研究者詳細 - 井原　邦夫

Microevolution toward loss of photosynthesis: Mutations promoting dark-heterotrophic growth and suppressing photosynthetic growth in cyanobacteria

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Shintaro Hida, Marie Nishio, Kazuma Uesaka, Mari Banba, Nobuyuki Takatani, Shinichi Takaichi, Haruki Yamamoto, Kunio Ihara, Yuichi Fujita

2024年4月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Cold Spring Harbor Laboratory

Summary

The prevalence of parasitic plants suggests frequent evolution of photosynthetic capacity loss in the natural environment. However, no studies have observed such evolutionary events as a loss of photosynthetic capacity. Herein, we report mutations that lead to a loss or decrease in photosynthetic growth capacity of dark-adapted variants of the cyanobacteriumLeptolyngbya boryana, which can grow heterotrophically even in the dark. We isolated 28 dark-adapted variants through long-term cultivation (7–49 months) under dark-heterotrophic conditions. All variants showed significantly faster dark-heterotrophic growth than the parental strains, accompanied by the loss of photosynthetic growth capacity in 15 variants. Genome resequencing of the variants revealed that 19 of the 28 variants carried various mutations in a common single gene (LBDG_21500) encoding a protein phosphatase 2C (PP2C) RsbU that is involved in the partner switching system (PSS). Phenotypic and transcriptomic analyses of aLBDG_21500-knockout mutant suggested that the PSS, including LBDG_21500, is involved in the global transcriptional regulation of various genes under both photoautotrophic and dark-heterotrophic conditions. We propose the renaming ofLBDG_21500tophsP(phototrophic–heterotrophic switching phosphatase). Our results imply that mutations in the global transcriptional regulatory system serve as the first evolutionary step leading to the loss of photosynthetic capacity.

Importance

Photosynthetic organisms that grow using minimal resources: light, water, and CO2, support most heterotrophic organisms as producers on the Earth. When photosynthetic organisms thrive over long generations under environments where organic compounds are readily available, they may lose the photosynthetic capacity because of the relief of selective pressure to maintain photosynthesis. The prevalence of parasitic plants in the natural environment supports this idea. However, there have been no actual observations of evolutionary processes leading to a loss of photosynthetic growth capacity. The significance of our research is in observing microevolution of a cyanobacterium through a long-term cultivating under dark heterotrophic conditions. In particular, the high frequency of mutations to a gene involved in the global transcriptional regulatory system suggests that such mutations in regulatory systems are regarded as an example of the initial evolutionary processes toward complete loss of photosynthesis.

DOI： 10.1101/2024.04.08.588626

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Identification of plb1 mutation that extends longevity via activating Sty1 MAPK in Schizosaccharomyces pombe. 査読有り国際誌 Open Access

Maekawa Y, Matsui K, Okamoto K, Shimasaki T, Ohtsuka H, Tani M, Ihara K, Aiba H

Molecular genetics and genomics : MGG 299 巻 ( 1 ) 頁： 20 - 20 2024年2月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Molecular Genetics and Genomics

To understand the lifespan of higher organisms, including humans, it is important to understand lifespan at the cellular level as a prerequisite. So, fission yeast is a good model organism for the study of lifespan. To identify the novel factors involved in longevity, we are conducting a large-scale screening of long-lived mutant strains that extend chronological lifespan (cell survival in the stationary phase) using fission yeast. One of the newly acquired long-lived mutant strains (No.98 mutant) was selected for analysis and found that the long-lived phenotype was due to a missense mutation (92Phe → Ile) in the plb1+ gene. plb1+ gene in fission yeast is a nonessential gene encoding a homolog of phospholipase B, but its functions under normal growth conditions, as well as phospholipase B activity, remain unresolved. Our analysis of the No.98 mutant revealed that the plb1 mutation reduces the integrity of the cellular membrane and cell wall and activates Sty1 via phosphorylation.

DOI： 10.1007/s00438-024-02107-8

Deciphering the genomes of motility-deficient mutants of Vibrio alginolyticus 138-2. 査読有り国際誌 Open Access

Uesaka K, Inaba K, Nishioka N, Kojima S, Homma M, Ihara K

PeerJ 12 巻頁： e17126 2024年

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：PeerJ

The motility of Vibrio species plays a pivotal role in their survival and adaptation to diverse environments and is intricately associated with pathogenicity in both humans and aquatic animals. Numerous mutant strains of Vibrio alginolyticus have been generated using UV or EMS mutagenesis to probe flagellar motility using molecular genetic approaches. Identifying these mutations promises to yield valuable insights into motility at the protein structural physiology level. In this study, we determined the complete genomic structure of 4 reference specimens of laboratory V. alginolyticus strains: a precursor strain, V. alginolyticus 138-2, two strains showing defects in the lateral flagellum (VIO5 and YM4), and one strain showing defects in the polar flagellum (YM19). Subsequently, we meticulously ascertained the specific mutation sites within the 18 motility-deficient strains related to the polar flagellum (they fall into three categories: flagellar-deficient, multi-flagellar, and chemotaxis-deficient strains) by whole genome sequencing and mapping to the complete genome of parental strains VIO5 or YM4. The mutant strains had an average of 20.6 (±12.7) mutations, most of which were randomly distributed throughout the genome. However, at least two or more different mutations in six flagellar-related genes were detected in 18 mutants specifically selected as chemotaxis-deficient mutants. Genomic analysis using a large number of mutant strains is a very effective tool to comprehensively identify genes associated with specific phenotypes using forward genetics.

