Characterization of the membrane vesicle fraction from Acetobacter sp. WSS15.

A bacterium that produces membrane vesicles (MVs), strain WSS15, was isolated from a traditional vinegar in Japan called Kurozu. A phylogenetic analysis of 16S rRNA gene sequences indicated that this bacterium belongs to the genus Acetobacter. MVs and peptidoglycan-associated lipoprotein (Pal) were detected in the MV fraction of strain WSS15. In the presence of the WSS15 MV fraction, murine macrophages produced the pro-inflammatory cytokine interleukin-6 (IL-6) via the recognition by superficial Toll-like receptor 2 (TLR2). WSS15 MVs adhered to the cell surface of macrophages. The macrophages secreted IL-6 through the TLR2 recognition of an acylated N-terminal peptide of Pal. We elucidated the mode of action of WSS15 MVs on immune cells and identified the Pal peptide from strain WSS15 as an agonist of TLR2.

Enhancing extracellular membrane vesicle productivity of Shewanella vesiculosa HM13, a prospective host for vesiculation-mediated protein secretion, by weakening outer membrane-peptidoglycan linkage.

Shewanella vesiculosa HM13, a psychrotrophic gram-negative bacterium isolated from the intestinal contents of horse mackerel, produces abundant extracellular membrane vesicles (EMVs) by budding the outer membrane. The EMVs of this bacterium carry a single major cargo protein, P49, of unknown function, which may be useful as a carrier for the secretory production of heterologous proteins as cargoes of EMVs. In this study, to increase the utility of S. vesiculosa HM13 as a host for EMV-mediated protein production, we improved its EMV productivity by weakening the linkage between the outer membrane and underlying peptidoglycan layer. In gram-negative bacteria, the outer membrane is connected to peptidoglycans predominantly through Braun's lipoprotein (Lpp), and the formation of this linkage is catalyzed by an l,d-transpeptidase (Ldt). We constructed gene-disrupted mutants of Lpp and Ldt and assessed their EMV productivity. The EMVs of the lpp- and ldt-disrupted mutants grown at 18 °C were evaluated using nanoparticle tracking analysis, and their morphologies were observed using transmission electron microscopy. As a result, an approximately 2.5-fold increase in EMV production was achieved, whereas the morphology of the EMVs of these mutants remained almost identical to that of the parent strain. In accordance with the increase in EMV production, the mutants secreted approximately 2-fold higher amounts of P49 than the parent strain into the culture broth as the EMV cargo. These findings will contribute to the development of an EMV-based secretory production system for heterologous proteins using S. vesiculosa HM13 as a host.

Intramolecular Electron Transfer in Multi-Redox Systems Based on Cyclic [3]Spirobifluorenylene Compound.

Cyclic [3]spirobifluorenylene with bulky alkyl groups at the ends (1) was designed and synthesized to investigate the electron transfer phenomena in a π-conjugated system including orthogonal π-conjugated chains. The three bifluorenyl units in 1 are conjugated to each other via spiro-conjugation, resulting in the splitting of the HOMO levels to a small extent. Therefore, the SOMO-HOMO gap of the radical cation species is small, which is considered to allow the facile intramolecular electron transfer. The electronic properties of 1 and its partial structures were characterized by absorption and fluorescence measurements and electrochemical analysis. From the electrochemical oxidation, the interchain Coulombic repulsion was observed. In the TD-DFT calculations for the radical cation species of 1, the geometry-featured interchain electronic transitions were visualized by NTO calculations. The radical cation species of 1 generated by chemical oxidation with SbCl5 exhibited a broadened and lower-energy NIR absorption band exceeding 2000 nm. Considering the results of the TD-DFT calculations, the NIR band of the radical cation of 1 was attributed to the intramolecular electron transfer processes among the bifluorenyl units in the macrocycle. ESR experiments also indicated the delocalization of a spin of 1⋅+ in the whole molecule via hole hopping in the ESR time scale at room temperature. This work demonstrates the usefulness of spiro-conjugation as a bridging unit in molecular wires to facilitate smooth electron transfer.

