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Structural studies of acetan, an exopolysaccharide elaborated by Acetobacter xylinum.

The exopolysaccharide acetan, elaborated by Acetobacter xylinum, has been investigated. The polysaccharide and a heptasaccharide, obtained on enzymic hydrolysis, corresponding to the repeating unit were characterised by sugar and methylation analysis and by NMR spectroscopy and MS. It is concluded that the polysaccharide is composed of repeating units with the following structure. [formula: see text] The polysaccharide further contains approximately two O-acetyl groups per repeating unit, which have not been assigned, but it appears that they are on primary locations.

Carbohydrate Conformation↗

NMR studies of acetan and the related bacterial polysaccharide, CR1/4, produced by a mutant strain of Acetobacter xylinum.

Acetan is a bacterial polysaccharide produced by Acetobacter xylinum NRRL B42. Chemical mutagenesis of A.xylinum allowed selection of a mutant strain which produced a new polysaccharide, CR1/4. 2D NMR methods have been used to assign the 1H and 13C spectra of the two polysaccharides and to determine that CR1/4 has the structure shown below. The total number of O-acetyl groups is slightly less than two per repeating unit. [formula: see text] The pentasaccharide side chain of acetan is truncated to a disaccharide unit in CR1/4, but the structures are otherwise identical. In particular, the degree of acetylation is about the same and the O-acetyl groups are located at the same position in both polysaccharides.

Carbohydrate Sequence↗

Modification of crystallinity and crystalline structure of Acetobacter xylinum cellulose in the presence of water-soluble beta-1,4-linked polysaccharides: 13C-NMR evidence.

Cellulose produced by Acetobacter xylinum in medium containing 0.5% xyloglucan or glucomannan showed altered crystallinities and shifted I alpha/I beta ratios when analysed by solid-state 13C-NMR. By estimating the spectra of cellulose components in each composite, a decreased I alpha content was shown to be countered by increased I beta content in cellulose aggregated in the presence of xyloglucan, causing minimal loss of crystallinity. However, the I alpha decrease was linked primarily to increased disordered content in cellulose produced in medium containing glucomannan. These results are considered in the light of two models for the morphological disposition of the I alpha phase: (i) a series model, proposed on the basis of electron diffraction measurements for an algal cellulose, in which regions of I alpha and I beta alternate along the length of a microfibril, and (ii) a superlattice model, in which the I alpha and I beta domains co-exist throughout the cross-section of each microfibril and form as a result of hierarchical aggregation. The latter model offers clearer insight into the role of the polysaccharides in inhibiting the formation of I alpha crystalline regions. In this superlattice model, polysaccharides adsorbed on surfaces of the most elementary aggregates are displaced to varying degrees during subsequent aggregation, with the presence of these polysaccharides altering the extent of I alpha production at interfaces.

Cellulose↗

Genetic analysis of the acetan biosynthetic pathway in Acetobacter xylinum.

We have identified, cloned and sequenced an 8422 base pair fragment of Acetobacter xylinum genomic DNA containing part of the acetan biosynthetic gene cluster. Computer analysis of the nucleotide sequence data generated revealed the presence of six open reading frames. Comparison of the translated sequences of putative genes to the amino acid sequences of genes from other organisms was used to assign functions to the aceA, aceC and manB genes. These genes were predicted to encode a UDP-glycosyl transferase, a GDP-mannosyl transferase and a phosphomannose isomerase/GDP-mannose pyrophosphorylase, respectively.

Bacterial Proteins↗

Biosynthesis of a novel polysaccharide by Acetobacter xylinum.

An Acetobacter xylinum adapted to a medium containing N-acetylglucosamine (GlcNAc) has been used to prepare a novel polysaccharide containing residual GlcNAc in cellulose. The maximum amount of incorporation was found to be 4 mol% in cellulose, when a mixed medium containing 1.4% glucose (Glc) and 0.6% GlcNAc was used for the culture of A. xylinum. The resulting polysaccharide was lysozyme-susceptible. The aminosugar residue incorporated into bacterial cellulose was found to be only GlcNAc, even if galactosamine (GalN) and glucosamine (GlcN) were applied, whereas there was little effect by mannosamine (ManN). As the major component of the resulting polysaccharide was Glc residues, even if the only carbon source in the culture medium was GlcNAc, it was suggested that there must be several enzyme systems to convert GlcNAc into Glc in the bacteria. Several ammonium salts were also found to be effective for the incorporation of GlcNAc residues when the incubation system was converted to rotatory and aerobic incubation from static incubation. The amount of residual GlcNAc was remarkably increased by the addition of lysozyme-susceptible phosphoryl-chitin (P-chitin) and increased slightly with addition of P-chitin that was less lysozyme-susceptible. However, little effect was found on addition of highly substituted P-chitin.

