Showing papers by "J.G. Kuenen published in 1985"
TL;DR: At saturating levels of pyrrolo-quinoline quinone, glucose was the most effective electron donor in E. coli, and glucose oxidation supported secondary transport at even higher rates than oxidation of reduced phenazine methosulfate.
Abstract: The coupling of membrane-bound glucose dehydrogenase (EC 1.1.99.17) to the respiratory chain has been studied in whole cells, cell-free extracts, and membrane vesicles of gram-negative bacteria. Several Escherichia coli strains synthesized glucose dehydrogenase apoenzyme which could be activated by the prosthetic group pyrrolo-quinoline quinone. The synthesis of the glucose dehydrogenase apoenzyme was independent of the presence of glucose in the growth medium. Membrane vesicles of E. coli, grown on glucose or succinate, oxidized glucose to gluconate in the presence of pyrrolo-quinoline quinone. This oxidation led to the generation of a proton motive force which supplied the driving force for uptake of lactose, alanine, and glutamate. Reconstitution of glucose dehydrogenase with limiting amounts of pyrrolo-quinoline quinone allowed manipulation of the rate of electron transfer in membrane vesicles and whole cells. At saturating levels of pyrrolo-quinoline quinone, glucose was the most effective electron donor in E. coli, and glucose oxidation supported secondary transport at even higher rates than oxidation of reduced phenazine methosulfate. Apoenzyme of pyrrolo-quinoline quinone-dependent glucose dehydrogenases with similar properties as the E. coli enzyme were found in Acinetobacter calcoaceticus (var. lwoffi) grown aerobically on acetate and in Pseudomonas aeruginosa grown anaerobically on glucose and nitrate.
133 citations
TL;DR: It is concluded that in C. utilis NADPH can be used in dissimilatory processes for the generation of ATP as evidenced by high respiratory control values and variations in the ratio of NADH and NADPH oxidase activities indicate that separate systems exist for the oxidation of exogenous redox equivalents by mitochondria of C.utilis.
Abstract: Mitochondria were isolated from Candida utilis CBS 621 grown in carbon-limited continuous cultures on glucose, gluconate, xylose, ethanol or acetate as the carbon source and ammonia or nitrate as the nitrogen source. In all cases mitochondria were isolated which could oxidize exogenous NADH and NADPH via a cyanide- and antimycin A-sensitive but rotenone-insensitive respiratory chain. Oxidation of NADH and NADPH was coupled to energy conservation as evidenced by high respiratory control values. Different respiratory control values of mitochondria with NADH and NADPH as well as variations in the ratio of NADH and NADPH oxidase activities indicate that separate systems exist for the oxidation of exogenous redox equivalents by mitochondria of C. utilis.
Variation of the NADPH requirement for biomass formation by applying differnt growth conditions did not result in significant changes in NADPH oxidase activities of mitochondria. It is concluded that in C. utilis NADPH can be used in dissimilatory processes for the generation of ATP.
41 citations
TL;DR: Aerobic denitrification would be of survival value in an environment where the ability to grow rapidly while denitrifying is important, but where limiting amounts of oxygen may be available.
Abstract: Thiosphaerapantotropha (Robertson and Kuenen, 1983), a new species isolated from a denitrifying, sulphide-oxidizing wastewater treatment plant, is capable of active denitrification in the presence of substantial amounts of oxygen. This is contrary to eurreaat belief. During studies on its denitrification system, Thiosphaera pantotropha was found to be capable of immediate denitrification after aerobic growth (Robertson and Kuenen, 1983). This suggested that the organism might have constitutive nitrate-reducing enzymes. Subsequent experiments using Kluyver flasks to ensure thorough aeration, and incorporating an oxygen electrode, showed that with the dissolved oxygen at at least 80~ of air saturation the cultures receiving nitrate grew faster than those without. They also gave a protein yield intermediate between those obtained with aerobic cultures lacking nitrate and anaerobic cultures provided with nitrate. A similar effect was observed when nitrite was used. Both the dissimilatory nitrate and nitrite reductases were present in cells grown on nitrate, but only nitrite reduetase levels were significant in cells grown on nitrite or without a nitrogen oxide. Sufficient nitrate had disappeared from the culture to account for half of the acetate oxidized to CO2. The remainder of the acetate must have been oxidized via oxygen. During these experiments, nitrite was found in the acetate cultures not supplied with nitrate or nitrite. Even in cultures supplied with 5 mM nitrite, the nitrite level rose, reaching a peak just before the end of the logarithmic phase. After this, the nitrite concentration rapidly fell. This phenomenon, known as heterotrophic nitrification, may account for the nitrite reductase found in cells grown without a nitrogen oxide. Its physiological significance remains to be studied. Aerobic denitrification would be of survival value in an environment where the ability to grow rapidly while denitrifying is important, but where limiting amounts of oxygen may be available.
