Paralimibaculum aggregatum gen. nov. sp. nov. and Biformimicrobium ophioploci gen. nov. sp. nov., two novel heterotrophs from brittle star Ophioplocus japonicus.

Two novel Gram-stain-negative, strictly aerobic, halophilic and non-motile bacterial strains, designated NKW23T and NKW57T, were isolated from a brittle star Ophioplocus japonicus collected from a tidal pool in Wakayama, Japan. The results of phylogenetic analysis based on 16S rRNA gene sequences indicated that NKW23T represented a member of the family Paracoccaceae, with Limibaculum halophilum CAU 1123T as its closest relative (94.4% sequence identity). NKW57T was identified as representing a member of the family Microbulbiferaceae, with up to 94.9% sequence identity with its closest relatives. Both strains displayed average nucleotide identity (ANI) and digital DNA-DNA hybridisation (dDDH) values below the species delimitation threshold against their closest relatives. Additionally, amino acid identity (AAI) values of both strains fell below the genus-defining threshold. Phylogenetic trees based on genome sequences indicated that NKW23T formed a novel lineage, branching deeply prior to the divergence of the genera Limibaculum and Thermohalobaculum, with an evolutionary distance (ED) of 0.31-0.32, indicative of genus-level differentiation. NKW57T similarly formed a distinct lineage separate from the species of the genus Microbulbifer. The major respiratory quinones of NKW23T and NKW57T were ubiquinone-10 (Q-10) and Q-8, respectively. The genomic DNA G+C contents of NKW23T and NKW57T were 71.4 and 58.8%, respectively. On the basis of the physiological and phylogenetic characteristics, it was proposed that these strains should be classified as novel species representing two novel genera: Paralimibaculum aggregatum gen. nov., sp. nov., with strain NKW23T (=JCM 36220T=KCTC 8062T) as the type strain, and Biformimicrobium ophioploci gen. nov., sp. nov., with strain NKW57T (=JCM 36221T=KCTC 8063T) as the type strain.

Tuning the Viscoelasticity of Peptide Vesicles by Adjusting Hydrophobic Helical Blocks Comprising Amphiphilic Polypeptides.

Amphiphilic block polypeptides of poly(sarcosine)-b-(l-Val-Aib)6 and poly(sarcosine)-b-(l-Leu-Aib)6 and their stereoisomers were self-assembled in water. Three kinds of binary systems of poly(sarcosine)-b-(l-Leu-Aib)6 with poly(sarcosine)-b-poly(d-Leu-Aib)6, poly(sarcosine)-b-poly(l-Val-Aib)6, or poly(sarcosine)-b-(d-Val-Aib)6 generated vesicles of ca. 200 nm diameter. The viscoelasticity of the vesicle membranes was evaluated by the nanoindentation method using AFM in water. The elasticity of the poly(sarcosine)-b-(l-Leu-Aib)6/poly(sarcosine)-b-poly(d-Leu-Aib)6 vesicle was 11-fold higher than that of the egg yolk liposome but decreased in combinations of the Leu- and Val-based amphiphilic polypeptides. The membrane elasticity is found to be adjustable by a suitable combination of helical blocks in terms of stereocomplex formation and the interdigitation of side chains among helices in the molecular assemblies.

Electronic properties of tetrathiafulvalene-modified cyclic-ß-peptide nanotube.

Cyclic tri-ß-peptide having tetrathiafulvalene (TTF) at the side chain was synthesized to prepare a peptide nanotube aligning TTF side chains along the nanotube. The polarized light microscopic observations revealed crystallization of the cyclic peptide by the vapor diffusion method. Fourier-transform infrared and electron diffraction measurements of the crystals clarified formation of homogeneous hydrogen bonds making a columnar structure with a layer spacing of 4.9 Å. Electronic measurements of the peptide crystals on a gold mica substrate were carried out by the current sensing AFM. The current-voltage curves showed a rectification behavior, whose profile was consistent with a metal and p-type semiconductor junction. The p-type property is supported by the first principle calculations, which showed the HOMO orbital delocalizing fully over the plane of the TTF ring with the energy level of -5.1 eV. © 2016 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 106: 275-282, 2016.