Activation of the toll-like receptor 2 signaling pathway by GAPDH from bacterial strain RD055328.

We characterized the membrane vesicle fraction (RD-MV fraction) from bacterial strain RD055328, which is related to members of the genus Companilactobacillus and Lactiplantibacillus plantarum. RD-MVs and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were detected in the RD-MV fraction. Immunoglobulin A (IgA) was produced by Peyer's patch cells following the addition of the RD-MV fraction. In the presence of the RD-MV fraction, RAW264 cells produced the pro-inflammatory cytokine IL-6. Recombinant GAPDH probably induced the production of IL-6 by RAW264 cells via superficial toll-like receptor 2 (TLR2) recognition. A confocal laser scanning microscopy image analysis indicated that RD-MVs and GAPDH were taken up by RAW264 cells. GAPDH wrapped around RAW264 cells. We suggest that GAPDH from strain RD055328 enhanced the production of IgA by acquired immune cells via the production of IL-6 by innate immune cells through TLR2 signal transduction.

Enhancement of IgA production by membrane vesicles derived from Bifidobacterium longum subsp. infantis.

Immunoglobulin A (IgA) is involved in the maintenance of gut homeostasis. Although the oral administration of bifidobacteria increases the amount of fecal IgA, the effects of bifidobacteria on intestinal immunity remain unclear. We found and characterized membrane vesicles (MVs) derived from Bifidobacterium longum subsp. infantis toward host immune cells. Bifidobacterium infantis MVs consisted of a cytoplasmic membrane, and extracellular solute-binding protein (ESBP) was specifically detected. In the presence of B. infantis MVs or recombinant ESBP, RAW264 cells produced the pro-inflammatory cytokine IL-6. IgA was produced by Peyer's patches cells following the addition of B. infantis MVs. Therefore, ESBP of B. infantis MVs is involved in the production of IgA by acquired immune cells via the production of IL-6 by innate immune cells.

The BcsD subunit of type I bacterial cellulose synthase interacts dynamically with the BcsAB catalytic core complex.

Cellulose synthase has two distinct functions: synthesis of the cellulose molecule (polymerization) and assembling the synthesized cellulose chains into the crystalline microfibril (crystallization). In the type I bacterial cellulose synthase (Bcs) complex, four major subunits - BcsA, BcsB, BcsC and BcsD - work in a coordinated manner. This study showed that the crystallization subunit BcsD interacts with the polymerization complex BcsAB in two modes: direct protein-protein interactions and indirect interactions through the product cellulose. We hypothesized that the former and latter modes represent the basal and active states of type I bacterial cellulose synthase, respectively, and this dynamic behaviour of the BcsD protein regulates the crystallization process of cellulose chains.

Characterization of extracellular vesicles from Lactiplantibacillus plantarum.

We investigated the characteristics and functionalities of extracellular vesicles (EVs) from Lactiplantibacillus plantarum (previously Lactobacillus plantarum) towards host immune cells. L. plantarum produces EVs that have a cytoplasmic membrane and contain cytoplasmic metabolites, membrane and cytoplasmic proteins, and small RNAs, but not bacterial cell wall components, namely, lipoteichoic acid and peptidoglycan. In the presence of L. plantarum EVs, Raw264 cells inducibly produced the pro-inflammatory cytokines IL-1ß and IL-6, the anti-inflammatory cytokine IL-10, and IF-γ and IL-12, which are involved in the differentiation of naive T-helper cells into T-helper type 1 cells. IgA was produced by PP cells following the addition of EVs. Therefore, L. plantarum EVs activated innate and acquired immune responses. L. plantarum EVs are recognized by Toll-like receptor 2 (TLR2), which activates NF-κB, but not by other TLRs or NOD-like receptors. N-acylated peptides from lipoprotein19180 (Lp19180) in L. plantarum EVs were identified as novel TLR2 ligands. Therefore, L. plantarum induces an immunostimulation though the TLR2 recognition of the N-acylated amino acid moiety of Lp19180 in EVs. Additionally, we detected a large amount of EVs in the rat gastrointestinal tract for the first time, suggesting that EVs released by probiotics function as a modulator of intestinal immunity.

