Effect of major nutrient elements on the growth and population homogeneity of the R, S and M dissociants of Pseudomonas aeruginosa and the glucose oxidation and fermentation pathways

The population homogeneity of the stationary-phase monocultures of Pseudomonas aeruginosa dissociants was studied as a function of the initial content of major nutrient elements (C, N, and P) in the cultivation medium. The monocultures of the dissociants remained homogeneous during cultivation if the initial concentrations of the major nutrient elements were either sufficiently high or, conversely, very low, but became heterogeneous during cultivation in unbalanced (with respect to the major nutrient elements) media. At the initial concentration of nitrate in the medium equal to 0.07% or phosphate equal to 0.004-0.014%, the initially homogeneous population of R dissociant cultivated to the stationary growth phase turned out to contain 30-40% of S-type cells, whereas the initially homogeneous population of S dissociant was found to contain 50-80% of M-type cells. The population of M dissociant remained homogeneous throughout the cultivation period. R dissociant grew better at sufficiently high concentrations of glucose, nitrate, and phosphate in the medium, whereas M dissociant grew better when the initial concentrations of these nutrients were low. During the cultivation of R dissociant, the pH of the medium changed insignificantly, and the C/P ratio (the ratio of the carbon and phosphorus consumed during growth) was minimal (among the three dissociants), indicating that the R dissociant accomplishes the oxidative pathway of glucose metabolism. During the cultivation of the M dissociant, the pH of the medium dropped to 3.4-3.9, and the C/P ratio was maximal, indicating that this dissociant accomplishes the fermentative pathway of glucose metabolism. During the cultivation of the S dissociant, the pH of the medium and the C/P ratio exhibited variations, indicating that this dissociant triggers its pathways of glucose metabolism.     

The Effect of Major Nutrient Elements on the Growth and Population Homogeneity of the R,S, and M Dissociants of Pseudomonas aeruginosa and the Glucose Oxidation and Fermentation Pathways

The population homogeneity of the stationary-phase monocultures of Pseudomonas aeruginosa dissociants was studied as a function of the initial content of major nutrient elements (C, N, and P) in the cultivation medium. The monocultures of the dissociants remained homogeneous during cultivation if the initial concentrations of the major nutrient elements were either sufficiently high or, conversely, very low, but became heterogeneous during cultivation in unbalanced (with respect to the major nutrient elements) media. At an initial concentration of nitrate in the medium equal to 0.07% or phosphate equal to 0.004–0.014%, the initially homogeneous population of R dissociant cultivated to the stationary growth phase turned out to contain 30–40% of S-type cells, whereas the initially homogeneous population of S dissociant was found to contain 50–80% of M-type cells. The population of M dissociant remained homogeneous throughout the cultivation period. R dissociant grew better at sufficiently high concentrations of glucose, nitrate, and phosphate in the medium, whereas M dissociant grew better when the initial concentrations of these nutrients were low. During the cultivation of R dissociant, the pH of the medium changed insignificantly, and the C/P ratio (the ratio of the carbon and phosphorus consumed during growth) was minimal (among the three dissociants), indicating that the R dissociant accomplishes the oxidative pathway of glucose metabolism. During the cultivation of the M dissociant, the pH of the medium dropped to 3.4–3.9, and the C/P ratio was maximal, indicating that this dissociant accomplishes the fermentative pathway of glucose metabolism. During the cultivation of the S dissociant, the pH of the medium and the C/P ratio exhibited variations, indicating that this dissociant triggers its pathways of glucose metabolism.     

Effect of carbon, nitrogen and phosphorus nutrition on the R, S, and M dissociants of Pseudomonas aeruginosa in mixed cultures

The effect of glucose, nitrate, and phosphate on the stationary-phase growth characteristics of R, S, and M dissociants of the hydrocarbon-oxidizing strain P. aeruginosa K-2 was studied. The optimal concentrations of glucose and phosphate providing for at least 90% of the maximal culture density were found to be 2-7% glucose and 0.02-0.12% phosphate. The main factor that determined the proportion of dissociants in bacterial populations was the initial concentration of phosphate. The fraction of R dissociant in populations increased linearly with the concentration of glucose and varied nonlinearly with the concentration of phosphate in the growth medium. The fraction of M dissociant depended solely on the concentration of phosphate in a manner inverse to that typical of R dissociant. In glucose-deficient media containing sufficient amounts of phosphorus, S dissociant prevailed over R dissociant.     