28 citations
TL;DR: It is concluded that, for the quality of mitochondrial preparations from yeasts, the distribution of NADH dehydrogenase among the cell fractions is a more reliable measure than that of the usual marker enzymes.
Abstract: The successive steps in the isolation of mitochondria from chemostat-grown Candida utilis have systematically been investigated for their effects on organelle integrity. Growth rate had a profound effect on the susceptibility of carbon-limited cells towards Zymolyase, whereas the nature of the carbon source had no effect. Stabilization of spheroplasts with at least 2M-sorbitol was required to prevent premature lysis. This was concluded from the amounts of glucose-6-phosphate dehydrogenase liberated during Zymolyase treatments. The influence of the method for disruption of spheroplasts on the quality of the mitochondria was analysed with particular emphasis on respiratory control values and the distribution of marker enzymes among the cell fractions. Disruption by osmotic shock resulted in mitochondria without respiratory control and a high degree of solubilization of NADH and NADPH dehydrogenase activities. Only a gradual decrease of the osmotic value of the medium, preferably by dialysis against a hypotonic buffer, in combination with mechanical disruption with a Potter-Elvehjem homogenizer yielded mitochondria with high respiratory control values and a high retention of NADH dehydrogenase in the organelle. It is concluded that, for the quality of mitochondrial preparations from yeasts, the distribution of NADH dehydrogenase among the cell fractions is a more reliable measure than that of the usual marker enzymes.
18 citations
TL;DR: A continuous culture study on the physiology of this organism with special attention to the regulation of GDH synthesis, 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.
Abstract: 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.
4 citations
TL;DR: To study the functional coupling of GDH to the respiratory chain, a procedure for the isolation of membrane vesicles from bacteria grown under well-defined conditions was developed and the rate of alanine uptake when energized with GDH was comparable with that of the artificial electron donor system ascorbate phenazine methosulphate.
Abstract: 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).
4 citations
TL;DR: Growth and respiration studies involving denitrification inhibitors have provided further evidence that Thiosphaera pantotropha denitrifies aerobically and N20 accumulates in aerobic cultures with acetylene.
Abstract: Growth and respiration studies involving denitrification inhibitors have provided further evidence that Thiosphaera pantotropha denitrifies aerobically. The reduction in total oxygen uptake caused by nitrate is nullified by agents such as antimycin a. Furthermore, N20 accumulates in aerobic cultures with acetylene. Aerobic denitrification has also been observed in the chemostat. Interpretation of the results has been complicated by this organism's ability to nitrify heterotrophically.
4 citations
TL;DR: The red-pigmented marine coccoid cyanobacteria, often present in large concentrations ranging from 103 to 10 s cells, form an important, newly recognized, component of oceanic primary productivity.
Abstract: The red-pigmented marine coccoid cyanobacteria, often present in large concentrations ranging from 103 to 10 s cells.m1-1 in tropical waters, form an important, newly recognized, component of oceanic primary productivity. Being small in size (1.0 p.m diam), they were easily washed away through filters of larger pore size, thus escaping attention and analysis. Their relative role is thought to be greater in oligotrophic than in more nutrient-rich inshore waters. Most of these small red-pigmented cyanobacteria resemble Synechococcus DC2, which was studied in light-limited continuous cultures. This species showed a distinct peak at 545 nm in the in vivo absorption spectrum, due to the presence of a unique 'phycoerythrin'. This red pigment in small coccoid marine cyanobacteria may well differ in structure and function from the 'normal' phycoerythrin found in other red-pigmented phytoplankters, since the latter has its absorption maximum at a higher wavelength (565 nm). The unique phycoerythrin proved to be an ideal pigment marker, allowing the use of the peak height at 545 nm in the in vivo absorption spectra as a fast tracer of small red-pigmented Synechococcus-type cyanobacteria in marine phytoplankton communities. The abundance of these species in the Banda Sea (Indonesia) was examined during the first cruise of the 'Pelagic Systems', Theme 3 of the Snellius II Expedition, in August 1984 when 'upwelling' (nutrient-rich) conditions were expected. The survey embraced 34 stations that were located at 50-miles distances. Small coccoid cyanobacteria were found at 75% of the Banda Sea stations, ranging from 104 to 10 s cells, ml1, and with a preference for the offshore stations where phytoplankton biomass concentrations were relatively low, and for the deeper layers where light intensities were low. It was hypothesized that cyanobacteria tended to move to deeper layers during the course of the day as a result of the relatively high total daily irradiances. The data indicated further that upwelling was not at its maximum level.
