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PubMed2000

Location and limitation of cellulose production by Acetobacter xylinum established from oxygen profiles.

The static fermentation of coconut water sucrose by Acetobacter xylinum was carried out at initial pH's of 3.0, 4.0, 5.0 or 6.0. Cellulose was produced at the surface, and its production was most favourable at pH's 4.0 and 5.0. These pH values also allowed for optimal bacterial growth. Oxygen concentration profiles were measured with microelectrodes at different cultivation stages, and steep profiles were obtained with penetration depths between 50 and 100 microm. A substrate penetration depth analysis confirmed the hypothesis that the first stage of the fermentation is entirely oxygen controlled. Diffusion calculations showed, however, that at a later stage sucrose becomes a limiting substrate also, which was confirmed by the decrease in cellulose production rate over time. The effective diffusion coefficient of oxygen in deactivated cellulose pellicles was measured with microelectrodes, and a value of 1.4 x 10(-9) m2/s was obtained under all investigated conditions. The oxygen flux was 5.9 x 10(-6) mol/m2.s, while a significantly higher value of 9.1 x 10(-6) mol/m2.s was obtained at pH 4.0.

Journal Article↗
PubMed2003

Quinoprotein alcohol dehydrogenase is involved in catabolic acetate production, while NAD-dependent alcohol dehydrogenase in ethanol assimilation in Acetobacter pasteurianus SKU1108.

The relationship between quinoprotein alcohol dehydrogenase (ADH) and NAD-dependent ADH was studied by constructing quinoprotein ADH-deficient mutants. Quinoprotein ADH-deficient mutants were successfully constructed from Acetobacter pasteurianus SKU1108 by N-methyl-N'-nitro-N-nitrosoguanidine (NTG) mutagenesis and also by adhA gene disruption with a kanamycin cassette. The NTG mutant exhibited a complete loss of its acetate-producing ability and acetic acid resistance, while the disruptant also exhibited a loss of its acetic acid resistance but retained a weak ADH activity. The immunoblot analysis of quinoprotein ADH indicated that there are no appreciable ADH subunits in the membranes of both mutant strains. The NTG mutant grew better than the wild-type strain in ethanol-containing medium, despite the absence of quinoprotein ADH. In the mutant, the activities of two NAD-dependent ADHs, present in a small amount in the wild-type strain, markedly increased in the cytoplasm when cultured in a medium containing ethanol, concomitant to the increase in the activities of the key enzymes in TCA and glyoxylate cycles. The disruptant showed a poorer growth than the wild-type strain, producing a lower amount of acetic acid in ethanol culture, and it induced one of the two NAD-dependent ADHs and some of the acetate-assimilating enzymes induced in the NTG mutant. This study clearly showed that quinoprotein ADH is extensively involved in acetic acid production, while NAD-dependent ADH only in ethanol assimilation through the TCA and glyoxylate cycles in acetic acid bacteria. The differences between the NTG mutant and the disruptant are also discussed.

Journal Article↗
PubMed2004

Cloning and characterization of the dnaKJ operon in Acetobacter aceti.

The dnaKJ operon of Acetobacter aceti was cloned and sequenced. The profile of the gene configuration was similar to that of other alpha-proteobacteria. In the DnaK and DnaJ proteins of A. aceti, the characteristic domains/motifs reported in other organisms were well conserved. This operon was transcribed in response to a temperature shift and exposure to ethanol/acetic acid. The overexpression of this operon in A. aceti resulted in improved growth compared to the control strain at high temperature or in the presence of ethanol, suggesting a correlation to resistance against stressors present during fermentation, although the overexpression did not increase the resistance to acetic acid.

Journal Article↗
PubMed1999

Effects of pH and dissolved oxygen on cellulose production by Acetobacter xylinum BRC5 in agitated culture.

Acetobacter xylinum BRC5 was cultivated in a jar fermentor using glucose as the sole carbon source. Strain BRC5 oxidized almost all of the glucose to gluconic acid; thereafter, it biosynthesized cellulose by utilizing gluconic acid accumulated in the broth. The optimal pH for metabolizing glucose to gluconic acid was 4.0, while a pH of 5.5 was preferred for cell growth and cellulose production from the accumulated gluconic acid in the medium. Shifting the pH from 4.0 to 5.5 during the cellulose production phase in batch cultures improved cellulose production and reduced the total fermentation time, compared to batch cultures at constant pH. In constant fed-batch culture, 10 g/l of cellulose was obtained from 40 g/l of glucose, a yield which was approximately 2-fold higher than in batch culture with the same initial glucose concentration, even without control of the level of dissolved oxygen. The highest cellulose yield was obtained in fed-batch cultures in which the dissolved oxygen concentration was controlled at 10% saturation. Control of pH and dissolved oxygen to optimal levels was effective for improving the production rate and yield of cellulose, to achieve a high cellulose productivity of 0.3 g cellulose/l x h. Approximately 15 g/l of cellulose was considered to be the highest yield obtainable using conventional fermentors because the culture broth then became too viscous to allow satisfactory aeration.

