Molecular phylogeny of fucoxanthin-chlorophyll a/c proteins from Chaetoceros gracilis and Lhcq/Lhcf diversity.

Diatoms adapt to various aquatic light environments and play major roles in the global carbon cycle using their unique light-harvesting system, i.e. fucoxanthin chlorophyll a/c binding proteins (FCPs). Structural analyses of photosystem II (PSII)-FCPII and photosystem I (PSI)-FCPI complexes from the diatom Chaetoceros gracilis have revealed the localization and interactions of many FCPs; however, the entire set of FCPs has not been characterized. Here, we identify 46 FCPs in the newly assembled genome and transcriptome of C. gracilis. Phylogenetic analyses suggest that these FCPs can be classified into five subfamilies: Lhcr, Lhcf, Lhcx, Lhcz, and the novel Lhcq, in addition to a distinct type of Lhcr, CgLhcr9. The FCPs in Lhcr, including CgLhcr9 and some Lhcqs, have orthologous proteins in other diatoms, particularly those found in the PSI-FCPI structure. By contrast, the Lhcf subfamily, some of which were found in the PSII-FCPII complex, seems to be diversified in each diatom species, and the number of Lhcqs differs among species, indicating that their diversification may contribute to species-specific adaptations to light. Further phylogenetic analyses of FCPs/light-harvesting complex (LHC) proteins using genome data and assembled transcriptomes of other diatoms and microalgae in public databases suggest that our proposed classification of FCPs is common among various red-lineage algae derived from secondary endosymbiosis of red algae, including Haptophyta. These results provide insights into the loss and gain of FCP/LHC subfamilies during the evolutionary history of the red algal lineage.

Ten Years of Collaborative Progress in the Quest for Orthologs.

Accurate determination of the evolutionary relationships between genes is a foundational challenge in biology. Homology-evolutionary relatedness-is in many cases readily determined based on sequence similarity analysis. By contrast, whether or not two genes directly descended from a common ancestor by a speciation event (orthologs) or duplication event (paralogs) is more challenging, yet provides critical information on the history of a gene. Since 2009, this task has been the focus of the Quest for Orthologs (QFO) Consortium. The sixth QFO meeting took place in Okazaki, Japan in conjunction with the 67th National Institute for Basic Biology conference. Here, we report recent advances, applications, and oncoming challenges that were discussed during the conference. Steady progress has been made toward standardization and scalability of new and existing tools. A feature of the conference was the presentation of a panel of accessible tools for phylogenetic profiling and several developments to bring orthology beyond the gene unit-from domains to networks. This meeting brought into light several challenges to come: leveraging orthology computations to get the most of the incoming avalanche of genomic data, integrating orthology from domain to biological network levels, building better gene models, and adapting orthology approaches to the broad evolutionary and genomic diversity recognized in different forms of life and viruses.

The physiological potential of anammox bacteria as revealed by their core genome structure.

We present here the second complete genome of anaerobic ammonium oxidation (anammox) bacterium, Candidatus (Ca.) Brocadia pituitae, along with those of a nitrite oxidizer and two incomplete denitrifiers from the anammox bacterial community (ABC) metagenome. Although NO2- reduction to NO is considered to be the first step in anammox, Ca. B. pituitae lacks nitrite reductase genes (nirK and nirS) responsible for this reaction. Comparative genomics of Ca. B. pituitae with Ca. Kuenenia stuttgartiensis and six other anammox bacteria with nearly complete genomes revealed that their core genome structure contains 1,152 syntenic orthologues. But nitrite reductase genes were absent from the core, whereas two other Brocadia species possess nirK and these genes were horizontally acquired from multiple lineages. In contrast, at least five paralogous hydroxylamine oxidoreductase genes containing candidate ones (hao2 and hao3) encoding another nitrite reductase were observed in the core. Indeed, these two genes were also significantly expressed in Ca. B. pituitae as in other anammox bacteria. Because many nirS and nirK genes have been detected in the ABC metagenome, Ca. B. pituitae presumably utilises not only NO supplied by the ABC members but also NO and/or NH2OH by self-production for anammox metabolism.

