Language：Japanese   Publishing type：Research paper (scientific journal)   Publisher：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

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Springer Science and Business Media LLC  

Abstract

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

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Other Link： https://www.nature.com/articles/s41598-022-06947-5

Language：Japanese   Publishing type：Research paper (scientific journal)   Publisher：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

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Language：Japanese   Publishing type：Research paper (scientific journal)   Publisher：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

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Language：Japanese   Publishing type：Research paper (scientific journal)   Publisher：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

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：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

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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

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATL ACAD SCIENCES  

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 mech-anisms, 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 pro-ceed along both CS and IF pathways with the flux ratio depending on conditions such as ligand concentration. However, the struc-tural and energetic basis of such complex reactions remains poorly understood. Therefore, we used experimental, theoretical, and computational approaches to explore structural and energetic as-pects 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 accumula-tion 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 displace-ment of the reaction "route" on the free energy surface, which was consistent with a flux analysis.

DOI： 10.1073/pnas.1914349117

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Public Library of Science (PLoS)  

DOI： 10.1371/journal.pgen.1008814

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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

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© 2020 The Authors. 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

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DOI： 10.1080/09168451.2019.1676695

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DOI： 10.1074/jbc.RA118.007340

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DOI： 10.1038/s41598-019-40746-9

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DOI： 10.1186/s12864-018-5375-5

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DOI： 10.1007/s00394-018-1873-0

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DOI： 10.1021/acs.biochem.8b00631

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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

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DOI： 10.1002/1873-3468.13079

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DOI： 10.1002/rmb2.12085

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DOI： 10.1038/s42003-018-0124-5

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DOI： 10.1099/ijsem.0.001941

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DOI： 10.1128/genomeA.01745-16

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DOI： : 10.15761/BGG.1000123

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DOI： 10.1093/femsle/fnw257

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DOI： 10.1093/gerona/glw074

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DOI： 10.1016/j.bbabio.2016.09.006.

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DOI： 10.1099/ijsem.0.001513.

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DOI： 10.1038/ncomms13471.

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DOI： 10.1128/genomeA.00868-16.

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DOI： 10.1371/journal.pone.0159011.

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DOI： 10.1021/acs.biochem.6b00277.

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DOI： 10.1002/mbo3.340.

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DOI： 10.1093/pcp/pcw071.

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DOI： 10.1093/pcp/pcv150

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DOI： 10.1038/ng.3241

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Although cyanobacteria are photoautotrophs, they have heterotrophic metabolism that enables them to survive in their natural habitat. However, cyanobacterial species that grow heterotrophically in the dark are rare. It remains largely unknown how cyanobacteria regulate heterotrophic activity. The cyanobacterium Leptolyngbya boryana grows heterotrophically with glucose in the dark. A dark-adapted variant dg5 isolated from the wild-type (WT) exhibits enhanced heterotrophic growth in the dark. We sequenced the genomes of dg5 and WT to identify the mutation(s) of dg5. The WT genome consists of a circular chromosome (6,176,364 bp), a circular plasmid pLBA (77,793 bp), and two linear plasmids pLBX (504,942 bp) and pLBY (44,369 bp). Genome comparison revealed three mutation sites. Phenotype analysis of mutants isolated from WT by introducing these mutations individually revealed that the relevant mutation is a single adenine insertion causing a frameshift of cytM encoding cytochrome cM. The respiratory oxygen consumption of the cytM-lacking mutant grown in the dark was significantly higher than that of WT. We isolated a cytM-lacking mutant, ΔcytM, from another cyanobacterium Synechocystis sp. PCC 6803, and ΔcytM grew in the dark with a doubling time of 33 h in contrast to no growth of WT. The respiratory oxygen consumption of ΔcytM grown in the dark was about 2-fold higher than that of wild type. These results suggest a suppressive role(s) of cytochrome cM in regulation of heterotrophic activity.