Degradation and synthesis of ß-glucans by a Magnaporthe oryzae endotransglucosylase, a member of the glycoside hydrolase 7 family.

BACKGROUND: Plant pathogens secrete enzymes that degrade plant cell walls to enhance infection and nutrient acquisition. RESULTS: A novel endotransglucosylase catalyzes cleavage and transfer of ß-glucans and decreases the physical strength of plant cell walls. CONCLUSION: Endotransglucosylation causes depolymerization and polymerization of ß-glucans, depending on substrate molecular size. SIGNIFICANCE: Enzymatic degradation of plant cell walls is required for wall loosening, which enhances pathogen invasion. A Magnaporthe oryzae enzyme, which was encoded by the Mocel7B gene, was predicted to act on 1,3-1,4-ß-glucan degradation and transglycosylation reaction of cellotriose after partial purification from a culture filtrate of M. oryzae cells, followed by liquid chromatography-tandem mass spectrometry. A recombinant MoCel7B prepared by overexpression in M. oryzae exhibited endo-typical depolymerization of polysaccharides containing ß-1,4-linkages, in which 1,3-1,4-ß-glucan was the best substrate. When cellooligosaccharides were used as the substrate, the recombinant enzyme generated reaction products with both shorter and longer chain lengths than the substrate. In addition, incorporation of glucose and various oligosaccharides including sulforhodamine-conjugated cellobiose, laminarioligosaccharides, gentiobiose, xylobiose, mannobiose, and xyloglucan nonasaccharide into ß-1,4-linked glucans were observed after incubation with the enzyme. These results indicate that the recombinant enzyme acts as an endotransglucosylase (ETG) that cleaves the glycosidic bond of ß-1,4-glucan as a donor substrate and transfers the cleaved glucan chain to another molecule functioning as an acceptor substrate. Furthermore, ETG treatment caused greater extension of heat-treated wheat coleoptiles. The result suggests that ETG functions to induce wall loosening by cleaving the 1,3-1,4-ß-glucan tethers of plant cell walls. On the other hand, use of cellohexaose as a substrate for ETG resulted in the production of cellulose II with a maximum length (degree of polymerization) of 26 glucose units. Thus, ETG functions to depolymerize and polymerize ß-glucans, depending on the size of the acceptor substrate.

Proton-dependent coniferin transport, a common major transport event in differentiating xylem tissue of woody plants.

Lignin biosynthesis is an essential physiological activity of vascular plants if they are to survive under various environmental stresses on land. The biosynthesis of lignin proceeds in the cell wall by polymerization of precursors; the initial step of lignin polymerization is the transportation of lignin monomers from the cytosol to the cell wall, which is critical for lignin formation. There has been much debate on the transported form of the lignin precursor, either as free monolignols or their glucosides. In this study, we performed biochemical analyses to characterize the membrane transport mechanism of lignin precursors using angiosperms, hybrid poplar (Populus sieboldii × Populus grandidentata) and poplar (Populus sieboldii), as well gymnosperms, Japanese cypress (Chamaecyparis obtusa) and pine (Pinus densiflora). Membrane vesicles prepared from differentiating xylem tissues showed clear ATP-dependent transport activity of coniferin, whereas less than 4% of the coniferin transport activity was seen for coniferyl alcohol. Bafilomycin A1 and proton gradient erasers markedly inhibited coniferin transport in hybrid poplar membrane vesicles; in contrast, vanadate had no effect. Cis-inhibition experiments suggested that this transport activity was specific for coniferin. Membrane fractionation of hybrid poplar microsomes demonstrated that transport activity was localized to the tonoplast- and endomembrane-rich fraction. Differentiating xylem of Japanese cypress exhibited almost identical transport properties, suggesting the involvement of a common endomembrane-associated proton/coniferin antiport mechanism in the lignifying tissues of woody plants, both angiosperms and gymnosperms.