Effect of carbon, nitrogen, and phosphorus sources of nutrition on the growth of R-, S- and M-dissociants of Pseudomonas aeruginosa]

The effect of glucose, nitrate, and phosphate on the stationary-phase growth parameters of the R, S, and M dissociants of the hydrocarbon-oxidizing bacterium Pseudomonas aeruginosa K-2 was studied. The data obtained were analyzed in terms of the Mitscherlich equation. S dissociant required less glucose than other dissociants, whereas M dissociant required less nitrogen and phosphorus. These findings were confirmed by the results of investigation of the combined action of glucose, nitrate, and phosphate in a 3 x 3 x 3 factor experiment. It is anticipated that M dissociant must prevail under conditions of nitrogen and phosphorus deficiency, and S dissociant must be dominant in the case of optimally chosen proportions between the biogenic elements studied.     

Effect of reduced concentrations of carbon, nitrogen, and phosphorus in a medium on growth of three dissociants of Pseudomonas aeruginosa]

The effect of lowered concentrations of carbon, nitrogen, and phosphorus sources in the medium on the specific growth rate mu of the R, S, and M dissociants of the hydrocarbon-oxidizing strain Pseudomonas aeruginosa K-2, culture pH, and the population composition was studied. Within the first 16 hours of cultivation in all of the four media tested, the R, S, and M dissociants had virtually identical mu. The maximal values of mu were reached by the 20th h of growth in the basal medium (R and S dissociants) and in the carbon-deficient medium containing 0.4% glucose (M dissociant). The R and M dissociants showed the most rapid decrease in mu in the nitrogen-deficient medium containing 0.55% NaNO3. By the end of cultivation in the basal medium, the pH of the R, S, and M cultures decreased to 6.3, 5.3, and 3.3, respectively. In the case of the carbon-deficient medium, the drop in the culture pH was lower. After a 2.5-day incubation of the S dissociant in the phosphorus-deficient medium containing 0.028% NaH2PO4.2H2O and of the M dissociant in the basal medium supplemented with chalk powder, these dissociants were completely displaced from the media.     

Development and population structure of mixed (S + M) Pseudomonas aeruginosa cultures in the late stationary growth phase

Population growth, the ratio between dissociants, pH, and levels of reducing sugars in the medium were monitored during prolonged (375 h) batch cultivation of Pseudomonas aeruginosa S and M dissociants on mineral medium with glucose. During the stationary growth phase (100-375 h), two scenarios were possible. The first one included extensive cell autolysis coupled to alkalinization of the medium and an increased ratio of the M dissociant. In the second case, acidification of the medium was coupled to the oscillating secondary growth, mostly of the M dissociant; the dynamics of cell numbers of this dissociant correlated with the dynamics of the culture optical density. In this scenario, periodical appearance of reducing sugars in the medium was detected; it was in the opposite phase with the changes of the M dissociant cell numbers. The differences between scenarios of P. aeruginosa growth in the late stationary phase were probably due to the physiological and biochemical characteristics of the S and M dissociants, including different pathways of glucose utilization (respiration or fermentation), resistance to acidification, synthetic (proteolytic) activity, and productivity of autoinducers.     

