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.

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.

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.

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 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.

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.

Mitochondrially-targeted bacterial phosphatidylethanolamine methyltransferase sustained phosphatidylcholine synthesis of a Saccharomyces cerevisiae Δpem1 Δpem2 double mutant without exogenous choline supply.

In eukaryotic cells, phospholipids are synthesized exclusively in the defined organelles specific for each phospholipid species. To explain the reason for this compartmental specificity in the case of phosphatidylcholine (PC) synthesis, we constructed and characterized a Saccharomyces cerevisiae strain that lacked endogenous phosphatidylethanolamine (PE) methyltransferases but had a recombinant PE methyltransferase from Acetobacter aceti, which was fused with a mitochondrial targeting signal from yeast Pet100p and a 3×HA epitope tag. This fusion protein, which we named as mitopmt, was determined to be localized to the mitochondria by fluorescence microscopy and subcellular fractionation. The expression of mitopmt suppressed the choline auxotrophy of a double deletion mutant of PEM1 and PEM2 (pem1Δpem2Δ) and enabled it to synthesize PC in the absence of choline. This growth suppression was observed even if the Kennedy pathway was inactivated by the repression of PCT1 encoding CTP:phosphocholine cytidylyltransferase, suggesting that PC synthesized in the mitochondria is distributed to other organelles without going through the salvage pathway. The pem1Δpem2Δ strain deleted for PSD1 encoding the mitochondrial phosphatidylserine decarboxylase was able to grow because of the expression of mitopmt in the presence of ethanolamine, implying that PE from other organelles, probably from the ER, was converted to PC by mitopmt. These results suggest that PC could move out of the mitochondria, and raise the possibility that its movement is not under strict directional limitations.

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.

A novel biomaterial: bacterial cellulose and its new era applications.

Bacterial cellulose (BC) is a promising natural polymer that is produced by bacteria and that has unique and desirable structural, physical, and chemical properties. From the time when the remarkable properties of BC were found 15 years ago compared with plant cellulose, interest has grown in BC and it has become an article of trade in diverse applications. Following this trend, this paper reviews the progress of relevant studies, including general information about cellulose, production by microorganisms as well as BC cultivation, and its properties. The applications reviewed in the present article comprise biological and nonbiological fields. The latest use of BC in the biomedical, environmental, agricultural, electronic, food, and industrial fields is discussed with its applications in composite form. The present article attempts to amass the assorted uses of BC under one umbrella. Thus, recent advances in BC applications in different fields are thoroughly reviewed. This article concludes with the need for future research of BC to make it commercialized as vital biomaterial.

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.

Geometrical separation method for lipoproteins using bioformulated-fiber matrix electrophoresis: size of high-density lipoprotein does not reflect its density.

The increasing number of patients with metabolic syndrome is a critical global problem. In this study, we describe a novel geometrical electrophoretic separation method using a bioformulated-fiber matrix to analyze high-density lipoprotein (HDL) particles. HDL particles are generally considered to be a beneficial component of the cholesterol fraction. Conventional electrophoresis is widely used but is not necessarily suitable for analyzing HDL particles. Furthermore, a higher HDL density is generally believed to correlate with a smaller particle size. Here, we use a novel geometrical separation technique incorporating recently developed nanotechnology (Nata de Coco) to contradict this belief. A dyslipidemia patient given a 1-month treatment of fenofibrate showed an inverse relationship between HDL density and size. Direct microscopic observation and morphological observation of fractionated HDL particles confirmed a lack of relationship between particle density and size. This new technique may improve diagnostic accuracy and medical treatment for lipid related diseases.

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.

Taxonomic structure and stability of the bacterial community in belgian sourdough ecosystems as assessed by culture and population fingerprinting.

A total of 39 traditional sourdoughs were sampled at 11 bakeries located throughout Belgium which were visited twice with a 1-year interval. The taxonomic structure and stability of the bacterial communities occurring in these traditional sourdoughs were assessed using both culture-dependent and culture-independent methods. A total of 1,194 potential lactic acid bacterium (LAB) isolates were tentatively grouped and identified by repetitive element sequence-based PCR, followed by sequence-based identification using 16S rRNA and pheS genes from a selection of genotypically unique LAB isolates. In parallel, all samples were analyzed by denaturing gradient gel electrophoresis (DGGE) of V3-16S rRNA gene amplicons. In addition, extensive metabolite target analysis of more than 100 different compounds was performed. Both culturing and DGGE analysis showed that the species Lactobacillus sanfranciscensis, Lactobacillus paralimentarius, Lactobacillus plantarum, and Lactobacillus pontis dominated the LAB population of Belgian type I sourdoughs. In addition, DGGE band sequence analysis demonstrated the presence of Acetobacter sp. and a member of the Erwinia/Enterobacter/Pantoea group in some samples. Overall, the culture-dependent and culture-independent approaches each exhibited intrinsic limitations in assessing bacterial LAB diversity in Belgian sourdoughs. Irrespective of the LAB biodiversity, a large majority of the sugar and amino acid metabolites were detected in all sourdough samples. Principal component-based analysis of biodiversity and metabolic data revealed only little variation among the two samples of the sourdoughs produced at the same bakery. The rare cases of instability observed could generally be linked with variations in technological parameters or differences in detection capacity between culture-dependent and culture-independent approaches. Within a sampling interval of 1 year, this study reinforces previous observations that the bakery environment rather than the type or batch of flour largely determines the development of a stable LAB population in sourdoughs.

