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PubMed1994

Characterisation of plasmids from diketogluconic acid producing strains of Gluconobacter oxydans.

Gluconobacter oxydans ATCC 9937, which produces 2,5-diketogluconic acid, an intermediate in vitamin C synthesis, has three plasmids of sizes 27.7 kb (pVJ1), 12.3 kb (pVJ2) and 18 kb (pVJ4). A restriction map was constructed of pVJ1. A potential glucose dehydrogenase gene was located on pVJ1 using the polymerase chain reaction with heterologous primers. Two other G. oxydans strains had no detectable plasmid DNA (IFO 12258) and a plasmid (pVJ3) of 9.4 kb (IFO 3293), respectively.

Base Sequence↗
PubMed1996

FET-microbial sensor for xylose detection based on Gluconobacter oxydans cells.

A potentiometric biosensor for xylose was devised utilizing Gluconobacter oxydans whole cells. Immobilization methods based on physical adsorption were used for G. oxydans cells and extracellular pH changes resulting from xylose dehydrogenation were monitored by a field effect transistor (FET). The G. oxydans, FET-based sensor detected xylose at a lower limit of 0.5 mM. From 5.0 to 30 mM xylose, the response of the sensor was linear. Expectedly, output signals were significantly suppressed by buffer (Tris-HCl). Responses were essentially stable for at least four weeks of storage and showed only a slight loss of initial xylose sensitivity. Xylitol exerted an insignificant influence on the sensor's response to xylose. However, the response to glucose was 5 times higher in relation to that of xylose at the same concentration (1 mM). For xylose determinations in the presence of glucose, a two-step assay is discussed.

Acetobacteraceae↗
PubMed1999

The quinohemoprotein alcohol dehydrogenase of Gluconobacter suboxydans has ubiquinol oxidation activity at a site different from the ubiquinone reduction site.

Alcohol dehydrogenase (ADH) of acetic acid bacteria functions as the primary dehydrogenase of the ethanol oxidase respiratory chain, where it donates electrons to ubiquinone. In addition to the reduction of ubiquinone, ADHs of Gluconobacter suboxydans and Acetobacter aceti were shown to have a novel function in the oxidation of ubiquinol. The oxidation activity of ubiquinol was detected as an ubiquinol:ferricyanide oxidoreductase activity, which can be monitored by selected wavelength pairs at 273 and 298 nm with a dual-wavelength spectrophotometer. The ubiquinol oxidation activity of G. suboxydans ADH was shown to be two times higher in 'inactive ADH', whose ubiquinone reductase activity is 10 times lower, than with normal 'active' ADH. No activity could be detected in the isolated subunit II or subunit I/III complex, but activity was detectable in the reconstituted ADH complex. Inactive and active ADHs exhibited a 2-3-fold difference in their affinity to ubiquinol despite having the same affinity to ubiquinone. Furthermore, the ubiquinol oxidation site in ADH could be distinguished from the ubiquinone reduction site by differences in their sensitivity to ubiquinone-related inhibitors and by their substrate specificity with several ubiquinone analogues. Thus, the results strongly suggest that the reactions occur at different sites. Furthermore, in situ reconstitution experiments showed that ADH is able to accept electrons from ubiquinol present in Escherichia coli membranes, suggesting the ubiquinol oxidation activity of ADH has a physiological function. Thus, ADH of acetic acid bacteria, which has ubiquinone reduction activity, was shown to have a novel ubiquinol oxidation activity, of which the physiological function in the respiratory chain of the organism is also discussed.

Acetobacter↗
PubMed2001

Monitoring of dihydroxyacetone production during oxidation of glycerol by immobilized Gluconobacter oxydans cells with an enzyme biosensor.

