Yasuhiro Tanizawa

DFAST: a flexible prokaryotic genome annotation pipeline for faster genome publication

Summary: We developed a prokaryotic genome annotation pipeline, DFAST, that also supports genome submission to public sequence databases. DFAST was originally started as an on‐line annotation server, and to date, over 7000 jobs have been processed since its first launch in 2016. Here, we present a newly implemented background annotation engine for DFAST, which is also available as a standalone command‐line program. The new engine can annotate a typical‐sized bacterial genome within 10 min, with rich information such as pseudogenes, translation exceptions and orthologous gene assignment between given reference genomes. In addition, the modular framework of DFAST allows users to customize the annotation workflow easily and will also facilitate extensions for new functions and incorporation of new tools in the future. Availability and implementation: The software is implemented in Python 3 and runs in both Python 2.7 and 3.4—on Macintosh and Linux systems. It is freely available at https://github.com/nigyta/dfast_core/under the GPLv3 license with external binaries bundled in the software distribution. An on‐line version is also available at https://dfast.nig.ac.jp/. Contact: yn@nig.ac.jp Supplementary information: Supplementary data are available at Bioinformatics online.

The effect of rapamycin on biodiesel-producing protist Euglena gracilis.

Rapamycin induces autophagy with lipid remodeling in yeast and mammalian cells. To investigate the lipid biosynthesis of Euglena gracilis, rapamycin was supplemented in comparison with two model algae, Chlamydomonas reinhardtii and Cyanidioschyzon merolae. In Euglena, rapamycin induced the reduction of chlorophylls and the accumulation of neutral lipids without deterring its cell proliferation. Its lipidomic profile revealed that the fatty acid composition did not alter by supplementing rapamycin. In Chlamydomonas, however, rapamycin induced serious growth inhibition as reported elsewhere. With a lower concentration of rapamycin, the alga accumulated neutral lipids without reducing chlorophylls. In Cyanidioschyzon, rapamycin did not increase neutral lipids but reduced its chlorophyll content. We also tested fatty acid elongase inhibitors such as pyroxasulfone or flufenacet in Euglena with no significant change in its neutral lipid contents. In summary, controlled supplementation of rapamycin can increase the yield of neutral lipids while the scheme is not always applicable for other algal species. Graphical Abstract Lipid profile of Euglena gracilis under supplementation of rapamycin. With <10u2009µM rapamycin, the neutral lipid contents increase without deterring cell growth.

Draft Sequencing of the Heterozygous Diploid Genome of Satsuma (Citrus unshiu Marc.) Using a Hybrid Assembly Approach

Satsuma (Citrus unshiu Marc.) is one of the most abundantly produced mandarin varieties of citrus, known for its seedless fruit production and as a breeding parent of citrus. De novo assembly of the heterozygous diploid genome of Satsuma (“Miyagawa Wase”) was conducted by a hybrid assembly approach using short-read sequences, three mate-pair libraries, and a long-read sequence of PacBio by the PLATANUS assembler. The assembled sequence, with a total size of 359.7 Mb at the N50 length of 386,404 bp, consisted of 20,876 scaffolds. Pseudomolecules of Satsuma constructed by aligning the scaffolds to three genetic maps showed genome-wide synteny to the genomes of Clementine, pummelo, and sweet orange. Gene prediction by modeling with MAKER-P proposed 29,024 genes and 37,970 mRNA; additionally, gene prediction analysis found candidates for novel genes in several biosynthesis pathways for gibberellin and violaxanthin catabolism. BUSCO scores for the assembled scaffold and predicted transcripts, and another analysis by BAC end sequence mapping indicated the assembled genome consistency was close to those of the haploid Clementine, pummel, and sweet orange genomes. The number of repeat elements and long terminal repeat retrotransposon were comparable to those of the seven citrus genomes; this suggested no significant failure in the assembly at the repeat region. A resequencing application using the assembled sequence confirmed that both kunenbo-A and Satsuma are offsprings of Kishu, and Satsuma is a back-crossed offspring of Kishu. These results illustrated the performance of the hybrid assembly approach and its ability to construct an accurate heterozygous diploid genome.