Journal Article↗
PubMed2001

Phosphodiesterase A1, a regulator of cellulose synthesis in Acetobacter xylinum, is a heme-based sensor.

The phosphodiesterase A1 protein of Acetobacter xylinum, AxPDEA1, is a key regulator of bacterial cellulose synthesis. This phosphodiesterase linearizes cyclic bis(3'-->5')diguanylic acid, an allosteric activator of the bacterial cellulose synthase, to the ineffectual pGpG. Here we show that AxPDEA1 contains heme and is regulated by reversible binding of O(2) to the heme. Apo-AxPDEA1 has less than 2% of the phosphodiesterase activity of holo-AxPDEA1, and reconstitution with hemin restores full activity. O(2) regulation is due to deoxyheme being a better activator than oxyheme. AxPDEA1 is homologous to the Escherichia coli direct oxygen sensor protein, EcDos, over its entire length and is homologous to the FixL histidine kinases over only a heme-binding PAS domain. The properties of the heme-binding domain of AxPDEA1 are significantly different from those of other O(2)-responsive heme-based sensors. The rate of AxPDEA1 autoxidation (half-life > 12 h) is the slowest observed so far for this type of heme protein fold. The O(2) affinity of AxPDEA1 (K(d) approximately 10 microM) is comparable to that of EcDos, but the rate constants for O(2) association (k(on) = 6.6 microM(-)(1) s(-)(1)) and dissociation (k(off) = 77 s(-)(1)) are 2000 times higher. Our results illustrate the versatility of signal transduction mechanisms for the heme-PAS class of O(2) sensors and provide the first example of O(2) regulation of a second messenger.

Amino Acid Sequence↗
PubMedDate not supplied

Enhancement of cellulose pellicle production by constitutively expressing vitreoscilla hemoglobin in Acetobacter xylinum.

Vitreoscilla hemoglobin (VHb) gene driven by the constitutive bla promoter was expressed in the cellulose-producing Acetobacter xylinum. The expressed VHb was biochemically active and could enhance cell growth in a shaken culture containing cellulase. VHb-expressing A. xylinum (VHb+) exhibited a specific growth rate 50% higher than that of the host strain (VHb-). Probably because of its faster growth rate, the size of tentacled cellulose beads produced by VHb+ was about 20% of that produced by VHb- after 2 days cultivation in a shake-flask. When cultured statically, the amount of cellulose pellicle produced by VHb+ could be 2-fold that produced by VHb-. Cellulose pellicle concentration of 11 g/L was obtained for VHb+, whereas 6 g/L was obtained for VHb- after 6 days of microaerobic incubation.

Bacterial Proteins↗
PubMed2002

Cultivation of Acetobacter xylinum for bacterial cellulose production in a modified airlift reactor.

Acetobacter xylinum for bacterial cellulose production was cultivated in a modified airlift reactor. Better results were obtained from the modified reactor than from a conventional bubble column. After 72 h of cultivation, the final concentration of bacterial cellulose was 7.72 g/l and the productivity was 0.107 g/l per h in the modified airlift reactor. The concentration of bacterial cellulose was about three times higher than that produced in the conventional bubble column. Moreover, the bacterial cellulose produced using the modified reactor formed a unique elliptical pellet (the average diameter was 10 mm), which is different from the fibrous form produced using the stirred-tank reactor. The modified airlift reactor with the suspended bacterial cellulose in pellet form had a higher volumetric oxygen-transfer coefficient and mixing capability than that with bacterial cellulose in fibrous form. The dissolved oxygen in the modified airlift reactor could be maintained above 35% throughout the cultivation.

Bioreactors↗
PubMed1968

The role of ubiquinone in the respiratory chain of Acetobacter xylinum.