Electrochemical and spectroscopic properties of twisted dibenzo[g,p]chrysene derivatives.

Dibenzo[g,p]chrysene (DBC), which consists of a twisted naphthalene core with four fused benzene rings, is a promising framework for organic electronic materials. Therefore, the research for structure-property relationships is important for the design of DBC-based materials. Here, the electrochemical and spectroscopic properties of DBC derivatives were investigated, and the effects of substituents and torsion of the naphthalene moiety were examined based on density functional theory (DFT) calculations. All the substituted DBC derivatives showed higher oxidation potentials than that for DBC-H, even for compounds that contained an electron-donating group such as DBC-Me and DBC-SMe. DFT calculations clearly indicate that these higher oxidation potentials are due to the ineffective conjugation of the MeO group, which is oriented perpendicular to the benzene ring because of the steric repulsion of substituents on both sides. More specifically, the inductive effect of the MeO group is dominant rather than the mesomeric effect when the substituent is located at both sides of the MeO group. Concerning the torsion of the naphthalene moiety, the twisting results in a slight increase in the HOMO and a slight lowering of the LUMO. The twisting effect is much smaller than the conjugation effect of the MeO group. Absorption spectra of all the substituted DBC derivatives also showed a red-shift as compared to that for DBC-H. Concerning the luminescence, a strong photoluminescence was observed for DBC-H and DBC-Si.

Ultrastructure and distribution of sensory receptors on the nonolfactory organs of the soldier caste in subterranean termite (Coptotermes spp.).

The soldier caste of termites uses sensilla to sense pheromonal, tactile, and vibrational cues to communicate inside and outside their nest. Although sensilla with many modalities on the antennae of subterranean termites have been well explored, there remains a lack of information regarding sensillum characteristics and distribution of the nonolfactory organs of the soldier caste in the Coptotermes genus. In this study, the ultrastructure of sensilla from the soldier caste of three Coptotermes spp. (Coptotermes formosanus, Coptotermes curvignathus, and Coptotermes gestroi) was observed by scanning and transmission electron microscopy, and the putative function of each type was deduced. Six total sensillum types were observed, with two mechanoreceptive sensillum types (hair and plate). The long flexible-peg mechanoreceptive sensilla may work as contact-chemoreceptive sensilla due to their elongated dendritic outer segments and uniporous characteristics. There was a significant depletion of mechano-chemoreceptive sensillum numbers in C. gestroi, which was compensated by a high density of short-peg mechanoreceptive sensilla on the pronotum. Finally, cuticular and innervation characteristics of thermo-/hygrosensitive sensilla were observed for the first time on the labrum of the soldier caste of Coptotermes.

Insights into the mechanism of diurnal variations in methane emission from the stem surfaces of Alnus japonica.

Recent studies have suggested that in certain environments, tree stems emit methane (CH4 ). This study explored the mechanism of CH4 emission from the stem surfaces of Alnus japonica in a riparian wetland. Stem CH4 emission rates and sap flux were monitored year-round, and fine-root anatomy was investigated. CH4 emission rates were estimated using a closed-chamber method. Sap flux was measured using Granier-type thermal dissipation probes. Root anatomy was studied using both optical and cryo-scanning electron microscopy. CH4 emissions during the leafy season exhibited a diurnally changing component superimposed upon an underlying continuum in which the diurnal variation was in phase with sap flux. We propose a model in which stem CH4 emission involves at least two processes: a sap flux-dependent component responsible for the diurnal changes, and a sap flux-independent component responsible for the background continuum. The contribution ratios of the two processes are season-dependent. The background continuum possibly resulted from the diffusive transport of gaseous CH4 from the roots to the upper trunk. Root anatomy analysis indicated that the intercellular space of the cortex and empty xylem cells in fine roots could serve as a passageway for transport of gaseous CH4 .

Identification of a Putative Sensor Protein Involved in Regulation of Vesicle Production by a Hypervesiculating Bacterium, Shewanella vesiculosa HM13.

