Published papers

The gut microbiota plays a crucial role in maintaining health. Monitoring the complex dynamics of its microbial population is, therefore, important. Here, we present a deep convolution network that can characterize the dynamic changes in the gut microbiota using low-resolution images of fecal samples. Further, we demonstrate that the microbial relative abundances, quantified via 16S rRNA amplicon sequencing, can be quantitatively predicted by the neural network. Our approach provides a simple and inexpensive method of gut microbiota analysis.

There have been a number of reports about the transcriptional regulatory networks in schizophrenia. However, most of these studies were based on a specific transcription factor or a single dataset, an approach that is inadequate to understand the diverse etiology and underlying common characteristics of schizophrenia. Here we reconstructed and compared the transcriptional regulatory network for lipid metabolism enzymes using 15 public transcriptome datasets of neural cells from schizophrenia patients. Since many of the well-known schizophrenia-related SNPs are in enhancers, we reconstructed a network including enhancer-dependent regulation and found that 53.3 % of the total number of edges (7,577 pairs) involved regulation via enhancers. By examining multiple datasets, we found common and unique transcriptional modes of regulation. Furthermore, enrichment analysis of SNPs that were connected with genes in the transcriptional regulatory networks by eQTL suggested an association with hematological cell counts and some other traits/diseases, whose relationship to schizophrenia was either not or insufficiently reported in previous studies. Based on these results, we suggest that in future studies on schizophrenia, information on genotype, comorbidities and hematological cell counts should be included, along with the transcriptome, for a more detailed genetic stratification and mechanistic exploration of schizophrenia.

Protein phosphorylation on serine, threonine, and tyrosine is well established as a crucial regulatory posttranslational modification in eukaryotes. With the recent whole-genome sequencing projects reporting the presence of serine/threonine kinases and two-component proteins both in prokaryotes and eukaryotes, the importance of protein phosphorylation in archaea and bacteria is gaining acceptance. While conventional biochemical methods failed to obtain a snapshot of the bacterial phosphoproteomes, advances in MS methods have paved the way for in-depth mapping of phosphorylation sites. Here, we present phosphoproteomes of two ecologically diverse non-enteric Gram-negative bacteria captured by a nanoLC-MS-based approach combined with a novel phosphoenrichment method. While the phosphoproteome data from the two species are not very similar, the results reflect high similarity to the previously published dataset in terms of the pathways the phosphoproteins belong to. This study additionally provides evidence to prior observations that protein phosphorylation is common in bacteria. Notably, phosphoproteins identified in Pseudomonas aeruginosa belong to motility, transport, and pathogenicity pathways that are critical for survival and virulence. We report, for the first time, that motility regulator A, probably acting via the novel secondary messenger cyclic diguanylate monophosphate, significantly affects protein phosphorylation in Pseudomonas putida. ©2009 Wiley-VCH Verlag GmbH & Co. KGaA.

Recent phosphoproteome analyses using mass spectrometry-based technologies have provided new insights into the extensive presence of protein phosphorylation in various species and have raised the interesting question of how this protein modification was gained evolutionarily on such a large scale. We investigated this issue by using human and mouse phosphoproteome data. We initially found that phosphoproteins followed a power-law distribution with regard to their number of phosphosites: most of the proteins included only a few phosphosites, but some included dozens of phosphosites. The power-law distribution, unlike more commonly observed distributions such as normal and log-normal distributions, is considered by the field of complex systems science to be produced by a specific rich-get-richer process called preferential attachment growth. Therefore, we explored the factors that may have promoted the rich-get-richer process during phosphosite evolution. We conducted a bioinformatics analysis to evaluate the relationship of amino acid sequences of phosphoproteins with the positions of phosphosites and found an overconcentration of phosphosites in specific regions of protein surfaces and implications that in many phosphoproteins these clusters of phosphosites are activated simultaneously. Multiple phosphosites concentrated in limited spaces on phosphoprotein surfaces may therefore function biologically as cooperative modules that are resistant to selective pressures during phosphoprotein evolution. We therefore proposed a hypothetical model by which the modularization of multiple phosphosites has been resistant to natural selection and has driven the rich-get-richer process of the evolutionary growth of phosphosite numbers. © 2009 by The American Society for Biochemistry and Molecular Biology, Inc.