1. Whole cells of Acetobacter xylinum were found to contain a quinone of the ubiquinone (coenzyme Q) group. The quinone was isolated from the cells and crystallized. It was identified by its physical, chemical and spectroscopic properties as a ubiquinone with 10 isoprene units (ubiquinone-10). No naphthaquinone was detected in the cells. 2. Cell-free extracts prepared by means of a French pressure cell were separated into three fractions by differential centrifugation. The ubiquinone was located predominantly in the particulate fraction sedimenting at 33000g, which also contained most of the NADH oxidase and malate oxidase activities. The concentration of ubiquinone-10 in extracts was similar to that of the flavoproteins and about three times the concentration of the individual cytochromes. 3. Aerobic incubations of crude extracts with either NADH or malate resulted in reduction of the endogenous ubiquinone-10 to steady-state concentrations of 55 and 40% of the total quinone respectively. In the presence of cyanide more than 95% of the endogenous ubiquinone-10 was reduced by either NADH or malate. 4. The initial rate of reduction of endogenous ubiquinone-10 by malate and the rate of ubiquinol oxidation, in A. xylinum extracts, were found to be compatible with the overall rate of malate oxidation with oxygen. 5. The effects of various respiratory inhibitors on the oxidation-reduction reactions of the endogenous quinone indicate that its position on the respiratory chain is between the malate flavoprotein dehydrogenase and the cytochrome chain.

Alcohol Oxidoreductases↗
PubMed1969

Factors afecting the activity of pyruvate kinase of Acetobacter xylinum.

1. Extracts of Acetobacter xylinum were found to contain the glycolytic enzymes involved in the conversion of triose phosphate into pyruvate. Pyruvate kinase had the lowest relative activity. Phosphofructokinase activity was not detected in the extracts. 2. Only slight differences in the activity of pyruvate kinase were observed between cells grown on glucose and those grown on intermediates of the tricarboxylic acid cycle. 3. Pyruvate kinase, partially purified from ultrasonic extracts by ammonium sulphate fractionation, required Mg(2+) ions for activity. It was not activated by K(+) or NH(4) (+) ions. 4. The plots representing the relationship between initial velocity and phosphoenolpyruvate concentration were sigmoidal, suggesting a co-operative effect for phosphoenolpyruvate. The Hill coefficient (n) for phosphoenolpyruvate was 2. The rate of the reaction changed with increasing ADP concentrations according to normal Michaelis-Menten kinetics. 5. The enzyme was inhibited by ATP (K(i)0.9x10(-3)m). The inhibition was competitive with regard to ADP but not with regard to phosphoenolpyruvate. It was not relieved by excess of Mg(2+) ions. 6. The possible relationship of the properties of pyruvate kinase to regulatory mechanisms for controlling gluconeogenesis and carbohydrate oxidation in A. xylinum is discussed.

Adenine Nucleotides↗
PubMed1973

The structure of novel C35 pentacyclic terpenes from Acetobacter xylinum.

A novel C(35) terpene and its monounsaturated analogue were isolated from cultures of Acetobacter xylinum, together with traces of their C(36) homologues. These substances were found to be hopane derivatives substituted by a five-carbon chain bearing four vicinal hydroxyl groups. For the parent hydrocarbon the term bacteriohopane is proposed. The elucidation of the structures utilized high-resolution mass spectrometry of the terpenes, degradation to C(32) hydrocarbons and detailed mass-spectrometric comparison of these with C(32) hydrocarbons synthesized from known pentacyclic triterpenes. High-resolution mass-spectral data of the terpenes are presented. N.m.r. data are in agreement with the proposed structures, which are further supported by the isolation from the same organism of 22-hydroxyhopane and derivative hopene(s).

Chromatography, Gas↗
PubMed1974

Regulation of hexose phosphate metabolism in Acetobacter xylinum.

The metabolism of glucose and fructose was studied in resting succinate-grown cells of Acetobacter xylinum. From fructose only cellulose and CO(2) were formed by the cells, whereas from glucose, gluconate was formed much more rapidly than these two products. The molar ratio of sugar converted into cellulose to sugar converted into CO(2) was significantly greater than unity for both hexoses. The pattern of label retention in the cellulose formed by the cells from specifically (14)C-labelled glucose, fructose or gluconate corresponded to that of hexose phosphate in a pentose cycle. On the other hand, the isotopic configuration of cellulose arising from variously singly (14)C-labelled pyruvate did not agree with the operation of a pentose cycle on gluconeogenic hexose phosphate. Readily oxidizable tricarboxylic acid-cycle intermediates such as acetate, pyruvate or succinate promoted cellulose synthesis from fructose and gluconate although retarding their oxidation to CO(2). The incorporation into cellulose of C-1 of fructose was greatly increased in the presence of these non-sugar substrates, although its oxidation to CO(2) was greatly diminished. It is suggested that the flow of hexose phosphate carbon towards cellulose or through the pentose cycle in A. xylinum is regulated by an energy-linked control mechanism.

