B.J. Van Schie

PQQ-Dependent Production of Gluconic Acid by Acinetobacter, Agrobacterium and Rhizobium Species

SUMMARY: Acinetobacter Iwoffi, Azotobacter vinelandii, Agrobacterium and Rhizobium species contain quinoprotein glucose dehydrogenase apoenzyme (EC 1.1.99.17). Addition to whole cells of pyrrolo-quinoline quinone (PQQ), the prosthetic group of this enzyme, resulted in the production of gluconic acid from glucose. The in vivo reconstitution of apo-glucose dehydrogenase with PQQ was dependent on the presence of Ca2+ or Mg2+. Optimal conditions for reconstitution allowed maximal glucose dehydrogenase activity in the presence of 1-10 nmol PQQ 1−1. Synthesis of the apoenzyme of glucose dehydrogenase was not dependent on glucose in the growth media. The physiological significance of the synthesis of apo-glucose dehydrogenase, as found in a variety of bacteria, is discussed.

An in vivo analysis of the energetics of aldose oxidation by acinetobacter calcoaceticus

SummaryA continuous culture study was made of the energetics of oxidation of various aldose sugars by Acinetobacter calcoaceticus LMD 79.41. The consumption of aldoses during carbon- and energy-limited growth of the organism on mixtures of acetate and an aldose was independent of the pH of the culture. Acid production, however, was strongly dependent on this parameter. It is shown that aldose consumption without concurrent acid production is due to formation of the corresponding lactone, the hydrolysis of which is pH-dependent.The cell yield of A. calcoaceticus on mixtures of acetate and glucose or xylose was much higher than during growth on acetate alone. This increase in cell yield was, however, dependent on the pH of the culture. Only at pH values which permitted a high rate of lactone hydrolysis an enhancement of the cell yield was observed. These results suggest that lactone hydrolysis has an important bearing on the efficiency of periplasmic oxidation of aldoses in bacteria.

GLUCOSE-DEHYDROGENASE-MEDIATED SOLUTE TRANSPORT AND ATP SYNTHESIS IN ACINETOBACTER-CALCOACETICUS

SUMMARY: Evidence is presented that in Acinetobacter calcoaceticus oxidation of glucose to gluconate by the periplasmic quinoprotein glucose dehydrogenase (EC 1.1.99.17) leads to energy conservation. Membrane vesicles prepared from cells grown in carbon-limited chemostat culture exhibited (1) a high rate of glucose-dependent oxygen consumption and gluconate production, (2) glucose-mediated cytochrome reduction, (3) uncoupler sensitive, glucose-dependent generation of a membrane potential and (4) glucose-driven accumulation of amino acids. Furthermore, oxidation of glucose to gluconate by whole cells was associated with ATP synthesis. These results confirm and extend previous observations that periplasmic glucose oxidation can act as a driving force for energy-requiring processes. It is therefore concluded that the incomplete oxidation of glucose by bacteria may serve as an auxiliary energy-generating system.

Role of quinoprotein glucose-dehydrogenase in gluconic acid production by Acinetobacter calcoaceticus

Methanopterin is probably the first coenzyme involved in the reduction of CO 2 to CH 4 by Methanobacterium thermoautotrophicum. It is reduced and labelled with 14C02 in whole cells (Daniels and Zeikus, 1978; Keltjens et al., 1982) and it stimulates the formation of CH 4 in resolved cell-free extracts (J. Leigh, personal communication). We developed a quantitative, anaerobic and non-destructive method to measure the conversion of methanopterin in cell-free extracts with reversed-phase HPLC and a gradient of 0-25% methanol in 25 mM acetate buffer (pH 6.0). The conversion of methanopterin and the production of methane proceeded under identical conditions and required the presence of Mg 2+ plus ATP, hydrogen and methylcoenzyme M. The main products formed during the enzymatic conversion of methanopterin were two reduced pterins, which had lost the major part of the side chain of methanopterin, the side chain itself and a fourth unidentified reaction product. Chemical reduction of methanopterin with H 2 and a Pd/C catalyst yielded the same products as found with the enzymatic conversion, except for the fourth reaction product. Methanopterin and a number of degradation products were purified and analysed. Detailed NM R studies showed that methanopterin from M. thermoautotrophicum contains ribitol, ribose-5-phosphate, a glutarate derivative, a pterin substituted at the C 6and C7-positions and a second chromophore, which is probably an aniline derivative.