Bacteria secrete and utilize nanoparticles, called extracellular membrane vesicles (EMVs), for survival in their growing environments. Therefore, the amount and components of EMVs should be tuned in response to the environment. However, how bacteria regulate vesiculation in response to the extracellular environment remains largely unknown. In this study, we identified a putative sensor protein, HM1275, involved in the induction of vesicle production at high lysine concentration in a hypervesiculating Gram-negative bacterium, Shewanella vesiculosa HM13. This protein was predicted to possess typical sensing and signaling domains of sensor proteins, such as methyl-accepting chemotaxis proteins. Comparison of vesicle production between the hm1275-disrupted mutant and the parent strain revealed that HM1275 is involved in lysine-induced hypervesiculation. Moreover, HM1275 has sequence similarity to a biofilm dispersion protein, BdlA, of Pseudomonas aeruginosa PAO1, and hm1275 disruption increased the amount of biofilm. Thus, this study showed that the induction of vesicle production and suppression of biofilm formation in response to lysine concentration are under the control of the same putative sensor protein.

Probing rapid carbon fixation in fast-growing seaweed Ulva meridionalis using stable isotope 13C-labelling.

The high growth rate of Ulva seaweeds makes it a potential algal biomass resource. In particular, Ulva meridionalis grows up to fourfold a day. Here, we demonstrated strong carbon fixation by U. meridionalis using 13C stable isotope labelling and traced the 13C flux through sugar metabolites with isotope-ratio mass spectrometry (IR-MS), Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS), 13C-nuclear magnetic resonance spectrometry (13C-NMR), and gas chromatography-mass spectrometry (GC-MS). U. meridionalis was first cultured in 13C-labelled enriched artificial seawater for 0-12 h, and the algae were collected every 4 h. U. meridionalis grew 1.8-fold (dry weight), and the 13C ratio reached 40% in 12 h, whereas 13C incorporation hardly occurred under darkness. At the beginning of the light period, 13C was incorporated into nucleic diphosphate (NDP) sugars in 4 h, and 13C labelled peaks were identified using FT-ICR-MS spectra. Using semiquantitative 13C-NMR measurements and GC-MS, 13C was detected in starch and matrix polysaccharides after the formation of NDP sugars. Moreover, the 14:10 light:dark regime resulted into 85% of 13C labelling was achieved after 72 h of cultivation. The rapid 13C uptake by U. meridionalis shows its strong carbon fixation capacity as a promising seaweed biomass feedstock.

Genetic characterization and functional implications of the gene cluster for selective protein transport to extracellular membrane vesicles of Shewanella vesiculosa HM13.

A hyper-vesiculating Gram-negative bacterium, Shewanella vesiculosa HM13, secretes a protein of unknown function (P49) as a major cargo of the extracellular membrane vesicles (EMVs). Here, we analyzed the transport mechanism of P49 to EMVs. The P49 gene is found in a gene cluster containing the genes encoding homologs of surface glycolipid biosynthesis proteins (Wza, WecA, LptA, and Wzx), components of type II secretion system (T2SS), glycerophosphodiester phosphodiesterase (GdpD), and nitroreductase (NfnB). We disrupted the genes in this cluster and analyzed the productivity and morphology of EMVs and the localization of P49. EMV production and morphology were only moderately affected by gene disruption, demonstrating that these gene products are not essential for EMV synthesis. In contrast, the localization of P49 was significantly affected by gene disruption. The lack of homologs of the T2SS components resulted in deficiency in secretion of P49. When gdpD, wzx, lptA, and nfnB were disrupted, P49 was released to the extracellular space without being loaded to the EMVs. These results suggest that P49 is translocated across the outer membrane through the T2SS-like machinery and subsequently loaded onto EMVs through interaction with surface glycolipids of EMVs.

Rhamnogalacturonan I galactosyltransferase: Detection of enzyme activity and its hyperactivation.