A bi-enzymatic biosensor for monitoring of dihydroxyacetone production during oxidation of glycerol by bacterial cells of Gluconobacter oxydans is presented. Galactose oxidase oxidizes dihydroxyacetone efficiently producing hydrogen peroxide, which reacts with co-immobilized peroxidase and ferrocene pre-adsorbed on graphite electrode. This mediator-based bi-enzymatic biosensor possesses very high sensitivity (4.7 µA/mM in phosphate buffer), low detection limit (0.8 µM, signal/noise = 3), short response time (22 s, 95% of steady-state) and broad linear range (0.002-0.55 mM in phosphate buffer). The effect of pH, temperature, type of buffer, as well as different stabilizers (combinations of a polyelectrolyte and a polyol) on the sensor performance were carefully optimized and discussed. Dihydroxyacetone produced during a batch conversion of glycerol by the pectate-immobilized bacteria in an air-lift reactor was determined by the biosensor and by reference spectrophotometric method. Both methods were compared and were in a very good correlation. The main advantage of the biosensor is a very short time needed for sample analysis (less than 1 min).

Journal Article↗
PubMed2000

Cloning of a gene for D-sorbitol dehydrogenase from Gluconobacter oxydans G624 and expression of the gene in Pseudomonas putida IFO3738.

We have cloned a novel gene for d-sorbitol dehydrogenase (SLDH), which efficiently converted D-sorbitol to L-sorbose, from Gluconobacter oxydans G624 (FERM BP-4415). A cosmid library of the genomic DNA was screened by assaying SLDH activity. The inserted DNA from a positive clone was downsized by subcloning into charomid and pUCP plasmid, successively. Sequencing analysis of the DNA responsible for SLDH activity revealed an open reading frame of 1455 bp coding for 485 amino acid residues with a calculated molecular mass of 53,642 Da. The amino acid sequence showed 42.2% identity with a NAD+-dependent mannitol dehydrogenase (MDH), which catalyzed conversion of d-sorbitol to d-fructose, from Pseudomonas fluorescens DSM50106. Since the intact SLDH was found to be very unstable during isolation and purification, this SLDH fused to 6 x His-tag was expressed in Pseudomonas putida IFO3738 and purified by immobilized metal affinity chromatography using cobalt-based resins. The 6 x His-tag SLDH catalyzed the oxidation of D-sorbitol to L-sorbose and exhibited 15 times higher activity in the presence of NADP+ than that of NAD+. These results indicate that the SLDH is a novel kind of dehydrogenase distinct from MDH previously reported.

Journal Article↗
PubMed1985

Identification of the covalently bound flavins of D-gluconate dehydrogenases from Pseudomonas aeruginosa and Pseudomonas fluorescens and of 2-keto-D-gluconate dehydrogenase from Gluconobacter melanogenus.

An improved method is presented for the purification of 8 alpha-(N1-histidyl)riboflavin, 8 alpha-(N3-histidyl)riboflavin and their 2',5'-anhydro forms, which permits the isolation of sizeable quantities of each of these compounds from a synthetic mixture in pure form. Flavin peptides were isolated from the D-gluconate dehydrogenases of Pseudomonas aeruginosa and Pseudomonas fluorescens and from the 2-keto-D-gluconate dehydrogenase of Gluconobacter melanogenus. After conversion into the aminoacyl-riboflavin, the flavin in all three enzymes was identified as 8 alpha-(N3-histidyl)riboflavin. By sequential treatment with nucleotide pyrophosphatase and alkaline phosphatase, the flavin in each enzyme was shown to be in the dinucleotide form.

Carbohydrate Dehydrogenases↗
PubMed1998

Molecular cloning and mutational analysis of the ddsA gene encoding decaprenyl diphosphate synthase from Gluconobacter suboxydans.