Development and population structure of mixed (S + M) <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> cultures in the late stationary growth phase

Population growth, the ratio between dissociants, pH, and levels of reducing sugars in the medium were monitored during prolonged (375 h) batch cultivation of Pseudomonas aeruginosa S and M dissociants on mineral medium with glucose. During the stationary growth phase (100–375 h), two scenarios were possible. The first one included extensive cell autolysis coupled to alkalinization of the medium and an increased ratio of the M dissociant. In the second case, acidification of the medium was coupled to the oscillating secondary growth, mostly of the M dissociant; the dynamics of cell numbers of this dissociant correlated with the dynamics of the culture optical density. In this scenario, periodical appearance of reducing sugars in the medium was detected; it was in the opposite phase with the changes of the M dissociant cell numbers. The differences between scenarios of P. aeruginosa growth in the late stationary phase were probably due to the physiological and biochemical characteristics of the S and M dissociants, including different pathways of glucose utilization (respiration or fermentation), resistance to acidification, as well as synthetic (proteolytic) activity and productivity of autoinducers.     

Activity of pentose phosphate pathway enzymes in alkane-oxidizing yeast cells]

The activity of the key enzymes of the pentose phosphate pathway (glucose 6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, transketolase) was determined in cell-free homogenates of Candida lipolytica 695 and Candida tropicalis 303 growing on different carbon sources. The activity of these enzymes remained almost the same in the course of growth of both cultures. The activity of the enzymes differed only slightly in the cells metabolizing hexadecane and glucose. The activity of glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase in the cell-free homogenates of C. tropicalis 303 was twice as high as in the cells of C. lipolytica 695. The activity of transketolase was the same in both cultures. The main role of the pentose phosphate pathway is presumed to consist not in catabolism of the carbon source, but in biosynthesis of pentoses and other important intermediates.     

A biochemical study of the intermediary carbon metabolism of Shewanella putrefaciens.

Cell extracts were used to determine the enzymes involved in the intermediary carbon metabolism of several strains of Shewanella putrefaciens. Enzymes of the Entner-Doudoroff pathway (6-phosphogluconate dehydratase and 2-keto-3-deoxy-6-phosphogluconate aldolase) were detected, but those of the Embden-Meyerhof-Parnas pathway were not. While several tricarboxylic acid cycle enzymes were present under both aerobic and anaerobic conditions, two key enzymes (2-oxoglutarate dehydrogenase and pyruvate dehydrogenase) were greatly diminished under anaerobic conditions. Extracts of cell grown anaerobically on formate as the sole source of carbon and energy were positive for hydroxypyruvate reductase, the key enzyme of the serine pathway in other methylotrophs, while no hexulose synthase activity was seen.     

The Effect of Lowered Concentrations of Carbon,Nitrogen and Phosphorus Sources on the Growth Dynamics of the R,S, and M Dissociants of Pseudomonas aeruginosa

The effect of lowered concentrations of carbon, nitrogen, and phosphorus sources in the medium on the specific growth rate of the R, S, and M dissociants of the hydrocarbon-oxidizing strain Pseudomonas aeruginosaK-2, culture pH, and the population composition was studied. Within the first 16 hours of cultivation in all of the four media tested, the R, S, and M dissociants have virtually identical . The maximal values of were reached by the 20th h of growth in the basal medium (R and S dissociants) and in the carbon-deficient medium containing 0.4% glucose (M dissociant). The R and M dissociants showed the most rapid decrease in in the nitrogen-deficient medium containing 0.55% NaNO₃. By the end of cultivation in the basal medium, the pH of the R, S, and M cultures decreased to 6.3, 5.3, and 3.3, respectively. In the case of the carbon-deficient medium, the drop in the culture pH was lower. After a 2.5-day incubation of the S dissociant in the phosphorus- deficient medium containing 0.028% NaH₂PO₄· 2H₂O and of the M dissociant in the basal medium supplemented with chalk powder, these dissociants were completely displaced from the media.     

Regulation of alternate peripheral pathways of glucose catabolism during aerobic and anaerobic growth of Pseudomonas aeruginosa.