Morphology control between twisted ribbon, helical ribbon, and nanotube self-assemblies with his-containing helical peptides in response to pH change.

pH-Responsive molecular assemblies with a variation in morphology ranging from a twisted ribbon, a helical ribbon, to a nanotube were prepared from a novel A3B-type amphiphilic peptide having three hydrophilic poly(sarcosine) (A block) chains, a hydrophobic helical dodecapeptide (B block), and two histidine (His) residues between the A3 and B blocks. The A3B-type peptide adopted morphologies of the twisted ribbon at pH 3.0, the helical ribbon at pH 5.0, and the nanotube at pH 7.4, depending upon the protonation states of the two His residues. On the other hand, another A3B-type peptide having one His residue between the A3 and B blocks showed a morphology change only between the helical ribbon and the relatively planar sheets with pH variation in this range. The morphology change is thus induced by one- or two-charge generation at the linking site of the hydrophilic and hydrophobic blocks of the component amphiphiles but in different ways.

Facile and precise formation of unsymmetric vesicles using the helix dipole, stereocomplex, and steric effects of peptides.

Unsymmetrical vesicular membranes were prepared from a binary mixture of the A3B-type and the AB-type host polypeptides, which were composed of the hydrophilic block (A) and the hydrophobic helical block (B) but with a different helix sense between the two host polypeptides. TEM and DLS revealed the formation of vesicles with ca. 100 nm diameter. The molecular assembly was driven by hydrophobic interaction, stereocomplex formation, and dipole-dipole interaction between hydrophobic helices. Furthermore, the A3B-type host polypeptide extended the hydrophilic block to the outer surface of vesicles as a result of the steric effect, resulting in the formation of unsymmetrical vesicular membranes. As a result, a functionalized AB-type guest polypeptide having the same helix sense with the A3B-type host polypeptide exposed the hydrophilic block to the outer surface. In contrast, an AB-type guest polypeptide having the same helix sense with the AB-type host polypeptide oriented the hydrophilic block to the inner surface. Functionalization of either the outer or inner surface of the binary vesicle is therefore facile to achieve when using either the right- or the left-handed helix of the functionalized guest polypeptide.

Functional reconstitution of cellulose synthase in Escherichia coli.

Cellulose is a high molecular weight polysaccharide of ß1 → 4-d-glucan widely distributed in nature-from plant cell walls to extracellular polysaccharide in bacteria. Cellulose synthase, together with other auxiliary subunit(s) in the cell membrane, facilitates the fibrillar assembly of cellulose polymer chains into a microfibril. The gene encoding the catalytic subunit of cellulose synthase is cesA and has been identified in many cellulose-producing organisms. Very few studies, however, have shown that recombinant CesA protein synthesizes cellulose polymer, but the mechanism by which CesA protein synthesizes cellulose microfibrils is not known. Here we show that cellulose-synthesizing activity is successfully reconstituted in Escherichia coli by expressing the bacterial cellulose synthase complex of Gluconacetobacter xylinus: CesA and CesB (formerly BcsA and BcsB, respectively). Cellulose synthase activity was, however, only detected when CesA and CesB were coexpressed with diguanyl cyclase (DGC), which synthesizes cyclic-di-GMP (c-di-GMP), which in turn activates cellulose-synthesizing activity in bacteria. Direct observation by electron microscopy revealed extremely thin fibrillar structures outside E. coli cells, which were removed by cellulase treatment. This fiber structure is not likely to be the native crystallographic form of cellulose I, given that it was converted to cellulose II by a chemical treatment milder than ever described. We thus putatively conclude that this fine fiber is an unprecedented structure of cellulose. Despite the inability of the recombinant enzyme to synthesize the native structure of cellulose, the system described in this study, named "CESEC (CEllulose-Synthesizing E. Coli)", represents a useful tool for functional analyses of cellulose synthase and for seeding new nanomaterials.

Formation of highly twisted ribbons in a carboxymethylcellulase gene-disrupted strain of a cellulose-producing bacterium.

