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Yasukazu Nakamura

Publications and source records attributed to Yasukazu Nakamura.

At least 19 recordsLinked to original sources

Microbiome Datahub: an open-access platform integrating environmental metadata, taxonomy, and functional annotation for comprehensive metagenome-assembled genome datasets.

BACKGROUND: Metagenome-assembled genomes (MAGs) provide crucial insights into the genomic diversity of uncultured microbes. However, MAG datasets deposited in public repositories such as INSDC are often difficult to reuse due to heterogeneous quality, inconsistent taxonomic and functional annotations, and insufficiently curated environmental metadata. While secondary MAG databases such as MGnify, IMG/M, and SPIRE provide standardized resources, they reconstruct MAGs de novo from public metagenomic reads and therefore do not represent the original MAGs reported in publications. RESULTS: To address this gap, we developed Microbiome Datahub, an open-access platform that systematically aggregates and re-annotates original MAGs from INSDC. We collected 214,427 MAGs, predicted genes by DFAST, performed quality assessment with CheckM, standardized taxonomic assignments with GTDB-Tk, inferred 27 phenotypic traits using Bac2Feature, assigned proteins to MBGD ortholog clusters and KEGG Orthology IDs using PZLAST, and annotated environmental metadata with the Metagenome and Microbes Environmental Ontology. Across these MAGs, the average completeness was 80.5% and contamination 1.8%; notably, the most frequent values were&#x2009;>95% completeness and&#x2009;<1% contamination, indicating that the majority of MAGs are of high quality. Comparative analyses showed that Microbiome Datahub provides phylogenetically and environmentally diverse MAGs: while the majority originated from vertebrate gut environments, a substantial number were also recovered from other habitats such as groundwater, including nearly 10,000 MAGs from the Patescibacteria. Inference of 27 phenotypic traits, including optimum growth temperature, further revealed ecological differentiation across phyla. Protein clustering revealed 56 million identity 40% clusters, with the majority unique compared with MGnify and GlobDB, and&#x2009;~19% of proteins unassigned to MBGD ortholog clusters, underscoring their novelty. CONCLUSIONS: Microbiome Datahub integrates MAG genome sequences, gene and protein predictions, quality metrics, environmental and taxonomic annotations, ortholog cluster assignments, and phenotype predictions, all accessible via a web interface, API, and bulk downloads. By combining original MAGs with curated metadata and functional annotations, Microbiome Datahub constitutes a comprehensive and reusable resource that will accelerate microbiome and microbial genomics research. Video Abstract.

Metagenome↗

Chromosome-scale assembly with improved annotation provides insights into breed-wide genomic structure and diversity in domestic cats.

INTRODUCTION: Comprehensive genomic resources offer insights into biological features, including traits/disease-related genetic loci. The current reference genome assembly for the domestic cat (Felis catus), Felis_Catus_9.0 (felCat9), derived from sequences of the Abyssinian cat, may inadequately represent the general cat population, limiting the extent of deducible genetic variations. OBJECTIVES: The goal was to develop Anicom American Shorthair 1.0 (AnAms1.0), a reference-grade chromosome-scale cat genome assembly. METHODS: In contrast to prior assemblies relying on Abyssinian cat sequences, AnAms1.0 was constructed from the sequences of more popular American Shorthair breed, which is related to more breeds than the Abyssinian cat. By combining advanced genomics technologies, including PacBio long-read sequencing and Hi-C- and optical mapping data-based sequence scaffolding, we compared AnAms1.0 to existing Felidae genome assemblies (20 scaffolds, scaffolds N50&#xa0;>&#xa0;150 Mbp). Homology-based and ab initio gene annotation through Iso-Seq and RNA-Seq was used to identify new coding genes and splice variants. RESULTS: AnAms1.0 demonstrated superior contiguity and accuracy than existing Felidae genome assemblies. Using AnAms1.0, we identified over 1.5 thousand structural variants and 29 million repetitions compared to felCat9. Additionally, we identified > 1,600 novel protein-coding genes. Notably, olfactory receptor structural variants and cardiomyopathy-related variants were identified. CONCLUSION: AnAms1.0 facilitates the discovery of novel genes related to normal and disease phenotypes in domestic cats. The analyzed data are publicly accessible on Cats-I (https://cat.annotation.jp/), which we established as a platform for accumulating and sharing genomic resources to discover novel genetic traits and advance veterinary medicine.

Animals↗

Large-scale genome analysis of bovine commensal Escherichia coli reveals that bovine-adapted E. coli lineages are serving as evolutionary sources of the emergence of human intestinal pathogenic strains.