DOI： 10.7717/peerj.17126

The GGDEF protein Dgc2 suppresses both motility and biofilm formation in the filamentous cyanobacterium Leptolyngbya boryana. 査読有り Open Access

Toida K, Kushida W, Yamamoto H, Yamamoto K, Ishii K, Uesaka K, Kanaly RA, Kutsuna S, Ihara K, Fujita Y, Iwasaki H

Microbiology spectrum 11 巻 ( 5 ) 頁： e0483722 2023年9月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Microbiology Spectrum

Colony pattern formations of bacteria with motility manifest complicated morphological self-organization phenomena. Leptolyngbya boryana is a filamentous cyanobacterium, which has been used as a genetic model organism for studying metabolism including photosynthesis and nitrogen fixation. A widely used type strain [wild type (WT) in this article] of this species has not been reported to show any motile activity. However, we isolated a spontaneous mutant strain that shows active motility (gliding activity) to give rise to complicated colony patterns, including comet-like wandering clusters and disk-like rotating vortices on solid media. Whole-genome resequencing identified multiple mutations in the genome of the mutant strain. We confirmed that inactivation of the candidate gene dgc2 (LBDG_02920) in the WT background was sufficient to give rise to motility and morphologically complex colony patterns. This gene encodes a protein containing the GGDEF motif which is conserved at the catalytic domain of diguanylate cyclase (DGC). Although DGC has been reported to be involved in biofilm formation, the dgc2 mutant significantly facilitated biofilm formation, suggesting a role for the dgc2 gene in suppressing both gliding motility and biofilm formation. Thus, Leptolyngbya is expected to be an excellent genetic model for studying dynamic colony pattern formation and to provide novel insights into the role of DGC family genes in biofilm formation.

DOI： 10.1128/spectrum.04837-22

Two Trk/Ktr/HKT-type potassium transporters, TrkG and TrkH, perform distinct functions in Escherichia coli K-12. 査読有り Open Access

Tanudjaja E, Hoshi N, Yamamoto K, Ihara K, Furuta T, Tsujii M, Ishimaru Y, Uozumi N

The Journal of biological chemistry 299 巻 ( 2 ) 頁： 102846 2023年2月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Journal of Biological Chemistry

Escherichia coli K-12 possesses two versions of Trk/Ktr/HKT-type potassium ion (K+) transporters, TrkG and TrkH. The current paradigm is that TrkG and TrkH have largely identical characteristics, and little information is available regarding their functional differences. Here, we show using cation uptake experiments with K+ transporter knockout mutants that TrkG and TrkH have distinct ion transport activities and physiological roles. K+-transport by TrkG required Na+, whereas TrkH-mediated K+ uptake was not affected by Na+. An aspartic acid located five residues away from a critical glycine in the third pore-forming region might be involved in regulation of Na+-dependent activation of TrkG. In addition, we found that TrkG but not TrkH had Na+ uptake activity. Our analysis of K+ transport mutants revealed that TrkH supported cell growth more than TrkG; however, TrkG was able to complement loss of TrkH-mediated K+ uptake in E. coli. Furthermore, we determined that transcription of trkG in E. coli was downregulated but not completely silenced by the xenogeneic silencing factor H-NS (histone-like nucleoid structuring protein or heat-stable nucleoid-structuring protein). Taken together, the transport function of TrkG is clearly distinct from that of TrkH, and TrkG seems to have been accepted by E. coli during evolution as a K+ uptake system that coexists with TrkH.

DOI： 10.1016/j.jbc.2022.102846

Existence of two substates in the O intermediate of the bacteriorhodopsin photocycle. 査読有り Open Access

Kouyama T, Ihara K

Biochimica et biophysica acta. Biomembranes 1864 巻 ( 10 ) 頁： 183998 2022年10月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Biochimica et Biophysica Acta - Biomembranes

The proton pumping cycle of bacteriorhodopsin (bR) is initiated when the retinal chromophore with the 13-trans configuration is photo-isomerized into the 13-cis configuration. To understand the recovery processes of the initial retinal configuration that occur in the late stage of the photocycle, we have performed a comprehensive analysis of absorption kinetics data collected at various pH levels and at different salt concentrations. The result of analysis revealed the following features of the late stages of the trans photocycle. i) Two substates occur in the O intermediate. ii) The visible absorption band of the first substate (O1) appears at a much shorter wavelength than that of the late substate (O2). iii) O1 is in rapid equilibrium with the preceding state (N), but O1 becomes less stable than N when an ionizable residue (X1) with a pKa value of 6.5 (in 2 M KCl) is deprotonated. iv) At a low pH and at a low salt concentration, the decay time constant of O2 is longer than those of the preceding states, but the relationship between these time constants is altered when the medium pH or the salt concentration is increased. On the basis of the present observations and previous studies on the structure of the chromophore in O, we suspect that the retinal chromophore in O1 takes on a distorted 13-cis configuration and the O1-to-O2 transition is accompanied by cis-to-trans isomerization about C13[dbnd]C14 bond.