Structural basis for assembly and function of a diatom photosystem I-light-harvesting supercomplex.

Photosynthetic light-harvesting complexes (LHCs) play a pivotal role in collecting solar energy for photochemical reactions in photosynthesis. One of the major LHCs are fucoxanthin chlorophyll a/c-binding proteins (FCPs) present in diatoms, a group of organisms having important contribution to the global carbon cycle. Here, we report a 2.40-Å resolution structure of the diatom photosystem I (PSI)-FCPI supercomplex by cryo-electron microscopy. The supercomplex is composed of 16 different FCPI subunits surrounding a monomeric PSI core. Each FCPI subunit showed different protein structures with different pigment contents and binding sites, and they form a complicated pigment-protein network together with the PSI core to harvest and transfer the light energy efficiently. In addition, two unique, previously unidentified subunits were found in the PSI core. The structure provides numerous insights into not only the light-harvesting strategy in diatom PSI-FCPI but also evolutionary dynamics of light harvesters among oxyphototrophs.

A simple and practical workflow for genotyping of CRISPR-Cas9-based knockout phenotypes using multiplexed amplicon sequencing.

Founder animals carrying high proportions of somatic mutation induced by CRISPR-Cas9 enable a rapid and scalable strategy for the functional screening of numerous target genes in vivo. In this functional screening, genotyping using pooled amplicons with next-generation sequencing is the most suitable approach for large-scale management of multiple samples and accurate evaluation of the efficiency of Cas9-induced somatic mutations at target sites. Here, we present a simple workflow for genotyping of multiple CRISPR-Cas9-based knockout founders by pooled amplicon sequencing. Using custom barcoded primers, pooled amplicons from multiple individuals can be run in a single-indexed library on the Illumina MiSeq platform. Additionally, a user-friendly web tool, CLiCKAR, is available to simultaneously perform demultiplexing of pooled sequence data and evaluation of somatic mutation in each phenotype. CLiCKAR provides users with practical reports regarding the positions of insertions/deletions, as well as the frameshift ratio and tables containing mutation sequences, and read counts of each phenotype, with just a few clicks by the implementation of demultiplexing for pooled sample data and calculation of the frameshift ratio. This genotyping workflow can be harnessed to evaluate genotype-phenotype correlations in CRISPR-Cas9-based loss-of-function screening of numerous target genes in various organisms.

RNA-Seq Analysis Reveals Genes Related to Photoreception, Nutrient Uptake, and Toxicity in a Noxious Red-Tide Raphidophyte Chattonella antiqua.

Aquaculture industries are under threat from noxious red tides, but harm can be mitigated by precautions such as early harvesting and restricting fish feeding to just before the outbreak of a red tide. Therefore, accurate techniques for forecasting red-tide outbreaks are strongly needed. Omics analyses have the potential to expand our understanding of the eco-physiology of these organisms at the molecular level, and to facilitate identification of molecular markers for forecasting their population dynamics and occurrence of damages to fisheries. Red tides of marine raphidophytes, especially Chattonella species, often extensively harm aquaculture industries in regions with a temperate climate around the world. A red tide of Chattonella tends to develop just after an input of nutrients along the coast. Chattonella displays diurnal vertical migration regulated by a weak blue light, so it photosynthesizes in the surface layer during the daytime and takes up nutrients in the bottom layer during the nighttime. Superoxide produced by Chattonella cells is a strong candidate for the cause of its toxicity to bacteria and fishes. Here we conducted mRNA-seq of Chattonella antiqua to identify genes with functions closely related to the dynamics of the noxious red tide, such as photosynthesis, photoreception, nutrient uptake, and superoxide production. The genes related to photosynthetic pigment biosynthesis and nutrient uptake had high similarity with those of model organisms of plants and algae and other red-tide microalgae. We identified orthologous genes of photoreceptors such as aureochrome (newly five genes), the cryptochrome/photolyase (CRY/PHR) family (6-4PHR, plant CRY or cyclobutane pyrimidine dimer [CPD] Class III, CPD Class II, and CRY-DASH), and phytochrome (four genes), which regulate various physiological processes such as flagellar motion and cell cycle in model organisms. Six orthologous genes of NADPH oxidase, which produces superoxide on the cell membrane, were found and divided into two types: one with 5-6 transmembrane domains and another with 11 transmembrane domains. The present study should open the way for analyzing the eco-physiological features of marine raphidophytes at the molecular level.