In bacterial circular chromosomes and most plasmids, the replication is known to be terminated when either of the following occurs: the forks progressing in opposite directions meet at the distal end of the chromosome or the replication forks become trapped by Tus proteins bound to Ter sites. Most bacterial genomes have various polarities in their genomic structures. The most notable feature is polar genomic compositional asymmetry of the bases G and C in the leading and lagging strands, called GC skew. This asymmetry is caused by replication-associated mutation bias, and this "footprint" of the replication machinery suggests that, in contrast to the two known mechanisms, replication termination occurs near the chromosome dimer resolution site dif. To understand this difference between the known replication machinery and genomic compositional bias, we undertook a simulation study of genomic mutations, and we report here how different replication termination models contribute to the generation of replication-related genomic compositional asymmetry. Contrary to naive expectations, our results show that a single finite termination site at dif or at the GC skew shift point is not sufficient to reconstruct the genomic compositional bias as observed in published sequences. The results also show that the known replication mechanisms are sufficient to explain the position of the GC skew shift point. © 2012 Kono et al.

Aconitase B (AcnB) and isocitrate dehydrogenase (ICDH) catalyze the sequential reaction in the Krebs cycle. Since each enzyme was characterized as an independent protein in a diluted condition, the catalytic performance within the cellular metabolism remains unclear. In particular, high macromolecular concentration in the cytosol promotes weak interactions, which affects structure and function of the proteins. We found that the two bacterial enzymes exhibit variable catalytic performance of the sequential reaction, depending on the oligomerization state. The small-angle solution X-ray scattering and the chemical crosslinking analyses revealed that not only the two enzymes but also the fusion protein of the two enzymes assume homodimers in solution. Interestingly, the fusion protein maintains the homodimeric architecture of ICDH, but not AcnB. Instead, one of the two monomeric AcnB regions associates with the homodimeric ICDH region. The fusion protein displayed different catalytic performance of the sequential reaction from that observed in the mixture of the AcnB and ICDH proteins in an equimolar ratio. Connecting the two proteins by a flexible linker yielded a locally high concentration to promote the weak protein-protein interaction. The versatile architecture of AcnB may alter the metabolic process involving the Krebs cycle. © 2008 Elsevier B.V. All rights reserved.

The oriC DNA replication origin in bacterial chromosomes, the location of which appears to be physically identified, is genetically regulated by relevant molecular machinery. In contrast, the location of the terminus remains obscure for many bacterial replicons, except for terC, the proposed and well-studied chromosome termination site in certain bacteria. The terC locus, which is composed of specific sequences for its binding protein, is located at a site opposite from oriC, exhibiting a symmetric structure around the oriC-terC axis. Here, we investigated Bacillus subtilis 168 strains whose axes were hindered and found that the native terC function was robust. However, eradication of terminus region specific binding protein resulted in the natural terC sites not being used for termination; instead, new termini were selected at a location exactly opposite to oriC. We concluded that replication generally terminates at the loci where the two approaching replisomes meet. This site was automatically selected, and two replisomes moving at the same rate supported symmetrical chromosome structures relative to oriC. The rule, which was even validated by artificial chromosomes irrespective of oriC, should be general for replicons administered by two replisomes. © 2014 The Authors.

Molecular heterogeneity of individual molecules within single cells has been recently shown to be crucial for cell fate diversifications. However, on a global scale, the effect of molecular variability for embryonic developmental stages is largely underexplored. Here, to understand the origins of transcriptome-wide variability of oocytes to blastocysts in human and mouse, we examined RNA-Seq datasets. Evaluating Pearson correlation, Shannon entropy and noise patterns (•2 vs.μ), our investigations reveal a phase transition from low to saturating levels of diversity and variability of transcriptome-wide expressions through the development stages. To probe the observed behaviour further, we utilised a stochastic transcriptional model to simulate the global gene expressions pattern for each development stage. From the model, we concur that transcriptome-wide regulation initially begins from 2-cell stage, and becomes strikingly variable from 8-cell stage due to amplification and quantal transcriptional activity.

