Comprehensive analysis of insertion sequences within rRNA genes of CPR bacteria and biochemical characterization of a homing endonuclease encoded by these sequences.

The Candidate Phyla Radiation (CPR) represents an extensive bacterial clade comprising primarily uncultured lineages and is distinguished from other bacteria by a significant prevalence of insertion sequences (ISs) within their rRNA genes. However, our understanding of the taxonomic distribution and characteristics of these ISs remains limited. In this study, we used a comprehensive approach to systematically determine the nature of the rRNA ISs in CPR bacteria. The analysis of hundreds of rRNA gene sequences across 65 CPR phyla revealed that ISs are present in 48% of 16S rRNA genes and 82% of 23S rRNA genes, indicating a broad distribution across the CPR clade, with exceptions in the 16S and 23S rRNA genes of Candidatus (Ca.) Saccharibacteria and the 16S rRNA genes of Ca. Peregrinibacteria. Over half the ISs display a group-I-intron-like structure, whereas specific 16S rRNA gene ISs display features reminiscent of group II introns. The ISs frequently encode proteins with homing endonuclease (HE) domains, centered around the LAGLIDADG motif. The LAGLIDADG HE (LHE) proteins encoded by the rRNA ISs of CPR bacteria predominantly have a single-domain structure, deviating from the usual single- or double-domain configuration observed in typical prokaryotic LHEs. Experimental analysis of one LHE protein, I-ShaI from Ca. Shapirobacteria, confirmed that its endonuclease activity targets the DNA sequence of its insertion site, and chemical cross-linking experiments demonstrated its capacity to form homodimers. These results provide robust evidence supporting the hypothesis that the explosive proliferation of rRNA ISs in CPR bacteria was facilitated by mechanisms involving LHEs. IMPORTANCE: Insertion sequences (ISs) in rRNA genes are relatively limited and infrequent in most bacterial phyla. With a comprehensive bioinformatic analysis, we show that in CPR bacteria, these ISs occur in 48% of 16S rRNA genes and 82% of 23S rRNA genes. We also report the systematic and biochemical characterization of the LAGLIDADG homing endonucleases (LHEs) encoded by these ISs in the first such analysis of the CPR bacteria. This study significantly extends our understanding of the phylogenetic positions of rRNA ISs within CPR bacteria and the biochemical features of their LHEs.

Features of smaller ribosomes in candidate phyla radiation (CPR) bacteria revealed with a molecular evolutionary analysis.

The candidate phyla radiation (CPR) is a large bacterial group consisting mainly of uncultured lineages. They have small cells and small genomes, and they often lack ribosomal proteins uL1, bL9, and/or uL30, which are basically ubiquitous in non-CPR bacteria. Here, we comprehensively analyzed the genomic information on CPR bacteria and identified their unique properties. The distribution of protein lengths in CPR bacteria peaks at around 100-150 amino acids, whereas the position of the peak varies in the range of 100-300 amino acids in free-living non-CPR bacteria, and at around 100-200 amino acids in most symbiotic non-CPR bacteria. These results show that the proteins of CPR bacteria are smaller, on average, than those of free-living non-CPR bacteria, like those of symbiotic non-CPR bacteria. We found that ribosomal proteins bL28, uL29, bL32, and bL33 have been lost in CPR bacteria in a taxonomic lineage-specific manner. Moreover, the sequences of approximately half of all ribosomal proteins of CPR differ, in part, from those of non-CPR bacteria, with missing regions or specifically added regions. We also found that several regions in the 16S, 23S, and 5S rRNAs of CPR bacteria are lacking, which presumably caused the total predicted lengths of the three rRNAs of CPR bacteria to be smaller than those of non-CPR bacteria. The regions missing in the CPR ribosomal proteins and rRNAs are located near the surface of the ribosome, and some are close to one another. These observations suggest that ribosomes are smaller in CPR bacteria than those in free-living non-CPR bacteria, with simplified surface structures.