Structural basis for energy harvesting and dissipation in a diatom PSII-FCPII supercomplex.

Light-harvesting antenna systems in photosynthetic organisms harvest solar energy and transfer it to the photosynthetic reaction centres to initiate charge-separation and electron-transfer reactions. Diatoms are one of the important groups of oxyphototrophs and possess fucoxanthin chlorophyll a/c-binding proteins (FCPs) as light harvesters. The organization and association pattern of FCP with the photosystem II (PSII) core are unknown. Here we solved the structure of PSII-FCPII supercomplexes isolated from a diatom, Chaetoceros gracilis, by single-particle cryoelectron microscopy. The PSII-FCPII forms a homodimer. In each monomer, two FCP homotetramers and three FCP monomers are associated with one PSII core. The structure reveals a highly complicated protein-pigment network that is different from the green-type light-harvesting apparatus. Comparing these two systems allows the identification of energy transfer and quenching pathways. These findings provide structural insights into not only excitation-energy transfer mechanisms in the diatom PSII-FCPII, but also changes of light harvesters between the red- and green-lineage oxyphototrophs during evolution.

A comprehensive reference transcriptome resource for the Iberian ribbed newt Pleurodeles waltl, an emerging model for developmental and regeneration biology.

Urodele newts have unique biological properties, notably including prominent regeneration ability. The Iberian ribbed newt, Pleurodeles waltl, is a promising model amphibian distinguished by ease of breeding and efficient transgenic and genome editing methods. However, limited genetic information is available for P. waltl. We conducted an intensive transcriptome analysis of P. waltl using RNA-sequencing to build and annotate gene models. We generated 1.2 billion Illumina reads from a wide variety of samples across 12 different tissues/organs, unfertilized egg, and embryos at eight different developmental stages. These reads were assembled into 1,395,387 contigs, from which 202,788 non-redundant ORF models were constructed. The set is expected to cover a large fraction of P. waltl protein-coding genes, as confirmed by BUSCO analysis, where 98% of universal single-copy orthologs were identified. Ortholog analyses revealed the gene repertoire evolution of urodele amphibians. Using the gene set as a reference, gene network analysis identified regeneration-, developmental-stage-, and tissue-specific co-expressed gene modules. Our transcriptome resource is expected to enhance future research employing this emerging model animal for regeneration research as well as for investigations in other areas including developmental biology, stem cell biology, and cancer research. These data are available via our portal website, iNewt (http://www.nibb.ac.jp/imori/main/).

MBGD update 2018: microbial genome database based on hierarchical orthology relations covering closely related and distantly related comparisons.

The Microbial Genome Database for Comparative Analysis (MBGD) is a database for comparative genomics based on comprehensive orthology analysis of bacteria, archaea and unicellular eukaryotes. MBGD now contains 6318 genomes. To utilize the database for both closely related and distantly related genomes, MBGD previously provided two types of ortholog tables: the standard ortholog table containing one representative genome from each genus covering the entire taxonomic range and the taxon specific ortholog tables for each taxon. However, this approach has a drawback in that the standard ortholog table contains only genes that are conserved in the representative genomes. To address this problem, we developed a stepwise procedure to construct ortholog tables hierarchically in a bottom-up manner. By using this approach, the new standard ortholog table now covers the entire gene repertoire stored in MBGD. In addition, we have enhanced several functionalities, including rapid and flexible keyword searching, profile-based sequence searching for orthology assignment to a user query sequence, and displaying a phylogenetic tree of each taxon based on the concatenated core gene sequences. For integrative database searching, the core data in MBGD are represented in Resource Description Framework (RDF) and a SPARQL interface is provided to search them. MBGD is available at http://mbgd.genome.ad.jp/.