Effects of growth rate and oxygen tension on glucose dehydrogenase activity in Acinetobacter calcoaceticus LMD 79.41

The regulation of the synthesis of the quinoprotein glucose dehydrogenase (EC 1.1.99.17) has been studied inAcinetobacter calcoaceticus LMD 79.41, an organism able to oxidize glucose to gluconic acid, but unable to grow on both compounds. Glucose dehydrogenase was synthesized constitutively in both batch and carbon-limited chemostat cultures on a variety of substrates. In acetate-limited chemostat cultures glucose dehydrogenase levels and the glucose-oxidizing capacity of whole cells were dependent on the growth rate. They strongly increased at low growth rates at which the maintenance requirement of the cells had a pronounced effect on biomass yield.Cultures grown on a mixture of acetate and glucose in carbon and energy-limited chemostat cultures oxidized glucose quantitatively to gluconic acid. However, during oxygen-limited growth on this mixture glucose was not oxidized and only very low levels of glucose dehydrogenase were detected in cell-free extracts. After introduction of excess oxygen, however, cultures or washed cell suspensions almost instantaneously gained the capacity to oxidize glucose at a high rate, by an as yet unknown mechanism.

Selection of Glucose-Assimilating Variants of Acinetobacter-Calcoaceticus Lmd-7941 in Chemostat Culture

Glucose metabolism has been studied in two strains ofAcinetobacter calcoaceticus. Strain LMD 82.3, was able to grow on glucose and possessed glucose dehydrogenase (EC 1.1.99.17). Glucose oxidation by whole cells was stimulated by PQQ, the prosthetic group of glucose dehydrogenase. PQQ not only increased the rate of glucose oxidation and gluconic acid production but also shortened the lag phase for growth on glucose.Strain LMD 79.41 also possessed glucose dehydrogenase but was unable to grow on glucose. Batch cultures and carbon-limited chemostat cultures growing on acetate in the presence of glucose oxidized the sugar to gluconic acid, which was not further metabolized. However, after prolonged cultivation on mixtures of acetate and glucose, carbon-limited chemostat cultures suddenly acquired the capacity to utilize gluconate. This phenomenon was accompanied by the appearance of gluconate kinase and a repression of isocitrate lyase synthesis. In contrast to the starter culture, cells from chemostats which had been fully adapted to gluconate utilization, were able to utilize glucose as a sole carbon and energy source in liquid and solid media.

Energization of Solute Transport by Pqq-Dependent Glucose-Dehydrogenase in Membrane-Vesicles of Acinetobacter Species

Most Acinetobacter strains can not grow on glucose or gluconic acid as a sole carbon source. However, when glucose is added to the growth medium, it is quantitatively oxidized to gluconic acid. The enzyme responsible for this reaction is a membrane-bound glucose dehydrogenase (GDH, EC 1.1.99.17) which contains PQQ as a prosthetic group. GDH synthesis is constitutive in A. calcoaceticus. Strains of A. lwoffi, on the other hand, are unable to oxidize glucose but nevertheless contain apo-GDH under all growth conditions. Addition of the prosthetic group PQQ is required to restore GDH activity in A. lwoffi (Van Schie et al., 1984). In order to study the functional coupling of GDH to the respiratory chain we developed a procedure for the isolation of membrane vesicles from bacteria grown under well-defined conditions. Cells were collected at 4 ~ from an acetate-limited continuous culture. The method for the isolation of membrane vesicles was a modification of the procedure described for Pseudomonas aeruginosa by Stinnett et al. (1973). Membrane vesicles prepared from carbon-limited chemostat cultures of A. calcoaceticus LMD 79.41 and A. lwoffi LMD 83.25 exhibited active transport of alanine energized by GDH. In case of A. lwoffi both oxidation of glucose and active transport energized by glucose oxidation were dependent on the presence of PQQ. The rate of alanine uptake when energized with GDH was comparable with the rate of uptake energized by the artificial electron donor system ascorbate phenazine methosulphate (Table l).

