Chemical Synthesis of Acetobacter pasteurianus Lipid A with a Unique Tetrasaccharide Backbone and Evaluation of Its Immunological Functions.

Lipopolysaccharide (LPS), a cell surface component of Gram-negative bacteria, activates innate immunity. Its active principle is the terminal glycolipid lipid A. Acetobacter pasteurianus is a Gram-negative bacterium used in the fermentation of traditional Japanese black rice vinegar (kurozu). In this study, we focused on A. pasteurianus lipid A, which is a potential immunostimulatory component of kurozu. The active principle structure of A. pasteurianus lipid A has not yet been identified. Herein, we first systematically synthesized three types of A. pasteurianus lipid As containing a common and unique tetrasaccharide backbone. We developed an efficient method for constructing the 2-trehalosamine skeleton utilizing borinic acid-catalyzed glycosylation to afford 1,1'-α,α-glycoside in high yield and stereoselectivity. A common tetrasaccharide intermediate with an orthogonal protecting group pattern was constructed via [2+2] glycosylation. After introducing various fatty acids, all protecting groups were removed to achieve the first chemical synthesis of three distinct types of A. pasteurianus lipid As. After evaluating their immunological function using both human and murine cell lines, we identified the active principles of A. pasteurianus LPS. We also found the unique anomeric structure of A. pasteurianus lipid A contributes to its high chemical stability.

Bacterial cellulose: Biopolymer with novel medical applications.

Due to the growing importance of green chemistry, the search for alternatives to cellulose has begun, leading to the rediscovery of bacterial cellulose (BC). The material is produced by Gluconacetobacter and Acetobacter bacteria, mainly Komagataeibacter xylinus. It is a pure biopolymer, without lignin or hemicellulose, forming a three-dimensional mesh, showing much lower organization than its plant counterpart. Thanks to its design, it has proven itself in completely unprecedented applications - especially in the field of biomedical sciences. Coming in countless forms, it has found use in applications such as wound dressings, drug delivery systems, or tissue engineering. The review article focuses on discussing the main structural differences between plant and bacterial cellulose, methods of bacterial cellulose synthesis, and the latest trends in BC applications in biomedical sciences.

The Peculiar Structure of Acetobacter pasteurianus CIP103108 LPS Core Oligosaccharide.

Acetobacter pasteurianus, a member of the Alphaproteobacteria, is an acetic acid-producing bacterium present on sugar-rich substrates such as such as fruits, flowers and vegetables and traditionally used in the production of fermented food. The preferred living habitat associated with acid conditions makes the structure of the bacterial cell wall interesting to study, due to expected uncommon features. We have used a combination of chemical, analytical and NMR spectroscopy approaches to define the complete structure of the core oligosaccharide from A. pasteurianus CIP103108 LPS. Interestingly, the core oligosaccharide displays a high concentration of negatively charged groups, structural features that might contribute to reinforcing the bacterial membrane.

Investigation of lipid profile in Acetobacter pasteurianus Ab3 against acetic acid stress during vinegar production.

Elucidation of the acetic acid resistance (AAR) mechanisms of Acetobacter pasteurianus is significant for vinegar production. In this study, cell membrane lipid profile of A. pasteurianus Ab3 was investigated by gas chromatography-mass spectrometer (GC-MS) and high performance liquid chromatography-electrospray ionization (HPLC-ESI) combined with high resolution accurate mass/mass spectrometry (HRAM/MS). We observed that cell remodeled the membrane physical state by decreasing the ratio of saturated fatty acids (SFAs)/unsaturated fatty acids (UFAs), and increasing the chain length of fatty acids (FAs) and the content of cyclopropane FAs in response to extreme acid stress. Noticeably, the content of octadecadienoic acid (C18:2) elevated remarkably. Moreover, a continuous reduction in cell membrane fluidity and a "V-type" variance in permeability were discovered. The content of glycerophospholipid and ceramide increased significantly in cells harvested from culture with acidity of 75 g/L and 95 g/L compared to that with acidity of 30 g/L. Among the identified lipid species, the content of phosphatidylcholine (e.g. PC 19:0/18:2 and 19:1/18:0), ceramide (e.g. Cer d18:0/16:1 and d18:0/16:1 + O), and dimethylphosphatidylethanolamine (e.g. dMePE 19:1/16:1) increased notably with increasing acidity. Collectively, these findings refresh our current understanding of the AAR mechanisms in A. pasteurianus Ab3, and should direct future strain breeding and vinegar fermentation.