Artificial acceleration of mammalian cell reprogramming by bacterial proteins.

The molecular mechanisms of cell reprogramming and differentiation involve various signaling factors. Small molecule compounds have been identified to artificially influence these factors through interacting cellular proteins. Although such small molecule compounds are useful to enhance reprogramming and differentiation and to show the mechanisms that underlie these events, the screening usually requires a large number of compounds to identify only a very small number of hits (e.g., one hit among several tens of thousands of compounds). Here, we show a proof of concept that xenospecific gene products can affect the efficiency of cell reprogramming to pluripotency. Thirty genes specific for the bacterium Wolbachia pipientis were forcibly expressed individually along with reprogramming factors (Oct4, Sox2, Klf4 and c-Myc) that can generate induced pluripotent stem cells in mammalian cells, and eight were found to affect the reprogramming efficiency either positively or negatively (hit rate 26.7%). Mechanistic analysis suggested one of these proteins interacted with cytoskeleton to promote reprogramming. Our results raise the possibility that xenospecific gene products provide an alternative way to study the regulatory mechanism of cell identity.

Ortholog Identification and Comparative Analysis of Microbial Genomes Using MBGD and RECOG.

Comparative genomics is becoming an essential approach for identification of genes associated with a specific function or phenotype. Here, we introduce the microbial genome database for comparative analysis (MBGD), which is a comprehensive ortholog database among the microbial genomes available so far. MBGD contains several precomputed ortholog tables including the standard ortholog table covering the entire taxonomic range and taxon-specific ortholog tables for various major taxa. In addition, MBGD allows the users to create an ortholog table within any specified set of genomes through dynamic calculations. In particular, MBGD has a "My MBGD" mode where users can upload their original genome sequences and incorporate them into orthology analysis. The created ortholog table can serve as the basis for various comparative analyses. Here, we describe the use of MBGD and briefly explain how to utilize the orthology information during comparative genome analysis in combination with the stand-alone comparative genomics software RECOG, focusing on the application to comparison of closely related microbial genomes.

SPANG: a SPARQL client supporting generation and reuse of queries for distributed RDF databases.

BACKGROUND: Toward improved interoperability of distributed biological databases, an increasing number of datasets have been published in the standardized Resource Description Framework (RDF). Although the powerful SPARQL Protocol and RDF Query Language (SPARQL) provides a basis for exploiting RDF databases, writing SPARQL code is burdensome for users including bioinformaticians. Thus, an easy-to-use interface is necessary. RESULTS: We developed SPANG, a SPARQL client that has unique features for querying RDF datasets. SPANG dynamically generates typical SPARQL queries according to specified arguments. It can also call SPARQL template libraries constructed in a local system or published on the Web. Further, it enables combinatorial execution of multiple queries, each with a distinct target database. These features facilitate easy and effective access to RDF datasets and integrative analysis of distributed data. CONCLUSIONS: SPANG helps users to exploit RDF datasets by generation and reuse of SPARQL queries through a simple interface. This client will enhance integrative exploitation of biological RDF datasets distributed across the Web. This software package is freely available at http://purl.org/net/spang .

An acid-tolerant ammonia-oxidizing γ-proteobacterium from soil.

