Effect of Type of Carbohydrate on Amino Acid Accumulation and Utilization by Tetrahymena

Both concentration and pattern of free amino acids in Tetrahymena pyriformis, as well as utilization, synthesis, and excretion of amino acids, were affected by type of carbohydrate in growth media. With glucose, cell pool concentrations were higher than with dextrin, and alanine and glycine together accounted for an average of 45% of the total pool. These two amino acids accumulated in similar proportions when their synthesis from essential amino acids was a prerequisite, as when they were provided by growth media, but they constituted only 15 to 18% of the free amino acids in cells from media with dextrin. Alanine, glycine, and glutamic acid were excreted into the medium regardless of whether they were provided by starting media; hence, they appear to be end products of the required metabolism of some of the essential amino acids. Both accumulation and excretion of the above non-essential amino acids were nearly twice as extensive in media providing glucose as the only carbohydrate as when the carbohydrate supplied was dextrin or dextrin plus glucose. Thus, the observed differences are not attributable to differences in osmolarity of monosaccharide and polysaccharide media. Free amino acid differences do not appear to be the immediate cause of the different growth-stimulating properties of such carbohydrates.     

Utilization of amino acids by Trypanosoma brucei in culture: L-threonine as a precursor for acetate.

The amino acid compositions of several culture media have been analysed and compared. The utilization and excretion of amino acids and other metabolites have been followed during growth of Trypanosoma brucei S42 in a defined medium. All of the added L-threonine was metabolized by the cells, even when it was present at elevated concentrations. Glucose was consumed throughout the growth cycle: glutamine was consumed more rapidly than glutamic acid, which was itself used at about the same rate as proline. Threonine was cleaved to form glycine and acetate, both of which accumulated in the medium. Alanine and succinate were excreted together with a small amount of pyruvate, but these three products accounted for less than half of the glucose used. CO2 production from glucose was not measured, but insignificant amounts of CO2 were produced from threonine. Tetraethylthiuram disulphide blocked the cleavage of threonine and was a potent inhibitor of trypanosome growth.     

Amino acid-dependent transport of sugars by Fusobacterium nucleatum ATCC 10953.

Resting cells of Fusobacterium nucleatum 10953 (grown previously in a medium containing glucose) failed to accumulate glucose under aerobic or anaerobic conditions. However, the addition of glutamic acid, lysine, or histidine to anaerobic suspensions of cells caused the immediate and rapid accumulation of glucose. Except for the amino acid-dependent transport of galactose and fructose (the latter being transported at approximately one-third the rate of glucose), no other sugars tested were accumulated by the resting cells. Amino acid-dependent uptake of sugar(s) by F. nucleatum was abolished by exposure of cells to air, and under aerobic conditions the rates of fermentation of glutamic acid and lysine were less than 15% of the rates determined anaerobically. The energy necessary for active transport of the sugars (acetyl phosphate and ATP) is derived from the anaerobic fermentation of glutamic acid, lysine, or histidine. Competition studies revealed that glucose and galactose were mutual and exclusive inhibitors of transport, and it is suggested that the two sugars (Km = 14 microM) are translocated via a common carrier. The products of amino acid-dependent sugar transport were recovered from resting cells as ethanol-precipitable, high-molecular-weight polymers. Polymer formation by F. nucleatum, during growth in medium containing glucose or galactose, was confirmed by electron microscopy.     

Amino Acid Requirements for the Growth of Aedes albopictus Clone C6/36 Cells and for the Production of Dengue and Chikungunya Viruses in the Infected Cells

Amino acid requirements for the growth of Aedes albopictus, clone C6/36, cells and for the production of dengue (DEN) and Chikungunya (CHIK) viruses were examined by growing the cells or the viruses in media which were deprived of one of the 20 amino acids. Cell growth was markedly inhibited when cystine was omitted from the medium, and to a lesser extent by arginine deprivation. On the other hand, omission of alanine, asparagine, aspartic acid, and glutamic acid at the same time did not affect cell growth. Marked accumulation of alanine was observed in the medium when the cells were grown for 8 days in complete medium, with concomitant depletion of aspartic acid and glutamic acid. The production of CHIK virus was inhibited markedly by omission of cystine from the medium after virus infection, while the production of DEN viruses was more affected by glycine deprivation, although cystine deprivation also inhibited virus production to a lesser extent. On the other hand, production of CHIK and DEN viruses was not affected when alanine, asparagine, aspartic acid, and glutamic acid were omitted from the medium at the same time.     