Rhamnogalacturonan I (RG-I), one of the pectic components of the plant cell wall, is composed of a backbone of repeating disaccharide units of rhamnose and galacturonic acid, and side chains, such as galactans, arabinans, and arabinogalactans. The activity of RG-I galactosyltransferase, which transfers galactosyl residues to rhamnosyl residues in the RG-I backbone, has not been detected until now. Here, we detected galactosyltransferase activity in azuki bean epicotyls using fluorogenic RG-I oligosaccharide acceptors. This enzyme prefers oligosaccharides with a degree of polymerization more than 9. The enzyme activity was detected in the Golgi apparatus, which is the site of pectin synthesis. In vitro hyperactivation of this enzyme was also observed. Moreover, enzyme activity was increased up to 40-fold in the presence of cationic surfactants or polyelectrolytes.

Selenite uptake by outer membrane porin ExtI and its involvement in the subcellular localization of rhodanese-like lipoprotein ExtH in Geobacter sulfurreducens.

Selenite reduction is a key step in the biogeochemical cycle of selenium-an essential trace element for life. A variety of bacteria can transform selenite into elemental selenium nanoparticles on the cell surface via anaerobic respiration or detoxification processes. However, the proteins associated with the uptake of selenite for these processes are poorly understood. In this study, we investigated the role of an outer membrane porin-like protein, ExtI, in selenite permeation in Geobacter sulfurreducens. We demonstrated that selenite uptake and selenium nanoparticle formation were impaired in an extI-deficient strain. A putative rhodanese-like lipoprotein is encoded by an extH gene located immediately upstream of extI in the genome. We showed that ExtH is translocated into inner and outer membranes and that extI deficiency exclusively affects the localization of ExtH in the outer membrane. Coelution of ExtI and ExtH during gel filtration analysis of the outer membrane fraction of wild-type cells suggests a direct protein-protein interaction between them. Taken together, these results lead us to propose a physiological role for ExtI as a selenite channel associated with ExtH in the outer membrane.

Sterical Recognition at Helix-Helix Interface of Leu-Aib-Based Polypeptides with and without a GxxxG-Motif.

An amphiphilic polypeptide, poly(sarcosine)- b-(l-Leu-Aib)8 (SL16), was reported to self-assemble into vesicles. A GxxxG motif, which is known to induce helix dimerization, is incorporated into the hydrophobic helical block of SL16 to synthesize poly(sarcosine)- b-(l-Leu-Aib)2-(Gly-Aib-l-Leu-Aib-Gly-Aib)-(l-Leu-Aib)3 (SG16). SG16 shows helix association in ethanol at a high concentration and low temperatures, which is not observed with SL16. SG16 self-assembles into vesicles, but are found to be more susceptible to rupture by the addition of Triton X-100 than SL16 vesicles. A mixture of SL16 and SG16 self-assembles into small sheets and micelles likely because of mismatch of the modes of helix association arising from sterical accommodation of iso-butyl groups at the helix-helix interface.

Observation of in vitro cellulose synthesis by bacterial cellulose synthase with time-resolved small angle X-ray scattering.

Cellulose synthase is the enzyme that produces cellulose in the living organisms like plant, and has two functions: polymerizing glucose residues (polymerization) and assembling these polymerized molecules into a crystalline microfibril with a "cellulose I" crystallographic structure (crystallization). Many studies, however, have shown that an in vitro reaction of cellulose synthase produces aggregates of a non-native crystallographic structure "cellulose II", despite the remaining polymerizing activity. This is partial denaturation or loss of crystallization function in cellulose synthase, which needs to be resolved to reconstitute its native activity. To this end, we aimed to clarify the process of cellulose II formation by bacterial cellulose synthase in vitro, using in situ small angle X-ray scattering (SAXS). An increase in scattering specific to synthesis was observed around two distinct regions of q (0.2-0.4 nm-1 and <0.1 nm-1) by time-resolved SAXS measurement. The scattering at higher q-region appears prior to lower-q scattering at beginning of the reaction, indicating the existence of smaller primitive aggregations at the initiation stage. This study demonstrates the use of in situ SAXS measurement to decipher the dynamics of biosynthesized cellulose chains, which is a remarkable example of polymer assembly in ambient conditions.

Isolation of a Novel Bacterial Strain Capable of Producing Abundant Extracellular Membrane Vesicles Carrying a Single Major Cargo Protein and Analysis of Its Transport Mechanism.