DOI： 10.1016/j.bbamem.2022.183998

Two substates in the O intermediate of the light-driven proton pump archaerhodopsin-2. 査読有り Open Access

Kouyama T, Ihara K

Biochimica et biophysica acta. Biomembranes 1864 巻 ( 7 ) 頁： 183919 2022年7月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Biochimica et Biophysica Acta - Biomembranes

The proton pumping cycle of archaerhodopsin-2 (aR2) was investigated over a wide pH range and at different salt concentrations. We have found that two substates, which are spectroscopically and kinetically distinguishable, occur in the O intermediate. The first O-intermediate (O1) absorbs maximumly at ~580 nm, whereas the late O-intermediate (O2) absorbs maximumly at 605 nm. At neutral pH, O1 is in rapid equilibrium with the N intermediate. When the medium pH is increased, O1 becomes less stable than N and, in proportion to the amount of O1 in the dynamic equilibrium between N and O1, the formation rate of O2 decreases. By contrast, the decay rate of O2 increases ~100 folds when the pH of a low-salt membrane suspension is increased from 5.5 to 7.5 or when the salt concentration is increased to 2 M KCl. Together with our recent study on two substates in the O intermediate of bacteriorhodopsin (bR), the present study suggests that the thermally activated re-isomerization of the retinylidene chromophore into the initial all-trans configuration takes place in the O1-to-O2 transition; that is, O1 contains a distorted 13-cis chromophore. It is also found that the pKa value of the key ionizable residue (Asp101aR2, Asp96bR) in the proton uptake channel is elevated in the O1 state of aR2 as compared to the O1 state of bR. This implies that the structural property of O1 in the aR2 photocycle can be investigated over a wide pH range.

DOI： 10.1016/j.bbamem.2022.183919

A slight bending of an α-helix in FliM creates a counterclockwise-locked structure of the flagellar motor in Vibrio. 査読有り

Mutations in the stator protein PomA affect switching of rotational direction in bacterial flagellar motor. 査読有り Open Access

Terashima H, Hori K, Ihara K, Homma M, Kojima S

Scientific reports 12 巻 ( 1 ) 頁： 2979 2022年2月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Scientific Reports

The flagellar motor rotates bi-directionally in counter-clockwise (CCW) and clockwise (CW) directions. The motor consists of a stator and a rotor. Recent structural studies have revealed that the stator is composed of a pentameric ring of A subunits and a dimer axis of B subunits. Highly conserved charged and neighboring residues of the A subunit interacts with the rotor, generating torque through a gear-like mechanism. The rotational direction is controlled by chemotaxis signaling transmitted to the rotor, with less evidence for the stator being involved. In this study, we report novel mutations that affect the switching of the rotational direction at the putative interaction site of the stator to generate rotational force. Our results highlight an aspect of flagellar motor function that appropriate switching of the interaction states between the stator and rotor is critical for controlling the rotational direction.

DOI： 10.1038/s41598-022-06947-5

その他リンク： https://www.nature.com/articles/s41598-022-06947-5

Takekawa N, Nishikino T, Yamashita T, Hori K, Onoue Y, Ihara K, Kojima S, Homma M, Imada K

Journal of biochemistry 170 巻 ( 4 ) 頁： 531 - 538 2021年12月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Journal of Biochemistry

Many bacteria swim by rotating flagella. The chemotaxis system controls the direction of flagellar rotation. Vibrio alginolyticus, which has a single polar flagellum, swims smoothly by rotating the flagellar motor counterclockwise (CCW) in response to attractants. In response to repellents, the motor frequently switches its rotational direction between CCW and clockwise (CW). We isolated a mutant strain that swims with a CW-locked rotation of the flagellum, which pulls rather than pushes the cell. This CW phenotype arises from a R49P substitution in FliM, which is the component in the C-ring of the motor that binds the chemotaxis signalling protein, phosphorylated CheY. However, this phenotype is independent of CheY, indicating that the mutation produces a CW conformation of the C-ring in the absence of CheY. The crystal structure of FliM with the R49P substitution showed a conformational change in the N-terminal α-helix of the middle domain of FliM (FliMM). This helix should mediates FliM-FliM interaction. The structural models of wild type and mutant C-ring showed that the relatively small conformational change in FliMM induces a drastic rearrangement of the conformation of the FliMM domain that generates a CW conformation of the C-ring.