H2DB: a heritability database across multiple species by annotating trait-associated genomic loci

H2DB (http://tga.nig.ac.jp/h2db/), an annotation database of genetic heritability estimates for humans and other species, has been developed as a knowledge database to connect trait-associated genomic loci. Heritability estimates have been investigated for individual species, particularly in human twin studies and plant/animal breeding studies. However, there appears to be no comprehensive heritability database for both humans and other species. Here, we introduce an annotation database for genetic heritabilities of various species that was annotated by manually curating online public resources in PUBMED abstracts and journal contents. The proposed heritability database contains attribute information for trait descriptions, experimental conditions, trait-associated genomic loci and broad- and narrow-sense heritability specifications. Annotated trait-associated genomic loci, for which most are single-nucleotide polymorphisms derived from genome-wide association studies, may be valuable resources for experimental scientists. In addition, we assigned phenotype ontologies to the annotated traits for the purposes of discussing heritability distributions based on phenotypic classifications.

Fructophilic Lactic Acid Bacteria, a Unique Group of Fructose-Fermenting Microbes

Fructophilic lactic acid bacteria (FLAB) are a recently discovered group, consisting of a few Fructobacillus and Lactobacillus species. Because of their unique characteristics, including poor growth on glucose and preference of oxygen, they are regarded as “unconventional” lactic acid bacteria (LAB). ABSTRACT Fructophilic lactic acid bacteria (FLAB) are a recently discovered group, consisting of a few Fructobacillus and Lactobacillus species. Because of their unique characteristics, including poor growth on glucose and preference of oxygen, they are regarded as “unconventional” lactic acid bacteria (LAB). Their unusual growth characteristics are due to an incomplete gene encoding a bifunctional alcohol/acetaldehyde dehydrogenase (adhE). This results in the imbalance of NAD/NADH and the requirement of additional electron acceptors to metabolize glucose. Oxygen, fructose, and pyruvate are used as electron acceptors. FLAB have significantly fewer genes for carbohydrate metabolism than other LAB, especially due to the lack of complete phosphotransferase system (PTS) transporters. They have been isolated from fructose-rich environments, including flowers, fruits, fermented fruits, and the guts of insects that feed on plants rich in fructose, and are separated into two groups on the basis of their habitats. One group is associated with flowers, grapes, wines, and insects, and the second group is associated with ripe fruits and fruit fermentations. Species associated with insects may play a role in the health of their host and are regarded as suitable vectors for paratransgenesis in honey bees. Besides their impact on insect health, FLAB may be promising candidates for the promotion of human health. Further studies are required to explore their beneficial properties in animals and humans and their applications in the food industry.

Lactobacillus silagincola sp. nov. and Lactobacillus pentosiphilus sp. nov., isolated from silage

Three Gram-stain positive, non-motile, non-spore-forming, catalase-negative and rod-shaped bacterial strains (IWT5T, IWT25T and IWT140), isolated from silage, were investigated by using a polyphasic taxonomic approach. Strains IWT5T and IWT25T grew at 10-37u2009°Cu2009and 30-37u2009°C, and at pH 4.0-7.5 and 4.0-7.0, respectively. The G+C contents of genomic DNA of strains IWT5T and IWT25T were 43.2 and 44.4u2009mol%, respectively. Strains IWT5T and IWT25T contained C16u200a:u200a0, C18u200a:u200a1u2009ω9c and summed feature 7 (unknown 18.846/C19u200a:u200a1u2009ω6c/C19u200a:u200a0cyclo ω10c) as the major fatty acids. Strain IWT5T was most closely related to the type strains of Lactobacillus mixtipabuli (99.9u200a% 16S rRNA gene sequence similarity) and Lactobacillus silagei (99.5u200a%). For IWT25T, the 16S rRNA gene sequence similarities with the closely related neighbour type strains L. mixtipabuli and L. silagei were 99.5 and 99.5u200a%, respectively. The 16S rRNA gene sequence similarities among the three novel isolates were 99.5-99.9u200a%. The average nucleotide identities of strains IWT5T and IWT25T to other neighbours of the genus Lactobacillus were less than 82u200a% and the genomes of IWT25T and IWT140 shared 97.3u200a% average nucleotide identity, demonstrating that the three strains were allocated to two different novel species of the genus Lactobacillus. Together with multilocus sequence analysis, phenotypic and chemotaxonomic characteristics, strains IWT5T (=JCM 31144T=DSM 102973T) and IWT25T (=JCM 31145T=DSM 102974T) are proposed as the type strains of novel species of the genus Lactobacillus, with the names Lactobacillus silagincola sp. nov. and Lactobacillus pentosiphilus sp. nov., respectively.