4 citations
TL;DR: Growth and respiration studies involving denitrification inhibitors have provided further evidence that Thiosphaera pantotropha denitrifies aerobically and N20 accumulates in aerobic cultures with acetylene.
Abstract: Growth and respiration studies involving denitrification inhibitors have provided further evidence that Thiosphaera pantotropha denitrifies aerobically. The reduction in total oxygen uptake caused by nitrate is nullified by agents such as antimycin a. Furthermore, N20 accumulates in aerobic cultures with acetylene. Aerobic denitrification has also been observed in the chemostat. Interpretation of the results has been complicated by this organism's ability to nitrify heterotrophically.
2 citations
TL;DR: In this paper, a chemostat enrichment on dimethyl sulphoxide (DMSO), which is metabolized via DMS (De Bont et al., 1981), was used to obtain a mixed culture that was able to oxidize not only DMSO but also DMS and several other reduced sulphur compounds as well as some Ca-compounds.
Abstract: Dimethyl sulphide (DMS) is an extremely toxic, volatile compound with a very offensive odour. It is assumed to play an important role in the global sulphur cycle in transporting reduced sulphur compounds from aquatic to terrestrial enviromnents. It is also one of the major compounds implicated in the volatilization of sulphur from soil. Furthermore, high quantities of volatile sulphur compounds are produced by the paper industry, in oil refineries and, to a lesser extent, in municipal sewage treatment plants. Since this may cause serious environmental pollution problems, an attempt was made to enrich for, and isolate, DMS-oxidizing bacteria, which in a later stage may be used for the biological treatment of DMS-containing wastes. In our hands, enrichments for bacteria able to use DMS as an energy and/or carbon source have been unsuccessful because of its extreme toxicity and volatility. However, by chemostat enrichment on dimethyl sulphoxide (DMSO), which is metabolized via DMS (De Bont et al., 1981), we obtained a mixed culture that was able to oxidize not only DMSO but also DMS and several other reduced sulphur compounds as well as some Ca-compounds (Table 1). In order to obtain information on the possible constituents of the mixed culture, activities of the key-enzymes of several metabolic pathways were measured in the mixed culture (Table 2). These indicated that an important part of the culture consisted of methylotrophs, whereas there was little or no autotrophic growth in the culture. Plate counts (in which a viability of 67~ was achieved) indicated that the majority of the culture (96%) was made up of a Ityphomicrobium species able to grow on several Cl-compounds (Hyphomicrobium EG).
1 citations
TL;DR: Inactivation experiments were performed with cyclopropanot, using dimeric as well as monomeric methanol dehydrogenase (the latter enzyme form was used in the experiments by Mincey et al., 1981), and the following conclusions can be drawn.