DOI： 10.1093/jb/mvab074

Identification of ksg1 mutation showing long-lived phenotype in fission yeast. 査読有り Open Access

Matsui K, Okamoto K, Hasegawa T, Ohtsuka H, Shimasaki T, Ihara K, Goto Y, Aoki K, Aiba H

Genes to cells : devoted to molecular & cellular mechanisms 26 巻 ( 12 ) 頁： 967 - 978 2021年12月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Genes to Cells

Fission yeast is a good model organism for the study of lifespan. To elucidate the mechanism, we screened for long-lived mutants. We found a nonsense mutation in the ksg1+ gene, which encodes an ortholog of mammalian PDK1 (phosphoinositide-dependent protein kinase). The mutation was in the PH domain of Ksg1 and caused defect in membrane localization and protein stability. Analysis of the ksg1 mutant revealed that the reduced amounts and/or activity of the Ksg1 protein are responsible for the increased lifespan. Ksg1 is essential for growth and known to phosphorylate multiple substrates, but the substrate responsible for the long-lived phenotype of ksg1 mutation is not yet known. Genetic analysis showed that deletion of pck2 suppressed the long-lived phenotype of ksg1 mutant, suggesting that Pck2 might be involved in the lifespan extension caused by ksg1 mutation.

DOI： 10.1111/gtc.12897

Comprehensive investigation of the gene expression system regulated by an Aspergillus oryzae transcription factor XlnR using integrated mining of gSELEX-Seq and microarray data. 査読有り

Identification of sur2 mutation affecting the lifespan of fission yeast. 査読有り国際誌 Open Access

Kurauchi T, Matsui K, Shimasaki T, Ohtsuka H, Tsubouchi S, Ihara K, Tani M, Aiba H

FEMS microbiology letters 368 巻 ( 12 ) 2021年6月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：FEMS Microbiology Letters

Yeast is a suitable model system to analyze the mechanism of lifespan. In this study, to identify novel factors involved in chronological lifespan, we isolated a mutant with a long chronological lifespan and found a missense mutation in the sur2+ gene, which encodes a homolog of Saccharomyces cerevisiae sphingolipid C4-hydroxylase in fission yeast. Characterization of the mutant revealed that loss of sur2 function resulted in an extended chronological lifespan. The effect of caloric restriction, a well-known signal for extending lifespan, is thought to be dependent on the sur2+ gene.

DOI： 10.1093/femsle/fnab070

Loss of cell wall integrity genes cpxA and mrcB causes flocculation in Escherichia coli. 査読有り国際誌 Open Access

Sugawara K, Toyoda H, Kimura M, Hayasaka S, Saito H, Kobayashi H, Ihara K, Ida T, Akaike T, Ando E, Hyodo M, Hayakawa Y, Hamamoto S, Uozumi N

The Biochemical journal 478 巻 ( 1 ) 頁： 41 - 59 2021年1月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Biochemical Journal

Flocculation has been recognized for hundreds of years as an important phenomenon in brewing and wastewater treatment. However, the underlying molecular mechanisms remain elusive. The lack of a distinct phenotype to differentiate between slow-growing mutants and floc-forming mutants prevents the isolation of floc-related gene by conventional mutant screening. To overcome this, we performed a two-step Escherichia coli mutant screen. The initial screen of E. coli for mutants conferring floc production during high salt treatment yielded a mutant containing point mutations in 61 genes. The following screen of the corresponding single-gene mutants identified two genes, mrcB, encoding a peptidoglycan-synthesizing enzyme and cpxA, encoding a histidine kinase of a two-component signal transduction system that contributed to salt tolerance and flocculation prevention. Both single mutants formed flocs during high salt shock, these flocs contained cytosolic proteins. ΔcpxA exhibited decreased growth with increasing floc production and addition of magnesium to ΔcpxA suppressed floc production effectively. In contrast, the growth of ΔmrcB was inconsistent under high salt conditions. In both strains, flocculation was accompanied by the release of membrane vesicles containing inner and outer membrane proteins. Of 25 histidine kinase mutants tested, ΔcpxA produced the highest amount of proteins in floc. Expression of cpxP was up-regulated by high salt in ΔcpxA, suggesting that high salinity and activation of CpxR might promote floc formation. The finding that ΔmrcB or ΔcpxA conferred floc production indicates that cell envelope stress triggered by unfavorable environmental conditions cause the initiation of flocculation in E. coli.

DOI： 10.1042/BCJ20200723

Randomized phase II study comparing the efficacy and safety of SOX versus mFOLFOX6 as neoadjuvant chemotherapy without radiotherapy for locally advanced rectal cancer (KSCC1301). 査読有り Open Access

Miwa K, Oki E, Enomoto M, Ihara K, Ando K, Fujita F, Tominaga M, Mori S, Nakayama G, Shimokawa M, Saeki H, Baba H, Mori M, Akagi Y

BMC cancer 21 巻 ( 1 ) 頁： 23 2021年1月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：BMC Cancer