Acetylglucosamine↗

Physicochemical characterization of an acetan variant secreted by Acetobacter xylinum strain CR1/4.

Chemical mutagenesis has been used to produce mutants of Acetobacter xylinum NRRL B42 that are cellulose-negative and that produce variants of the acetan structure deficient in the side-chain sugar residues. The product of A. xylinum strain CR1/4 has been shown to possess a tetrasaccharide repeat unit with the side chain terminating in glucuronic acid. X-ray diffraction studies of oriented fibres suggest that the polysaccharide CR1/4 forms a fivefold helix with a pitch of 4.8 nm. Light-scattering studies on CR1/4 solutions suggest a molecular weight of 1.2 x 10(6) with radii of gyration values of 86 nm (aqueous solution) and 67 nm (0.1 M NaCl solution). The magnitude of the measured radii of gyration and the shape of the Holtzer plots suggest that CR1/4 can be described as a stiff coil. Preliminary differential scanning calorimetry data show melting behaviour consistent with order-disorder transitions of a charged helical structure. Rheological studies have revealed new synergistic interactions of CR1/4 with locust bean gum. Comparative studies of acetan and CR1/4 show that decreasing the length of the side chain enhances the solution viscosity.

Calorimetry, Differential Scanning↗

Production of cellulose II by Acetobacter xylinum in the presence of 2,6-dichlorobenzonitrile.

This report provides X-ray diffraction and Raman spectral evidence that, when 2,6-dichlorobenzonitrile is present in the culture medium, Acetobacter xylinum, which is a model system for investigation of the biosynthesis of native cellulose, produces cellulose II, as well as cellulose I. The significance of the observations with respect to the mechanism of biosynthesis of cellulose is discussed briefly.

Cellulose↗

Construction of a brewer's yeast having alpha-acetolactate decarboxylase gene from Acetobacter aceti ssp. xylinum integrated in the genome.

alpha-Acetolactate decarboxylase (ALDC) gene from Acetobacter aceti ssp. xylinum has several possible initiation codons in the N-terminus. To determine the initiation codon of the ALDC giving the highest expression levels, glyceraldehyde-3-phosphate dehydrogenase (GPD) promoter was linked just upstream of each possible initiation codon. The ALDC whose translation starts 130 bp downstream from the first ATG codon had the highest activity in yeast cells. When expression levels of the ALDC gene were compared using three strong yeast promoters of glycolytic genes, alcohol dehydrogenase I (ADC1), phosphoglycerate kinase (PGK) and GPD, the GPD promoter was the strongest. The ALDC gene was integrated in a ribosomal RNA gene of a brewer's yeast by co-transformation with an expression plasmid of G418-resistance gene. The laboratory-scale growth test confirmed that the total diacetyl concentration was reduced in wort.

Base Sequence↗

Cellulose synthesis by Acetobacter xylinum. I. Low molecular weight compounds present in the region of synthesis.

An analysis has been made of the low molecular weight fraction present in the region of cellulose synthesis in Acetobacter xylinum suspensions. A number of nucleic acid bases, nucleosides and nucleotides, together with alpha-glucose 1-phosphate and UDPG, were detected in various extracts of washed cells supplied with glucose. Since glucose-6-P could be detected in extracts of ultrasonically disrupted cells, but not in extracts of whole cells, it was concluded that separate pools of hexose phosphate exist in A. xylinum. Preferential release of alpha-glucose-1-P, UDPG and nucleotides was observed during ethanol and EDTA treatment of bacteria. Electron microscopic examination of treated and untreated cells revealed that extensive modification of the cell wall region occurred during such treatments. The results support the proposal that alpha-glucose-1-P, UDPG and nucleotide pools are localised in the cell envelope region, possibly in the periplasm, and that A. xylinum possesses a second permeability barrier outside the cytoplasmic membrane. Nucleic acid bases and nucleosides were observed to diffuse freely through the cell wall and accumulate in the medium, probably as the result of nucleic acid breakdown. The results imply that the effects of cell damage caused by the isolation of the bacteria from the surface pellicle of the culture medium, together with nutrient deprivation, should be considered in work using the non-proliferating system. A stydy of the variation in concentration with time of alpha-glucose-1-P and UDPG, during cellulose synthesis, indicated that both components may play an immediate role in cellulose synthesis. Glycosylated lipid compounds were detected in both cell wall extracts and supernatant fluid, but it is not certain whether these compounds are constituents of the supernatant fluid in vivo.