Nitrification, the microbial oxidation of ammonia to nitrate via nitrite, occurs in a wide range of acidic soils. However, the ammonia-oxidizing bacteria (AOB) that have been isolated from soil to date are acid-sensitive. Here we report the isolation and characterization of an acid-adapted AOB from an acidic agricultural soil. The isolated AOB, strain TAO100, is classified within the Gammaproteobacteria based on phylogenetic characteristics. TAO100 can grow in the pH range of 5-7.5 and survive in highly acidic conditions until pH 2 by forming cell aggregates. Whereas all known gammaproteobacterial AOB (γ-AOB) species, which have been isolated from marine and saline aquatic environments, are halophiles, TAO100 is not phenotypically halophilic. Thus, TAO100 represents the first soil-originated and non-halophilic γ-AOB. The TAO100 genome is considerably smaller than those of other γ-AOB and lacks several genes associated with salt tolerance which are unnecessary for survival in soil. The ammonia monooxygenase subunit A gene of TAO100 and its transcript are higher in abundance than those of ammonia-oxidizing archaea and betaproteobacterial AOB in the strongly acidic soil. These results indicate that TAO100 plays an important role in the nitrification of acidic soils. Based on these results, we propose TAO100 as a novel species of a new genus, Candidatus Nitrosoglobus terrae.

Complete genome sequence and expression profile of the commercial lytic enzyme producer Lysobacter enzymogenes M497-1.

Lysobacter enzymogenes M497-1 is a producer of commercialized achromopeptidase and is expected to harbour genes encoding various other antimicrobial enzymes. Here, we present the complete sequence of the genome of M497-1 and the expression profiles of the genes for various antimicrobial enzymes. Of the 117 peptidase-encoding genes found in the 6.1-Mb genome of M497-1, 15 genes (aside from the gene encoding the achromopeptidase) were expressed at a level higher than that of the average ribosomal protein genes in the 24-h culture. Thus, the strain was found more valuable than hitherto considered. In addition, M497-1 harbours 98 genes involved in the biosynthesis of various natural products, 16 of which are M497-1-specific across 4 Lysobacter species. A gene cluster starting at LEN_2603 through LEN_2673 among the 98 genes closely resembled the lysobactin biosynthesis gene cluster of Lysobacter sp. ATCC 53042. It is likely that M497-1 may produce lysobactin or related antibacterial compounds. Furthermore, comparative genomic analysis of M497-1 and four other Lysobacter species revealed that their core genome structure comprises 3,737 orthologous groups. Our findings are expected to advance further biotechnological application of Lysobacter spp. as a promising source of natural bioactive compounds.

Genome sequence and analysis of the Japanese morning glory Ipomoea nil.

Ipomoea is the largest genus in the family Convolvulaceae. Ipomoea nil (Japanese morning glory) has been utilized as a model plant to study the genetic basis of floricultural traits, with over 1,500 mutant lines. In the present study, we have utilized second- and third-generation-sequencing platforms, and have reported a draft genome of I. nil with a scaffold N50 of 2.88 Mb (contig N50 of 1.87 Mb), covering 98% of the 750 Mb genome. Scaffolds covering 91.42% of the assembly are anchored to 15 pseudo-chromosomes. The draft genome has enabled the identification and cataloguing of the Tpn1 family transposons, known as the major mutagen of I. nil, and analysing the dwarf gene, CONTRACTED, located on the genetic map published in 1956. Comparative genomics has suggested that a whole genome duplication in Convolvulaceae, distinct from the recent Solanaceae event, has occurred after the divergence of the two sister families.

Draft Genome Sequence of Helicobacter suis Strain SNTW101, Isolated from a Japanese Patient with Nodular Gastritis.

We present here the draft whole-genome shotgun sequence of an uncultivated strain SNTW101 of Helicobacter suis, which has been maintained in the stomachs of mice. This strain was originally isolated from gastric biopsy specimens of a urea breath test-negative Japanese patient suffering from nodular gastritis.

A Novel Approach to Helicobacter pylori Pan-Genome Analysis for Identification of Genomic Islands.