Extracellular membrane vesicles (EMVs) play an important role in various bacterial activities. EMVs have potential for use as vaccines, drug-delivery vehicles, platforms for extracellular production of recombinant proteins, and so on. In this study, we newly isolated a cold-adapted bacterium, Shewanella vesiculosa HM13, which abundantly produces EMVs, characterized them, and analyzed their cargo transport mechanism. S. vesiculosa HM13, isolated from the intestine of a horse mackerel as a prospective host for a low-temperature secretory protein expression system, produced a single major secretory protein, P49, of unknown function in the culture supernatant. Analysis using sucrose density gradient ultracentrifugation indicated that P49 is a cargo protein carried by EMVs. S. vesiculosa HM13 displayed extensive blebbing on the surface of the outer membrane, and the size of blebs was comparable to that of EMVs. These blebs are thought to be precursors of the EMVs. Disruption of the P49 gene resulted in only a marginal decrease in the EMV production, indicating that the EMVs are produced even in the absence of the major cargo protein. Whole genome sequencing of S. vesiculosa HM13 revealed that this bacterium has a gene cluster coding for a non-canonical type II protein secretion system (T2SS) homolog in addition to a gene cluster coding for canonical T2SS. The P49 gene was located downstream of the former gene cluster. To examine the role of the putative non-canonical T2SS-like translocon, we disrupted the gene coding for a putative outer membrane channel of the translocon, named GspD2. The gspD2 disruption lead to disappearance of P49 in the EMV fraction, whereas the production of EMVs was not significantly affected by this mutation. These results are indicative that the T2SS-like machinery functions as a novel type of protein translocon responsible for selective cargo loading to the EMVs. We also found that GFP fused to the C-terminus of P49 expressed in S. vesiculosa HM13 was transported to EMVs, indicating that P49 is useful as a carrier to deliver the fusion partner to EMVs. These findings deepen our understanding of the mechanism of biogenesis of EMVs and facilitate their applications.

Compartmentalized host spaces accommodating guest aromatic molecules in a chiral way in a helix-peptide-aromatic framework.

A novel host molecular assembly of a free-standing flat nanosheet with compartmentalized spaces was prepared using a bolaamphiphilic peptide composed of two amphiphilic helical peptides and an oligo(naphthaleneethynylene) (ONE) unit at the center of the molecule. The nanosheet possesses void host spaces that can accommodate two mol-equivalent ONE groups to form columns of ONE groups in a right-handed helical way and ONE channels over a long distance. The present molecular system therefore can provide a chiral pore channel for relatively large molecules.

Two one-dimensional arrays of naphthyl and anthryl groups along peptide nanotubes prepared from cyclic peptides comprising α- and ß-amino acids.

A novel cyclic hexapeptide composed of l-α-naphthylalanine, d-α-anthrylalanine, and four ß-alanines (CP6) is synthesized and its molecular assembly into peptide nanotubes (PNTs) and the electronic properties arising from one-dimensional arrays of aromatic groups along the PNTs were investigated. CP6 with a combination of l- and d-α-amino acids is designed to self-assemble into PNTs with them stacking on top of each other under the constraint of maximizing the number of intermolecular hydrogen bonds between the cyclic peptides. Upon PNT formation, the respective side chains of l- and d-α-amino acids are aligned in line along the PNTs. The topological arrangement of the anthryl groups being in close proximity in the CP6 PNT is supported by higher photo-excited energy transfer, appearance of the induced Cotton effects, and the promoted photo-dimerization reaction upon PNT formation. AFM observations reveal that PNT bundles with diameters 5-15 nm are dielectric microcrystals having a piezoelectric coefficient of 2-6 pC N-1. Kelvin force microscopy observations show the generation of surface potentials over 100 mV owing to the one-dimensional array of the anthryl groups along PNTs. Incorporation of α-amino acids with opposite chirality into cyclic ß-peptides is therefore an effective molecular design for the nano-architecture of PNTs displaying one-dimensional arrays of chromophores along PNTs.