Decaprenyl diphosphate (decaprenyl-PP) synthase catalyzes the consecutive condensation of isopentenyl diphosphate with allylic diphosphates to produce decaprenyl-PP, which is used for the side chain of ubiquinone (Q)-10. We have cloned the synthase gene, designated ddsA, from Gluconobacter suboxydans and expressed it in Escherichia coli. Sequence analysis revealed the presence of an ORF of 948 bp capable of encoding a 33,898-Da polypeptide that displays high similarity (30-50%) to other prenyl diphosphate synthases. Expression of the ddsA gene complemented the lethality resulting from a defect in the octaprenyl diphosphate synthase gene of E. coli and produced Q-10, indicating that Q-10 can substitute for the function of Q-8. The His-tagged DdsA protein was purified to characterize its enzymatic properties. This enzyme required detergent (0.05% Triton X-100) and 10 mM Mg2+, for full activity. The Michaelis constants for geranyl diphosphate, all-E-farnesyl diphosphate and all-E-geranylgeranyl diphosphate were 7.00, 0.50 and 0.32 microM, respectively. Nine single-amino-acid substitutions were introduced upstream of conserved region II or VI. Most of the mutants showed a considerable decrease in catalytic activity or shortening of the ultimate chain length. However, the A70G mutant produced a longer-chain-length product than wild-type decaprenyl-PP synthase, and the A70Y mutant completely abolished the decaprenyl-PP synthase function, indicating that Ala70 is important for enzyme activity and the determination of the chain-length properties of DdsA.

Acetobacteraceae↗
PubMed1996

Function of multiple heme c moieties in intramolecular electron transport and ubiquinone reduction in the quinohemoprotein alcohol dehydrogenase-cytochrome c complex of Gluconobacter suboxydans.

Alcohol dehydrogenase (ADH) of acetic acid bacteria functions as the primary dehydrogenase of the ethanol oxidase respiratory chain, where it donates electrons to ubiquinone. ADH is a membrane-bound quinohemoprotein-cytochrome c complex which consists of subunits I (78 kDa), II (48 kDa), and III (14 kDa) and contains several hemes c as well as pyrroloquinoline quinone as prosthetic groups. To understand the role of the heme c moieties in the intramolecular electron transport and the ubiquinone reduction, the ADH complex of Gluconobacter suboxydans was separated into a subunit I/III complex and subunit II, then reconstituted into the complex. The subunit I/III complex, probably subunit I, contained 1 mol each of pyrroloquinoline quinone and heme c and exhibited significant ferricyanide reductase, but no Q1 reductase activities. Subunit II was a triheme cytochrome c and had no enzyme activity, but it enabled the subunit I/III complex to reproduce the Q1 and ferricyanide reductase activities. Hybrid ADH consisting of the subunit I/III complex of G. suboxydans ADH and subunit II of Acetobacter aceti ADH was constructed and it had showed a significant Q1 reductase activity, indicating that subunit II has a ubiquinone-binding site. Inactive ADH from G. suboxydans exhibiting only 10% of the Q1 and ferricyanide reductase activities of the active enzyme has been isolated separately from active ADH (Matsushita, K., Yakushi, T., Takaki, Y., Toyama, H., and Adachi, O (1995) J. Bacteriol. 177, 6552-6559). Using these active and inactive ADHs and also isolated subunit I/III complex, we performed kinetic studies which suggested that ADH contains four ferricyanide-reacting sites, one of which was detected in subunit I and the others in subunit II. One of the three ferricyanide-reacting sites in subunit II was defective in inactive ADH. The ferricyanide-reacting site remained inactive even after alkali treatment of inactive ADH and also after reconstituting the ADH complex from the subunits, in contrast to the restoration of Q1 reductase activity and the other ferricyanide reductase activities. Thus, the data suggested that the heme c in subunit I and two of the three heme c moieties in subunit II are involved in the intramolecular electron transport of ADH into ubiquinone, where one of the two heme c sites may work at, or close to, the ubiquinone-reacting site and another between that and the heme c site in subunit I. The remaining heme c moiety in subunit II may have a function other than the electron transfer from ethanol to ubiquinone in ADH.

Acetobacter↗
PubMed1989

Reactivity with ubiquinone of quinoprotein D-glucose dehydrogenase from Gluconobacter suboxydans.