DOI： 10.1093/pcp/pcu165

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Cruxrhodopsin-3 (cR3), a retinylidene protein found in the claret membrane of Haloarcula vallismortis, functions as a light-driven proton pump. In this study, the membrane fusion method was applied to crystallize cR3 into a crystal belonging to space group P321. Diffraction data at 2.1 Å resolution show that cR3 forms a trimeric assembly with bacterioruberin bound to the crevice between neighboring subunits. Although the structure of the proton-release pathway is conserved among proton-pumping archaeal rhodopsins, cR3 possesses the following peculiar structural features: 1) The DE loop is long enough to interact with a neighboring subunit, strengthening the trimeric assembly; 2) Three positive charges are distributed at the cytoplasmic end of helix F, affecting the higher order structure of cR3; 3) The cytoplasmic vicinity of retinal is more rigid in cR3 than in bacteriorhodopsin, affecting the early reaction step in the proton-pumping cycle; 4) the cytoplasmic part of helix E is greatly bent, influencing the proton uptake process. Meanwhile, it was observed that the photobleaching of retinal, which scarcely occurred in the membrane state, became significant when the trimeric assembly of cR3 was dissociated into monomers in the presence of an excess amount of detergent. On the basis of these observations, we discuss structural factors affecting the photostabilities of ion-pumping rhodopsins.

DOI： 10.1371/journal.pone.0108362. eCollection 2014.

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Special xylem tissue called compression wood is formed on the lower side of inclined stems
when gymnosperms grow on a slope. We investigated the molecular mechanism of compression
wood formation. Transcriptome analysis by next-generation sequencing (NGS) was applied to the
xylem of Chamaecyparis obtusa to develop a catalog of general gene expression in differentiating
xylem during compression and normal wood formation. The sequencing output generated
234,924,605 reads and 40,602 contigs (mean size = 529 bp). Based on a sequence similarity
search with known proteins, 54.2% (22,005) of the contigs showed homology with sequences in
the databases. Of these annotated contigs, 19,293 contigs were assigned to Gene Ontology categories.
Differential gene expression between the compression and normal wood libraries was
analyzed by mapping the reads from each library to the assembled contigs. In total, 2875 contigs
were identified as differentially expressed, including 1207 that were up-regulated and 1668 that
were down-regulated in compression wood. We selected 30 genes and compared the transcript
abundance between compression and normal wood by quantitative polymerase chain reaction
analysis to validate the NGS results. We found that 27 of the 30 genes showed the same expression
patterns as the original NGS results.

DOI： 10.4236/ajps.2014.518295

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DOI： 10.1371/journal.pone.0096271

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DOI： 10.1093/jb/mvt042

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DOI： 10.1186/1471-2164-14-248

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DOI： 10.1128/JB.01850-12

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DOI： 10.1128/JB.01850-12.

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DOI： 10.1016/j.jmb.2011.08.021.

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In many prokaryotic species, 16S rRNA genes are present in multiple copies, and their sequences in general do not differ significantly owing to the concerted evolution. The genus Haloarcula of the family Halobacteriaceae comprises nine validly published species, and all of them possess two to four, highly heterogeneous, 16S rRNA genes. Existence of heterogeneous multiple 16S rRNA genes makes it difficult to reconstruct a biological phylogenetic tree using their sequences data. If the orthologous gene is able to be discriminated from paralogous genes, a tree reconstructed from orthologous genes will reflect a simple biological phylogenetic relationship. At present, however, we have no means to distinguish the orthologous rRNA operon from paralogous one in the members of Halobacteriaceae.In this paper, we found that dihydroorotate oxidase gene, pyrD, was present in the immediate upstream of one 16S rRNA gene, in ten strains of Halobacteriaceae whose genome sequences have been determined, and the direction of pyrD was the opposite of that of the 16S rRNA genes. In two other strains whose genome sequences have been determined, the pyrD was present in far separated positions. We designed PCR primer sets to amplify DNA fragments encompassing from the conserved region of pyrD to a conserved region of tRNA-Ala genes or 23S rRNA genes to determine the 16S rRNA gene sequences preceded by pyrD, and to see if the pyrD is conserved in the immediate upstream of rRNA operon(s) in the type strains of the types species of 28 genera of Halobacteriaceae. Seventeen type strains, including the ten strains mentioned above, gave amplified DNA fragments of approximately 4,000 bp, while eleven type strains including the two strains mentioned above, did not give any PCR products. These eleven strains are members of the Clade I, which was originally defined by Walsh et al. (2004) and expanded by Minegishi et al. (2010). Analysis of contig sequences of three strains belonging to the Clade I also revealed the absence of the pyrD in the immediate upstream of any 16S rRNA genes. It may be scientifically sound to hypothesize that during the evolution of members of Halobacteriaceae, pyrD transposition event happened in one group, and this was followed by subsequent speciation processes in each group, yielding species/genera of the Clade I group and "the rest" of the present family Halobacteriaceae.