Silver nanoparticle/bacterial nanocellulose paper composites for paste-and-read SERS detection of pesticides on fruit surfaces.

This study aimed to develop an eco-friendly flexible surface-enhanced Raman scattering (SERS) substrate for in-situ detection of pesticides using biodegradable bacterial nanocellulose (BNC). Plasmonic silver nanoparticle- bacterial nanocellulose paper (AgNP-BNCP) composites were prepared by vacuum-assisted filtration. After loading AgNPs into BNC hydrogel, AgNPs were trapped firmly in the network of nanofibrous BNCP upon ambient drying process, resulting in 3D SERS hotspots within a few-micron depth on the substrate. The fabricated AgNP-BNCPs exhibited high SERS activity with good reproducibility and stability as demonstrated by the detection of 4-aminothiophenol and methomyl pesticide. Due to the optical transparency of BNCP, a direct and rapid detection of methomyl on fruit peels using AgNP-BNCPs can be achieved, demonstrating a simple and effective 'paste-and-read' SERS approach. These results demonstrate potential of AgNP-BNCP composites for user-friendly in-situ SERS analysis.

Surface engineering of spongy bacterial cellulose via constructing crossed groove/column micropattern by low-energy CO2 laser photolithography toward scar-free wound healing.

Bacterial cellulose (BC) is a bio-derived polymer, and it has been considered as an excellent candidate material for tissue engineering. In this study, a crossed groove/column micropattern was constructed on spongy, porous BC using low-energy CO2 laser photolithography. Applying the targeted immobilization of a tetrapeptide consisting of Arginine-Glycine-Aspartic acid-Serine (H-Arg-Gly-Asp-Ser-OH, RGDS) as a fibronectin onto the column platform surface, the resulting micropatterned BC (RGDS-MPBC) exhibited dual affinities to fibroblasts and collagen. Material characterization of RGDS-MPBC revealed that the micropattern was built by the column part with size of ~100 × 100 µm wide and ~100 µm deep, and the groove part with size of ~150 µm wide. Hydrating the MPBC did not result in the collapse of the integrity of the micropattern, suggesting its potential application in a highly hydrated wound environment. Cell culture assays revealed that the RGDS-MPBC exhibited an improved cytotoxicity to mouse fibroblasts L929, as compared to the pristine BC. Meanwhile, it was observed that the RGDS-MPBC was able to guide the ordered aggregation of human skin fibroblast (HSF) cells on the column platform surface, and no HSF cells were found in the groove channels. Over time, it was found that a dense network of collagen was gradually established across the groove channels. Furthermore, the in-vivo animal study preliminarily demonstrated the scar-free healing potential of the micropatterned BC materials. Therefore, this RGDS-MPBC material exhibited its advantages in guiding cell migration and collagen distribution, which could present a prospect in the establishment of "basket-woven" organization of collagen in normal skin tissue against the formation of dense, parallel aggregation of collagen fibers in scar tissue toward scar-free wound healing outcome.

Cobalamin is produced by Acetobacter pasteurianus DSM 3509.