Acetates↗
PubMed1976

Factors affecting the activity of citrate synthase of Acetobacter xylinum and its possible regulatory role.

The citrate synthase activity of Acetobacter xylinum cells grown on glucose was the same as of cells grown on intermediates of the tricarboxylic acid cycle. The activity of citrate synthase in extracts is compatible with the overall rate of acetate oxidation in vivo. The enzyme was purified 47-fold from sonic extracts and its molecular weight was determined to be 280000 by gel filtration. It has an optimum activity at pH 8.4. Reaction rates with the purified enzyme were hyperbolic functions of both acetyl-CoA and oxaloacetate. The Km for acetyl-CoA is 18 mum and that for oxaloacetate 8.7 mum. The enzyme is inhibited by ATP according to classical kinetic patterns. This inhibition is competitive with respect to acetyl-CoA (Ki = 0.9 mM) and non-competitive with respect to oxaloacetate. It is not affected by changes in pH and ionic strength and is not relieved by an excess of Mg2+ ions. Unlike other Gram-negative bacteria, the A. xylinum enzyme is not inhibited by NADH, but is inhibited by high concentrations of NADPH. The activity of the enzyme varies with energy charge in a manner consistent with its role in energy metabolism. It is suggested that the flux through the tricarboxylic acid cycle in A. xylinum is regulated by modulation of citrate synthase activity in response to the energy state of the cells.

Acetyl Coenzyme A↗
PubMed1976

Activities of citrate synthase and other enzymes of Acetobacter xylinum in situ and in vitro.

The activities of a number of enzymes, extracted from Acetobacter xylinum, that are involved in carbohydrate metabolism may be accounted for in situ in permeabilized cells. The kinetic properties of citrate synthase and glycerokinase observed in vitro are also retained in situ. So is the regulatory sensitivity of these enzymes. Both in vitro and in situ, (a) citrate synthase, in contrast with the enzyme for other Gram-negative bacteria, is inhibited by ATP and is insensitive to NADH, and (b) glycerokinase is inhibited by fructose diphosphate and the ratio of its activities towards glycerol and dihydroxyacetone is the same.

Cell Membrane Permeability↗
PubMed1997

Studies on recombinant Acetobacter xylinum alpha-phosphoglucomutase.

The phosphoglucomutase (PGM) from Acetobacter xylinum, which had been cloned and expressed in Escherichia coli, has been studied. After expression, the enzyme was purified from the E. coli in a three-step process consisting of (NH4)2SO4 precipitation, gel filtration and anion-exchange chromatography. The purified enzyme gave one band on gel electrophoresis and was judged essentially free of impurities, although it was unstable when diluted without the addition of 15 microM BSA. The isoelectric point for A. xylinum PGM was 4.8 and the molar absorbance was 3.9 x 10(4) M-1.cm-1. The enzyme was reasonably heat-stable below 50 degrees C and was stable throughout the pH 5.5-7.4 range, but was 70% inactivated at pH 10.0 and completely inactivated after standing for 10 min at pH 3.0 or at pH 12.4. When isolated, the recombinant enzyme was fully active without the addition of extra Mg2+. The Km for glucose 1-phosphate was much higher than that of other PGM species reported, which accords with the production of extracellular cellulose in A. xylinum. Glucose 1,6-diphosphate is not considered to be a substrate or coenzyme but an activating cofactor like Mg2+. The following kinetic constants were determined: Vmax 81.1 units/mg; kcat and the turnover rate 135 s-1; Km (glucose 1,6-diphosphate) 0.2 microM; Km (glucose 1-phosphate) 2.6 mM; kcat/Km (glucose 1-phosphate) 5.2 x 10(4) M-1.s-1. The recombinant enzyme is considered to follow a characteristic substituted enzyme or Ping Pong reaction mechanism.

Anions↗
PubMed1980

Cellulose biogenesis: Polymerization and crystallization are coupled processes in Acetobacter xylinum.

Calcofluor White ST, stilbene derivative used commerically as an optical brightener for cellulose, increased the rate of glucose polymerization into cellulose by resting cells of the gram-negative bacterium Acetobacter xylinum. This bacterium normally produces a ribbon of cellulose that is a composite of crystalline microfibrils. In concentrations above 0.1 mM, Calcofluor disrupts the assembly of crystalline cellulose I microfibrils and their integration into a composite ribbon by stoichiometric binding to glucose residues of newly polymerized glucan chains. Under these conditions, the rate of glucose polymerization increases up to 4 times the control rate, whereas oxygen uptake increases only 10-15%. These observed effects are readily reversible. If free Calcofluor is washed away or depleted below the threshold value by binding to cellulose as polymerization continues, ribbon production and the normal rate of polymerization resume. It is concluded that polymerization and crystallization are cell-directed, coupled processes and that the rate of crystallization determines the rate of polymerization. It is suggested that coupling must be maintained for biogenesis of crystalline cellulose I.