Cellulases are enzymes that normally digest cellulose; however, some are known to play essential roles in cellulose biosynthesis. Although some endogenous cellulases of plants and cellulose-producing bacteria are reportedly involved in cellulose production, their functions in cellulose production are unknown. In this study, we demonstrated that disruption of the cellulase (carboxymethylcellulase) gene causes irregular packing of de novo-synthesized fibrils in Gluconacetobacter xylinus, a cellulose-producing bacterium. Cellulose production was remarkably reduced and small amounts of particulate material were accumulated in the culture of a cmcax-disrupted G. xylinus strain (F2-2). The particulate material was shown to contain cellulose by both solid-state (13)C nuclear magnetic resonance analysis and Fourier transform infrared spectroscopy analysis. Electron microscopy revealed that the cellulose fibrils produced by the F2-2 cells were highly twisted compared with those produced by control cells. This hypertwisting of the fibrils may reduce cellulose synthesis in the F2-2 strains.

Quantitative morphological transformation of vascular bundles in the culm of moso bamboo (Phyllostachys pubescens).

Vascular bundles of bamboo are determinants for mechanical properties of bamboo material and for physiological properties of living bamboo. The morphology of vascular bundles reflecting mechanical and physiological functions differs not only within internode tissue but also among different internodes in the culm. Although the distribution of vascular bundle fibers has received much attention, quantitative evaluation of the morphological transformation of vascular bundles associated with spatial distribution patterns has been limited. In this study deep learning models were used to determine quantitative changes in the distribution and morphology of vascular bundles in the culms of moso bamboo (Phyllostachys pubescens). A precise model for extracting vascular bundles from cross-sectional images was constructed using the U-Net model. Analyses of extracted vascular bundles from different internodes showed significant changes in vascular bundle distribution and morphology among internodes. Vascular bundles in lower internodes showed outer relative position and larger area than those in upper internodes. Aspect ratio and eccentricity indicate that vascular bundles in internodes near the base have more elliptical morphology, with a long axis in the radial direction. The variational autoencoder model using extracted vascular bundles enabled simulation of the morphological transformation of vascular bundles along with radial direction. These deep learning models enabled highly accurate quantification of vascular bundle morphologies, and will contribute to a further understanding of bamboo development as well as evaluation of the mechanical and physiological properties of bamboo.

Maturation stress generation in poplar tension wood studied by synchrotron radiation microdiffraction.

Tension wood is widespread in the organs of woody plants. During its formation, it generates a large tensile mechanical stress called maturation stress. Maturation stress performs essential biomechanical functions such as optimizing the mechanical resistance of the stem, performing adaptive movements, and ensuring the long-term stability of growing plants. Although various hypotheses have recently been proposed, the mechanism generating maturation stress is not yet fully understood. In order to discriminate between these hypotheses, we investigated structural changes in cellulose microfibrils along sequences of xylem cell differentiation in tension and normal wood of poplar (Populus deltoides × Populus trichocarpa 'I45-51'). Synchrotron radiation microdiffraction was used to measure the evolution of the angle and lattice spacing of crystalline cellulose associated with the deposition of successive cell wall layers. Profiles of normal and tension wood were very similar in early development stages corresponding to the formation of the S1 layer and the outer part of the S2 layer. Subsequent layers were found with a lower microfibril angle (MFA), corresponding to the inner part of the S2 layer of normal wood (MFA approximately 10°) and the G layer of tension wood (MFA approximately 0°). In tension wood only, this steep decrease in MFA occurred together with an increase in cellulose lattice spacing. The relative increase in lattice spacing was found close to the usual value of maturation strains. Analysis showed that this increase in lattice spacing is at least partly due to mechanical stress induced in cellulose microfibrils soon after their deposition, suggesting that the G layer directly generates and supports the tensile maturation stress in poplar tension wood.

Self-assemblies of triskelion A2B-type amphiphilic polypeptide showing pH-responsive morphology transformation.

A pH-responsive rolled-sheet morphology was prepared from a triskelion A(2)B-type amphiphilic polypeptide having a histidine residue as a pH-responsive unit. The dimensions of the rolled sheet were 85 nm diameter and 210 nm length with a sheet turn number of 2.0 at pH 7.4. Upon decreasing the pH from 7.4 to 5.0, the layer spacing of the rolled sheets was widened from ca. 9 to ca. 19 nm due to electrostatic repulsion caused by histidine protonation. This morphology change occurred reversibly with a pH change between 7.4 and 5.0. The molecular packing in the rolled sheets was shown to be loosened at pH 5.0 on the basis of electron diffraction measurements. The tightness of the rolled sheets was thus controlled reversibly by a pH change due to a single protonation in the amphiphilic polypeptide.