Microbiome analysis of the restricted bacteria in radioactive element-containing water at the Fukushima Daiichi Nuclear Power Station.

A major incident occurred at the Fukushima Daiichi Nuclear Power Station following the tsunami triggered by the Tohoku-Pacific Ocean Earthquake in March 2011, whereby seawater entered the torus room in the basement of the reactor building. Here, we identify and analyze the bacterial communities in the torus room water and several environmental samples. Samples of the torus room water (1 × 109 Bq137Cs/L) were collected by the Tokyo Electric Power Company Holdings from two sampling points between 30 cm and 1 m from the bottom of the room (TW1) and the bottom layer (TW2). A structural analysis of the bacterial communities based on 16S rRNA amplicon sequencing revealed that the predominant bacterial genera in TW1 and TW2 were similar. TW1 primarily contained the genus Limnobacter, a thiosulfate-oxidizing bacterium. γ-Irradiation tests on Limnobacter thiooxidans, the most closely related phylogenetically found in TW1, indicated that its radiation resistance was similar to ordinary bacteria. TW2 predominantly contained the genus Brevirhabdus, a manganese-oxidizing bacterium. Although bacterial diversity in the torus room water was lower than seawater near Fukushima, ~70% of identified genera were associated with metal corrosion. Latent environment allocation-an analytical technique that estimates habitat distributions and co-detection analyses-revealed that the microbial communities in the torus room water originated from a distinct blend of natural marine microbial and artificial bacterial communities typical of biofilms, sludge, and wastewater. Understanding the specific bacteria linked to metal corrosion in damaged plants is important for advancing decommissioning efforts. IMPORTANCE: In the context of nuclear power station decommissioning, the proliferation of microorganisms within the reactor and piping systems constitutes a formidable challenge. Therefore, the identification of microbial communities in such environments is of paramount importance. In the aftermath of the Fukushima Daiichi Nuclear Power Station accident, microbial community analysis was conducted on environmental samples collected mainly outside the site. However, analyses using samples from on-site areas, including adjacent soil and seawater, were not performed. This study represents the first comprehensive analysis of microbial communities, utilizing meta 16S amplicon sequencing, with a focus on environmental samples collected from the radioactive element-containing water in the torus room, including the surrounding environments. Some of the identified microbial genera are shared with those previously identified in spent nuclear fuel pools in countries such as France and Brazil. Moreover, our discussion in this paper elucidates the correlation of many of these bacteria with metal corrosion.

Systematic Analysis of Diverse Polynucleotide Kinase Clp1 Family Proteins in Eukaryotes: Three Unique Clp1 Proteins of Trypanosoma brucei.

The Clp1 family proteins, consisting of the Clp1 and Nol9/Grc3 groups, have polynucleotide kinase (PNK) activity at the 5' end of RNA strands and are important enzymes in the processing of some precursor RNAs. However, it remains unclear how this enzyme family diversified in the eukaryotes. We performed a large-scale molecular evolutionary analysis of the full-length genomes of 358 eukaryotic species to classify the diverse Clp1 family proteins. The average number of Clp1 family proteins in eukaryotes was 2.3 ± 1.0, and most representative species had both Clp1 and Nol9/Grc3 proteins, suggesting that the Clp1 and Nol9/Grc3 groups were already formed in the eukaryotic ancestor by gene duplication. We also detected an average of 4.1 ± 0.4 Clp1 family proteins in members of the protist phylum Euglenozoa. For example, in Trypanosoma brucei, there are three genes of the Clp1 group and one gene of the Nol9/Grc3 group. In the Clp1 group proteins encoded by these three genes, the C-terminal domains have been replaced by unique characteristics domains, so we designated these proteins Tb-Clp1-t1, Tb-Clp1-t2, and Tb-Clp1-t3. Experimental validation showed that only Tb-Clp1-t2 has PNK activity against RNA strands. As in this example, N-terminal and C-terminal domain replacement also contributed to the diversification of the Clp1 family proteins in other eukaryotic species. Our analysis also revealed that the Clp1 family proteins in humans and plants diversified through isoforms created by alternative splicing.