Only a few cobalamin-producing bacterial species are known which are suitable for food fermentations. The strain of Acetobacter pasteurianus DSM 3509 was found to have the capability to synthesize cobalamin. A survival test and a preliminary genetic study of the gene of uroporphyrinogen-III synthase indicated the ability to synthesize cobalamin. By a modified microbiological assay based on Lactobacillus delbrueckii ssp. lactis DSM 20355, 4.57 ng/mL of cyanocorrinoids and 0.75 ng/mL of noncorrinoid growth factors were detected. The product extracted and isolated by immunoaffinity chromatography in its cyanide form had the similar UV spectrum as standard cyanocobalamin and Coα-[α-(7-adenyl)]-(Coß-cyano) cobamide also known as pseudovitamin B12 produced by Lactobacillus reuteri DSM 20016. The chromatographically separated product of A. pasteurianus was subjected to mass spectrometrical analysis. There, its fragmentation pattern turned out to be equivalent to that of cyanocobalamin also produced by Propionibacterium freudenreichii ssp. freudenreichii DSM 20271 and clearly differs from pseudovitamin B12. Due to the presence of this species in several food applications, there might be cobalamin residues in food fermented with these bacteria.

Structure and inflammatory activity of the LPS isolated from Acetobacter pasteurianus CIP103108.

Acetobacter pasteurianus is an acetic acid-producing Gram-negative bacterium commonly found associated with plants and plant products and widely used in the production of fermented foods, such as kefir and vinegar. Due to the acid conditions of the bacterium living habitat, uncommon structural features composing its cell envelope are expected. In the present work we have investigated the A. pasteurianus CIP103108 lipopolysaccharide (LPS) structure and immunoactivity. The structure of the lipid A and of two different O-polysaccharides was assessed. Furthermore, immunological studies with human cells showed a low immunostimulant activity of the isolated LPS, in addition to a slight capability to lower the NF-kB activation upon stimulation by toxic LPS.

Investigation of microorganisms involved in kefir biofilm formation.

Kefir is a natural fermentation agent composed of various microorganisms. To address the mechanism of kefir grain formation, we investigated the microbial role in forming kefir biofilms. The results showed that a biofilm could be formed in kefir-fermented milk and the biofilm forming ability reached the maximum at 13 days. The strains Kluyveromyces marxianus, Lactococcus lactis, Leuconostoc mesenteroides, Lactobacillus kefiri, Lactobacillus sunkii and Acetobacter orientalis were isolated from kefir biofilms by the streak-plate method. These microorganisms were analysed with respect to biofilm forming properties, including their surface characterisation (hydrophobicity and zeta potentials) and the microbial aggregation. The results indicated that Klu. marxianus possessed the strongest biofilm forming properties with the strongest hydrophobicity, lowest zeta potential and greatest auto-aggregation ability. When Klu. marxianus and Ac. orientalis were co-cultured with kefir LAB strains respectively, it was found that mixing Klu. marxianus with Lb. sunkii produced the highest co-aggregation ability. These results elucidated the mechanism of kefir biofilm formation and the microorganisms involved.

Characterization of outer membrane vesicles of Acetobacter pasteurianus NBRC3283.

Acetobacter pasteurianus is characterized as a fermenting bacterium of kurozu, which is a common traditional Japanese black vinegar. Recently, we separated acid-resistant and low Toll-like receptor 4 (TLR4)-stimulatory lipopolysaccharides (LPS) from A. pasteurianus. We also showed that their lipid A parts possessed a novel sugar backbone that is responsible for the low TLR4-stimulatory and acid-resistant properties of the LPS. Outer membrane vesicles (OMVs) are nano-sized spherical structures secreted from many gram-negative bacteria. OMVs contain LPS and act as immunomodulants such as vaccines. In this study, we investigated OMVs secreted from A. pasteurianus. OMV secretion from A. pasteurianus NBRC 3283 cells was observed after 2 days in culture by transmission electron microscopy imaging. Thus OMVs were separated from the culture supernatants by ultracentrifugation and then purified by OptiPrep density gradient centrifugation. The OMVs contained several proteins including outer membrane proteins, and several sugars as components of LPS. The OMVs weakly stimulated TLR4 in accordance with the activity of A. pasteurianus LPS. Additionally, the TLR2-stimulating activity of the OMVs was significantly potent, indicating the existence of lipoproteins. Furthermore OMV-like spherical particles were observed in kurozu. Some of these particles are probably derived from A. pasteurianus. These data suggest that A. pasteurianus produce OMVs that contain LPS and probably lipoproteins, and can modulate the innate immune system.