Cellulose↗

Cellulose synthesis by Acetobacter xylinum. II. Investigation into the relation between cellulose synthesis and cell envelope components.

Cell envelope fractions, capable of cellulose synthesis from uridine diphosphate glucose, alpha-glucose-1-phosphate, glucose-6-phosphate and glucose, have been isolated from Acetobacter xylinum suspensions and various enzymatic properties examined. Essential enzymes were found to be distributed throughout the cell envelope region, with both inner (cytoplasmic) and outer (cell wall) membranes contributing to cellulose synthesis. The central role of UDPG in cellulose synthesis was confirmed and the results indicated that the nucleoside diphosphate sugar functions solely in the cell envelope region of whole cells. A comparison of properties of the cell envelope system with those of different preparations used by other workers, suggested that the method of cell disruption may influence substrate specificity.

Cell Membrane↗

Cellulose synthesis by Acetobacter xylinum. III. Matrix, primer and lipid requirements and heat stability of the cellulose-forming enzymes.

The addition of soluble cellodextrins of increasing size to a cell envelope preparation of Acetobacter xylinum stimulated cellulose synthesis from UDPG. This stimulation was attributed to both acceptor and activator effects. Enzymes required for cellulose synthesis were found to be heat-unstable and those required for synthesis of glycosylated lipid components from UDPG, heat-stable. Both heat-inactivated envelope fragments and supernatant fluid from whole cells were necessary for cellulose synthesis from UDPG. Cellulose was not formed from UDPG in the presence of either supernatant fluid alone or heat-inactivated envelopes alone. The combined results of this and previous studies suggest that either the cell envelope is necessary for synthesis of a more immediate precursor to cellulose than UDPG, or that the synthesis from UDPG requires a matrix. The former suggestion and its possible link with lipid intermediate involvement was strengthened by the observation of inefficient glycosylated lipid formation by a celluloseless mutant strain of A. xylinum. The possible locations of various enzyme activities required for the synthesis of the cellulose precursor are indicated and a possible microfibril nucleation process is discussed.

Cell Wall↗

Characterization of chemically treated bacterial (Acetobacter xylinum) biopolymer: some thermo-mechanical properties.

Bacterial cellulose prepared from pellicles of Acetobacter xylinum (Gluconacetobacter xylinus) is a unique biopolymer in terms of its molecular structure, mechanical strength and chemical stability. The biochemical analysis revealed that various alkali treatment methods were effective in removing proteins and nucleic acids from native membrane resulting in pure cellulose membrane. The effect of various treatment regimens on thermo-mechanical properties of the material was investigated. The cellulose in the form of purified cellulose membranes was characterized by differential scanning calorimetry (DSC), thermo-gravimetric analysis (TGA) and dynamic mechanical thermal analysis (DMTA). The glass transition temperature (T(g)) of the native cellulose (untreated, compressed and dried pellicle) was found to be 13.94 degrees C, in contrast, the chemically treated cellulose membranes has higher T(g) values, ranging from 41.41 degrees C to 48.82 degrees C. Investigations on isothermal crystallization were carried out to study the bulk crystallization kinetics. Thermal decomposition pattern of the native as well as alkali treated cellulose was determined by obtaining thermo-gravimetric curves. At higher temperatures (>300 degrees C), the biopolymer was found to degrade. Nevertheless, the alkaline treated cellulose membrane was more stable (between 343.27 degrees C and 370.05 degrees C) in comparison to the native cellulose (298.07 degrees C). Further, the percentage weight loss in case of native cellulose was found to be 26.57%, in comparison to 6.45% for the treated material, at 300 degrees C. The DMTA revealed complex dynamic modulus of the material, at different temperatures and fixed shear stress, applied at a frequency of 5 Hz. The study delineated the effect of alkali treatment regimens, on the thermo-mechanical properties of bacterial cellulose for its application over a wide range of temperatures.

Alkalies↗

Intramolecular electron transport in quinoprotein alcohol dehydrogenase of Acetobacter methanolicus: a redox-titration study

Quinohemoprotein-cytochrome c complex alcohol dehydrogenase (ADH) of acetic acid bacteria consists of three subunits, of which subunit I contains pyrroloquinoline quinone (PQQ) and heme c, and subunit II contains three heme c components. The PQQ and heme c components are believed to be involved in the intramolecular electron transfer from ethanol to ubiquinone. To study the intramolecular electron transfer in ADH of Acetobacter methanolicus, the redox potentials of heme c components were determined with ADH complex and the isolated subunits I and II of A. methanolicus, as well as hybrid ADH consisting of the subunit I/III complex of Gluconobacter suboxydans ADH and subunit II of A. methanolicus ADH. The redox potentials of hemes c in ADH complex were -130, 49, 188, and 188 mV at pH 7.0 and 24, 187, 190, and 255 mV at pH 4.5. In hybrid ADH, one of these heme c components was largely changed in the redox potential. Reduced ADH was fully oxidized with potassium ferricyanide, while ubiquinone oxidized the enzyme partially. The results indicate that electrons extracted from ethanol at PQQ site are transferred to ubiquinone via heme c in subunit I and two of the three hemes c in subunit II. Copyright 1998 Elsevier Science B.V.