DOI： ijs.0.031708-0 [pii] 10.1099/ijs.0.031708-0

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Rhodopsins possess retinal chromophore surrounded by seven transmembrane alpha-helices, are widespread in prokaryotes and in eukaryotes, and can be utilized as optogenetic tools. Although rhodopsins work as distinctly different photo-receptors in various organisms, they can be roughly divided according to their two basic functions, light-energy conversion and light-signal transduction. In microbes, light-driven proton transporters functioning as light-energy converters have been modified by evolution to produce sensory receptors that relay signals to transducer proteins to control motility. In this study, we cloned and characterized two newly identified microbial rhodopsins from Haloquadratum walsbyi. One of them has photochemical properties and a proton pumping activity similar to the well known proton pump bacteriorhodopsin (BR). The other, named middle rhodopsin (MR), is evolutionarily transitional between BR and the phototactic sensory rhodopsin II (SRII), having an SRII-like absorption maximum, a BR-like photocycle, and a unique retinal composition. The wild-type MR does not have a light-induced proton pumping activity. On the other hand, a mutant MR with two key hydrogen-bonding residues located at the interaction surface with the transducer protein HtrII shows robust phototaxis responses similar to SRII, indicating that MR is potentially capable of the signaling. These results demonstrate that color tuning and insertion of the critical threonine residue occurred early in the evolution of sensory rhodopsins. MR may be a missing link in the evolution from type 1 rhodopsins (microorganisms) to type 2 rhodopsins (animals), because it is the first microbial rhodopsin known to have 11-cis-retinal similar to type 2 rhodopsins.

DOI： 10.1074/jbc.M110.190058

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Baliga et al. (2004) [1] reported the existence of a functionally unpredictable opsin gene, named xop2, in Haloarcula marismortui, a holophilic archaeon. Ihara et al. [38] performed molecular phylogenetic analysis and determined that the product of xop2 belonged to a new class of opsins in the sensory rhodopsins. This microbial rhodopsin was therefore named H. marismortui sensory rhodopsin III (HmSRIII). Here, we functionally expressed HmSRIII in Escherichia coli cell membranes to examine the photochemistry. The wavelength of maximum absorption (lambda(max)) for HmSRIII was 506 nm. We observed a very slow photocycle that completed in similar to 50 s. Intermediates were defined as M (lambda(max) similar to 380 nm), N (lambda(max) similar to 460 nm) and O (lambda(max) similar to 530 nm) 0.01 s after the flash excitation. The nomenclature for these intermediates was based on their locations along the absorption maxima of bacteriorhodopsin. Analysis of laser-flash-photolysis data in the presence and absence of azide gave the following results: (1) an equilibrium between N and 0 was attained, (2) the direct product of the M-decay was 0 but not N, and (3) the last photo-intermediate (HmSRIII') had a lambda(max) similar to that of the original, and its decay rate was very slow. Resonance Raman spectroscopy revealed that this N-intermediate had 13-cis retinal conformation. Proton uptake occurred during the course of M-decay, whereas proton release occurred during the course of O-decay (or exactly N-O equilibrium). Very weak proton-pumping activity was observed whose direction is the same as that of bacteriorhodopsin, a typical light-driven proton pump. (C) 2010 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.jphotobiol.2010.09.004

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Sensory rhodopsin I (SRI) functions as a dual receptor regulating both negative and positive phototaxis. It transmits light signals through changes in protein-protein interactions with its transducer protein, HtrI. The phototaxis function of Halobacterium salinarum SRI (HsSRI) has been well characterized using genetic and molecular techniques, whereas that of Salinibacter ruber SRI (SrSRI) has not. SrSRI has the advantage of high protein stability compared with HsSRI and, therefore, provided new information about structural changes and Cl(-) binding of SRI. However, nothing is known about the functional role of SrSRI in phototaxis behavior. In this study, we expressed a SRI homologue from the archaeon Haloarcula vallismortis (HvSRI) as a recombinant protein which uses all-trans-retinal as a chromophore. Functionally important residues of HsSRI are completely conserved in HvSRI (unlike in SrSRI), and HvSRI is extremely stable in buffers without Cl(-). Taking advantage of the high stability, we characterized the photochemical properties of HvSRI under acidic and basic conditions and observed the effects of Cl(-) on the protein under both conditions. Fourier transform infrared results revealed that the structural changes in HvSRI were quite similar to those in HsSRI and SrSRI. Thus, HvSRI can become a useful protein model for improving our understanding of the molecular mechanism of the dual photosensing by SRI.

