes of bivalves have often been used for comparative genomics and for resolving phylogenetic relationships. More than 100 bivalve complete mitochondrial genomes have been sequenced to date. However, few mitochondrial genomes have been reported for deep-sea chemosymbiotic bivalves, which belong to the subclasses Pteriomorphia and Heterodonta. In the present study, we sequenced the mitochondrial genomes of eight deep-sea chemosymbiotic bivalve species: three species of Bathymodiolus mussels (B. japonicus, B. platifrons, and B. septemdierum), four species of vesicomyid clams (Abyssogena mariana, A. phaseoliformis, Isorropodon fossajaponicum, and Phreagena okutanii, all of which were formerly classified in the genus Calyptogena), and one species of thyasirid clam (Conchocele cf. bisecta). With a few exceptions, these mitochondrial genomes contained genes that are typical of metazoans: 13 protein-coding genes, two rRNA genes, and 22 tRNA genes. The major non-coding region with a high A+T content of each genome, which contained tandem repeats and hairpins, was hypothesized to function as a control region. The phylogenetic trees of Pteriomorphia and Heterodonta were reconstructed based on the concatenated sequences of 14 shared genes. Bathymodiolus formed a monophyletic clade with asymbiotic Mytilidae mussels, the vesicomyid clams formed a monophyly that was sister to the Veneridae, and C. cf. bisecta branched basally in the Heterodonta. It is known that the gene orders of mitochondrial genomes vary among bivalves. To examine whether gene order variation exhibits phylogenetic signals, tree topologies based on the minimum number of gene rearrangements were reconstructed for two clades (superfamily Tellinoidea, which includes the Psammobiidae, Semelidae, Solecurtidae, and Tellinidae; and the clade comprising the Myidae, Mactridae, Arcticidae, Vesicomyidae, and Veneridae) with high statistical support in sequence-based phylogenies. The resulting tree topologies were almost identical to those of the sequence-based trees. Our present findings suggest that the evolution of bivalves could be precisely traced back through the analysis of mitochondrial genomes, and that such an analysis could contribute to understanding bivalve evolution and diversity.

Quest for Ancestors of Eukaryal Cells Based on Phylogenetic Analyses of Aminoacyl-tRNA Synthetases.

The three-domain phylogenetic system of life has been challenged, particularly with regard to the position of Eukarya. The recent increase of known genome sequences has allowed phylogenetic analyses of all extant organisms using concatenated sequence alignment of universally conserved genes; these data supported the two-domain hypothesis, which place eukaryal species as ingroups of the Domain Archaea. However, the origin of Eukarya is complicated: the closest archaeal species to Eukarya differs in single-gene phylogenetic analyses depending on the genes. In this report, we performed molecular phylogenetic analyses of 23 aminoacyl-tRNA synthetases (ARS). Cytoplasmic ARSs in 12 trees showed a monophyletic Eukaryotic branch. One ARS originated from TACK superphylum. One ARS originated from Euryarchaeota and three originated from DPANN superphylum. Four ARSs originated from different bacterial species. The other 8 cytoplasmic ARSs were split into two or three groups in respective trees, which suggested that the cytoplasmic ARSs were replaced by secondary ARSs, and the original ARSs have been lost during evolution of Eukarya. In these trees, one original cytoplasmic ARS was derived from Euryarchaeota and three were derived from DPANN superphylum. Our results strongly support the two-domain hypothesis. We discovered that rampant-independent lateral gene transfers from several archaeal species of DPANN superphylum have contributed to the formation of Eukaryal cells. Based on our phylogenetic analyses, we proposed a model for the establishment of Eukarya.

Birth of Archaeal Cells: Molecular Phylogenetic Analyses of G1P Dehydrogenase, G3P Dehydrogenases, and Glycerol Kinase Suggest Derived Features of Archaeal Membranes Having G1P Polar Lipids.

Bacteria and Eukarya have cell membranes with sn-glycerol-3-phosphate (G3P), whereas archaeal membranes contain sn-glycerol-1-phosphate (G1P). Determining the time at which cells with either G3P-lipid membranes or G1P-lipid membranes appeared is important for understanding the early evolution of terrestrial life. To clarify this issue, we reconstructed molecular phylogenetic trees of G1PDH (G1P dehydrogenase; EgsA/AraM) which is responsible for G1P synthesis and G3PDHs (G3P dehydrogenase; GpsA and GlpA/GlpD) and glycerol kinase (GlpK) which is responsible for G3P synthesis. Together with the distribution of these protein-encoding genes among archaeal and bacterial groups, our phylogenetic analyses suggested that GlpA/GlpD in the Commonote (the last universal common ancestor of all extant life with a cellular form, Commonote commonote) acquired EgsA (G1PDH) from the archaeal common ancestor (Commonote archaea) and acquired GpsA and GlpK from a bacterial common ancestor (Commonote bacteria). In our scenario based on this study, the Commonote probably possessed a G3P-lipid membrane synthesized enzymatically, after which the archaeal lineage acquired G1PDH followed by the replacement of a G3P-lipid membrane with a G1P-lipid membrane.