Behavioral and brain- transcriptomic synchronization between the two opponents of a fighting pair of the fish Betta splendens.

Conspecific male animals fight for resources such as food and mating opportunities but typically stop fighting after assessing their relative fighting abilities to avoid serious injuries. Physiologically, how the fighting behavior is controlled remains unknown. Using the fighting fish Betta splendens, we studied behavioral and brain-transcriptomic changes during the fight between the two opponents. At the behavioral level, surface-breathing, and biting/striking occurred only during intervals between mouth-locking. Eventually, the behaviors of the two opponents became synchronized, with each pair showing a unique behavioral pattern. At the physiological level, we examined the expression patterns of 23,306 brain transcripts using RNA-sequencing data from brains of fighting pairs after a 20-min (D20) and a 60-min (D60) fight. The two opponents in each D60 fighting pair showed a strong gene expression correlation, whereas those in D20 fighting pairs showed a weak correlation. Moreover, each fighting pair in the D60 group showed pair-specific gene expression patterns in a grade of membership analysis (GoM) and were grouped as a pair in the heatmap clustering. The observed pair-specific individualization in brain-transcriptomic synchronization (PIBS) suggested that this synchronization provides a physiological basis for the behavioral synchronization. An analysis using the synchronized genes in fighting pairs of the D60 group found genes enriched for ion transport, synaptic function, and learning and memory. Brain-transcriptomic synchronization could be a general phenomenon and may provide a new cornerstone with which to investigate coordinating and sustaining social interactions between two interacting partners of vertebrates.

Proteomic and metabolomic analyses uncover sex-specific regulatory pathways in mouse fetal germline differentiation†.

Regulatory mechanisms of germline differentiation have generally been explained via the function of signaling pathways, transcription factors, and epigenetic regulation; however, little is known regarding proteomic and metabolomic regulation and their contribution to germ cell development. Here, we conducted integrated proteomic and metabolomic analyses of fetal germ cells in mice on embryonic day (E)13.5 and E18.5 and demonstrate sex- and developmental stage-dependent changes in these processes. In male germ cells, RNA processing, translation, oxidative phosphorylation, and nucleotide synthesis are dominant in E13.5 and then decline until E18.5, which corresponds to the prolonged cell division and more enhanced hyper-transcription/translation in male primordial germ cells and their subsequent repression. Tricarboxylic acid cycle and one-carbon pathway are consistently upregulated in fetal male germ cells, suggesting their involvement in epigenetic changes preceding in males. Increased protein stability and oxidative phosphorylation during female germ cell differentiation suggests an upregulation of aerobic energy metabolism, which likely contributes to the proteostasis required for oocyte maturation in subsequent stages. The features elucidated in this study shed light on the unrevealed mechanisms of germ cell development.

Distinct requirements for energy metabolism in mouse primordial germ cells and their reprogramming to embryonic germ cells.

Primordial germ cells (PGCs), undifferentiated embryonic germ cells, are the only cells that have the ability to become gametes and to reacquire totipotency upon fertilization. It is generally understood that the development of PGCs proceeds through the expression of germ cell-specific transcription factors and characteristic epigenomic changes. However, little is known about the properties of PGCs at the metabolite and protein levels, which are directly responsible for the control of cell function. Here, we report the distinct energy metabolism of PGCs compared with that of embryonic stem cells. Specifically, we observed remarkably enhanced oxidative phosphorylation (OXPHOS) and decreased glycolysis in embryonic day 13.5 (E13.5) PGCs, a pattern that was gradually established during PGC differentiation. We also demonstrate that glycolysis and OXPHOS are important for the control of PGC reprogramming and specification of pluripotent stem cells (PSCs) into PGCs in culture. Our findings about the unique metabolic property of PGCs provide insights into our understanding of the importance of distinct facets of energy metabolism for switching PGC and PSC status.