How pathogens evolve their virulence to humans in nature is a scientific issue of great medical and biological importance. Shiga toxin (Stx)-producing Escherichia coli (STEC) and enteropathogenic E. coli (EPEC) are the major foodborne pathogens that can cause hemolytic uremic syndrome and infantile diarrhea, respectively. The locus of enterocyte effacement (LEE)-encoded type 3 secretion system (T3SS) is the major virulence determinant of EPEC and is also possessed by major STEC lineages. Cattle are thought to be the primary reservoir of STEC and EPEC. However, genome sequences of bovine commensal E. coli are limited, and the emerging process of STEC and EPEC is largely unknown. Here, we performed a large-scale genomic comparison of bovine commensal E. coli with human commensal and clinical strains, including EPEC and STEC, at a global level. The analyses identified two distinct lineages, in which bovine and human commensal strains are enriched, respectively, and revealed that STEC and EPEC strains have emerged in multiple sublineages of the bovine-associated lineage. In addition to the bovine-associated lineage-specific genes, including fimbriae, capsule, and nutrition utilization genes, specific virulence gene communities have been accumulated in stx- and LEE-positive strains, respectively, with notable overlaps of community members. Functional associations of these genes probably confer benefits to these E. coli strains in inhabiting and/or adapting to the bovine intestinal environment and drive their evolution to highly virulent human pathogens under the bovine-adapted genetic background. Our data highlight the importance of large-scale genome sequencing of animal strains in the studies of zoonotic pathogens.

Animals↗

Genome-wide analysis of ATP-binding cassette (ABC) proteins in a model legume plant, Lotus japonicus: comparison with Arabidopsis ABC protein family.

ATP-binding cassette (ABC) proteins constitute a large family in plants with more than 120 members each in Arabidopsis and rice, and have various functions including the transport of auxin and alkaloid, as well as the regulation of stomata movement. In this report, we carried out genome-wide analysis of ABC protein genes in a model legume plant, Lotus japonicus. For analysis of the Lotus genome sequence, we devised a new method 'domain-based clustering analysis', where domain structures like the nucleotide-binding domain (NBD) and transmembrane domain (TMD), instead of full-length amino acid sequences, are used to compare phylogenetically each other. This method enabled us to characterize fragments of ABC proteins, which frequently appear in a draft sequence of the Lotus genome. We identified 91 putative ABC proteins in L. japonicus, i.e. 43 'full-size', 40 'half-size' and 18 'soluble' putative ABC proteins. The characteristic feature of the composition is that Lotus has extraordinarily many paralogs similar to AtMRP14 and AtPDR12, which are at least six and five members, respectively. Expression analysis of the latter genes performed with real-time quantitative reverse transcription-PCR revealed their putative involvement in the nodulation process.

ATP-Binding Cassette Transporters↗

Comprehensive structural analysis of the genome of red clover (Trifolium pratense L.).

With the aim of establishing the basic knowledge and resources needed for applied genetics, we investigated the genome structure of red clover Trifolium pratense L. by a combination of cytological, genomic and genetic approaches. The deduced genome size was approximately 440 Mb, as estimated by measuring the nuclear DNA content by flow cytometry. Seven chromosomes could be distinguished by microscopic observation of DAPI stained prometaphase chromosomes and fluorescence in situ hybridization using 28S and 5S rDNA probes and bacterial artificial chromosome probes containing microsatellite markers with known positions on a genetic linkage map. The average GC content of the genomes of chloroplast, mitochondrion and nucleus were shown to be 33.8, 42.9 and 34.2%, respectively, by the analysis of 1.4 Mb of random genomic sequences. A total of 26,356 expressed sequence tags (ESTs) that were grouped into 9339 non-redundant sequences were collected, and 78% of the ESTs showed sequence similarity to registered genes, mainly of Arabidopsis thaliana and rice. To facilitate basic and applied genetics in red clover, we generated a high-density genetic linkage map with gene-associated microsatellite markers. A total of 7159 primer pairs were designed to amplify simple sequence repeats (SSRs) identified in four different types of libraries. Based on sequence similarity, 82% of the SSRs were likely to be associated with genes. Polymorphism was examined using two parent plants, HR and R130, and 10 F(1) progeny by agarose gel electrophoresis, followed by genotyping for the primer pairs showing polymorphisms using 188 F(1) plants from the mapping population. The selected 1305 microsatellite markers as well as the previously developed 167 restriction fragment length polymorphism markers were subjected to linkage analysis. A total of 1434 loci detected by 1399 markers were successfully mapped onto seven linkage groups totaling 868.7 cM in length; 405 loci (28%) were bi-parental, 611 (43%) were specific to HR and 418 (29%) were specific to R130. Each genetic linkage group was linked to a corresponding chromosome by FISH analysis using seven microsatellite markers specific to each of the linkage groups as probes. Transferability of the developed microsatellite markers to other germplasms was confirmed by testing 268 selected markers on 88 red clover germplasms. Macrosynteny at the segmental level was observed between the genomes of red clover and two model legumes, Lotus japonicus and Medicago truncatula, strongly suggesting that the genome information for the model legumes is transferable to red clover for genetic investigations and experimental breeding.