Extremotolerant tardigrade genome and improved radiotolerance of human cultured cells by tardigrade-unique protein.

Tardigrades, also known as water bears, are small aquatic animals. Some tardigrade species tolerate almost complete dehydration and exhibit extraordinary tolerance to various physical extremes in the dehydrated state. Here we determine a high-quality genome sequence of Ramazzottius varieornatus, one of the most stress-tolerant tardigrade species. Precise gene repertoire analyses reveal the presence of a small proportion (1.2% or less) of putative foreign genes, loss of gene pathways that promote stress damage, expansion of gene families related to ameliorating damage, and evolution and high expression of novel tardigrade-unique proteins. Minor changes in the gene expression profiles during dehydration and rehydration suggest constitutive expression of tolerance-related genes. Using human cultured cells, we demonstrate that a tardigrade-unique DNA-associating protein suppresses X-ray-induced DNA damage by ∼40% and improves radiotolerance. These findings indicate the relevance of tardigrade-unique proteins to tolerability and tardigrades could be a bountiful source of new protection genes and mechanisms.

Investigation of the Interplanetary Transfer of Microbes in the Tanpopo Mission at the Exposed Facility of the International Space Station.

UNLABELLED: The Tanpopo mission will address fundamental questions on the origin of terrestrial life. The main goal is to test the panspermia hypothesis. Panspermia is a long-standing hypothesis suggesting the interplanetary transport of microbes. Another goal is to test the possible origin of organic compounds carried from space by micrometeorites before the terrestrial origin of life. To investigate the panspermia hypothesis and the possible space origin of organic compounds, we performed space experiments at the Exposed Facility (EF) of the Japanese Experiment Module (JEM) of the International Space Station (ISS). The mission was named Tanpopo, which in Japanese means dandelion. We capture any orbiting microparticles, such as micrometeorites, space debris, and terrestrial particles carrying microbes as bioaerosols, by using blocks of silica aerogel. We also test the survival of microbial species and organic compounds in the space environment for up to 3 years. The goal of this review is to introduce an overview of the Tanpopo mission with particular emphasis on the investigation of the interplanetary transfer of microbes. The Exposed Experiment Handrail Attachment Mechanism with aluminum Capture Panels (CPs) and Exposure Panels (EPs) was exposed on the EF-JEM on May 26, 2015. The first CPs and EPs will be returned to the ground in mid-2016. Possible escape of terrestrial microbes from Earth to space will be evaluated by investigating the upper limit of terrestrial microbes by the capture experiment. Possible mechanisms for transfer of microbes over the stratosphere and an investigation of the effect of the microbial cell-aggregate size on survivability in space will also be discussed. KEY WORDS: Panspermia-Astrobiology-Low-Earth orbit. Astrobiology 16, 363-376.

Epistasis effects of multiple ancestral-consensus amino acid substitutions on the thermal stability of glycerol kinase from Cellulomonas sp. NT3060.

Thermostable variants of the Cellulomonas sp. NT3060 glycerol kinase have been constructed by through the introduction of ancestral-consensus mutations. We produced seven mutants, each having an ancestral-consensus amino acid residue that might be present in the common ancestors of both bacteria and of archaea, and that appeared most frequently at the position of 17 glycerol kinase sequences in the multiple sequence alignment. The thermal stabilities of the resulting mutants were assessed by determining their melting temperatures (Tm), which was defined as the temperature at which 50% of the initial catalytic activity is lost after 15 min of incubation, as well as when the half-life of the catalytic activity occurs at a temperature of 60°C (t1/2). Three mutants showed increased stabilities compared to the wild-type protein. We then produced five more mutants with multiple amino acid substitutions. Some of the resulting mutants showed thermal stabilities much greater than those expected given the stabilities of the respective mutants with single mutations. Therefore, the effects of mutations are not always simply additive and some amino acid substitutions, which do not affect or only slightly improve stability when individually introduced into the protein, show substantial stabilizing effects in combination with other mutations.

Robustness of predictions of extremely thermally stable proteins in ancient organisms.

A number of studies have addressed the environmental temperatures experienced by ancient life. Computational studies using a nonhomogeneous evolution model have estimated ancestral G + C contents of ribosomal RNAs and the amino acid compositions of ancestral proteins, generating hypotheses regarding the mesophilic last universal common ancestor. In contrast, our previous study computationally reconstructed ancestral amino acid sequences of nucleoside diphosphate kinases using a homogeneous model and then empirically resurrected the ancestral proteins. The thermal stabilities of these ancestral proteins were equivalent to or greater than those of extant homologous thermophilic proteins, supporting the thermophilic universal ancestor theory. In this study, we reinferred ancestral sequences using a dataset from which hyperthermophilic sequences were excluded. We also reinferred ancestral sequences using a nonhomogeneous evolution model. The newly reconstructed ancestral proteins are still thermally stable, further supporting the hypothesis that the ancient organisms contained thermally stable proteins and therefore that they were thermophilic.