Microstructured Multilevel Bacterial Cellulose Allows the Guided Growth of Neural Stem Cells.

Repeated photolithographic and etching processes allow the production of multileveled polymer microstructures that can be used as templates to produce bacterial cellulose with defined surfaces on demand. By applying this approach, the bacterial cellulose surface obtains new properties and its use for culturing neural stem cells cellulose substrate topography influences the cell growth in a defined manner.

Characterization of a Novel d-Glycero-d-talo-oct-2-ulosonic acid-substituted Lipid A Moiety in the Lipopolysaccharide Produced by the Acetic Acid Bacterium Acetobacter pasteurianus NBRC 3283.

Acetobacter pasteurianus is an aerobic Gram-negative rod that is used in the fermentation process used to produce the traditional Japanese black rice vinegar kurozu. Previously, we found that a hydrophobic fraction derived from kurozu stimulates Toll-like receptors to produce cytokines. LPSs, particularly LPS from A. pasteurianus, are strong candidates for the immunostimulatory component of kurozu. The LPS of A. pasteurianus remains stable in acidic conditions during the 2 years of the abovementioned fermentation process. Thus, we hypothesized that its stability results from its structure. In this study, we isolated the LPS produced by A. pasteurianus NBRC 3283 bacterial cells and characterized the structure of its lipid A component. The lipid A moiety was obtained by standard weak acid hydrolysis of the LPS. However, the hydrolysis was incomplete because a certain proportion of the LPS contained acid-stable d-glycero-d-talo-oct-2-ulosonic acid (Ko) residues instead of the acid-labile 3-deoxy-d-manno-oct-2-ulosonic acid residues that are normally found in typical LPS. Even so, we obtained a Ko-substituted lipid A with a novel sugar backbone, α-Man(1-4)[α-Ko(2-6)]ß-GlcN3N(1-6)α-GlcN(1-1)α-GlcA. Its reducing end GlcN(1-1)GlcA bond was also found to be quite acid-stable. Six fatty acids were attached to the backbone. Both the whole LPS and the lipid A moiety induced TNF-α production in murine cells via Toll-like receptor 4, although their activity was weaker than those of Escherichia coli LPS and lipid A. These results suggest that the structurally atypical A. pasteurianus lipid A found in this study remains stable and, hence, retains its immunostimulatory activity during acetic acid fermentation.

Superior hybrid hydrogels of polyacrylamide enhanced by bacterial cellulose nanofiber clusters.

Hybrid polyacrylamide/bacterial cellulose nanofiber clusters (PAM/BC) hydrogels with high strength, toughness and recoverability were synthesized by in situ polymerization of acrylamide monomer in BC nanofiber clusters suspension. The hybrid gels exhibited an extremely large elongation at break of 2200%, and a high fracture stress of 1.35MPa. Additionally, the original length of hydrogels could be recovered after releasing the tensile force. Compressive results showed that the PAM/BC hybrid gels could reach a strain of about 99% without break, and was able to completely recover its original shape immediately after releasing the compression force. The compressive stress at 99% reached as high as 30MPa. Nearly no hysteresis in cyclic compressive tests was observed with these hybrid gels. The FT-IR, XRD and TGA analysis showed that hydrogen bonds between the PAM chains and BC nanofiber clusters mainly contributed to the superior mechanical properties of hybrid hydrogels. The cell viability results suggested that PAM/BC hybrid hydrogel was benign for biomedical application. These PAM/BC hydrogels offer a great promise as biomaterials such as bone and cartilage repair materials.