The extremely thermophilic bacterium Thermus thermophilus is a model organism for structural biology and systems biology, and the so-called "Structural and Functional Whole-Cell Project for T. thermophilus HB8" is in progress. The released genomic sequence of the strain HB8 is composed of chromosome, pTT27 megaplasmid, and pTT8 plasmid. In this paper, however, a third plasmid was demonstrated and its sequence was determined. Although this plasmid pVV8 had been reported before, limited information and an unfortunate dropout in the substrain, whose genomic sequence was determined, would have prevented the plasmid from coming to public attention. The intrinsic circular plasmid, which was estimated to be six to ten copies in a cell, is 81151 bp and its G + C content is 68%. Among the identified 91 ORFs, a single gene has been experimentally analyzed before and is known as xylose isomerase. The phnCDEGHIJKLMX operon related to phosphonate metabolism, alkaline phosphatase, putative transcriptional regulators, several sets of toxin-antitoxin system, and transposase-like ORFs are also encoded on the pVV8 plasmid. Although association with cell aggregation was the one phenotypic characteristic of the plasmid that had been reported, it was never confirmed. Comparison of T. thermophilus HB8 strains suggests that the pVV8 is nonessential for growth. © 2011 Springer.

Background: Transcription promoters are fundamental genomic cis-elements controlling gene expression. They can be classified into two types by the degree of imprecision of their transcription start sites: peak promoters, which initiate transcription from a narrow genomic region; and broad promoters, which initiate transcription from a wide-ranging region. Eukaryotic transcription initiation is suggested to be associated with the genomic positions and modifications of nucleosomes. For instance, it has been recently shown that histone with H3K9 acetylation (H3K9ac) is more likely to be distributed around broad promoters rather than peak promoters; it can thus be inferred that there is an association between histone H3K9 and promoter architecture.Results: Here, we performed a systematic analysis of transcription promoters and gene expression, as well as of epigenetic histone behaviors, including genomic position, stability within the chromatin, and several modifications. We found that, in humans, broad promoters, but not peak promoters, generally had significant associations with nucleosome positioning and modification. Specifically, around broad promoters histones were highly distributed and aligned in an orderly fashion. This feature was more evident with histones that were methylated or acetylated; moreover, the nucleosome positions around the broad promoters were more stable than those around the peak ones. More strikingly, the overall expression levels of genes associated with broad promoters (but not peak promoters) with modified histones were significantly higher than the levels of genes associated with broad promoters with unmodified histones.Conclusion: These results shed light on how epigenetic regulatory networks of histone modifications are associated with promoter architecture. © 2011 Nozaki et al; licensee BioMed Central Ltd.

Despite the widespread availability of genome sequences and the systematic assignment of individual open reading frames, many of the activities of metabolic enzymes are still veiled. It is difficult to assign biochemical functions to enzymes and uncover the in vivo regulation of their enzymatic activities, but this is crucial to understand cellular functions. The development of metabolomics and metabolite profiling, which can globally analyze intracellular and extracellular metabolites, has provided some valuable tools for systems biology. This report reviews the recent metabolomics approaches to elucidate unidentified metabolic systems while focusing on their enzymatic activities, including their biochemical and biological functions, and their regulation in response to environmental perturbations in microorganisms. © 2010 Elsevier Ltd. All rights reserved.

Background: During transcription, the nontranscribed DNA strand becomes single-stranded DNA (ssDNA), which can form secondary structures. Unpaired bases in the ssDNA are less protected from mutagens and hence experience more mutations than do paired bases. These mutations are called transcription-associated mutations. Transcription-associated mutagenesis is increased under stress and depends on the DNA sequence. Therefore, selection might significantly influence protein-coding sequences in terms of the transcription-associated mutability per transcription event under stress to improve the survival of Escherichia coli. Methodology/Principal Findings: The mutability index (MI) was developed by Wright et al. to estimate the relative transcription-associated mutability of bases per transcription event. Using the most stable fold of each ssDNA that have an average length n, MI was defined as (the number of folds in which the base is unpaired)/n ×(highest -ΔG of all n folds in which the base is unpaired), where ΔG is the free energy. The MI values show a significant correlation with mutation data under stress but not with spontaneous mutations in E. coli. Protein sequence diversity is preferred under stress but not under favorable conditions. Therefore, we evaluated the selection pressure on MI in terms of the protein sequence diversity for all the protein-coding sequences in E. coli. The distributions of the MI values were lower at bases that could be substituted with each of the other three bases without affecting the amino acid sequence than at bases that could not be so substituted. Start codons had lower distributions of MI values than did nonstart codons. Conclusions/Significance: Our results suggest that the majority of protein-coding sequences have evolved to promote protein sequence diversity and to reduce gene knockout under stress. Consequently, transcription-associated mutagenesis increases protein sequence diversity more effectively than does random mutagenesis under stress. Nonrandom transcription-associated mutagenesis under stress should improve the survival of E. coli. © 2010 Kim et al.