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The light-driven chloride pump halorhodopsin from Natronomonas pharaonis (phR) crystallised into the monoclinic space group C2, with a phR trimer per the asymmetric unit. Diffraction data at 2.0-A resolution showed that the carotenoid bacterioruberin binds to crevices between adjacent protein subunits in the trimeric assembly. Besides seven transmembrane helices (A to G) that characterise archaeal rhodopsins, the phR protomer possesses an amphipathic alpha-helix (A') at the N-terminus. This helix, together with a long loop between helices B and C, forms a hydrophobic cap that covers the extracellular surface and prevents a rapid ion exchange between the active centre and the extracellular medium. The retinal bound to Lys256 in helix G takes on an all-trans configuration with the Schiff base being hydrogen-bonded to a water molecule. The Schiff base also interacts with Asp252 and a chloride ion, the latter being fixed by two polar groups (Thr126 and Ser130) in helix C. In the anion uptake pathway, four ionisable residues (Arg123, Glu234, Arg176 and His100) and seven water molecules are aligned to form a long hydrogen-bonding network. Conversely, the cytoplasmic half is filled mostly by hydrophobic residues, forming a large energetic barrier against the transport of anion. The height of this barrier would be lowered substantially if the cytoplasmic half functions as a proton/HCl antiporter. Interestingly, there is a long cavity extending from the main-chain carbonyl of Lys256 to Thr71 in helix B. This cavity, which is commonly seen in halobacterial light-driven proton pumps, is one possible pathway that is utilised for a water-mediated proton transfer from the cytoplasmic medium to the anion, which is relocated to the cytoplasmic channel during the photocycle. Copyright (c) 2009. Elsevier Ltd. All rights reserved.

Language：English   Publishing type：Research paper (scientific journal)  

Three halophilic isolates, strains Halo-G*T, AUS-1 and Naxos II, were compared. Halo-G* was isolated from an evaporitic salt crystal from Baja California, Mexico, whereas AUS-1 and Naxos II were isolated from salt pools in Western Australia and the Greek island of Naxos, respectively. Halo-G*T had been exposed previously to conditions of outer space and survived 2 weeks on the Biopan facility. Chemotaxonomic and molecular comparisons suggested high similarity between the three strains. Phylogenetic analysis based on the 16S rRNA gene sequences revealed that the strains clustered with Halorubrum species, showing sequence similarities of 99.2-97.1%. The DNA-DNA hybridization values of strain Halo-G*T and strains AUS-1 and Naxos II are 73 and 75%, respectively, indicating that they constitute a single species. The DNA relatedness between strain Halo-G*T and the type strains of 13 closely related species of the genus Halorubrum ranged from 39 to 2%, suggesting that the three isolates constitute a different genospecies. The G+C content of the DNA of the three strains was 65.5-66.5 mol%. All three strains contained C20C20 derivatives of diethers of phosphatidylglycerol, phosphatidylglyceromethylphosphate and phosphatidylglycerolsulfate, together with a sulfated glycolipid. On the basis of these results, a novel species that includes the three strains is proposed, with the name Halorubrum chaoviator sp. nov. The type strain is strain Halo-G*T (=DSM 19316T=NCIMB 14426T=ATCC BAA-1602T).

Language：English   Publishing type：Research paper (scientific journal)  

Sensory rhodopsin I (SRI) is one of the most interesting photo- sensory receptors because of its function in using the photochromic reaction to mediate opposing signals which depend on the color of light. It was initially thought that SRI exists only in the archaea, but we recently reported for the first time a newly functional SRI from a eubacterium, Salinibacter ruber (SrSRI). The amino acid sequence of SrSRI shows 43% identity with the well-known SRI (HsSRI) and contains most of the amino acid residues identified as necessary for SRI function. The photochemical properties of SrSRI are similar to those of HsSRI. In addition, SrSRI is a highly stable protein, even in dilute salt conditions. Thus, SrSRI could be a key protein for characterizing its association with the SrSRI transducer protein, SrHtrI, and for elucidating structural changes of SRI and HtrI that occur during their function. Recently, new approaches to manipulate cellular functions with rhodopsins have been established. SRI can activate and deactivate a kinase, CheA, by the photochromic reaction. Kinases are key molecules for signal transduction in various organisms, and SRI could potentially manipulate their cellular functions.