Base Composition↗

A nucleoporin is required for induction of Ca2+ spiking in legume nodule development and essential for rhizobial and fungal symbiosis.

Nuclear-cytoplasmic partitioning and traffic between cytoplasmic and nuclear compartments are fundamental processes in eukaryotic cells. Nuclear pore complexes mediate transport of proteins, RNAs and ribonucleoprotein particles in and out of the nucleus. Here we present positional cloning of a plant nucleoporin gene, Nup133, essential for a symbiotic signal transduction pathway shared by Rhizobium bacteria and mycorrhizal fungi. Mutation of Nup133 results in a temperature sensitive nodulation deficient phenotype and absence of mycorrhizal colonization. Root nodules developing with reduced frequency at permissive temperatures are ineffective and electron microscopy show that Rhizobium bacteria are not released from infection threads. Measurement of ion fluxes using a calcium-sensitive dye show that Nup133 is required for the Ca2+ spiking normally detectable within minutes after application of purified rhizobial Nod-factor signal molecules to root hairs. Localization of NUP133 in the nuclear envelope of root cells and root hair cells shown with enhanced yellow fluorescent protein fusion proteins suggests a novel role for NUP133 nucleoporins in a rapid nuclear-cytoplasmic communication after host-plant recognition of symbiotic microbes. Our results identify a component of an intriguing signal process requiring interaction at the cell plasma membrane and at intracellular nuclear and plastid organelle-membranes to induce a second messenger.

Alleles↗

Activation tagging approach in a model legume, Lotus japonicus.

We constructed T-DNA insertional lines of a model legume, Lotus japonicus, using a multifunctional vector for gene and exon activation tagging. The vector had the CaMV 35S promoter together with two additional enhancer elements, the start codon, and splice donor and acceptor sites facing the left border, in anticipation of the activation of T-DNA flanking genes and forced expression of flanking exons. The improved transformation technique yielded more than 3,500 lines, including 45 dominant mutant candidates with abnormal phenotypes with respect to aerial parts, nodules, and roots. Among the 44 selected lines, one copy of T-DNA was inserted into the genome of 37 lines (84%). The T-DNA flanking regions of seven lines were isolated by thermal asymmetric interlaced (TAIL)-PCR or reverse transcription (RT)-PCR, and the corresponding genomic clones were analyzed. The transcripts of four genes adjacent to T-DNA out of 11 genes tested were increased in the T(1) generation, demonstrating that gene and exon activation effects by the newly developed tagging vector are heritable. The T-DNA insertional population of L. japonicus will provide legume-specific dominant mutants.

Base Sequence↗

Promoter trapping in Lotus japonicus reveals novel root and nodule GUS expression domains.

Agrobacterium-based transformation was used to introduce a promoter-less glucuronidase uidA gene (beta-glucuronidase; GUS) into Lotus japonicus. Transgenic plants were screened for GUS activation at different stages after inoculation with its symbiont, Mesorhizobium loti. Functional GUS fusion frequencies ranged from about 2 to 5% of the total number of transgenic lines. These lines provide excellent histological markers for tissue ontogeny analysis. Some of the activations generated GUS expression patterns that correspond to well-known tissue types, such as lateral root and nodule primordia, root tips and developing nodules (line CHEETAH). Others generated GUS activation associated with predictable but previously unknown (i) tissue types, such as the vascular bundle of the nodule (line VASCO); or (ii) expression domains, such as pericycle, nodule primordia, nodule and flower connective/vascular tissue (line FATA MORGANA) or inner root cortex cells in the vicinity of a curled root hair, nodule primordia and nodule cortex (line TIMPA). Putative members of two gene superfamilies, EH (Esp homolog) and AAA ATPase (ATPase associated with various cellular activities), were located next to the CHEETAH and VASCO insertions, respectively, and a nodulin gene, LjENOD40-2, was located next to the FATA MORGANA insertion. We utilized promoter GUS fusions to investigate the genetic regulation of LjENOD40-2 and FATA MORGANA GUS. The LjENOD40-2 promoter defined a novel expression domain and the FATA MORGANA nodule expression was reiterated by the 2 kb sequence upstream of the T-DNA insertion.