Journal Article↗
PubMed1982

Requirement for a membrane potential for cellulose synthesis in intact cells of Acetobacter xylinum.

The marked lability in cell-free preparations of the enzyme system involved in cellulose biosynthesis in most organisms studied led us to investigate factors responsible for loss of activity on cellular disruption. Previous studies have led to the suggestion that the existence of a transmembrane electrical potential (DeltaPsi) may be one factor responsible for maintaining an active system in intact cells. In this report, we show that dissipation of the DeltaPsi in metabolizing cells of Acetobacter xylinum results in severe inhibition of cellulose synthesis. The effect can be reversed by restoration of the DeltaPsi. Inhibition of cellulose biosynthesis by dissipation of the DeltaPsi can be observed under conditions in which no substantial impairment of energy metabolism occurs-i.e., under conditions in which a transmembrane pH gradient is of sufficient magnitude to maintain an adequate overall protonmotive force across the membrane. The inhibition of cellulose biosynthesis is specifically related to changes in the DeltaPsi, since the process can proceed normally in the absence of the pH gradient. These results support the suggestion that loss of the DeltaPsi on cellular disruption may be one of the factors responsible for the low capacity for cellulose synthesis in isolated membrane preparations and also raise the possibility that modulation of the DeltaPsi could be one means of regulating the rate of cellulose synthesis in vivo.

Journal Article↗
PubMed1982

Achievement of high rates of in vitro synthesis of 1,4-beta-D-glucan: activation by cooperative interaction of the Acetobacter xylinum enzyme system with GTP, polyethylene glycol, and a protein factor.

Regulatory properties of a cellulose synthase (UDP-forming)(UDPglucose:1,4-beta-D-glucan 4-beta-D-glucosyltransferase, EC 2.4.1.12) have been demonstrated by using enzyme preparations derived from cells of Acetobacter xylinum. Preparation of a particulate fraction in the presence of 20% (wt/vol) polyethylene glycol-4000 (PEG-4000) yields enzyme with activity 3- to 10-fold higher than that previously reported. The enzyme prepared in this fashion also shows a further marked, specific activation by GTP. The Ka for GTP is 34 microM. Guanosine 5'-[gamma-thio]triphosphate, an analog of GTP, is even more effective than GTP (Ka for guanosine 5'-[gamma-thio]triphosphate = 17 microM). A large number of other nucleotides and nucleotide derivatives were tested with no effect. Enzyme prepared in the absence of PEG-4000 does not respond to GTP because it lacks a protein factor necessary for GTP activation. PEG-4000 promotes the interaction of the protein factor with the enzyme. The factor itself has no synthase activity nor does it stimulate activity of the enzyme in the absence of GTP. In the presence of GTP, protein factor, and PEG-4000, initial rates of enzyme activity 200 times greater than those previously reported can be achieved. Such rates exceed 40% of the in vivo rate of cellulose synthesis from glucose.

Bacterial Proteins↗
PubMed1987

In vitro synthesis of cellulose II from a cytoplasmic membrane fraction of Acetobacter xylinum.

The cytoplasmic and outer membranes of Acetobacter xylinum (ATCC 53582) were isolated by discontinuous sucrose density ultracentrifugation. Both lysozyme (EC 3.2.1.17) and trypsin (EC 3.4.21.4) were required for efficient crude membrane separation. Primary dehydrogenases and NADH oxidase were used as cytoplasmic membrane markers, and 2-keto-3-deoxyoctulosonic acid was used to identify the outer membranes. Cellulose synthetase (UDP-glucose:1,4-beta-D-glucan 4-beta-D-glucosyltransferase; EC 2.4.1.12) activity was assayed as the conversion of radioactivity from UDP-[(14)C]glucose into an alkali-insoluble beta-1,4-D-[(14)C]glucan. This activity was predominantly found in the cytoplasmic membrane. The cellulose nature of the product was demonstrated by (i) enzymatic hydrolysis followed by TLC, (ii) methylation analysis followed by TLC, and (iii) GC/MS. Further, the weight-average and number-average degree of polymerization of the in vitro product, determined by high-performance gel permeation chromatography, were 4820 and 5270, respectively. In addition, x-ray diffraction analysis indicated that the in vitro product is cellulose II, which is in contrast to the in vivo product-namely, cellulose I.

Journal Article↗