Expansion of Noncanonical V-Arm-Containing tRNAs in Eukaryotes.

Transfer RNA (tRNA) is essential for the translation of genetic information into proteins, and understanding its molecular evolution is important if we are to understand the genetic code. In general, long variable-arm (V-arm) structures form in tRNA(Leu), tRNA(Ser), and bacterial and organellar tRNA(Tyr). However, as we have previously reported, noncanonical V-arms occur in nematode tRNA(Gly) and tRNA(Ile), and potentially affect translational fidelity. Here, we comprehensively analyzed 69 eukaryotic genome sequences and examined the evolutionary divergence of the V-arm-containing tRNAs. In total, 253 V-arm-containing tRNAs, with neither leucine nor serine anticodons, were identified in organisms ranging from nematodes to fungi, plants, and vertebrates. We defined them as "noncanonical V-arm-containing tRNAs" (nov-tRNAs). Moreover, 2,415 nov-tRNA-like sequences lacking some of the conserved features of tRNAs were also identified, largely in vertebrate genomes. These nov-tRNA/nov-tRNA-like sequences can be categorized into three types, based on differences in their possible evolutionary origins. The type A nov-tRNAs in nematodes probably evolved not only from tRNA(Leu) but also from tRNA(Ser) and other isotypes on several independent occasions. The type B nov-tRNAs are dispersed abundantly throughout vertebrate genomes, and seem to have originated from retrotransposable elements. The type C nov-tRNAs may have been acquired from plant chloroplasts or from bacteria through horizontal transfer. Our findings provide unexpected insight into the evolution of the tRNA molecule, which was diverse and occurred independently in nematodes, vertebrates, and plants.

Identification, expression, and molecular evolution of microRNAs in the "living fossil" Triops cancriformis (tadpole shrimp).

MicroRNAs have been identified and analyzed in various model species, but an investigation of miRNAs in nonmodel species is required for a more complete understanding of miRNA evolution. In this study, we investigated the miRNAs of the nonmodel species Triops cancriformis (tadpole shrimp), a "living fossil," whose morphological form has not changed in almost 200 million years. Dramatic ontogenetic changes occur during its development. To clarify the evolution of miRNAs, we comparatively analyzed its miRNAs and the components of its RNAi machinery. We used deep sequencing to analyze small RNA libraries from the six different developmental stages of T. cancriformis (egg, first-fourth instars, and adult), and also analyzed its genomic DNA with deep sequencing. We identified 180 miRNAs (87 conserved miRNAs and 93 novel candidate miRNAs), and deduced the components of its RNAi machinery: the DICER1, AGO1-3, PIWI, and AUB proteins. A comparative miRNA analysis of T. cancriformis and Drosophila melanogaster showed inconsistencies in the expression patterns of four conserved miRNAs. This suggests that although the miRNA sequences of the two species are very similar, their roles differ across the species. An miRNA conservation analysis revealed that most of the conserved T. cancriformis miRNAs share sequence similarities with those of arthropods, although T. cancriformis is called a "living fossil." However, we found that let-7 and DICER1 of T. cancriformis are more similar to those of the vertebrates than to those of the arthropods. These results suggest that miRNA systems of T. cancriformis have evolved in a unique fashion.

Systematic characterization of artificial small RNA-mediated inhibition of Escherichia coli growth.