Glucose may be converted to 6-phosphogluconate by alternate pathways in Pseudomonas aeruginosa. Glucose is phosphorylated to glucose-6-phosphate, which is oxidized to 6-phosphogluconate during anaerobic growth when nitrate is used as respiratory electron acceptor. Mutant cells lacking glucose-6-phosphate dehydrogenase are unable to catabolize glucose under these conditions. The mutant cells utilize glucose as effectively as do wild-type cells in the presence of oxygen; under these conditions, glucose is utilized via direct oxidation to gluconate, which is converted to 6-phosphogluconate. The membrane-associated glucose dehydrogenase activity was not formed during anaerobic growth with glucose. Gluconate, the product of the enzyme, appeared to be the inducer of the gluconate transport system, gluconokinase, and membrane-associated gluconate dehydrogenase. 6-Phosphogluconate is probably the physiological inducer of glucokinase, glucose-6-phosphate dehydrogenase, and the dehydratase and aldolase of the Entner-Doudoroff pathway. Nitrate-linked respiration is required for the anaerobic uptake of glucose and gluconate by independently regulated transport systems in cells grown under denitrifying conditions.     

The requirements of Pseudomonas aeruginosa dissociants for carbon, nitrogen, and phosphorus

Quantitative data on the nutritional requirements of microorganisms are necessary to predict the behavior of bacterial populations and to control their cultivation. The requirements of the R, S, and M dissociants of Pseudomonas aeruginosa for carbon, nitrogen, and phosphorus were derived from the results of 88 cultivation experiments. For each of the dissociants, we derived a coefficient that relates the optical density and the number of cells in the dissociant culture, determined the time when the cultures entered the stationary growth phase, studied cultural changes induced by transfer to the stationary phase, and determined what nutrients limit the growth of particular dissociants. The nutritional requirements of the dissociants are discussed in relation to our earlier data.     

The Requirements of Pseudomonas aeruginosa Dissociants for Carbon,Nitrogen, and Phosphorus

Quantitative data on the nutritional requirements of microorganisms are necessary to predict the behavior of bacterial populations and to control their cultivation. The requirements of the R, S, and M dissociants of P. aeruginosa for carbon, nitrogen, and phosphorus were derived from the results of 88 cultivation experiments. For each of the dissociants, we derived a coefficient that relates the optical density and the number of cells in the dissociant culture, determined the time when the cultures entered the stationary growth phase, studied cultural changes induced by transfer to the stationary phase, and determined what nutrients limit the growth of particular dissociants. The nutritional requirements of the dissociants are discussed in relation to our earlier data.     

Inhibition of 6-phosphogluconate dehydrogenase (decarboxylating) by glucose 1,6-bisphosphate.

Glucose 1,6-bisphosphate, the powerful common regulator of several key enzymes in carbohydrate metabolism, was found to exert a potent inhibitory effect on the activity of 6-phosphogluconate dehydrogenase (decarboxylating) from yeast and several rat tissues. These findings suggest that glucose 1,6-bisphosphate may have a regulatory influence on the pentose phosphate pathway.     

Metabolism of C2-C6-substrates under mixotrophic growth of Acinetobacter sp. B-7005 and B-7005 (1HG) strains

Activities of the key enzymes of C2-C6-metabolism were assayed under cultivation of Acinetobacter sp. B-7005 and B-7005 (1Hgamma) strains on ethanol and glucose mixture. Under mixotrophic growth of bacteria the enzymes activity of ethanol metabolism (NAD+ -dependent alcohol dehydrogenase, NADP+ -dependent acetaldehyde dehydrogenase, acetyl-KoA-synthetase) and glucose metabolism (6-phosphofructokinase and 6-phosphogluconate dehydratase) was lower than that on corresponding monosubstrates. The activity of isocitrate lyase and malate synthase in cells grown on the substrate mixture declined to an even greater extent, indicating that the role of the glyoxylate cycle in such cells is insignificant. The simultaneous functioning of the glyoxylate cycle and pyruvate carboxylase reaction, increasing of phosphoenolpyruvate synthetase activity testify to the gluconeogenesis intensification under mixotrophic growth of Acinetobacter sp. B-7005 and B-7005 (1Hgamma).     

Oxidation of C1-compounds in Pseudomonas C.

