Takeshi Ara

Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection

We have systematically made a set of precisely defined, single‐gene deletions of all nonessential genes in Escherichia coli K‐12. Open‐reading frame coding regions were replaced with a kanamycin cassette flanked by FLP recognition target sites by using a one‐step method for inactivation of chromosomal genes and primers designed to create in‐frame deletions upon excision of the resistance cassette. Of 4288 genes targeted, mutants were obtained for 3985. To alleviate problems encountered in high‐throughput studies, two independent mutants were saved for every deleted gene. These mutants—the ‘Keio collection’—provide a new resource not only for systematic analyses of unknown gene functions and gene regulatory networks but also for genome‐wide testing of mutational effects in a common strain background, E. coli K‐12 BW25113. We were unable to disrupt 303 genes, including 37 of unknown function, which are candidates for essential genes. Distribution is being handled via GenoBase (http://ecoli.aist‐nara.ac.jp/).

MassBank: a public repository for sharing mass spectral data for life sciences.

MassBank is the first public repository of mass spectra of small chemical compounds for life sciences (<3000 Da). The database contains 605 electron-ionization mass spectrometry (EI-MS), 137 fast atom bombardment MS and 9276 electrospray ionization (ESI)-MS(n) data of 2337 authentic compounds of metabolites, 11 545 EI-MS and 834 other-MS data of 10,286 volatile natural and synthetic compounds, and 3045 ESI-MS(2) data of 679 synthetic drugs contributed by 16 research groups (January 2010). ESI-MS(2) data were analyzed under nonstandardized, independent experimental conditions. MassBank is a distributed database. Each research group provides data from its own MassBank data servers distributed on the Internet. MassBank users can access either all of the MassBank data or a subset of the data by specifying one or more experimental conditions. In a spectral search to retrieve mass spectra similar to a query mass spectrum, the similarity score is calculated by a weighted cosine correlation in which weighting exponents on peak intensity and the mass-to-charge ratio are optimized to the ESI-MS(2) data. MassBank also provides a merged spectrum for each compound prepared by merging the analyzed ESI-MS(2) data on an identical compound under different collision-induced dissociation conditions. Data merging has significantly improved the precision of the identification of a chemical compound by 21-23% at a similarity score of 0.6. Thus, MassBank is useful for the identification of chemical compounds and the publication of experimental data.

KaPPA-View4: a metabolic pathway database for representation and analysis of correlation networks of gene co-expression and metabolite co-accumulation and omics data

Correlations of gene-to-gene co-expression and metabolite-to-metabolite co-accumulation calculated from large amounts of transcriptome and metabolome data are useful for uncovering unknown functions of genes, functional diversities of gene family members and regulatory mechanisms of metabolic pathway flows. Many databases and tools are available to interpret quantitative transcriptome and metabolome data, but there are only limited ones that connect correlation data to biological knowledge and can be utilized to find biological significance of it. We report here a new metabolic pathway database, KaPPA-View4 (http://kpv.kazusa.or.jp/kpv4/), which is able to overlay gene-to-gene and/or metabolite-to-metabolite relationships as curves on a metabolic pathway map, or on a combination of up to four maps. This representation would help to discover, for example, novel functions of a transcription factor that regulates genes on a metabolic pathway. Pathway maps of the Kyoto Encyclopedia of Genes and Genomes (KEGG) and maps generated from their gene classifications are available at KaPPA-View4 KEGG version (http://kpv.kazusa.or.jp/kpv4-kegg/). At present, gene co-expression data from the databases ATTED-II, COXPRESdb, CoP and MiBASE for human, mouse, rat, Arabidopsis, rice, tomato and other plants are available.

Complete Nucleotide Sequence of Plasmid Rts1: Implications for Evolution of Large Plasmid Genomes

Rts1, a large conjugative plasmid originally isolated from Proteus vulgaris, is a prototype for the IncT plasmids and exhibits pleiotropic thermosensitive phenotypes. Here we report the complete nucleotide sequence of Rts1. The genome is 217,182 bp in length and contains 300 potential open reading frames (ORFs). Among these, the products of 141 ORFs, including 9 previously identified genes, displayed significant sequence similarity to known proteins. The set of genes responsible for the conjugation function of Rts1 has been identified. A broad array of genes related to diverse processes of DNA metabolism were also identified. Of particular interest was the presence of tus-like genes that could be involved in replication termination. Inspection of the overall genome organization revealed that the Rts1 genome is composed of four large modules, providing an example of modular evolution of plasmid genomes.