A new screening system for artificial small RNAs (sRNAs) that inhibit the growth of Escherichia coli was constructed. In this system, we used a plasmid library to express RNAs of ∼120 nucleotides, each with a random 30-nucleotide sequence that can recognize its target mRNA(s). After approximately 60,000 independent colonies were screened, several plasmids that inhibited bacterial growth were isolated. To understand the inhibitory mechanism, we focused on one sRNA, S-20, that exerted a strong inhibitory effect. A time-course analysis of the proteome of S-20-expressing E. coli and a bioinformatic analysis were used to identify potential S-20 target mRNAs, and suggested that S-20 binds the translation initiation sites of several mRNAs encoding enzymes such as peroxiredoxin (osmC), glycyl-tRNA synthetase α subunit (glyQ), uncharacterized protein ygiM, and tryptophan synthase ß chain (trpB). An in vitro translation analysis of chimeric luciferase-encoding mRNAs, each containing a potential S-20 target sequence, indicated that the translation of these mRNAs was inhibited in the presence of S-20. A gel shift analysis combined with the analysis of a series of S-20 mutants suggested that S-20 targets multiple mRNAs that are responsible for inhibiting E. coli growth. These data also suggest that S-20 acts like an endogenous sRNA and that E. coli can utilize artificial sRNAs.

Precise mapping and dynamics of tRNA-derived fragments (tRFs) in the development of Triops cancriformis (tadpole shrimp).

BACKGROUND: In a deep sequencing analysis of small RNAs prepared from a living fossil, the tadpole shrimp Triops cancriformis, a 32-nt small RNA was specifically detected in the adult stage. A nucleotide sequence comparison between the 32-nt small RNA and predicted tRNA sequences in the draft nuclear genomic DNA showed that the small RNA was derived from tRNA(Gly)(GCC). To determine the overall features of the tRNA-derived fragments (tRFs) of T. cancriformis, the small RNA sequences in each of the six developmental stages (egg, 1st-4th instar larvae, and adult) were compared with the mitochondrial and nuclear tRNA sequences. RESULTS: We found that the tRFs were derived from mitochondrial and nuclear tRNAs corresponding to 16 and 39 anticodons, respectively. The total read number of nuclear tRFs was approximately 400 times larger than the number of mitochondrial tRFs. Interestingly, the main regions in each parental tRNA from which these tRFs were derived differed, depending on the parental anticodon. Mitochondrial tRF(Ser)(GCU)s were abundantly produced from the 5' half regions of the parental tRNA, whereas mitochondrial tRF(Val)(UAC)s were mainly produced from the 3' end regions. Highly abundant nuclear tRFs, tRF(Gly)(GCC)s, tRF(Gly)(CCC)s, tRF(Glu)(CUC)s, and tRF(Lys)(CUU)s were derived from the 5' half regions of the parental tRNAs. Further analysis of the tRF read counts in the individual developmental stages suggested that the expression of mitochondrial and nuclear tRFs differed during the six stages. Based on these data, we precisely summarized the positions of the tRFs in their parental tRNAs and their expression changes during development. CONCLUSIONS: Our results reveal the entire dynamics of the tRFs from both the nuclear and mitochondrial genomes of T. cancriformis and indicate that the majority of tRFs in the cell are derived from nuclear tRNAs. This study provides the first examples of developmentally expressed mitochondrial tRFs.

Effect of codon adaptation on codon-level and gene-level translation efficiency in vivo.

BACKGROUND: There is a significant difference between synonymous codon usage in many organisms, and it is known that codons used more frequently generally showed efficient decoding rate. At the gene level, however, there are conflicting reports on the existence of a correlation between codon adaptation and translation efficiency, even in the same organism. RESULTS: To resolve this issue, we cultured Escherichia coli under conditions designed to maintain constant levels of mRNA and protein and subjected the cells to ribosome profiling (RP) and mRNA-seq analyses. We showed that the RP results correlated more closely with protein levels generated under similar culture conditions than with the mRNA abundance from the mRNA-seq. Our result indicated that RP/mRNA ratio could be used as a measure of translation efficiency at gene level. On the other hand, the RP data showed that codon-specific ribosome density at the decoding site negatively correlated with codon usage, consistent with the hypothesis that preferred codons display lower ribosome densities due to their faster decoding rate. However, highly codon-adapted genes showed higher ribosome densities at the gene level, indicating that the efficiency of translation initiation, rather than higher elongation efficiency of preferred codons, exerted a greater effect on ribosome density and thus translation efficiency. CONCLUSIONS: These findings indicate that evolutionary pressure on highly expressed genes influenced both codon bias and translation initiation efficiency and therefore explains contradictory findings that codon usage bias correlates with translation efficiency of native genes, but not with the artificially created gene pool, which was not subjected to evolution pressure.