Background: Preoperative chemoradiotherapy (CRT), the current standard of care for locally advanced rectal cancer (LARC), is associated with many radiotherapy (RT)-related side effects. We aimed to evaluate whether S-1 and oxaliplatin (SOX) or folinic acid, 5-FU, and oxaliplatin (mFOLFOX6) can be as effective as neoadjuvant chemotherapy (NAC) regimens for LARC without RT. Methods: Patients with untreated resectable LARC were randomly assigned to receive SOX or mFOLFOX6. The NAC protocol period was 3 months. The primary endpoint was 3-year disease-free survival (DFS), and the secondary endpoints included pathological effects, surgical completion rate, 3-year survival, and safety. Results: From September 2013 to October 2015, 56 and 54 patients were enrolled in the SOX and mFOLFOX6 arms, respectively. The 3-year DFS rates were 69.4% (95% confidence interval [CI] 54.9–83.6) and 73.4% (95% CI 58.7–83.6) in the SOX and mFOLFOX6 arms, respectively; no significant differences were found between the arms (log-rank test; P = 0.5315, hazard ratio: 0.808, 95% CI 0.414–1.578). The 3-year survival rates were 92.3 and 91.8% in the SOX and mFOLFOX6 arms, respectively. The surgical completion rate was 98.1% overall, 100% in the SOX arm, and 96.0% in the mFOLFOX6 arm. The incidences of pathological response rates ≥grade 1b were 41.5 and 43.8% in the SOX and mFOLFOX6 arms, respectively. Both treatments were manageable and tolerable. Conclusion: We demonstrated the effectiveness and safety of SOX and mFOLFOX6, both of which may be new neoadjuvant treatment candidates in previously untreated LARC cases. Trial registration: Date of enrolment of the first participant to the trial: 3rd Oct 2013; This study was registered in the UMIN clinical trials registry on 14th Aug, 2013. (Prospectively registered, UMIN-CTR number UMIN000011486). https://upload.umin.ac.jp/cgi-open-bin/ctr/ctr.cgi?function=brows&recptno=R000013441&language=J

DOI： 10.1186/s12885-020-07766-5

Haloarcula mannanilytica sp. nov., a galactomannan-degrading haloarchaeon isolated from commercial salt. 査読有り Open Access

Enomoto S, Shimane Y, Ihara K, Kamekura M, Itoh T, Ohkuma M, Takahashi-Ando N, Fukushima Y, Yoshida Y, Usami R, Takai K, Minegishi H

International journal of systematic and evolutionary microbiology 70 巻 ( 12 ) 頁： 6331 - 6337 2020年12月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：International Journal of Systematic and Evolutionary Microbiology

A mannan-degrading halophilic archaeal strain, MD130-1T, was isolated from a commercial salt sample. Cells were motile, rod-shaped, and stained Gram-negative. Colonies were pink pigmented. Strain MD130-1T was able to grow at 1.5–4.6 M NaCl (optimum, 3.6 M) at pH 6.0–8.0 (optimum, pH 7.0) and at 25–50 °C (optimum, 40 °C). The DNA G+C content was 62.1 mol% (genome). The orthologous 16S rRNA gene sequence showed the highest similarity (99.4%) to those of Haloarcula japonica JCM 7785T and Haloarcula hispanica JCM 8911T. The values of genome relatedness between strain MD130-1T and Haloarcula species were 84.33–85.96% in ANIb and 30.4–32.9% using GGDC formula 2. The polar lipids of strain MD130-1T were phosphatidylg-lycerol, phosphatidylglycerol phosphate methyl ester and triglycosyl diether-2. Based on the results of phenotypic and phylogenetic analyses, the strain represents a new species of the genus Haloarcula, for which the name Haloarcula mannanilytica sp. nov. is proposed. The type strain is MD130-1T (=JCM 33835T=KCTC 4287T) isolated from commercial salt made in Ishikawa prefecture, Japan.

DOI： 10.1099/ijsem.0.004535

Energetics and kinetics of substrate analog-coupled staphylococcal nuclease folding revealed by a statistical mechanical approach. 査読有り Open Access

Mizukami T, Furuzawa S, Itoh SG, Segawa S, Ikura T, Ihara K, Okumura H, Roder H, Maki K

Proceedings of the National Academy of Sciences of the United States of America 117 巻 ( 33 ) 頁： 19953 - 19962 2020年8月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Proceedings of the National Academy of Sciences of the United States of America

Protein conformational changes associated with ligand binding, especially those involving intrinsically disordered proteins, are mediated by tightly coupled intra- and intermolecular events. Such reactions are often discussed in terms of two limiting kinetic mechanisms, conformational selection (CS), where folding precedes binding, and induced fit (IF), where binding precedes folding. It has been shown that coupled folding/binding reactions can proceed along both CS and IF pathways with the flux ratio depending on conditions such as ligand concentration. However, the structural and energetic basis of such complex reactions remains poorly understood. Therefore, we used experimental, theoretical, and computational approaches to explore structural and energetic aspects of the coupled-folding/binding reaction of staphylococcal nuclease in the presence of the substrate analog adenosine-3',5'-diphosphate. Optically monitored equilibrium and kinetic data, combined with a statistical mechanical model, gave deeper insight into the relative importance of specific and Coulombic protein-ligand interactions in governing the reaction mechanism. We also investigated structural aspects of the reaction at the residue level using NMR and all-atom replica-permutation molecular dynamics simulations. Both approaches yielded clear evidence for accumulation of a transient protein-ligand encounter complex early in the reaction under IF-dominant conditions. Quantitative analysis of the equilibrium/kinetic folding revealed that the ligand-dependent CS-to-IF shift resulted from stabilization of the compact transition state primarily by weakly ligand-dependent Coulombic interactions with smaller contributions from specific binding energies. At a more macroscopic level, the CS-to-IF shift was represented as a displacement of the reaction "route" on the free energy surface, which was consistent with a flux analysis.