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.

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.

Atomic-resolution crystal structure of thioredoxin from the acidophilic bacterium Acetobacter aceti.

The crystal structure of thioredoxin (AaTrx) from the acetic acid bacterium Acetobacter aceti was determined at 1 A resolution. This is currently the highest resolution crystal structure available for any thioredoxin. Thioredoxins facilitate thiol-disulfide exchange, a process that is expected to be slow at the low pH values encountered in the A. aceti cytoplasm. Despite the apparent need to function at low pH, neither the active site nor the surface charge distribution of AaTrx is notably different from that of Escherichia coli thioredoxin. Apparently the ancestral thioredoxin was sufficiently stable for use in A. aceti or the need to interact with multiple targets constrained the variation of surface residues. The AaTrx structure presented here provides a clear view of all ionizable protein moieties and waters, a first step in understanding how thiol-disulfide exchange might occur in a low pH cytoplasm, and is a basis for biophysical studies of the mechanism of acid-mediated unfolding. The high resolution of this structure should be useful for computational studies of thioredoxin function, protein structure and dynamics, and side-chain ionization.

Surface functional group dependent apatite formation on bacterial cellulose microfibrils network in a simulated body fluid.

The apatite forming ability of biopolymer bacterial cellulose (BC) has been investigated by soaking different BC specimens in a simulated body fluid (1.5 SBF) under physiological conditions, at 37 degrees C and pH 7.4, mimicking the natural process of apatite formation. From ATR-FTIR spectra and ICP-AES analysis, the crystalline phase nucleated on the BC microfibrils surface was calcium deficient carbonated apatite through initial formation of octacalcium phosphate (OCP) or OCP like calcium phosphate phase regardless of the substrates. Morphology of the deposits from SEM, FE-SEM, and TEM observations revealed the fine structure of thin film plates uniting together to form apatite globules of various size (from <1 mum to 3 mum) with respect to the substrates. Surface modification by TEMPO (2,2,6,6-tetramethylpyperidine-1-oxyl)-mediated oxidation, which can readily form active carboxyl functional groups upon selective oxidation of primary hydroxyl groups on the surface of BC microfibrils, enhanced the rate of apatite nucleation. Ion exchanged treatment with calcium chloride solution after TEMPO-mediated oxidation was found to be remarkably different from other BC substrates with the highest deposit weight and the smallest apatite globules size. The role of BC substrates to induce mineralization rate differs according to the nature of the BC substrates, which strongly influences the growth behavior of the apatite crystals.

Identification and characterization of lipopolysaccharide in acetic acid bacteria.

BACKGROUND: Lipopolysaccharide (LPS), a major component of the cell walls of Gram-negative bacteria, was one of the main components of Coley's vaccine and is known to have strong adjuvanticity. Though it is known that LPS exists in the digestive tract of organisms, the biological significance for the organism has not been clarified. In this study, the correlation between the structure and function of LPS was determined using acetic acid bacteria. These are Gram-negative bacteria consumed in human diets. MATERIALS AND METHODS: Extracts were obtained from a strain of acetic acid bacteria which is used for producing vinegar. Determination of the LPS neutralizing activity was carried out by the Limulus test. Tumor necrosis factor (TNF) and nitric oxide (NO) production were then observed after the addition of the extracts to murine monocyte macrophages (RAW264. 7), with or without Polymyxin B. TNF production in peritoneal macrophages derived from LPS-low responsive mice (C3H/HeJ) was studied after the addition of extracts. RESULTS: The extracts were shown to be positive only in LPS-specific Limulus test and were negative in the (1,3)-beta-D-glucan-specific Limulus test. Both extracts induced NO and TNF production in RAW264. 7 cells, but this was inhibited by the presence of Polymyxin B. TNF production was inhibited in peritoneal macrophages from LPS low-responsive mice (C3H/HeJ). CONCLUSION: LPS with macrophage-activating activity is present in acetic acid bacteria, routinely consumed by humans.