D-Glucose dehydrogenase is a pyrroloquinoline quinone-dependent oxidoreductase linked to the respiratory chain of a wide variety of bacteria. There is a controversy as to whether the glucose dehydrogenase is linked to the respiratory chain via ubiquinone or cytochrome b. In this study, it was shown that the glucose dehydrogenase of Gluconobacter suboxydans has the ability to react directly with ubiquinone. The enzyme purified from the membranes of G. suboxydans was able to react with ubiquinone homologues such as ubiquinone-1, -2, or -6 in detergent solution. Furthermore, in order to demonstrate the reactivity of the enzyme with native ubiquinone, ubiquinone-10, in the native membranous environment, the dehydrogenase was reconstituted together with cytochrome o, the terminal oxidase of the respiratory chain, into a phospholipid bilayer containing ubiquinone-10. The proteoliposomes thus reconstituted exhibited a reasonable glucose oxidase activity, the electron transfer reaction of which was able to generate a membrane potential and a pH gradient. Thus, D-glucose dehydrogenase of G. suboxydans has been demonstrated to donate electrons directly to ubiquinone in the respiratory chain.

Carbohydrate Dehydrogenases↗
PubMed1990

Evidence for electron transfer via ubiquinone between quinoproteins D-glucose dehydrogenase and alcohol dehydrogenase of Gluconobacter suboxydans.

Gluconobacter suboxydans contains membrane-bound D-glucose and alcohol dehydrogenases (GDH and ADH) as the primary dehydrogenases in the respiratory chain. These enzymes are known to be quinoproteins having pyrroloquinoline quinone as the prosthetic group. GDH reduces an artificial electron acceptor, ferricyanide, in the membrane, but not after solubilization with Triton X-100, while ADH can react with the electron acceptor even after solubilization and further purification. In this study, it has been shown that the ferricyanide reductase activity of GDH is restored by adding the supernatant solubilized with Triton X-100 to the residue, and also by incorporation of purified ADH into the membranes of an ADH-deficient strain. G. suboxydans var. alpha. In addition, the ferricyanide reductase activity of GDH was reconstituted in proteoliposomes from GDH, ADH, and ubiquinone-10. Thus, the results indicated that the electron transfer from GDH to ferricyanide was mediated by ubiquinone and ADH. The data also suggest that GDH and ADH transfer electrons mutually via ubiquinone in the respiratory chain.

Alcohol Dehydrogenase↗
PubMed1979

Occurrence of old yellow enzyme in Gluconobacter suboxydans, and the cyclic regeneration of NADP.

Old yellow enzyme system has been found in the cytosol fraction of Gluconobacter suboxydans. This is the first time that the enzyme has been found in organisms other than yeast cells. Old yellow enzyme [EC 1.6.99.1], D-glucose-6-phosphate dehydrogenase [EC 1.1.1.49], and catalase were isolated and crystallized separately from the organism. The old yellow enzyme from G. suboxydans showed catalytic and physicochemical properties almost identical with those of the enzyme from yeast cells. NADPH was specifically oxidized by the old yellow enzyme and the reduced enzyme was spontaneously reoxidized by atmospheric oxygen. The old yellow enzyme from G. suboxydans also contained FMN as a prosthetic group, and two mol of FMN were found per mol of enzyme (molecular weight, 88,000 as determined by gel filtration). In the oxidation of D-glucose-6-phosphate to 6-phospho-D-gluconate, cyclic regeneration of NADP occurred smoothly in the presence of D-glucose-6-phosphate dehydrogenase and catalase, even when a limited amount of NADP or NADPH was present in the reaction mixture.

Catalase↗
PubMed1997

Transposon induced mutation in Gluconobacter oxydans with special reference to its direct-glucose oxidation metabolism.

Transposons are important genetic tools for mutation studies and for location of genes in prokaryotes. However, very little published work is available on transposon mutagenesis in Gluconobacter oxydans. We report here Tn5-induced mutation in a keto acid-producing strain of G. oxydans ATCC 9937 with special reference to the direct-glucose oxidation pathway operative in this organism. In this study, a mutant deficient in glucose dehydrogenase (GDH) activity has been developed by Tn5 mutagenesis. The data of plasmid profiles in the wild-type and the GDH- mutant are indicative of transposition in the first instance on one of the three plasmids (pVJ1) harboured by the organism, resulting in rearrangement of the plasmid and finally stable transposition of Tn5 on the main genome. The final location of Tn5 on the genome has been established by DNA hybridisation studies.