DOI： 10.1073/pnas.1914349117

The role of ROC75 as a daytime component of the circadian oscillator in Chlamydomonas reinhardtii. 査読有り Open Access

Matsuo T, Iida T, Ohmura A, Gururaj M, Kato D, Mutoh R, Ihara K, Ishiura M

PLoS genetics 16 巻 ( 6 ) 頁： e1008814 - e1008814 2020年6月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：PLoS Genetics

The circadian clocks in chlorophyte algae have been studied in two model organisms, Chlamydomonas reinhardtii and Ostreococcus tauri. These studies revealed that the chlorophyte clocks include some genes that are homologous to those of the angiosperm circadian clock. However, the genetic network architectures of the chlorophyte clocks are largely unknown, especially in C. reinhardtii. In this study, using C. reinhardtii as a model, we characterized RHYTHM OF CHLOROPLAST (ROC) 75, a clock gene encoding a putative GARP DNAbinding transcription factor similar to the clock proteins LUX ARRHYTHMO (LUX, also called PHYTOCLOCK 1 [PCL1]) and BROTHER OF LUX ARRHYTHMO (BOA, also called NOX) of the angiosperm Arabidopsis thaliana. We observed that ROC75 is a day/subjective day-phase-expressed nuclear-localized protein that associates with some night-phased clock genes and represses their expression. This repression may be essential for the gating of reaccumulation of the other clock-related GARP protein, ROC15, after its light-dependent degradation. The restoration of ROC75 function in an arrhythmic roc75 mutant under constant darkness leads to the resumption of circadian oscillation from the subjective dawn, suggesting that the ROC75 restoration acts as a morning cue for the C. reinhardtii clock. Our study reveals a part of the genetic network of C. reinhardtii clock that could be considerably different from that of A. thaliana.

DOI： 10.1371/journal.pgen.1008814

Presynaptic MAST kinase controls opposing postsynaptic responses to convey stimulus valence in Caenorhabditis elegans. 査読有り国際誌 Open Access

Nakano S, Ikeda M, Tsukada Y, Fei X, Suzuki T, Niino Y, Ahluwalia R, Sano A, Kondo R, Ihara K, Miyawaki A, Hashimoto K, Higashiyama T, Mori I

Proceedings of the National Academy of Sciences of the United States of America 117 巻 ( 3 ) 頁： 1638 - 1647 2020年1月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Proceedings of the National Academy of Sciences of the United States of America

Presynaptic plasticity is known to modulate the strength of synaptic transmission. However, it remains unknown whether regulation in presynaptic neurons can evoke excitatory and inhibitory postsynaptic responses.We report here that the Caenorhabditis elegans homologs of MAST kinase, Stomatin, and Diacylglycerol kinase act in a thermosensory neuron to elicit in its postsynaptic neuron an excitatory or inhibitory response that correlates with the valence of thermal stimuli. By monitoring neural activity of the valence-coding interneuron in freely behaving animals, we show that the alteration between excitatory and inhibitory responses of the interneuron is mediated by controlling the balance of two opposing signals released from the presynaptic neuron. These alternative transmissions further generate opposing behavioral outputs necessary for the navigation on thermal gradients. Our findings suggest that valence-encoding interneuronal activity is determined by a presynaptic mechanism whereby MAST kinase, Stomatin, and Diacylglycerol kinase influence presynaptic outputs.

DOI： 10.1073/pnas.1909240117

gas1 mutation extends chronological lifespan via Pmk1 and Sty1 MAPKs in Schizosaccharomyces pombe 査読有り

Imai Yuki, Shimasaki Takafumi, Enokimura Chihiro, Ohtsuka Hokuto, Tsubouchi Satoshi, Ihara Kunio, Aiba Hirofumi

BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY 頁： 1-8 2019年10月

The mechanosensitive channel YbdG from Escherichia coli has a role in adaptation to osmotic up-shock. 査読有り

Amemiya S, Toyoda H, Kimura M, Saito H, Kobayashi H, Ihara K, Kamagata K, Kawabata R, Kato S, Nakashimada Y, Furuta T, Hamamoto S, Uozumi N

The Journal of biological chemistry 2019年6月

Cholecystokinin induces crowing in chickens 査読有り国際誌 Open Access

Shimmura, T; Tamura, M; Ohashi, S; Sasaki, A; TakamichiYamanaka; Nakao, N; Ihara, K; Okamura, S; TakashiYoshimura

SCIENTIFIC REPORTS 9 巻 ( 1 ) 頁： 3978 2019年3月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Scientific Reports

Animals that communicate using sound are found throughout the animal kingdom. Interestingly, in contrast to human vocal learning, most animals can produce species-specific patterns of vocalization without learning them from their parents. This phenomenon is called innate vocalization. The underlying molecular basis of both vocal learning in humans and innate vocalization in animals remains unknown. The crowing of a rooster is also innately controlled, and the upstream center is thought to be localized in the nucleus intercollicularis (ICo) of the midbrain. Here, we show that the cholecystokinin B receptor (CCKBR) is a regulatory gene involved in inducing crowing in roosters. Crowing is known to be a testosterone (T)-dependent behavior, and it follows that roosters crow but not hens. Similarly, T-administration induces chicks to crow. By using RNA-sequencing to compare gene expression in the ICo between the two comparison groups that either crow or do not crow, we found that CCKBR expression was upregulated in T-containing groups. The expression of CCKBR and its ligand, cholecystokinin (CCK), a neurotransmitter, was observed in the ICo. We also showed that crowing was induced by intracerebroventricular administration of an agonist specific for CCKBR. Our findings therefore suggest that the CCK system induces innate vocalization in roosters.