Genomes of a given bacterial species can show great variation in gene content and thus systematic analysis of the entire gene repertoire, termed the pan-genome, is important for understanding bacterial intra-species diversity, population genetics, and evolution. Here, we analyzed the pan-genome from 30 completely sequenced strains of the human gastric pathogen Helicobacter pylori belonging to various phylogeographic groups, focusing on 991 accessory (not fully conserved) orthologous groups (OGs). We developed a method to evaluate the mobility of genes within a genome, using the gene order in the syntenically conserved regions as a reference, and classified the 991 accessory OGs into five classes: Core, Stable, Intermediate, Mobile, and Unique. Phylogenetic networks based on the gene content of Core and Stable classes are highly congruent with that created from the concatenated alignment of fully conserved core genes, in contrast to those of Intermediate and Mobile classes, which show quite different topologies. By clustering the accessory OGs on the basis of phylogenetic pattern similarity and chromosomal proximity, we identified 60 co-occurring gene clusters (CGCs). In addition to known genomic islands, including cag pathogenicity island, bacteriophages, and integrating conjugative elements, we identified some novel ones. One island encodes TerY-phosphorylation triad, which includes the eukaryote-type protein kinase/phosphatase gene pair, and components of type VII secretion system. Another one contains a reverse-transcriptase homolog, which may be involved in the defense against phage infection through altruistic suicide. Many of the CGCs contained restriction-modification (RM) genes. Different RM systems sometimes occupied the same (orthologous) locus in the strains. We anticipate that our method will facilitate pan-genome studies in general and help identify novel genomic islands in various bacterial species.

Complete Genome Sequence of Aurantimicrobium minutum Type Strain KNCT, a Planktonic Ultramicrobacterium Isolated from River Water.

Aurantimicrobium minutum type strain KNC(T) is a planktonic ultramicrobacterium isolated from river water in western Japan. Strain KNC(T) has an extremely small, streamlined genome of 1,622,386 bp comprising 1,575 protein-coding sequences. The genome annotation suggests that strain KNC(T) has an actinorhodopsin-based photometabolism.

The Orthology Ontology: development and applications.

BACKGROUND: Computational comparative analysis of multiple genomes provides valuable opportunities to biomedical research. In particular, orthology analysis can play a central role in comparative genomics; it guides establishing evolutionary relations among genes of organisms and allows functional inference of gene products. However, the wide variations in current orthology databases necessitate the research toward the shareability of the content that is generated by different tools and stored in different structures. Exchanging the content with other research communities requires making the meaning of the content explicit. DESCRIPTION: The need for a common ontology has led to the creation of the Orthology Ontology (ORTH) following the best practices in ontology construction. Here, we describe our model and major entities of the ontology that is implemented in the Web Ontology Language (OWL), followed by the assessment of the quality of the ontology and the application of the ORTH to existing orthology datasets. This shareable ontology enables the possibility to develop Linked Orthology Datasets and a meta-predictor of orthology through standardization for the representation of orthology databases. The ORTH is freely available in OWL format to all users at http://purl.org/net/orth . CONCLUSIONS: The Orthology Ontology can serve as a framework for the semantic standardization of orthology content and it will contribute to a better exploitation of orthology resources in biomedical research. The results demonstrate the feasibility of developing shareable datasets using this ontology. Further applications will maximize the usefulness of this ontology.

Genome-wide survey of codons under diversifying selection in a highly recombining bacterial species, Helicobacter pylori.

Selection has been a central issue in biology in eukaryotes as well as prokaryotes. Inference of selection in recombining bacterial species, compared with clonal ones, has been a challenge. It is not known how codons under diversifying selection are distributed along the chromosome or among functional categories or how frequently such codons are subject to mutual homologous recombination. Here, we explored these questions by analysing genes present in >90% among 29 genomes of Helicobacter pylori, one of the bacterial species with the highest mutation and recombination rates. By a method for recombining sequences, we identified codons under diversifying selection (dN/dS> 1), which were widely distributed and accounted for ∼0.2% of all the codons of the genome. The codons were enriched in genes of host interaction/cell surface and genome maintenance (DNA replication,recombination, repair, and restriction modification system). The encoded amino acid residues were sometimes found adjacent to critical catalytic/binding residues in protein structures.Furthermore, by estimating the intensity of homologous recombination at a single nucleotide level, we found that these codons appear to be more frequently subject to recombination.We expect that the present study provides a new approach to population genomics of selection in recombining prokaryotes.