Metabolonote: a wiki-based database for managing hierarchical metadata of metabolome analyses

Metabolomics – technology for comprehensive detection of small molecules in an organism – lags behind the other “omics” in terms of publication and dissemination of experimental data. Among the reasons for this are difficulty precisely recording information about complicated analytical experiments (metadata), existence of various databases with their own metadata descriptions, and low reusability of the published data, resulting in submitters (the researchers who generate the data) being insufficiently motivated. To tackle these issues, we developed Metabolonote, a Semantic MediaWiki-based database designed specifically for managing metabolomic metadata. We also defined a metadata and data description format, called “Togo Metabolome Data” (TogoMD), with an ID system that is required for unique access to each level of the tree-structured metadata such as study purpose, sample, analytical method, and data analysis. Separation of the management of metadata from that of data and permission to attach related information to the metadata provide advantages for submitters, readers, and database developers. The metadata are enriched with information such as links to comparable data, thereby functioning as a hub of related data resources. They also enhance not only readers’ understanding and use of data but also submitters’ motivation to publish the data. The metadata are computationally shared among other systems via APIs, which facilitate the construction of novel databases by database developers. A permission system that allows publication of immature metadata and feedback from readers also helps submitters to improve their metadata. Hence, this aspect of Metabolonote, as a metadata preparation tool, is complementary to high-quality and persistent data repositories such as MetaboLights. A total of 808 metadata for analyzed data obtained from 35 biological species are published currently. Metabolonote and related tools are available free of cost at http://metabolonote.kazusa.or.jp/.

Wide-range screening of anti-inflammatory compounds in tomato using LC-MS and elucidating the mechanism of their functions

Obesity-induced chronic inflammation is a key factor in type 2 diabetes. A vicious cycle involving pro-inflammatory mediators between adipocytes and macrophages is a common cause of chronic inflammation in the adipose tissue. Tomato is one of the most popular vegetables and is associated with a reduced risk of diabetes. However, the molecular mechanism underlying the effect of tomato on diabetes is unclear. In this study, we focused on anti-inflammatory compounds in tomato. We found that the extract of tomato reduced plasma glucose and inflammatory markers in mice. We screened anti-inflammatory fractions in tomato using lipopolysaccharide-stimulated RAW264.7 macrophages, and active compounds were estimated by liquid chromatography-mass spectrometry over a wide range. Surprisingly, a large number of compounds including oxylipin and coumarin derivatives were estimated as anti-inflammatory compounds. Especially, 9-oxo-octadecadienoic acid and daphnetin suppressed pro-inflammatory cytokines in RAW264.7 macrophages inhibiting mitogen-activated protein kinase phosphorylation and inhibitor of kappa B α protein degradation. These findings suggest that tomato containing diverse anti-inflammatory compounds ameliorates chronic inflammation in obese adipose tissue.

Pathway-specific metabolome analysis with 18O2-labeled Medicago truncatula via a mass spectrometry-based approach

IntroductionOxygen from carbon dioxide, water or molecular oxygen, depending on the responsible enzyme, can lead to a large variety of metabolites through chemical modification.ObjectivesPathway-specific labeling using isotopic molecular oxygen (18O2) makes it possible to determine the origin of oxygen atoms in metabolites and the presence of biosynthetic enzymes (e.g., oxygenases). In this study, we established the basis of 18O2-metabolome analysis.Methods18O2 labeled whole Medicago truncatula seedlings were prepared using 18O2-air and an economical sealed-glass bottle system. Metabolites were analyzed using high-accuracy and high-resolution mass spectrometry. Identification of the metabolite was confirmed by NMR following UHPLC–solid-phase extraction (SPE).ResultsA total of 511 peaks labeled by 18O2 from shoot and 343 peaks from root were annotated by untargeted metabolome analysis. Additionally, we identified a new flavonoid, apigenin 4′-O-[2′-O-coumaroyl-glucuronopyranosyl-(1–2)-O-glucuronopyranoside], that was labeled by 18O2. To the best of our knowledge, this is the first report of apigenin 4′-glucuronide in M. truncatula. Using MSn analysis, we estimated that 18O atoms were specifically incorporated in apigenin, the coumaroyl group, and glucuronic acid. For apigenin, an 18O atom was incorporated in the 4′-hydroxy group. Thus, non-specific incorporation of an 18O atom by recycling during one month of labeling is unlikely compared with the more specific oxygenase-catalyzing reaction.ConclusionOur finding indicated that 18O2 labeling was effective not only for the mining of unknown metabolites which were biosynthesized by oxygenase-related pathway but also for the identification of metabolites whose oxygen atoms were derived from oxygenase activity.

An Improved MatchedIonsFinder Algorithm for Refining ion Feature Assignments among Chromatograms in Liquid Chromatography-Mass Spectrometry

Liquid chromatography-mass spectrometry (LC-MS) analysis is a well-known and powerful tool for metabolite analysis. To identify common or unique metabolites between samples, detected features of identical ions in LC-MS are assigned among chromatograms using analytical software. In general, most ion feature assignments between chromatograms are correct, but some parts of assignments must be corrected. To address this issue, we previously established an algorithm to refine feature assignments and provided a software tool MatchedIonsFinder. In this report, we describe an improved algorithm of MatchedIonsFinder for enhanced accuracy in feature assignments. The new algorithm optimizes MS/MS spectra to specify features of identical ions among chromatograms. Sample order dependency in the new algorithm was also eliminated. The effectiveness of the new algorithm was tested in a metabolome data set.