Language：English   Publishing type：Research paper (scientific journal)  

Halobacterium salinarum sensory rhodopsin I (HsSRI), a dual receptor regulating both negative and positive phototaxis in haloarchaea, transmits light signals through changes in protein-protein interactions with its transducer, halobacterial transducer protein I (HtrI). Haloarchaea also have another sensor pigment, sensory rhodopsin II (SRII), which functions as a receptor regulating negative phototaxis. Compared with HsSRI, the signal relay mechanism of SRII is well characterized because SRII from Natronomonus pharaonis (NpSRII) is much more stable than HsSRI and HsSRII, especially in dilute salt solutions and is much more resistant to detergents. Two genes encoding SRI homologs were identified from the genome sequence of the eubacterium Salinibacter ruber. Those sequences are distantly related to HsSRI ( approximately 40% identity) and contain most of the amino acid residues identified as necessary for its function. To determine whether those genes encode functional protein(s), we cloned and expressed them in Escherichia coli. One of them (SrSRI) was expressed well as a recombinant protein having all-trans retinal as a chromophore. UV-Vis, low-temperature UV-Vis, pH-titration, and flash photolysis experiments revealed that the photochemical properties of SrSRI are similar to those of HsSRI. In addition to the expression system, the high stability of SrSRI makes it possible to prepare large amounts of protein and enables studies of mutant proteins that will allow new approaches to investigate the photosignaling process of SRI-HtrI.

Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)  

The halorhodopsin (hR)-overproducing mutant strain KM-1 was isolated from the extremely haloalkaliphilic archaeon Natronomonas pharaonis type strain DSM2160(T). hR-enriched membranes were easily obtained by washing the cells with distilled water. The membranes were claret colored owing to two pigments: hR and bacterioruberin. The hR component in the absorption spectra changed from blue to purple upon the addition of Cl(-) and had a K(m) value of 1.7 mM. Overexpression of hR in strain KM-1 might be caused by the point mutation Asp324-->Asn in the bacteriorhodopsin activator homologues of N. pharaonis. The mutation changed the hR-expression pattern from inducible to constitutive in the late exponential phase.

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

Bacteriorhodopsin (BR) and halorhodopsin (HR) are light-driven proton and chloride ion pumps, respectively, in Halobacterium salinarum. The amino acid identity of these proteins is about 25%, suggesting that each has been optimized for their own functions during evolution. However, it is known that the BR mutants, D85T and D85S, can pump chloride ions. This fact implies that the Schiff base region is important in determining ionic selectivity. The X-ray crystallographic structure of D85S(Br(-)) showed the presence of a bromide ion in the Schiff base region (Facciotti, M. T., Cheung, V. S., Nguyen, D., Rouhani, S., and Glaeser, R. M. (2003) Biophys. J. 85, 451-458). In this article, we report on the study of hydrogen bonds of the Schiff base and water molecules in D85S in the absence and presence of various halides, assigning their N-D and O-D stretching vibrations in D(2)O, respectively, in low-temperature Fourier-transform infrared (FTIR) spectroscopy. We found that the hydrogen bond of the Schiff base in D85S(Cl(-)) is much stronger than that in HR, being as strong as that in wild-type BR. Similar halide dependence in D85S and in solution implies that the Schiff base forms a direct hydrogen bond with a halide, consistent with the X-ray structure. Photoisomerization causes a weakened hydrogen bond of the Schiff base, and halide dependence on the stretching frequency is lost. These spectral features are similar to those in the photocycle of proton-pumping BR, though the weakened hydrogen bond is more significant for BR. However, the spectral features of water bands in D85S are closer to chloride-pumping HR because O-D stretching vibrations of water are observed only at >2500 cm(-)(1). Unlike in BR, we did not observe strongly hydrogen-bonded water molecules for halide-pumping D85S mutants. This observation agrees with our recent hypothesis that strongly hydrogen-bonded water molecules are required for the proton-pumping activity of archaeal rhodopsins. Hydrogen-bond

Authorship：Lead author   Language：English  

Authorship：Lead author   Language：English  

Language：English   Publishing type：Research paper (scientific journal)  