Base Sequence↗

The sulfate transporter SST1 is crucial for symbiotic nitrogen fixation in Lotus japonicus root nodules.

Symbiotic nitrogen fixation (SNF) by intracellular rhizobia within legume root nodules requires the exchange of nutrients between host plant cells and their resident bacteria. Little is known at the molecular level about plant transporters that mediate such exchanges. Several mutants of the model legume Lotus japonicus have been identified that develop nodules with metabolic defects that cannot fix nitrogen efficiently and exhibit retarded growth under symbiotic conditions. Map-based cloning of defective genes in two such mutants, sst1-1 and sst1-2 (for symbiotic sulfate transporter), revealed two alleles of the same gene. The gene is expressed in a nodule-specific manner and encodes a protein homologous with eukaryotic sulfate transporters. Full-length cDNA of the gene complemented a yeast mutant defective in sulfate transport. Hence, the gene was named Sst1. The sst1-1 and sst1-2 mutants exhibited normal growth and development under nonsymbiotic growth conditions, a result consistent with the nodule-specific expression of Sst1. Data from a previous proteomic study indicate that SST1 is located on the symbiosome membrane in Lotus nodules. Together, these results suggest that SST1 transports sulfate from the plant cell cytoplasm to the intracellular rhizobia, where the nutrient is essential for protein and cofactor synthesis, including nitrogenase biosynthesis. This work shows the importance of plant sulfate transport in SNF and the specialization of a eukaryotic transporter gene for this purpose.

Amino Acid Sequence↗

Comparison of the transcript profiles from the root and the nodulating root of the model legume Lotus japonicus by serial analysis of gene expression.

We performed a comprehensive transcript analysis on the early stage of root nodulation in the model legume Lotus japonicus by serial analysis of gene expression (SAGE). SAGE libraries were made from uninfected roots and nodulating roots abundant in nodule primordia, and 85,482 and 80,233 SAGE tags were recovered, respectively. Comparison of the tag frequency identified 407 tag species that appeared in significantly greater numbers in the nodulating root than in the uninfected root, and the converse was found for 428 tag species. Gene identification of the tags was performed by matching them to L. japonicus expressed sequence tag sequences. We made several novel findings by applying SAGE to transcript analysis of legume root nodulation. A gene that showed the most significant increase in tag number upon nodulation has not been described previously. Different levels of transcription induction among leghemoglobin gene paralogs were found, indicating the effectiveness of SAGE in discriminating different gene family members. We identified genes for 44 unknown tags by means of reverse SAGE. We found 11 antisense tags that increased during nodulation, indicating that regulation of gene expression by antisense transcripts may occur in an organ-dependent manner.

Expressed Sequence Tags↗

Expression profiling-based identification of CO2-responsive genes regulated by CCM1 controlling a carbon-concentrating mechanism in Chlamydomonas reinhardtii.

Photosynthetic acclimation to CO2-limiting stress is associated with control of genetic and physiological responses through a signal transduction pathway, followed by integrated monitoring of the environmental changes. Although several CO2-responsive genes have been previously isolated, genome-wide analysis has not been applied to the isolation of CO2-responsive genes that may function as part of a carbon-concentrating mechanism (CCM) in photosynthetic eukaryotes. By comparing expression profiles of cells grown under CO2-rich conditions with those of cells grown under CO2-limiting conditions using a cDNA membrane array containing 10,368 expressed sequence tags, 51 low-CO2 inducible genes and 32 genes repressed by low CO2 whose mRNA levels were changed more than 2.5-fold in Chlamydomonas reinhardtii Dangeard were detected. The fact that the induction of almost all low-CO2 inducible genes was impaired in the ccm1 mutant suggests that CCM1 is a master regulator of CCM through putative low-CO2 signal transduction pathways. Among low-CO2 inducible genes, two novel genes, LciA and LciB, were identified, which may be involved in inorganic carbon transport. Possible functions of low-CO2 inducible and/or CCM1-regulated genes are discussed in relation to the CCM.

Acclimatization↗

Distinctive features of plant organs characterized by global analysis of gene expression in Arabidopsis.