Abstract: Bacterial alcohol oxidation frequently proceeds via respiratory-chain linked dehydrogenases which have pyrrolo-quinoline quinone (PQQ) as their coenzyme. These so-called quinoprotein alcohol dehydrogenases have originally been isolated from methylotrophic bacteria and in these cases they are known as methanol dehydrogenases (EC 1.1.99.8). In this report we describe the results of mechanistic studies using monomeric as well as dimeric enzyme forms. Recently Mincey et al. (1981) put forward a view on the mechanism of action which is quite different from the one reported by us (Duine and Frank, 1981). Their model is based on an enzyme form containing the semiquinone of PQQ, reacting with substrate and producing a three-electron reduced form of PQQ. In our view, only the fully oxidized enzyme form reacts with substrate. In order to settle this controversy, inactivation experiments were performed with cyclopropanot, using dimeric as well as monomeric methanol dehydrogenase (the latter enzyme form was used in the experiments by Mincey et al., 1981). From the results the following conclusions can be drawn: cyclopropanoI acts as a suicide substrate for both enzyme forms. only enzyme molecules in the fully oxidized form react with cyclopropanol. titration experiments reveal that there is no proton production and no electron acceptor consumption during the inactivation step. in fully inactivated enzyme, all PQQ is converted into an adduct with suicide substrate. in dimeric enzyme, the two catalytic sites act independently (50% inactivation is achieved with stoichiometric amounts ofcyclopropanol and enzyme). From these results and from evidence obtained from ESR experiments (De Beer et ak, I983) it is clear that reaction of substrate or suicide substrate only occurs with fully oxidized enzyme molecules. In this respect, no difference is observed between monomeric and dimeric enzyme preparations. All conclusions are in agreement with and substantiate our reported model for the catalytic cycle of the enzyme (Duine and Frank, 1981).
TL;DR: In this paper, the authors showed that Thiobaeillus strain Q can oxidize thiosulphate completely to sulphate and that the necessary enzymes are also present when grown on acetate alone and can be induced to full capacity in several hours.
Abstract: in soil and marine environments. Although much is known about the physiology of the obligately and facultativety autotrophic sulphide oxidizers very little is known of these heterotrophic sulphide oxidizers (chemolithoheterotrophs). Recent work of our group has indicated that the latter group of organisms would be particularly important in those environments where high turnover rates of organic compounds would be combined with low rates of reduced inorganic sulphur compounds. Indeed, enrichments using mixed feed of acetate (15 raM) and thiosulphate (10 mM) yielded a dominant culture ofa chemolithoheterotroph (Gottschal and Kuenen, 1980). In order to obtain a better understanding of this type of metabolism physiological studies were made on this organism, provisionally called Thiobaeillus strain Q. This organism is able to oxidize thiosulphate completely to sulphate. Thiosulphate-oxidizing.capacity measurements indicate that the necessary enzymes are also present when Thiobacillus Q is grown on acetate alone and can be induced to full capacity in several hours. The addition of thiosulphate (5 mM) to acetate-limited (10 mM) chemostat cultures resulted in an increase in yield from 60 to 85 mg protein per litre indicating that Thiobacillus Q can indeed make use of the energy liberated by thiosulphate oxidation. All attempts to grow this organism autotrophically have failed. This indicates that Thiobaeillus Q should be considered a chemolithoheterotroph, able to utilize energy derived from the oxidation of reduced sulphur compounds and unable to grow autotrophically.
TL;DR: The aim of these studies is to answer the question what the role of the carboxysomes is in CO2-fixation and to analyse the structure of these microbodies.
Abstract: Many chemolithotrophic bacteria like Thiobacillus neapolitanus contain polyhedral inclusion bodies (Shively et al., 1973) which contain ribulose-1, 5-bisphosphate carboxylase (RuBisCo). These microbodies are referred to as carboxysomes. In CO2qimited cultures of Th. neapolitanus the number of carboxysomes is higher than in any other culture. The degree of CO2-1imitation determines the amount of particulate RuBisCo (Beudeker et al., 1981). Beside RuBisCo, many other enzymes were reported to be present inside the carboxysomes: all the enzymes of the Calvin-cycle and a set of enzymes that made it possible for malate to act as reductant inside the microbodies (malate shuttle; Beudeker and Kuenen, 1981). These observations, however, could not be confirmed by other investigators (Cannon and Shively, 1983). The question what the role of the carboxysomes is in CO2-fixation is therefore still not answered. The aim of our studies is to answer this question and to analyse the structure of these microbodies. A new method was developed for purifying the carboxysomes resulting in full separation of the carboxysomes from other cell material. In these purified carboxysomes, in addition to RuBisCo, only phosphoglycerate kinase could be detected with low activities. Stimulation of CO2-fixation in vitro was observed after addition of ATP. The malate shuttle mechanism (Beudeker and Kuenen, 1981) could not be detected in our purified carboxysome preparations. Malate did not stimulate CO2-fixation and no malate dehydrogenase activity was found.