Nematode-specific tRNAs that decode an alternative genetic code for leucine.

Class II transfer RNAs (tRNAs), including tRNA(Leu) and tRNA(Ser), have an additional stem and loop structure, the long variable arm (V-arm). Here, we describe Class II tRNAs with a unique anticodon corresponding to neither leucine nor serine. Because these tRNAs are specifically conserved among the nematodes, we have called them 'nematode-specific V-arm-containing tRNAs' (nev-tRNAs). The expression of nev-tRNA genes in Caenorhabditis elegans was confirmed experimentally. A comparative sequence analysis suggested that the nev-tRNAs derived phylogenetically from tRNA(Leu). In vitro aminoacylation assays showed that nev-tRNA(Gly) and nev-tRNA(Ile) are only charged with leucine, which is inconsistent with their anticodons. Furthermore, the deletion and mutation of crucial determinants for leucylation in nev-tRNA led to a marked loss of activity. An in vitro translation analysis showed that nev-tRNA(Gly) decodes GGG as leucine instead of the universal glycine code, indicating that nev-tRNAs can be incorporated into ribosomes and participate in protein biosynthesis. Our findings provide the first example of unexpected tRNAs that do not consistently obey the general translation rules for higher eukaryotes.

X-ray structure of the fourth type of archaeal tRNA splicing endonuclease: insights into the evolution of a novel three-unit composition and a unique loop involved in broad substrate specificity.

Cleavage of introns from precursor transfer RNAs (tRNAs) by tRNA splicing endonuclease (EndA) is essential for tRNA maturation in Archaea and Eukarya. In the past, archaeal EndAs were classified into three types (α'2, α4 and α2ß2) according to subunit composition. Recently, we have identified a fourth type of archaeal EndA from an uncultivated archaeon Candidatus Micrarchaeum acidiphilum, referred to as ARMAN-2, which is deeply branched within Euryarchaea. The ARMAN-2 EndA forms an ε2 homodimer and has broad substrate specificity like the α2ß2 type EndAs found in Crenarchaea and Nanoarchaea. However, the precise architecture of ARMAN-2 EndA was unknown. Here, we report the crystal structure of the ε2 homodimer of ARMAN-2 EndA. The structure reveals that the ε protomer is separated into three novel units (αN, α and ßC) fused by two distinct linkers, although the overall structure of ARMAN-2 EndA is similar to those of the other three types of archaeal EndAs. Structural comparison and mutational analyses reveal that an ARMAN-2 type-specific loop (ASL) is involved in the broad substrate specificity and that K161 in the ASL functions as the RNA recognition site. These findings suggest that the broad substrate specificities of ε2 and α2ß2 EndAs were separately acquired through different evolutionary processes.

Homo-trimeric structure of the ribonuclease for rRNA processing, FAU-1, from Pyrococcus furiosus.