Continuous Culture Studies on the Regulation of Pqq-Dependent Glucose-Dehydrogenase in Acinetobacter-Calcoaceticus

Irrespective of the growth substrate A cinetobacter calcoaceticus cells are capable of oxidizing glucose. Addition of glucose to the growth medium does not enhance this capacity. One single enzyme, namely glucose dehydrogenase, is responsible for this oxidation. Addition of glucose to cultures of A. calcoaceticus results in its quantitative conversion to gluconic acid (De Bont et al., 1984). Glucose dehydrogenase (GDH) is a membrane-bound periplasmic enzyme. It contains PQQ as a prosthetic group which donates its electrons directly to the electron transport chain. Since A. calcoacetieus is a very versatile organism, capable of aerobic growth on at least 80 organic compounds, it is rather surprising that GDH is synthesized constitutively. Moreover, since A. calcoaeeticus LMD 79.41 is not capable of growth on glucose of gluconate, it is rather peculiar that GDH is synthesized at all. In order to gain more information on the possible role of this enzyme in energy metabolism of A. calcoaceticus we performed a continuous culture study on the physiology of this organism with special attention to the regulation of GDH synthesis. Cells were cultured in mineral medium with acetate as a sole carbon and energy source, on which A. calcoaceticus grows very rapidly (t/max of 1.26 h-J). Upon decreasing the dilution rate from 0.55 h l the influence of the maintenance energy became already apparent at dilution rates below 0.2 h -1. From a Pirt plot values for m e and ymax of 3.8 mmol acetate-g cells -~ .h -1 and 26 g cells, mol acetatel, respectively, could be calculated. This cell yield is rather low but comparable to that of other oxidase-negative (cytochrome c-lacking) bacteria. GDH activity was determined in cell-free extracts, using a PES/DCPIP spectrophotometric assay. The levels of this enzyme increased 5-10-fold with decreasing growth rate. Various growth conditions were tested at a dilution rate of 0.15 h l with acetate-grown cultures, including variations in culture pH and temperature, nitrogen limitation and oxygen limitation. Of these only oxygen limitation significantly affected GDH synthesis. During oxygen-limited growth the levels of GDH decreased 10-20-fold. These results are in agreement with the observation that under all growth conditions, except under oxygen limitation, inclusion of glucose into the growth medium resulted in the production of gluconate.

Experimental and Theoretical Discrepancies in Growth Yields of Acinetobacter-Calcoaceticus - a Correction of Published Data

Acinetobacter calcoaceticus can incompletely oxidize aldose sugars to the corresponding aldonic acids. This reaction can serve as an auxiliary energy source for the organism. An increase in biomass yields is observed in acetate-limited chemostat cultures grown in the presence of, for example, xylose. However, experimental and theoretical discrepancies exist with respect to the magnitude of the yield enhancement as a result of xylose addition. We previously observed increases in cell yields that were unexpectedly high. In contrast, other data were in agreement with the theoretical predictions. In this paper, evidence is presented indicating that this discrepancy is likely to be due to errors in the methodology used for our previous investigation, in particular with respect to the determination of biomass concentrations.

The Gluconic Acid-Producing Enzyme of Acinetobacters

The capacity to produce an aldonic acid from aldose sugars is widespread among acinetobacters. The enzyme responsible for this reaction is a membrane-bound, pyrrolo-quinoline quinone (PQQ)dependent aldose dehydrogenase (E.C. 1.1.99,17). Fermentor studies with Acinetobacter calcoacetieus strain LMD 79.41 showed that thealdose dehydrogenase is synthesiT~ constitutively,-Pr~minary results obtained with carbon-limited chemostat cultures of this organism revealed that the cell yield on mixtures of acetate and glucose was significantly higher than on acetate alone (molar growth yield (g. mole2) for acetate 14.6 versus 21.3 for acetate plus glucose). Since glucose is almost quantitatively oxidized to gluconic acid it follows that the aldose dehydrogenase may play a role in energy metabolism of acinetobacters. The activity of the enzyme and the rate of acid production from aldose sugars by whole cells show large differences in various Aeinetobacter strains, cultivated under the same conditions. However, a low activity of aldose dehydrogenase is not necessarily due to a low level of apo-enzyme. In various strains the addition of PQQ to cell suspensions resulted in an instantaneous enhancement of the rate of glucose oxidation. Efficient recombination of apo-enzyme and PQQ in such strains was als0 observed in vitro. It is concluded therefore that the apo-enzyme and coenzyme are not always synchronically synthesized in acinetobacters. Preliminary results show that this may also be true for a number of other genera including Pseudomonas and Rhodopseudomonas.