Electrically conductive nano graphite-filled bacterial cellulose composites.

A unique three dimensional (3D) porous structured bacterial cellulose (BC) can act as a supporting material to deposit the nanofillers in order to create advanced BC-based functional nanomaterials for various technological applications. In this study, novel nanocomposites comprised of BC with exfoliated graphite nanoplatelets (xGnP) incorporated into the BC matrix were prepared using a simple particle impregnation strategy to enhance the thermal properties and electrical conductivity of the BC. The flake-shaped xGnP particles were well dispersed and formed a continuous network throughout the BC matrix. The temperature at 10% weight loss, thermal stability and residual ash content of the nanocomposites increased at higher xGnP loadings. The electrical conductivity of the composites increased with increasing xGnP loading (attaining values 0.75 S/cm with the addition of 2 wt.% of xGnP). The enhanced conductive and thermal properties of the BC-xGnP nanocomposites will broaden applications (biosensors, tissue engineering, etc.) of BC and xGnP.

Comparative Proteome of Acetobacter pasteurianus Ab3 During the High Acidity Rice Vinegar Fermentation.

As a traditional Asian food for several centuries, vinegar is known to be produced by acetic acid bacteria. The Acetobacter species is the primary starter for vinegar fermentation and has evolutionarily acquired acetic acid resistance, in which Acetobacter pasteurianus Ab3 is routinely used for industrial production of rice vinegar with a high acidity (9 %, w/v). In contrast to the documented short-term and low acetic acid effects on A. pasteurianus, here we investigated the molecular and cellular signatures of long-term and high acetic acid responses by proteomic profiling with bidimensional gel electrophoresis and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI TOF/MS) analyses. Protein spots of interest were selected based on the threshold ANOVA p value of 0.05 and minimal twofold of differential expression, leading to the identification of 26 proteins that are functionally enriched in oxidoreductase activity, cell membrane, and metabolism. The alterations in protein functioning in respiratory chain and protein denaturation may underlay cellular modifications at the outer membrane. Significantly, we found that at higher acidity fermentation phase, the A. pasteurianus Ab3 cells would adapt to distinct physiological processes from that of an ordinary vinegar fermentation with intermediate acidity, indicating increasing energy requirement and dependency of membrane integrity during the transition of acetic acid production. Together, our study provided new insights into the adaptation mechanisms in A. pasteurianus to high acetic acid environments and yield novel regulators and key pathways during the development of acetic acid resistance.

Identification of Microbial Metabolites Elevating Vitamin Contents in Barley Seeds.

The current investigation analyzes metabolites of Acetobacter aceti to explore chemical compounds responsible for the induction of vitamins in barley seeds. A bioactivity guided assay of bacterial extracts and chromatographic analyses of barley produce revealed 13 chemical compounds, which were subjected to principal component analysis (PCA). PCA determined four chemical compounds (i.e., quinolinic acid, pyridoxic acid, p-aminobenzoate, and α-oxobutanoic acid) highly associated with increased quantities of vitamins. Further experimentations confirmed that quinolinic acid and p-aminobenzoate were the most efficient vitamin inducers. The results indicated chloroform/ethanol (4:1) as the best solvent system for the extraction of active compounds from crude metabolites of A. aceti. Significant quantities of mevalonic acid were detected in the extracted fraction, indicating the possible induction of the isoprenoid pathway. Altogether, the current investigation broadens the frontiers in plant-microbe interaction.

Preparation and characterization of transparent PMMA-cellulose-based nanocomposites.