DOI： 10.1038/s41598-019-40746-9

Oka H, Kojima T, Ihara K, Kobayashi T, Nakano H

BMC genomics 20 巻 ( 1 ) 頁： 16 2019年1月

Obesity and mental health improvement following nutritional education focusing on gut microbiota composition in Japanese women: a randomised controlled trial. 査読有り

Uemura M, Hayashi F, Ishioka K, Ihara K, Yasuda K, Okazaki K, Omata J, Suzutani T, Hirakawa Y, Chiang C, Aoyama A, Ohira T

European journal of nutrition 2018年12月

Three-Step Isomerization of the Retinal Chromophore during the Anion Pumping Cycle of Halorhodopsin. 査読有り国際誌

Kouyama T, Ihara K, Maki K, Chan SK

Biochemistry 57 巻 ( 41 ) 頁： 6013 - 6026 2018年10月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Biochemistry

The anion pumping cycle of halorhodopsin from Natronomonas pharaonis (pHR) is initiated when the all-trans/15-anti isomer of retinal is photoisomerized into the 13-cis/15-anti configuration. A recent crystallographic study suggested that a reaction state with 13-cis/15-syn retinal occurred during the anion release process, i.e., after the N state with the 13-cis/15-anti retinal and before the O state with all-trans/15-anti retinal. In this study, we investigated the retinal isomeric composition in a long-living reaction state at various bromide ion concentrations. It was found that the 13-cis isomer (csHR′), in which the absorption spectrum was blue-shifted by ∼8 nm compared with that of the trans isomer (taHR), accumulated significantly when a cold suspension of pHR-rich claret membranes in 4 M NaBr was illuminated with continuous light. Analysis of flash-induced absorption changes suggested that the branching of the trans photocycle into the 13-cis isomer (csHR′) occurs during the decay of an O-like state (O′) with 13-cis/15-syn retinal; i.e., O′ can decay to either csHR′ or O with all-trans/15-anti retinal. The efficiency of the branching reaction was found to be dependent on the bromide ion concentration. At a very high bromide ion concentration, the anion pumping cycle is described by the scheme taHR -(hν) → K → L1a ↔L1b ↔N ↔N′ ↔O′ ↔csHR′ ↔taHR. At a low bromide ion concentration, on the other hand, O′ decays into taHR via O.

DOI： 10.1021/acs.biochem.8b00631

Draft Genome Sequence of Burkholderia vietnamiensis Strain RS1, a Nitrogen-Fixing Endophyte Isolated from Sweet Potato. 査読有り国際誌 Open Access

Shinjo R, Uesaka K, Ihara K, Sakazaki S, Yano K, Kondo M, Tanaka A

Microbiology resource announcements 7 巻 ( 3 ) 2018年7月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Microbiology Resource Announcements

Burkholderia vietnamiensis strain RS1 is an endophytic bacterium with nitrogen-fixing ability that was isolated from tuberous roots of sweet potato. Here, we present its draft genome of 6,542,727 bases that contains a cluster of genes associated with nitrogen fixation. This genome sequence will provide important insights into the plant growth-promoting potential of endophytic bacteria.

DOI： 10.1128/MRA.00820-18

In vivo transposon tagging in the nonheterocystous nitrogen-fixing cyanobacterium Leptolyngbya boryana

Tomatsu Chie, Uesaka Kazuma, Yamakawa Hisanori, Tsuchiya Tohru, Ihara Kunio, Fujita Yuichi

FEBS LETTERS 592 巻 ( 10 ) 頁： 1634-1642 2018年5月

Co-expression of the calcitonin receptor gene in the hypothalamic kisspeptin neurons in female rats 査読有り

Assadullah, Nahoko Ieda, Narumi Kawai, Hirotaka Ishii, Kunio Ihara, Naoko Inoue, Yoshihisa Uenoyama, Hiroko Tsukamura

Reprod. Med. Biol. 2018年

SLO potassium channels antagonize premature decision making in C. elegans. 査読有り

Aoki I, Tateyama M, Shimomura T, Ihara K, Kubo Y, Nakano S, Mori I

Communications biology 1 巻頁： 123 2018年

Fulminant type 1 diabetes mellitus in Japanese children and adolescents: Multi-institutional joint research of the Japanese study group of insulin therapy for childhood and adolescent diabetes