Crystal structure of a ribonuclease for ribosomal RNA processing, FAU-1, from Pyrococcus furiosus was determined with the resolution of 2.57 Å in a homo-trimeric form. The monomer structure consists of two domains: N-terminal and C-terminal domains. C-terminal domain forms trimer and each N-terminal domain locates outside of the trimer core. In the obtained crystal, a dinucleotide, pApUp, was bound to the N-terminal domain, indicating that N-terminal domain has the RNA-binding ability. The affinities to RNA of FAU-1 and a fragment corresponding to the N-terminal domain, FAU-ΔC, were confirmed by polyacrylamide gel electrophoresis and nuclear magnetic resonance (NMR). Interestingly, well-dispersed NMR signals were observed at 318K, indicating that the FAU-ΔC-F18 complex form an ordered structure at higher temperature. As predicted in our previous works, FAU-1 and ribonuclease (RNase) E show a structural similarity in their RNA-binding regions. However, structural similarity between RNase E and FAU-1 could be found in the limited regions of the N-terminal domain. On the other hand, structural similarity between C-terminal domain and some proteins including a phosphatase was found. Thus, it is possible that the catalytic site is located in C-terminal domain.

A novel three-unit tRNA splicing endonuclease found in ultrasmall Archaea possesses broad substrate specificity.

tRNA splicing endonucleases, essential enzymes found in Archaea and Eukaryotes, are involved in the processing of pre-tRNA molecules. In Archaea, three types of splicing endonuclease [homotetrameric: α(4), homodimeric: α(2), and heterotetrameric: (αß)(2)] have been identified, each representing different substrate specificity during the tRNA intron cleavage. Here, we discovered a fourth type of archaeal tRNA splicing endonuclease (ε(2)) in the genome of the acidophilic archaeon Candidatus Micrarchaeum acidiphilum, referred to as ARMAN-2 and its closely related species, ARMAN-1. The enzyme consists of two duplicated catalytic units and one structural unit encoded on a single gene, representing a novel three-unit architecture. Homodimeric formation was confirmed by cross-linking assay, and site-directed mutagenesis determined that the conserved L10-pocket interaction between catalytic and structural unit is necessary for the assembly. A tRNA splicing assay reveal that ε(2) endonuclease cleaves both canonical and non-canonical bulge-helix-bulge motifs, similar to that of (αß)(2) endonuclease. Unlike other ARMAN and Euryarchaeota, tRNAs found in ARMAN-2 are highly disrupted by introns at various positions, which again resemble the properties of archaeal species with (αß)(2) endonuclease. Thus, the discovery of ε(2) endonuclease in an archaeon deeply branched within Euryarchaeota represents a new example of the coevolution of tRNA and their processing enzymes.

tRNADB-CE 2011: tRNA gene database curated manually by experts.

We updated the tRNADB-CE by analyzing 939 complete and 1301 draft genomes of prokaryotes and eukaryotes, 171 complete virus genomes, 121 complete chloroplast genomes and approximately 230 million sequences obtained by metagenome analyses of 210 environmental samples. The 287 102 tRNA genes in total, and thus two times of the tRNA genes compiled previously, are compiled, in which sequence information, clover-leaf structure and results of sequence similarity and oligonucleotide-pattern search can be browsed. In order to pool collective knowledge with help from any experts in the tRNA research field, we included a column to which comments can be added on each tRNA gene. By compiling tRNAs of known prokaryotes with identical sequences, we found high phylogenetic preservation of tRNA sequences, especially at a phylum level. Furthermore, a large number of tRNAs obtained by metagenome analyses of environmental samples had sequences identical to those found in known prokaryotes. The identical sequence group, therefore, can be used as phylogenetic markers to clarify the microbial community structure of an ecosystem. The updated tRNADB-CE provided functions, with which users can obtain the phylotype-specific markers (e.g. genus-specific markers) by themselves and clarify microbial community structures of ecosystems in detail. tRNADB-CE can be accessed freely at http://trna.nagahama-i-bio.ac.jp.

Insights into the evolution of Archaea and eukaryotic protein modifier systems revealed by the genome of a novel archaeal group.