Uptake and excretion of amino acids and utilization of glucose by Schistosoma japonicum eggs

The utilization of amino acids and glucose in the external nutrients and the excretion of nitrogenous compounds by Schistosoma japonicum eggs were investigated with the eggs cultured in a chemically defined medium (MEMSE-J). Of the 15 amino acids in MEMSE-J, arginine and glutamine markedly decreased in concentration during cultivation of S. japonicum eggs. The nitrogenous excretory products of developing eggs were demonstrated to be at least four amino acids (alanine, proline, glutamic acid and ornithine), urea and ammonia. Glucose was consumed at an estimated rate of 32 ng/living egg/day during the period of egg growth and differentiation. When 14C-labelled glucose was included in the culture medium, the radioactivity was incorporated into three amino acids (alanine, proline and glutamic acid), which were excreted by S. Japonicum eggs. The results were discussed with reference to the possible role in stimulating fibrosis in the granuloma of schistosomiasis.     

Influence of the nitrogen source on Saccharomyces cerevisiae anaerobic growth and product formation.

To prevent the loss of raw material in ethanol production by anaerobic yeast cultures, glycerol formation has to be reduced. In theory, this may be done by providing the yeast with amino acids, since the de novo cell synthesis of amino acids from glucose and ammonia gives rise to a surplus of NADH, which has to be reoxidized by the formation of glycerol. An industrial strain of Saccharomyces cerevisiae was cultivated in batch cultures with different nitrogen sources, i.e., ammonium salt, glutamic acid, and a mixture of amino acids, with 20 g of glucose per liter as the carbon and energy source. The effects of the nitrogen source on metabolite formation, growth, and cell composition were measured. The glycerol yields obtained with glutamic acid (0.17 mol/mol of glucose) or with the mixture of amino acids (0.10 mol/mol) as a nitrogen source were clearly lower than those for ammonium-grown cultures (0.21 mol/mol). In addition, the ethanol yield increased for growth on both glutamic acid (by 9%) and the mixture of amino acids (by 14%). Glutamic acid has a large influence on the formation of products; the production of, for example, alpha-ketoglutaric acid, succinic acid, and acetic acid, increased compared with their production with the other nitrogen sources. Cultures grown on amino acids have a higher specific growth rate (0.52 h-1) than cultures of both ammonium-grown (0.45 h-1) and glutamic acid-grown (0.33 h-1) cells. Although the product yields differed, similar compositions of the cells were attained. The NADH produced in the amino acid, RNA, and extracellular metabolite syntheses was calculated together with the corresponding glycerol formation. The lower-range values of the theoretically calculated yields of glycerol were in good agreement with the experimental yields, which may indicate that the regulation of metabolism succeeds in the most efficient balancing of the redox potential.     

The Composition and Attributes of Colletotrichum truncatum Spores Are Altered by the Nutritional Environment

Previous sporulation studies with Colletotrichum truncatum NRRL 13737, a fungal pathogen of the noxious weed Sesbania exaltata, showed that the carbon-to-nitrogen (CN) ratio of the conidiation medium influenced spore yield, morphology, and efficacy in inciting disease in S. exaltata. Spores produced in a medium with a CN ratio of 10:1 were more effective than were spores produced in a 30:1 or 80:1 ratio in causing disease in S. exaltata. With a basal salts medium supplemented with glucose and Casamino Acids, substrate utilization, spore production, biomass accumulation, and biomass and spore composition were compared in submerged cultures of C. truncatum grown in media with CN ratios of 80:1, 30:1, and 10:1. All cultures were sporulating by day 2, and spore concentrations in 5-day-old cultures were significantly different: 30:1 > 10:1 > 80:1. Amino acid and glucose utilization was balanced in cultures grown in media with a CN ratio of 10:1, whereas cultures grown in media with a CN ratio of 30:1 or 80:1 depleted amino acids prior to glucose. Conidia produced in media with a CN ratio of 10:1 contained significantly more protein (32% of dry weight) and less lipid (17% of dry weight) than conidia produced in media with a CN ratio of either 30:1 (15% protein, 33% lipid) or 80:1 (12% protein, 37% lipid). The higher lipid content of spores produced in media with a CN ratio of 30:1 or 80:1 was associated with the presence of increased numbers of lipid droplets. Optimization studies on conidia produced in media with CN ratios between 30:1 and 10:1 which compared yield, attributes, and efficacy in inciting disease in S. exaltata suggest that media with a CN ratio of 15:1 to 20:1 may be optimal for conidium production.     

Nutritional requirements of some non-pathogenic Neisseria grown in simple synthetic media.