Acetobacteraceae↗
PubMed1999

A mutant of gluconobacter oxydans deficient in gluconic acid dehydrogenase

Gluconobacter oxydans ATCC 9937 was subjected to transposon mutagenesis using Tn5. A non-pigmented mutant was shown to be defective in gluconic acid dehydrogenase and to produce gluconic acid from glucose, whereas the parent strain produced 2, 5-diketogluconic acid. Cloning and sequencing of the region containing the Tn5 insertion showed that the insertion point occurred in an open reading frame homologous (42% amino acid identity) to the ribF genes of Pseudomonas fluorescens and Escherichia coli. The resulting lack of a riboflavin cofactor would explain the loss of enzyme activity.

Journal Article↗
PubMed1977

Application of Oxygen-Enriched Aeration in the Conversion of Glycerol to Dihydroxyacetone by Gluconobacter melanogenus IFO 3293.

Gluconobacter melanogenus 3293 converts glycerol to dihydroxyacetone(DHA) during exponential growth on a yeast extract-phosphate medium at pH 7. The efficiency of this conversion in 25-liter batch fermentations has been found to increase over threefold, when oxygen tension is controlled by increasing the partial pressure of oxygen in the aeration. Conversion of glycerol to DHA does not occur under oxygen-limited fermentation conditions. When the dissolved oxygen tension was maintained at 0.05 atmospheres (using oxygen-enriched air), quantitative conversion of up to 100 g of glycerol/liter to DHA was obtained in 33 h. The amount of glycerol converted can be increased without increasing impeller speed or aeration rate. This increase is not the result of increased production of cell mass. The specific conversion of glycerol to DHA increased from 12.2 g of DHA/g of cell mass at the point of maximum conversion to 35.8 with oxygen enrichment. This increased specific production occurred even though the specific growth rate during the period of oxygen enrichment decreased from 0.23 to 0.06/h.

Journal Article↗
PubMed1995

Cloning and nucleotide sequencing of the membrane-bound L-sorbosone dehydrogenase gene of Acetobacter liquefaciens IFO 12258 and its expression in Gluconobacter oxydans.

Cloning and expression of the gene encoding Acetobacter liquefaciens IFO 12258 membrane-bound L-sorbosone dehydrogenase (SNDH) were studied. A genomic library of A. liquefaciens IFO 12258 was constructed with the mobilizable cosmid vector pVK102 (mob+) in Escherichia coli S17-1 (Tra+). The library was transferred by conjugal mating into Gluconobacter oxydans OX4, a mutant of G. oxydans IFO 3293 that accumulates L-sorbosone in the presence of L-sorbose. The transconjugants were screened for SNDH activity by performing a direct expression assay. One clone harboring plasmid p7A6 converted L-sorbosone to 2-keto-L-gulonic acid (2KGA) more rapidly than its host did and also converted L-sorbose to 2KGA with no accumulation of L-sorbosone. The insert (25 kb) of p7A6 was shortened to a 3.1-kb fragment, in which one open reading frame (1,347 bp) was found and was shown to encode a polypeptide with a molecular weight of 48,222. The SNDH gene was introduced into the 2KGA-producing strain G. oxydans IFO 3293 and its derivatives, which contained membrane-bound L-sorbose dehydrogenase. The cloned SNDH was correctly located in the membrane of the host. The membrane fraction of the clone exhibited almost stoichiometric formation of 2KGA from L-sorbosone and L-sorbose. Resting cells of the clones produced 2KGA very efficiently from L-sorbosone and L-sorbose, but not from D-sorbitol; the conversion yield from L-sorbosone was improved from approximately 25 to 83%, whereas the yield from L-sorbose was increased from 68 to 81%. Under fermentation conditions, cloning did not obviously improve the yield of 2KGA from L-sorbose.

Acetobacter↗
PubMed1997

Characterization of the genes encoding the three-component membrane-bound alcohol dehydrogenase from Gluconobacter suboxydans and their expression in Acetobacter pasteurianus.