Background: Phosphorylation is a ubiquitous and fundamental regulatory mechanism that controls signal transduction in living cells. The number of identified phosphoproteins and their phosphosites is rapidly increasing as a result of recent mass spectrometry-based approaches.Results: We analyzed time-course phosphoproteome data obtained previously by liquid chromatography mass spectrometry with the stable isotope labeling using amino acids in cell culture (SILAC) method. This provides the relative phosphorylation activities of digested peptides at each of five time points after stimulating HeLa cells with epidermal growth factor (EGF). We initially calculated the correlations between the phosphorylation dynamics patterns of every pair of peptides and connected the strongly correlated pairs to construct a network. We found that peptides extracted from the same intracellular fraction (nucleus vs. cytoplasm) tended to be close together within this phosphorylation dynamics-based network. The network was then analyzed using graph theory and compared with five known signal-transduction pathways. The dynamics-based network was correlated with known signaling pathways in the NetPath and Phospho.ELM databases, and especially with the EGF receptor (EGFR) signaling pathway. Although the phosphorylation patterns of many proteins were drastically changed by the EGF stimulation, our results suggest that only EGFR signaling transduction was both strongly activated and precisely controlled.Conclusions: The construction of a phosphorylation dynamics-based network provides a useful overview of condition-specific intracellular signal transduction using quantitative time-course phosphoproteome data under specific experimental conditions. Detailed prediction of signal transduction based on phosphoproteome dynamics remains challenging. However, since the phosphorylation profiles of kinase-substrate pairs on the specific pathway were localized in the dynamics-based network, our method will be a complementary strategy to explore new components of protein signaling pathways in combination with previous methods (including software) of predicting direct kinase-substrate relationships. © 2010 Imamura et al; licensee BioMed Central Ltd.

Transfer RNA (tRNA) is essential for decoding the genome sequence into proteins. In Archaea, previous studies have revealed unique multiple intron-containing tRNAs and tRNAs that are encoded on 2 separate genes, so-called split tRNAs. Here, we discovered 10 fragmented tRNA genes in the complete genome of the hyperthermoacidophilic Archaeon Caldivirga maquilingensis that are individually transcribed and further trans-spliced to generate all of the missing tRNAs encoding glycine, alanine, and glutamate. Notably, the 3 mature tRNAGly's with synonymous codons are created from 1 constitutive 3′ half transcript and 4 alternatively switching transcripts, representing tRNA made from a total of 3 transcripts named a "tri-split tRNA." Expression and nucleotide sequences of 10 split tRNA genes and their joined tRNA products were experimentally verified. The intervening sequences of split tRNA have high identity to tRNA intron sequences located at the same positions in ifntron-containing tRNAs in related Thermoproteales species. This suggests that an evolutionary relationship between intron-containing and split tRNAs exists. Our findings demonstrate the first example of split tRNA genes in a free-living organism and a unique tri-split tRNA gene that provides further insight into the evolution of fragmented tRNAs. © 2009 by The National Academy of Sciences of the USA.