Revealing the genomic differences between two subgroups in Lactobacillus gasseri

Being an autochthonous species in humans, Lactobacillus gasseri is widely used as a probiotic for fermented products. We thoroughly compared the gene contents of 75 L. gasseri genomes and identified two intraspecific groups by the average nucleotide identity (ANI) threshold of 94%. Group I, with 48 strains, possessed 53 group-specific genes including the gassericin T cluster (9 genes) and N-acyl homoserine lactone lactonase. Group II, with 27 strains, including the type strain ATCC 33323, possessed group-specific genes with plasmid- or phage-related annotations. The genomic differences provide evidences for demarcating a new probiotic group within L. gasseri.

Visualization of consensus genome structure without using a reference genome

BackgroundStandard graphical tools for whole genome comparison require a reference genome. However, any reference is also subject to annotation biases and rearrangements, and may not serve as the standard except for those of extensively studied model species. To fully exploit the rapidly accumulating sequence data from the recent sequencing technologies, genome comparison without any reference has been anticipated.ResultsWe introduce a circular genome visualizer to compare complete genomes of closely related species. This tool visualizes the position of orthologous gene clusters rather than actual sequences or their features, thereby achieving the comparative view without using a single reference genome. The essential information is the matrix of orthologous gene clusters whose positions (not sequences) are color-coded in circular graphics. As a demonstration, comparison of 14 Lactobacillus paracasei strains and one L. casei strain revealed not only large-scale rearrangements but also genomic islands that are strain-specific. Comparison of 73 Helicobacter pylori strains confirmed their genetic consistency and also revealed the three general patterns of large-scale genome inversions.ConclusionsFrom the ample sequence information in the GenBank/ENA/DDBJ repository, we can reconstruct a genomic consensus for particular species. By visualizing multiple strains at a glance, we can identify conserved as well as strain-specific regions in multiply sequenced genomes. Positional consistency for orthologous genes provides information orthogonal to major sequence features such as the GC content or sequence similarity of marker genes. The positional comparison is therefore useful for identifying large-scale genome rearrangements or gene transfers.

Lactobacillus paragasseri sp. nov., a sister taxon of Lactobacillus gasseri, based on whole-genome sequence analyses.

Three strains, JCM 5343T, JCM 5344 and JCM 1130, currently identified as Lactobacillus gasseri, were investigated using a polyphasic taxonomic approach. Although these strains shared high 16S rRNA gene sequence similarities with L. gasseri ATCC 33323T (99.9u200a%), they formed a clade clearly distinct from ATCC 33323T based on whole-genome relatedness. The average nucleotide identity and in silico DNA-DNA hybridization values of these three strains compared to L. gasseri ATCC 33323T were 93.4-93.7 and 53.1-54.1u200a%, respectively, and both were less than the widely accepted threshold to distinguish two species (95 and 70u200a%, respectively). The three strains were Gram-stain positive, non-motile, non-spore-forming, catalase-negative and rod-shaped bacteria. They grew at 25-45u2009°Cu2009and in the presence of 2.0u200a% (w/v) NaCl. The major fatty acids of the three strains were C16u200a:u200a0 and C18u200a:u200a1 ω9c. Based on phylogenetic analyses of the phenylalanyl-tRNA synthase alpha subunit and RNA polymerase alpha subunit genes, and on phenotypic and chemotaxonomic characteristics, strains JCM 5343T, JCM 5344 and JCM 1130 represent a novel species distinct from L. gasseri, for which we propose the name Lactobacillusparagasseri sp. nov. In addition, a large portion of genomes currently labelled as L. gasseri in the public sequence database should be reclassified as L. paragasseri based on whole-genome relatedness.

Genomic characterization reconfirms the taxonomic status of Lactobacillus parakefiri

Whole-genome sequencing was performed for Lactobacillus parakefiri JCM 8573T to confirm its hitherto controversial taxonomic position. Here, we report its first reliable reference genome. Genome-wide metrics, such as average nucleotide identity and digital DNA-DNA hybridization, and phylogenomic analysis based on multiple genes supported its taxonomic status as a distinct species in the genus Lactobacillus. The availability of a reliable genome sequence will aid future investigations on the industrial applications of L. parakefiri in functional foods such as kefir grains.