Archaerhodopsin-1 and -2 (aR-1 and aR-2) are light-driven proton pumps found in Halorubrum sp. aus-1 and -2, which share 55-58% sequence identity with bacteriorhodopsin (bR), a proton pump found in Halobacterium salinarum. In this study, aR-1 and aR-2 were crystallized into 3D crystals belonging to P4(3)2(1)2 (a = b = 128.1 A, c = 117.6 A) and C222(1) (a = 122.9 A, b = 139.5 A, c = 108.1 A), respectively. In both the crystals, the asymmetric unit contains two protein molecules with slightly different conformations. Each subunit is composed of seven helical segments as seen in bR but, unlike bR, aR-1 as well as aR-2 has a unique omega loop near the N terminus. It is found that the proton pathway in the extracellular half (i.e. the proton release channel) is more opened in aR-2 than in aR-1 or bR. This structural difference accounts for a large variation in the pKa of the acid purple-to-blue transition among the three proton pumps. All the aromatic residues surrounding the retinal polyene chain are conserved among the three proton pumps, confirming a previous argument that these residues are required for the stereo-specificity of the retinal isomerization. In the cytoplasmic half, the region surrounded by helices B, C and G is highly conserved, while the structural conservation is very low for residues extruded from helices E and F. Structural conservation of the hydrophobic residues located on the proton uptake pathway suggests that their precise arrangement is necessary to prevent a backward flow of proton in the presence of a large pH gradient and membrane potential. An empty cavity is commonly seen in the vicinity of Leu93 contacting the retinal C13 methyl. Existence of such a cavity is required to allow a large rotation of the side-chain of Leu93 at the early stage of the photocycle, which has been shown to accompany water translocation across the Schiff base.

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (bulletin of university, research institution)  

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

Authorship：Lead author   Language：Japanese   Publishing type：Research paper (scientific journal)  

Authorship：Lead author   Language：Japanese  

Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)  

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Language：English   Publishing type：Research paper (scientific journal)  

Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)  

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Language：Japanese

Event date： 2022.3

Presentation type：Poster presentation  

Event date： 2021.11

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Venue：オンライン（理化学研究所バイオリソースセンター）   Country：Japan  

Event date： 2021.3

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Venue：名古屋  

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Venue：オンライン(九州大学）  

Event date： 2021.3 - 2020.3

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Event date： 2021.3 - 2020.3

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Event date： 2019.3

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Venue：八王子市（東京都立大学）  

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Event date： 2017.9

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Venue：東北大学　川内北キャンパス   Country：Japan  

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Venue：慶應義塾大学　湘南藤沢キャンパス   Country：Japan  

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Venue：東洋大学　白山キャンパス　8号館7階　125記念ホール   Country：Japan  

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Event date： 2005.11

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Event date： 2004.9

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Event date： 2004.6

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Event date： 2003.11

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Event date： 2001.10

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Event date： 2001.8

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Venue：慶應義塾大学 湘南藤沢キャンパス  

researchmap

Language：Japanese   Presentation type：Poster presentation  

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Language：Japanese   Presentation type：Poster presentation  

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Venue：東北大学 川内北キャンパス  

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Audience： High school students

Type：Other

Audience： High school students

Type：Other

高校の先生と生徒、計36 名に対して、乳製品を持ち寄ってもらい、（１）乳製品の一部を牛乳に接種することでヨーグルトができること、（２）乳製品の一部をMRS寒天培地にまくことで菌のコロニーを作らせること、（３）乳製品から直接DNAを取り出して、PCRにより16S rRNA遺伝子を増幅し、塩基配列を決めることで、菌を同定すること。
を行い、研究の”考え方”を学んだ。

高校の先生と生徒、計20 名に遺伝子組換え大腸菌（GFPを導入）の作製と大腸菌で発現したGFPを精製する実験の指導を行った。同時に、組換えDNA実験の基本に関して学習した。

明和高校で大学の最先端研究の話を出来るだけ分かりやすく講義する。

Type：Lecture

高校生の自然科学に関する研究発表会に参加し、色々なコメント、議論を通じて論理的な考え方を養い、問題解決の為の実験計画に対してアドバイスをする。
次世代シーケンサーの話題に関して、基礎的な所から最先端の所までを簡単に講演した。

Type：University open house

小学生高学年、中学生を対象に、遺伝現象の担い手であるDNAが細胞のどこにあるのか？を顕微鏡で観察し、実際にDNAを取り出す作業をすることで、生命科学の片鱗に触れる。