Background: To elucidate the interaction of dynamics among modules that constitute biological systems, comprehensive datasets obtained from "omics" technologies have been used. In recent plant metabolomics approaches, the reconstruction of metabolic correlation networks has been attempted using statistical techniques. However, the results were unsatisfactory and effective data-mining techniques that apply appropriate comprehensive datasets are needed. Results: Using capillary electrophoresis mass spectrometry (CE-MS) and capillary electrophoresis diode-array detection (CE-DAD), we analyzed the dynamic changes in the level of 56 basic metabolites in plant foliage (Oryza sativa L. ssp. japonica) at hourly intervals over a 24-hr period. Unsupervised clustering of comprehensive metabolic profiles using Kohonen's self-organizing map (SOM) allowed classification of the biochemical pathways activated by the light and dark cycle. The carbon and nitrogen (C/N) metabolism in both periods was also visualized as a phenotypic linkage map that connects network modules on the basis of traditional metabolic pathways rather than pairwise correlations among metabolites. The regulatory networks of C/N assimilation/dissimilation at each time point were consistent with previous works on plant metabolism. In response to environmental stress, glutathione and spermidine fluctuated synchronously with their regulatory targets. Adenine nucleosides and nicotinamide coenzymes were regulated by phosphorylation and dephosphorylation. We also demonstrated that SOM analysis was applicable to the estimation of unidentifiable metabolites in metabolome analysis. Hierarchical clustering of a correlation coefficient matrix could help identify the bottleneck enzymes that regulate metabolic networks. Conclusion: Our results showed that our SOM analysis with appropriate metabolic time-courses effectively revealed the synchronous dynamics among metabolic modules and elucidated the underlying biochemical functions. The application of discrimination of unidentified metabolites and the identification of bottleneck enzymatic steps even to non-targeted comprehensive analysis promise to facilitate an understanding of large-scale interactions among components in biological systems. © 2008 Sato et al; licensee BioMed Central Ltd.

Hsp70 chaperones assist protein folding by cycling between the ATP-bound T state with low affinity for substrates and the ADP-bound R state with high affinity for substrates. The transition from the T to R state is catalyzed by the synergistic action of the substrate and DnaJ cochaperones. The reverse transition from the R state to the T state is accelerated by the nucleotide exchange factor GrpE. These two processes, T-to-R and R-to-T conversion, are affected differently by temperature change. Here we modeled Hsp70-mediated protein folding under permanent and transient heat shock based on published experimental data. Our simulation results were in agreement with in vitro wild-type Escherichia coli chaperone experimental data at 25°C and reflected R-to-T ratio dynamics in response to temperature effects. Our simulation results suggested that the chaperone system evolved naturally to maintain the concentration of active protein as high as possible during heat shock, even at the cost of recovered activity after return to optimal growth conditions. They also revealed that the chaperone system evolved to suppress ATP consumption at non-optimal high growing temperatures. © Springer Science + Business Media B.V. 2007.

Thymidine phosphorylase (TP), a rate-limiting enzyme in thymidine catabolism, plays a pivotal role in tumor progression; however, the mechanisms underlying this role are not fully understood. Here, we found that TP-mediated thymidine catabolism could supply the carbon source in the glycolytic pathway and thus contribute to cell survival under conditions of nutrient deprivation. In TP-expressing cells, thymidine was converted to metabolites, including glucose 6-phosphate, lactate, 5-phospho-α-D-ribose 1-diphosphate, and serine, via the glycolytic pathway both in vitro and in vivo. These thymidine-derived metabolites were required for the survival of cells under low-glucose conditions. Furthermore, activation of thymidine catabolism was observed in human gastric cancer. These findings demonstrate that thymidine can serve as a glycolytic pathway substrate in human cancer cells.

© 2018 Japan society for Bioscience, Biotechnology, and agrochemistry. Bacillus subtilis 168 was developed as a genome vector to manipulate large DNA fragments. The system is based on the inherent natural transformation (TF) activity. However, DNA size transferred by TF is limited up to approximately 100 kb. A conjugal transfer system capable of transferring DNA fragments considerably larger than those transferred by TF was developed. A well-defined oriT110 sequence and a cognate relaxase gene from the pUB110 plasmid were inserted into the xkdE gene of the B. subtilis genome. Transfer of antibiotic resistance markers distant from the oriT110 locus to the recipient B. subtilis occurred only in the presence of pLS20, a helper plasmid that provides a type IV secretion system. Marker transmission was consistent with the orientation of oriT110 and required a recA-proficient recipient. The first conjugal transfer system of genomic DNA should provide a valuable alternative genetic tool for editing the B. subtilis genome.