The crystalline phase of cellulose changes under developmental control in a marine chordate.

The native form of cellulose is a fibrillar composite of two crystalline phases, the triclinic I(α) and monoclinic I(ß) allomorphs. Allomorph ratios are species-specific, and this gives rise to natural structural variations in cellulose crystals. However, the mechanisms contributing to crystal formation remain unknown. We show that the two crystalline phases of cellulose are tailored to distinct structures during different developmental stages of the tunicate chordate Oikopleura dioica. Larval cellulose consisting of I(α) allomorph constitutes the body cuticle fin, whereas adult cellulose consisting of I(ß) allomorph frames a mucous filter-feeding device, the "house." Both structures are secreted from the epidermis in accordance with the mutually exclusive expression patterns of two distinct putative cellulose synthase genes. We discuss a possible linkage between structural variations of the crystalline phases of cellulose and the underlying evolutionary genetics of cellulose biosynthesis.

Intra-annual fluctuation in morphology and microfibril angle of tracheids revealed by novel microscopy-based imaging.

Woody cells, such as tracheids, fibers, vessels, rays etc., have unique structural characteristics such as nano-scale ultrastructure represented by multilayers, microfibril angle (MFA), micro-scale anatomical properties and spatial arrangement. Simultaneous evaluation of the above indices is very important for their adequate quantification and extracting the effects of external stimuli from them. However, it is difficult in general to achieve the above only by traditional methodologies. To overcome the above point, a new methodological framework combining polarization optical microscopy, fluorescence microscopy, and image segmentation is proposed. The framework was tested to a model softwood species, Chamaecyparis obtusa for characterizing intra-annual transition of MFA and tracheid morphology in a radial file unit. According our result, this framework successfully traced the both characteristics tracheid by tracheid and revealed the high correlation (|r| > 0.5) between S2 microfibril angles and tracheidal morphology (lumen radial diameter, tangential wall thickness and cell wall occupancy). In addition, radial file based evaluation firstly revealed their complex transitional behavior in transition and latewood. The proposed framework has great potential as one of the unique tools to provide detailed insights into heterogeneity of intra and inter-cells in the wide field of view through the simultaneous evaluation of cells' ultrastructure and morphological properties.

Limiting silicon supply alters lignin content and structures of sorghum seedling cell walls.

Sorghum has been recognized as a promising energy crop. The composition and structure of lignin in the cell wall are important factors that affect the quality of plant biomass as a bioenergy feedstock. Silicon (Si) supply may affect the lignin content and structure, as both Si and lignin are possibly involved in plant mechanical strength. However, our understanding regarding the interaction between Si and lignin in sorghum is limited. Therefore, in this study, we analyzed the lignin in the cell walls of sorghum seedlings cultured hydroponically with or without Si supplementation. Limiting the Si supply significantly increased the thioglycolic acid lignin content and thioacidolysis-derived syringyl/guaiacyl monomer ratio. At least part of the modification may be attributable to the change in gene expression, as suggested by the upregulation of phenylpropanoid biosynthesis-related genes under -Si conditions. The cell walls of the -Si plants had a higher mechanical strength and calorific value than those of the +Si plants. These results provide some insights into the enhancement of the value of sorghum biomass as a feedstock for energy production by limiting Si uptake.

Maturation stress generation in poplar tension wood studied by synchrotron radiation microdiffraction.