Extracts of Pseudomonas C grown on methanol as a sole carbon and energy source contain a methanol dehydrogenase activity which can be coupled to phenazine methosulfate. This enzyme catalyzes two reactions namely the conversion of methanol to formaldehyde (phenazine methosulfate coupled) and the oxidation of formaldehyde to formate (2,6-dichloroindophenol-coupled). Activities of glutathione-dependent formaldehyde dehydrogenase (NAD+) and formate dehydrogenase (NAD+) were also detected in the extracts. The addition of D-ribulose 5-phosphate to the reaction mixtures caused a marked increase in the formaldehyde-dependent reduction of NAD+ or NADP+. In addition, the oxidation of [14C]formaldehyde to CO2, by extracts of Pseudomonas C, increased when D-ribulose 5-phosphate was present in the assay mixtures. The amount of radioactivity found in CO2, was 6;8-times higher when extracts of methanol-grown Pseudomonas C were incubated for a short period of time with [1-14C]glucose 6-phosphate than with [U-14C]glucose 6-phosphate. These data, and the presence of high specific activities of hexulose phosphate synthase, phosphoglucoisomerase, glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase indicate that in methanol-grown Pseudomonas C, formaldehyde carbon is oxidized to CO2 both via a cyclic pathway which includes the enzymes mentioned and via formate as an oxidation intermediate, with the former predominant.     

Enzymes of glucose metabolism in Frankia sp.

Enzymes of glucose metabolism were assayed in crude cell extracts of Frankia strains HFPArI3 and HFPCcI2 as well as in isolated vesicle clusters from Alnus rubra root nodules. Activities of the Embden-Meyerhof-Parnas pathway enzymes glucokinase, phosphofructokinase, and pyruvate kinase were found in Frankia strain HFPArI3 and glucokinase and pyruvate kinase were found in Frankia strain HFPCcI2 and in the vesicle clusters. An NADP+-linked glucose 6-phosphate dehydrogenase and an NAD-linked 6-phosphogluconate dehydrogenase were found in all of the extracts, although the role of these enzymes is unclear. No NADP+-linked 6-phosphogluconate dehydrogenase was found. Both dehydrogenases were inhibited by adenosine 5-triphosphate, and the apparent Km's for glucose 6-phosphate and 6-phosphogluconate were 6.86 X 10(-4) and 7.0 X 10(-5) M, respectively. In addition to the enzymes mentioned above, an NADP+-linked malic enzyme was detected in the pure cultures but not in the vesicle clusters. In contrast, however, the vesicle clusters had activity of an NAD-linked malic enzyme. The possibility that this enzyme resulted from contamination from plant mitochondria trapped in the vesicle clusters could not be discounted. None of the extracts showed activities of the Entner-Doudoroff enzymes or the gluconate metabolism enzymes gluconate dehydrogenase or gluconokinase. Propionate- versus trehalose-grown cultures of strain HFPArI3 showed similar activities of most enzymes except malic enzyme, which was higher in the cultures grown on the organic acid. Nitrogen-fixing cultures of strain HFPArI3 showed higher specific activities of glucose 6-phosphate and 6-phosphogluconate dehydrogenases and phosphofructokinase than ammonia-grown cultures.     