The three-component membrane-bound alcohol dehydrogenase (ADH) of Gluconobacter suboxydans IFO12528 was purified, and the NH2-terminal amino acid sequence of each subunit was determined. On the basis of the amino acid sequences, the genes adhA, encoding the 72-kDa dehydrogenase, adhB, encoding the 44-kDa cytochrome c-553 (a CO-binding cytochrome c), and adhS, encoding a 15-kDa protein, were cloned and the amino acid sequences of their products were deduced from the nucleotide sequences. The dehydrogenase and cytochrome genes were clustered with the same transcription polarity, as is the case in species of Acetobacter, another genus of acetic acid bacteria. These AdhA and AdhB subunits showed similarity in amino acid sequence to those from Acetobacter spp., whereas AdhS showed no similarity to the corresponding subunit of the ADH complex of Acetobacter pasteurianus. Consistent with this, adhS of G. suboxydans could not complement a defect in the corresponding subunit of A. pasteurianus. When the adhA-adhB gene cluster of G. suboxydans was expressed in an ADH-deficient mutant of A. pasteurianus, the transformant showed distinct ADH activity. The ADH complex was purified to near homogeneity and consisted of two subunits, the dehydrogenase and the cytochrome c subunits derived from G. suboxydans, without any other subunit. These data suggested that AdhS, the smallest subunit of ADH, from G. suboxydans is not essential for ADH activity in A. pasteurianus, in contrast to the essential role of A. pasteurianus AdhS, which is required for correct assembly of the dehydrogenase and cytochrome c subunits on the membrane.

Acetobacter↗
PubMed1997

Characterization of an insertion sequence, IS12528, from Gluconobacter suboxydans.

A novel insertion sequence element, IS12528, was found to be associated with inactivation of the alcohol dehydrogenase by insertion in the adhA gene, which encodes the primary dehydrogenase subunit of the three-component membrane-bound alcohol dehydrogenase complex in Gluconobacter suboxydans. Cloning and sequencing analyses revealed that IS12528 was 905 bp in length and had a terminal inverted repeat of 18 bp. In addition, IS12528 was found to generate a 3-bp duplication (TMA, where M represents C or A) at the inserted site upon transposition. IS12528 encoded one long product of 274 amino acids that was rich in basic amino acids. This protein showed significant homology with putative transposases of the IS1031 family isolated from Acetobacter xylinum, which belongs to another genus of acetic acid bacteria. IS12528-like sequences were distributed in a wide variety of acetic acid bacteria, as determined by Southern hybridization and PCR. These observations suggest that IS12528 is one of the insertion sequences that are responsible for genetic instability leading to deficiencies in various physiological properties in a variety of acetic acid bacteria.

Acetobacter↗
PubMed1974

Metabolic consequences of a block in the synthesis of 5-keto-D-fructose in a mutant of Gluconobacter cerinus.

A mutant of Gluconobacter cerinus var. ammoniacus, IFO 3267, has been isolated which is deficient with respect to fructose 5-dehydrogenase, the enzyme catalyzing the oxidation of d-fructose to 5-keto-d-fructose (5 KF). Growth of this mutant on fructose as the sole carbon source was impaired unless the culture medium was supplemented with 5 KF. Significant randomization of the 1 and 6 positions of fructose has been reported previously for the wild-type organism during growth on this ketohexose. The pattern of (3)H incorporation into the C5 position of ribonucleic acid-ribose when the mutant was grown on [1-(3)H]fructose and [6-(3)H]fructose in the presence of 5 KF indicated that such randomization did not occur in this variant. The randomization observed in the wild type is, therefore, a consequence of the partial oxidation of fructose to the symmetrical 5 KF intermediate prior to its conversion to pentose. When the mutant was grown on [1-(3)H]fructose in the presence of unlabeled 5 KF, [5-(3)H]fructose appeared in the culture medium. Thus, 5 KF served as the oxidant for the nicotinamide adenine dinucleotide phosphate, reduced form, generated during growth on fructose.

Alcohol Oxidoreductases↗