Nanocomposites of polymethylmethacrylate (PMMA) and cellulose were made by a solution casting method using acetone as the solvent. The nanofiber networks were prepared using three different types of cellulose nanofibers: (i) nanofibrillated cellulose (NFC), (ii) cellulose nanocrystals (CNC) and (iii) bacterial cellulose from nata de coca (NDC). The loading of cellulose nanofibrils in the PMMA varied between 0.25 and 0.5 wt%. The mechanical properties of the composites were evaluated using a dynamic mechanical thermal analyzer (DMTA). The flexural modulus of the nanocomposites reinforced with NDC at the 0.5 wt% loading level increased 23% compared to that of pure PMMA. The NFC composite also exhibited a slightly increased flexural strength around 60 MPa while PMMA had a flexural strength of 57 MPa. The addition of NDC increased the storage modulus (11%) compared to neat PMMA at room temperature while the storage modulus of PPMA/CNC nanocomposite containing 0.25 and 0.5 wt% cellulose increased about 46% and 260% to that of the pure PMMA at the glass transition temperature, respectively. Thermogravimetric analysis (TGA) indicated that there was no significant change in thermal stability of the composites. The UV-vis transmittance of the CNF nanocomposites decreased by 9% and 27% with the addition of 0.25 wt% CNC and NDC, respectively. This work is intended to spur research and development activity for application of CNF reinforced PMMA nanocomposites in applications such as: packaging, flexible screens, optically transparent films and light-weight transparent materials for ballistic protection.

Protein profile of Acetobacter pasteurianus HSZ3-21.

Acetobacter pasteurianus plays an important role in the process of traditional vinegar production and is also essential for the fermentation of Zhenjiang aromatic vinegar. In this study, we utilized the proteomic approach to analyze the proteomic profile of A. pasteurianus HSZ3-21, and 258 proteins were successfully identified by MALDI-TOF-MS and database search. The hydropathy and GO analyse combined with COG results of the identified proteins revealed the molecular biological characteristics of A. pasteurianus proteins, that is, most proteins of A. pasteurianus were related to metabolic process, binding, catalytic or cellular response. Meanwhile, our results also showed that some proteins of A. pasteurianus may be responsible for acetic acid tolerance, thermotolerance, and stress response. Therefore, the identification of 258 proteins not only deciphers protein composition and functional classification of A. pasteurianus, but also provides useful information for improving quality of Zhenjiang aromatic vinegar.

Evolution of morphology of bacterial cellulose scaffolds during early culture.

Morphological characteristics of a fibrous tissue engineering (TE) scaffold are key parameters affecting cell behavior. However, no study regarding the evolution of morphology of bacterial cellulose (BC) scaffolds during the culture process has been reported to date. In this work, BC scaffolds cultured for different times starting from 0.5h were characterized. The results demonstrated that the formation of an integrated scaffold and its 3D network structure, porosity, fiber diameter, light transmittance, and the morphology of hydroxyapatite (HAp)-deposited BC scaffolds changed with culture time. However, the surface and crystal structure of BC fibers did not change with culture time and no difference was found in the crystal structure of HAp deposited on BC templates regardless of BC culture time. The findings presented herein suggest that proper selection of culture time can potentially enhance the biological function of BC TE scaffold by optimizing its morphological characteristics.

Synthesis of a novel acrylated abietic acid-g-bacterial cellulose hydrogel by gamma irradiation.

Acrylated abietic acid (acrylated AbA) and acrylated abietic acid-grafted bacterial cellulose pH sensitive hydrogel (acrylated AbA-g-BC) were prepared by a one-pot synthesis. The successful dimerization of acrylic acid (AA) and abietic acid (AbA) and grafting of the dimer onto bacterial cellulose (BC) was confirmed by 13C solid state NMR as well as FT-IR. X-ray diffraction analysis showed characteristic peaks for AbA and BC; further, there was no effect of increasing amorphous AA content on the overall crystallinity of the hydrogel. Differential scanning calorimetry revealed a glass transition temperature of 80°C. Gel fraction and swelling studies gave insight into the features of the hydrogel, suggesting that it was suitable for future applications such as drug delivery. Scanning electron microscopy observations showed an interesting interpenetrating network within the walls of hydrogel samples with the lowest levels of AA and gamma radiation doses. Cell viability test revealed that the synthesized hydrogel is safe for future use in biomedical applications.