Regulation of Glucose Metabolism in Thiobacillus intermedius

Glucose-yeast extract or glucose-casein hydrolysate-grown Thiobacillus intermedius cells, which use glucose for energy generation, possess high specific activities of the Entner-Doudoroff pathway and related enzymes, 6-phosphogluconate dehydrase, 2-keto-3-deoxy-6-phosphogluconate aldolase, glucokinase, and glucose-6-phosphate dehydrogenase, but low activities of enzymes unique to the pentose shunt and Embden-Meyerhof pathways. Although the synthesis of the latter enzymes remains largely unaffected by the growth environment, that of the former is stimulated by glucose. Radiorespirometric measurements demonstrate an early and parallel respiration of glucose carbon atoms one and four in glucose-casein hydrolysate broth. It is concluded that the Entner-Doudoroff pathway performs an energetic role in glucose metabolism by T. intermedius with the pentose shunt and Embden-Meyerhof pathways functioning mainly in biosynthesis. The presence of thiosulfate in the growth medium inhibits the synthesis of the Entner-Doudoroff pathway and related enzymes. In addition, both thiosulfate and glucose inhibit the synthesis of the Krebs cycle enzymes, nicotinamide adenine dinucleotide phosphate-linked isocitrate and alpha-ketoglutarate dehydrogenases. Thus, repression of enzymes is of significance in the adaptation of T. intermedius to its nutritional environment. The activity of glucose-6-phosphate dehydrogenase of T. intermedius is inhibited by adenosine triphosphate. Such a control could afford the organism a mechanism to regulate the flow of glucose into major energetic and biosynthetic routes.     

Oxidative Enzymes and Pathways of Hexose and Triose Metabolism in Chlorella

Cell-free preparations of Chlorella pyrenoidosa Chick, van Niel's strain, were assayed for oxidative enzymes, utilizing isotopic and spectrophotometric techniques. The enzyme activity of heterotrophic and autotrophic cells was compared. The study was divided into categories, one concerned with the spectrophotometric detection of enzymes involved in the initial reactions of glycolysis and the hexose monophosphate shunt, and the other with the direct oxidation of glucose as compared with that oxidized via glycolysis. The reduction of pyridine nucleotides in crude extracts was studied with glucose, glucose-6-phosphate, 6-phosphogluconate, and fructose-1-6-diphosphate as substrates. Enzymes detected in both heterotrophic and autotrophic cells were hexokinase, fructose-diphosphate-aldolase, NAD-linked 3-phosphoglyceraldchyde dehydrogenase, glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, and a NADP-linked 3-phosphoglyceraldchyde dehydrogenase. In addition to isotopic studies designed to make an appraisal of the hexose monophosphate shunt, a comparison of the rate of reduction of NADP by glucose-6-phosphate and 6-phosphogluconate in relation to the reduction of NAD by 3-phosphoglyceraldehyde was made in light- and dark-grown cells. The rate of reduction of NADP appeared to be lowered in the light-grown cells, suggesting, as did also the isotopic studies, that the hexose monophosphate shunt is less active in autotrophic metabolism than in heterotrophic metabolism.     

Glucose Metabolism in Neisseria gonorrhoeae

The metabolism of glucose was examined in several clinical isolates of Neisseria gonorrhoeae. Radiorespirometric studies revealed that growing cells metabolized glucose by a combination on the Entner-Doudoroff and pentose phosphate pathways. A portion of the glyceraldehyde-3-phosphate formed via the Entner-Doudoroff pathway was recycled by conversion to glucose-6-phosphate. Subsequent catabolism of this glucose-6-phosphate by either the Entner-Doudoroff or pentose phosphate pathways yielded CO(2) from the original C6 of glucose. Enzyme analyses confirmed the presence of all enzymes of the Entner-Doudoroff, pentose phosphate, and Embden-Meyerhof-Parnas pathways. There was always a high specific activity of glucose-6-phosphate dehydrogenase (EC 1.1.1.49) relative to that of 6-phosphogluconate dehydrogenase (EC 1.1.1.44). The glucose-6-phosphate dehydrogenase utilized either nicotinamide adenine dinucleotide phosphate or nicotinamide adenine dinucleotide as electron acceptor. Acetate was the only detectable nongaseous end product of glucose metabolism. Following the disappearance of glucose, acetate was metabolized by the tricarboxylic acid cycle as evidenced by the preferential oxidation of [1-(14)C]acetate over that of [2-(14)C]acetate. When an aerobically grown log-phase culture was subjected to anaerobic conditions, lactate and acetate were formed from glucose. Radiorespirometric studies showed that under these conditions, glucose was dissimilated entirely by the Entner-Doudoroff pathway. Further studies determined that this anaerobic dissimilation of glucose was not growth dependent.