The distinctive features of plant organs are primarily determined by organ-specific gene expression. We analyzed the expression specificity of 8809 genes in 7 organs of Arabidopsis using a cDNA macroarray system. Using relative expression (RE) values between organs, many known and unknown genes specifically expressed in each organ were identified. We also analyzed the organ specificity of various gene groups using the GRE (group relative expression) value, the average of the REs of all genes in a group. Consequently, we found that many gene groups even ribosomal protein genes, have strong organ-specific expression. Clustering of the expression profiles revealed that the 8809 genes were classified into 9 major categories. Although 3451 genes were clustered into the largest category, which showed constitutive gene expression, 266 and 1005 genes were found to be root- and silique-specific genes, respectively. By this clustering, particular gene groups which showed multi-organ-specific expression profiles, such as bud-flower-specific, stem-silique-specific or bud-flower-root-specific profiles, could be effectively identified. From these results, major features of plant organs could be characterized by their distinct profiles of global gene expression. These data of organ-specific gene expression are available at our web site: Arabidopsis thaliana Tissue-Specific Expression Database, ATTED (http://www.atted.bio.titech.ac.jp/).

Arabidopsis↗

Characteristics of the Lotus japonicus gene repertoire deduced from large-scale expressed sequence tag (EST) analysis.

To perform a comprehensive analysis of genes expressed in a model legume, Lotus japonicus, a total of 74472 3'-end expressed sequence tags (EST) were generated from cDNA libraries produced from six different organs. Clustering of sequences was performed with an identity criterion of 95% for 50 bases, and a total of 20457 non-redundant sequences, 8503 contigs and 11954 singletons were generated. EST sequence coverage was analyzed by using the annotated L. japonicus genomic sequence and 1093 of the 1889 predicted protein-encoding genes (57.9%) were hit by the EST sequence(s). Gene content was compared to several plant species. Among the 8503 contigs, 471 were identified as sequences conserved only in leguminous species and these included several disease resistance-related genes. This suggested that in legumes, these genes may have evolved specifically to resist pathogen attack. The rate of gene sequence divergence was assessed by comparing similarity level and functional category based on the Gene Ontology (GO) annotation of Arabidopsis genes. This revealed that genes encoding ribosomal proteins, as well as those related to translation, photosynthesis, and cellular structure were more abundantly represented in the highly conserved class, and that genes encoding transcription factors and receptor protein kinases were abundantly represented in the less conserved class. To make the sequence information and the cDNA clones available to the research community, a Web database with useful services was created at http://www.kazusa.or.jp/en/plant/lotus/EST/.

DNA, Complementary↗

Structural analysis of a Lotus japonicus genome. V. Sequence features and mapping of sixty-four TAC clones which cover the 6.4 mb regions of the genome.

We determined the nucleotide sequences of 64 TAC (transformation-competent artificial chromosome) clones selected from genomic libraries of Lotus japonicus accession Miyakojima MG-20 based on the sequence information of expressed sequence tags (ESTs), cDNAs, genes and DNA markers from L. japonicus and other legumes. The length of the DNA regions sequenced in this study was 6,370,255 bp, and the total length of the L. japonicus genome sequenced so far is 32,537,698 bp together with the nucleotide sequences of 256 TAC clones previously reported. Five hundred forty-eight potential protein-encoding genes with known or predicted functions, 127 gene segments and 224 pseudogenes were assigned to the newly sequenced regions by computer prediction and similarity searches against the sequences in protein and EST databases. Based on the nucleotide sequences of the clones, simple sequence repeat length polymorphism (SSLP) or derived cleaved amplified polymorphic sequence (dCAPS) markers were generated, and each clone was genetically localized onto the linkage map of two accessions of L. japonicus, MG-20 and Gifu B-129. The sequence data, gene information and mapping information are available through the World Wide Web at http://www.kazusa.or.jp/lotus/.

Base Sequence↗

Structural analysis of four large plasmids harboring in a unicellular cyanobacterium, Synechocystis sp. PCC 6803.

The genome of the unicellular cyanobacterium Synechocystis sp. PCC 6803 consists of a single chromosome and several plasmids of different sizes, and the nucleotide sequences of the chromosome and three small plasmids (5.2 kb, 2.4 kb, and 2.3 kb) have already been sequenced. We newly determined the nucleotide sequences of four large plasmids, which have been identified in our laboratory (pSYSM:120 kb, pSYSX:106 kb, pSYSA:103 kb, and pSYSG:44 kb). Computer-aided analysis was performed to explore the genetic information carried by these plasmids. A total of 397 potential protein-encoding genes were predicted, but little information was obtained about the functional relationship of plasmids to host cell, as a large portion of the predicted genes (77%) were of unknown function. The occurrence of the potential genes on plasmids was divergent, and parA was the only gene common to all four large plasmids. The distribution data of a Cyanobacterium-specific sequence (HIP1: 5'-GCGATCGC-3') suggested that respective plasmids could have originated from different cyanobacterial strains.

Base Sequence↗