Tension wood is widespread in the organs of woody plants. During its formation, it generates a large tensile mechanical stress, called maturation stress. Maturation stress performs essential biomechanical functions such as optimizing the mechanical resistance of the stem, performing adaptive movements, and ensuring long-term stability of growing plants. Although various hypotheses have recently been proposed, the mechanism generating maturation stress is not yet fully understood. In order to discriminate between these hypotheses, we investigated structural changes in cellulose microfibrils along sequences of xylem cell differentiation in tension and normal wood of poplar (Populus deltoides x Populus trichocarpa 'I45-51'). Synchrotron radiation microdiffraction was used to measure the evolution of the angle and lattice spacing of crystalline cellulose associated with the deposition of successive cell wall layers. Profiles of normal and tension wood were very similar in early development stages corresponding to the formation of the S1 and the outer part of the S2 layer. The microfibril angle in the S2 layer was found to be lower in its inner part than in its outer part, especially in tension wood. In tension wood only, this decrease occurred together with an increase in cellulose lattice spacing, and this happened before the G-layer was visible. The relative increase in lattice spacing was found close to the usual value of maturation strains, strongly suggesting that microfibrils of this layer are put into tension and contribute to the generation of maturation stress.

Temperature-triggered fusion of vesicles composed of right-handed and left-handed amphiphilic helical peptides.

Vesicles prepared from a mixture of (Sar)(25)-b-(L-Leu-Aib)(6) (SLL) and (Sar)(25)-b-(D-Leu-Aib)(6) (SDL) fused with themselves upon heating to 90 °C. The vesicles also fused with (Sar)(28)-b-(L-Leu-Aib)(8) vesicles upon heating to 90 °C. The temperature-triggered fusion was due to the phase transition of the mixed membrane of SLL and SDL at 90 °C and should be driven by the bending energy stored in the stereocomplex membrane upon taking a vesicular structure.

Enzymatic polymerization catalyzed by immobilized endoglucanase on gold.

Enzymatic polymerization was carried out on gold by immobilized genetically engineered endoglucanase II (EGII) from Trichoderma viride , and the polymerization behavior and the produced cellulose were analyzed in comparison with those by free enzymes. Mutant EGIIs were EGII(core2) and EGII(core2H), which consist of two sequential catalytic core domains with one His-tag (His6) on N-terminal and with totally two His-tags on both terminals, respectively. His-tagged EGIIs were immobilized via Ni chelators of nitrilotriacetic acid (NTA) introduced on gold surface. From SPR measurements, the affinity of EGII(core2H) to the surface was higher than that of EGII(core2), and the molecular occupation area of EGII(core2H) was larger than that of EGII(core2), indicating that EGII(core2H) was immobilized with utilizing two His-tags introduced on both terminals. The hydrolytic activity of the immobilized EGII(core2H) using cellohexaose as substrate was slightly lower than that of free EGII(core2H) when they were compared at the same amount in the hydrolytic system. Enzymatic polymerization catalyzed by both immobilized EGII(core2) and EGII(core2H) proceeded with producing highly crystalline cellulose in comparison with free enzyme. Immobilization of the endoglucanase is thus effective to obtain crystalline cellulose due to the high density of the catalytic domain on gold.

Rational design of peptide nanotubes for varying diameters and lengths.

Amphiphilic helical peptides (Sar)(m) -b-(L-Leu-Aib)(n) (m = 22-25; n = 7, 8, 10) with a hydrophobic block as a right-handed helix were synthesized and their mixtures with (Sar)(25) -b-(D-Leu-Aib)(6) containing the hydrophobic block as a left-handed helix were examined in their molecular assembly formation. The single component (Sar)(25) -b-(D-Leu-Aib)(6) forms peptide nanotubes of 70 nm diameter and 200 nm length. The two-component mixtures of (Sar)(25) -b-(D-Leu-Aib)(6) with (Sar)(24) -b-(L-Leu-Aib)(7) , (Sar)(22) -b-(L-Leu-Aib)(8) , and (Sar)(25) -b-(L-Leu-Aib)(10) yield peptide nanotubes of varying dimensions with 200 nm diameter and 400 nm length, 70 nm diameter and several micrometer length (maximum 30 µm), and 70 nm diameter and 100-600 nm length, respectively. The right-handed and the left-handed helix were thus found to be molecularly mixed due to the stereo-complex formation and to generate nanotubes of different sizes. When the mismatch of the hydrophobic helical length between the two components was of four residues, the longest nanotube was generated. Correspondingly, the hydrophobic helical segments have to interdigitate with an anti-parallel orientation at the hydrophobic core region of the nanotube.