Shiga K., Urakami T., Suzuki J., Igarashi Y., Tajima H., Amemiya S., Sugihara S., Takahashi I., Tanahashi Y., Matsuo K., Iwamoto J., Yatsuga S., Endo A., Takemoto K., Hamada J., Ihara K., Ohki Y., Takaya R., Mochizuki T., Kawamura T., Horita Y., Furujo M., Inoue M., Hasegawa K., Adachi M., Okajima M., Shimura K., Ohtsu S., Kitsuda K., Ito Y., Hori T., Ishii K., Kawada Y., Nakajima H., Ito I., Nakamura K., Miyagawa S., Jinno K., Horikawa R., Yoshida T., Fukami M., Mori T., Kobayashi K., Konda S., Sasaki N., Kikuchi T., Musha I., Aizu K., Mochizuki H., Hotsubo T., Sato K., Yamamoto Y., Kadowaki H., Nishii A., Matsui K., Yokota I., Miyoshi T., Takubo N., Hara Y., Matsuura N., Miyamoto S., Soneda S., Takahashi T., Tsubouchi K., Tatematsu T., Minamitani K., Ogawa E., Izumi Y., Sasaki G., Tachikawa E., Uchigata Y., Yasuda Y., Sasaki K., Kitanaka S., Isojima T., Fujiwara I., Kanno J., Kotani Y., Ayabe T., Shimura N., Ichikawa J., Hanaki K., Nagaishi J., Kanzaki S., Fujimoto M., Ito J., Mizuno H., Abe Y., Nagasaki K., Ogawa Y., Kadotani S., Kashihara Y., Okuno M., Ogata T., Ohtaka K., Miyako K., Kohno H., Fujisawa T., Suga S., Koike A.

Endocrine Journal 65 巻 ( 8 ) 頁： 795 - 803 2018年

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Endocrine Journal

Fulminant type 1 diabetes mellitus (FT1DM) is a subtype of type 1 diabetes mellitus characterized by a remarkably abrupt onset. In Japan, FT1DM accounts for approximately 20% of acute-onset adult type 1 diabetes mellitus cases; however, reports of pediatric-onset FT1DM are rare. We aimed to determine the frequency and clinical characteristics of FT1DM in Japanese children and adolescents by conducting a 2-phase questionnaire survey among the members of the Japanese Study Group of Insulin Therapy for Childhood and Adolescent Diabetes (JSGIT) regarding their clinical experience with FT1DM. Responses were obtained from 54 of the 79 participating hospitals (68.4%). Of these, 8 hospitals managed a total of 15 pediatric patients with FT1DM (4 patients in each of 2 hospitals, 2 patients in 1 hospital, and 1 patient in each of 5 hospitals). The distribution of patient age was biphasic, with peaks in children younger than 5 years and older than 8 years of age. The clinical characteristics of FT1DM in this population (such as the duration from onset of symptoms to diagnosis, severity of symptoms, preceding flu-like episodes, and abnormal laboratory data) did not differ from those of patients with adult-onset FT1DM. The frequency of pediatric-onset FT1DM is low compared with that of adult-onset FT1DM. The genetic background and susceptibility patterns of pediatric patients with FT1DM may differ from those typical of adults with FT1DM, but both age groups share similar clinical characteristics.

DOI： 10.1507/endocrj.EJ17-0029

Incidence rate and characteristics of symptomatic vitamin d deficiency in children: A nationwide survey in Japan Open Access

Kubota T., Nakayama H., Kitaoka T., Nakamura Y., Fukumoto S., Fujiwara I., Hasegawa Y., Ihara K., Kitanaka S., Koyama S., Kusuda S., Mizuno H., Nagasaki K., Oba K., Sakamoto Y., Takubo N., Shimizu T., Tanahashi Y., Hasegawa K., Tsukahara H., Yorifuji T., Michigami T., Ozono K.

Endocrine Journal 65 巻 ( 6 ) 頁： 593 - 599 2018年

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Endocrine Journal

There is concern that vitamin D deficiency is prevalent among children in Japan as well as worldwide. We conducted a nationwide epidemiologic survey of symptomatic vitamin D deficiency to observe its incidence rate among Japanese children. A questionnaire inquiring the number of new patients with vitamin D deficiency rickets and/or hypocalcemia for 3 years was sent to 855 randomly selected hospitals with a pediatrics department in Japan. In this survey, we found that 250 children were diagnosed with symptomatic vitamin D deficiency. The estimated number of patients with symptomatic vitamin D deficiency per year was 183 (95% confidence interval (CI): 145–222). The overall annual incidence rate among children under 15 years of age was 1.1 per 100,000 population (95% CI: 0.9–1.4). The second survey has provided detailed information on 89 patients with symptomatic vitamin D deficiency under 5 years of age in hospitals in the current research group. The nationwide and second surveys estimated the overall annual incidence rate of symptomatic vitamin D deficiency in children under 5 years of age to be 3.5 (2.7–4.2) per 100,000 population. The second survey revealed 83% had bowed legs, 88% had exclusive breastfeeding, 49% had a restricted and/or unbalanced diet and 31% had insufficient sun exposure among the 89 patients. This is the first nationwide survey on definitive clinical vitamin D deficiency in children in Japan. Elucidating the frequency and characteristics of symptomatic vitamin D deficiency among children is useful to develop preventative public health strategies.

DOI： 10.1507/endocrj.EJ18-0008

Open Access