Journal Article↗

Novel glycosyltransferase genes involved in the acetan biosynthesis of Acetobacter xylinum.

Novel aceQ and aceR genes involved in the acetan biosynthesis of Acetobacter xylinum were newly isolated. The homology search with DNA Data Bank of Japan indicated that aceQ and aceR were glycosyltransferases. Their gene-disrupted mutants were obtained by homologous recombination using the tetracycline resistance gene and the electroporation method. By NMR and ESI-MS analyses, aceQ-disrupted mutant DQ was found to secrete a water-soluble polysaccharide harboring the -Man-GlcUA side chain and the aceR-disrupted mutant DR was found to secrete an acetan analog, lacking the terminal Rha residue. These results suggested that aceQ and aceR encode a glucosyltransferase and a rhamnosyltransferase, respectively. It was indicated that acetan analogs harboring various side chains can be generated easily by genetic engineering.

Base Sequence↗

The production of a new water-soluble polysaccharide by Acetobacter xylinum NCI 1005 and its structural analysis by NMR spectroscopy.

A new water-soluble polysaccharide (WSP) was isolated from a culture of Acetobacter xylinum NCI 1005 grown on sucrose. The structure of the WSP was analysed by nuclear magnetic resonance spectroscopy and determined to be a beta-(2-->6)-linked polyfructan, which is structurally different from the polymer synthesized from glucose instead of sucrose by the same strain. The discovery of this new polysaccharide has revealed that the bacterium is able to synthesize two different kinds of water-soluble polysaccharides.

Carbohydrate Sequence↗

c-di-GMP-binding protein, a new factor regulating cellulose synthesis in Acetobacter xylinum.

A protein which specifically binds cyclic diguanylic acid (c-di-GMP), the reversible allosteric activator of the membrane-bound cellulose synthase system of Acetobacter xylinum, has been identified in membrane preparations of this organism. c-di-GMP binding is of high affinity (KD 20 nM), saturable and reversible. The equilibrium of the reaction is markedly and specifically shifted towards the binding direction by K+. The c-di-GMP binding protein, structurally associated with the cellulose synthase, appears to play a major role in modulating the intracellular concentration of free c-di-GMP and thus may constitute an essential factor in regulating cellulose synthesis in vivo.

Allosteric Regulation↗

Characterisation of the polysaccharide produced by Acetobacter xylinum strain CR1/4 by light scattering and atomic force microscopy.

The molecular weight of the extracellular polysaccharide (CR1/4) produced by Acetobacter xylinum strain CR1/4 has been shown to be dependent upon growth conditions. Under normal growth conditions a high molecular weight polysaccharide ( > 1 x 10(6) Da) is produced. Maintaining the pH at 5 results in an order of magnitude increase in the total yield of polysaccharide, but also an order of magnitude decrease in molecular weight. Analysis of the CR1/4 polysaccharides by the techniques of atomic force microscopy and static light scattering suggests that they are double helices. In solution the molecules behave as stiff coils with a Kuhn statistical segment length of 325 nm.

Carbohydrate Conformation↗

Insertion of an E. coli lacZ gene in Acetobacter xylinus for the production of cellulose in whey.

A mini-Tn10:lacZ:kan was inserted into a wild-type strain of Acetobacter xylinus by random transposon mutagenesis, generating a lactose-utilising and cellulose-producing mutant strain designated ITz3. Antibiotic selection plate assays and Southern hybridisation revealed that the lacZ gene was inserted once into the chromosome of strain ITz3 and was stably maintained in non-selective medium after more than 60 generations. The modified strain had, on the average, a 28-fold increase in cellulose production and a 160-fold increase in beta-galactosidase activity when grown in lactose medium. beta-Galactosidase activity is present in either lactose or sucrose medium indicating that the gene is constitutively expressed. Cellulose and beta-galactosidase production by the modified strain was also evaluated in pure and enriched whey substrates. Utilisation of lactose in whey substrate by ITz3 reached 17 g l(-1) after 4 days incubation.

Cellulose↗