The domain Archaea has historically been divided into two phyla, the Crenarchaeota and Euryarchaeota. Although regarded as members of the Crenarchaeota based on small subunit rRNA phylogeny, environmental genomics and efforts for cultivation have recently revealed two novel phyla/divisions in the Archaea; the 'Thaumarchaeota' and 'Korarchaeota'. Here, we show the genome sequence of Candidatus 'Caldiarchaeum subterraneum' that represents an uncultivated crenarchaeotic group. A composite genome was reconstructed from a metagenomic library previously prepared from a microbial mat at a geothermal water stream of a sub-surface gold mine. The genome was found to be clearly distinct from those of the known phyla/divisions, Crenarchaeota (hyperthermophiles), Euryarchaeota, Thaumarchaeota and Korarchaeota. The unique traits suggest that this crenarchaeotic group can be considered as a novel archaeal phylum/division. Moreover, C. subterraneum harbors an ubiquitin-like protein modifier system consisting of Ub, E1, E2 and small Zn RING finger family protein with structural motifs specific to eukaryotic system proteins, a system clearly distinct from the prokaryote-type system recently identified in Haloferax and Mycobacterium. The presence of such a eukaryote-type system is unprecedented in prokaryotes, and indicates that a prototype of the eukaryotic protein modifier system is present in the Archaea.

GTP-dependent RNA 3'-terminal phosphate cyclase from the hyperthermophilic archaeon Pyrococcus furiosus.

We discovered that the PF1549 gene in Pyrococcus furiosus encodes a very heat-stable RNA 3'-terminal phosphate cyclase (Pf-Rtc). Although all previously reported Rtc proteins are ATP-dependent enzymes, we found that Pf-Rtc requires GTP for its cyclase activity at 95 °C. Low-level activation of the enzyme was also observed in the presence of dGTP but not other dNTPs, indicating that the guanine base is very important for Pf-Rtc activity. We analyzed a series of GTP analogues and found that the conversion from GTP to GMP is important for Pf-Rtc activity and that an excess of GMP inhibits this activity. Gel-shift analysis clearly showed that the RNA-binding activity of Pf-Rtc is totally dependent on the linear form of the 3'-terminal phosphate, with an apparent K(d) value of 20 nm at 95°C. Furthermore, we found that Pf-Rtc may contribute to GTP-dependent RNA ligation activity through the PF0027 protein (a 2'-5' RNA ligase-like protein in P. furiosus). The possible roles of Pf-Rtc and the importance of terminal phosphate structures in RNA are discussed.

Metatranscriptomic analysis of microbes in an Oceanfront deep-subsurface hot spring reveals novel small RNAs and type-specific tRNA degradation.

Studies of small noncoding RNAs (sRNAs) have been conducted predominantly using culturable organisms, and the acquisition of further information about sRNAs from global environments containing uncultured organisms now is very important. In this study, hot spring water (57°C, pH 8.1) was collected directly from the underground environment at depths of 250 to 1,000 m in Yunohama, Japan, and small RNA sequences obtained from the environment were analyzed. A phylogenetic analysis of both archaeal and bacterial 16S rRNA gene sequences was conducted, and the results suggested the presence of unique species in the environment, corresponding to the Archaeal Richmond Mine Acidophilic Nanoorganisms (ARMAN) group and three new Betaproteobacteria. A metatranscriptomic analysis identified 64,194 (20,057 nonredundant) cDNA sequences. Of these cDNAs, 90% were either tRNAs, tRNA fragments, rRNAs, or rRNA fragments, whereas 2,181 reads (10%) were classified as previously uncharacterized putative candidate sRNAs. Among these, 15 were particularly abundant, 14 of which showed no sequence similarity to any known noncoding RNA, and at least six of which form very stable RNA secondary structures. The analysis of a large number of tRNA fragments suggested that unique relationships exist between the anticodons of the tRNAs and the sites of tRNA degradation. Previous bacterial tRNA degradation studies have been limited to specific organisms, such as Escherichia coli and Streptomyces coelicolor, and the current results suggest that specific tRNA decay occurs more frequently than previously expected.