Ten species of non-pathogenic Neisseria were grown in simple defined liquid media containing amino acids, biotin, nicotinic acid, calcium pantothenate, ferrous sulfate, magnesium sulfate, and potassium phosphate. Two of these Neisseria were induced to grow with glutamic acid as the carbon and nitrogen source. The remaining eight Neisseria grew in glutamic acid medium supplemented with from one to four additional amino acids, lactate, or lactate and glucose. A strain of N. flavescens grew in the absence of added growth factors whereas the remaining nine species of Neisseria required either biotin or nicotinic acid; pantothenate was required by two and was stimulatory for three of these species. Use of carbohydrates by the non-pathogenic Neisseria in synthetic medium was tested. Two strains failed to use any of the 14 carbohydrates tested; one strain used only glucose; the remaining seven strains used fructose, glucose, maltose, and sucrose to varying degrees.     

Optimization of Escherichia coli growth by controlled addition of glucose

During aerobic growth of Escherichia coli (recombinant K-12 and strain B) on protein hydrolysate (L-broth) and a carbon source (glucose), acetic acid is produced via glucose metabolism until the late log phase. At this point, the culture pH starts to increase and the growth rate decreases. In cultures without further glucose supplementation, these changes are associated with the accumulation of ammonia, the utilization of acetic acid, the depletion of amino acids, and the complete depletion of glucose. We hypothesize that, after depletion of the glucose, the bacteria catabolize amino acids for energy and carbon and give off the nitrogen as ammonia. Also contributing to the overall increase in pH is the depletion of the acetic acid produced earlier as it is metabolized upon exhaustion of glucose. However, there is a lag time of about 1 hour after the initial pH increase before the sustained accumulation of ammonia begins. This lag indicates that an unidentified factor, in addition to the increase in ammonia, contributes to the increase in pH. Advantage was taken of the turnaround from acid production to base production as reflected in the culture pH to implement the addition of glucose. In growth experiments during which the pH was controlled in the basic direction by glucose addition, the observed decrease in growth rate was significantly postponed and the pH change in the basic direction was reversed as a result of acid production by the cells from the newly added glucose. Furthermore, coll densities of twice that obtained without glucose feeding were demonstrated. Based on the media cost per unit cell density, the data indicate a 31% cost savings.     

Amino acid consumption by the parasitic, amoeboid protists Entamoeba histolytica and E. invadens

Abstract Amino acid consumption by Entamoeba histolytica and E. invadens has been measured in order to assess the possible roles of amino acids as energy substrates. Mixtures of amino acids enhanced the growth of the parasites in complex medium and their survival in simple medium. The consumption of several amino acids by the parasites suspended in simple media was greater when glucose was absent, suggesting that they may act as alternative energy sources. Under these conditions, asparagine was consumed extremely rapidly by E. histolytica in particular, and arginine, leucine and threonine were used greatly by both species. There was also a marked consumption of aspartate, but this occurred even when glucose was present. These five amino acids and phenylalanine were the ones consumed in greatest amounts during growth of E. histolytica in complex medium. Under the same growth conditions, E. invadens also used asparagine, arginine, leucine and threonine and in addition there was a large consumption of serine and especially glutamate. In contrast, the aspartate concentration in the complex medium increased and there was also a net increase in the concentration of some other amino acids. Alanine was produced by both species when the parasites were incubated in simple medium with glucose, and in greater amounts during growth in complex media, suggesting that it is an end product of energy metabolism. The findings provide support for the suggestion that energy generation through amino acid catabolism may be a characteristic feature of anaerobic parasitic protists.     

A kinetic study of the assimilation of 15N-labelled ammonium in rice seedling roots

The pathway of ammonium nitrogen assimilation, its incorporationinto amino acids and synthesis of protein was studied with theaid of nitrogen-15. The analysis of ¹⁵N involves the use ofoptical emission spectrometry. Kinetic analysis of nitrogen assimilation by the roots indicatesthat glutamine and glutamic acid were the primary products ofammonium assimilation. Possibly some of the amino acids, suchas aspartic acid and alanine received their amino nitrogen directlyfrom free ammonia in the roots. Amino groups were transformedinto other amino acids from these primary products, especiallyfrom glutamic acid through transamination. (Received April 1, 1974; )     

Studies on l-Glutamic Acid Fermentation by Microbacterium ammoniaphilum

The present investigation is concerned with the effects of biotin and glucose on glutamic acid fermenation by Microbacterium ammoniaphilum. Both optimal amounts of biotin necessary for maximum growth and maximum accumulation of glutamic acid were determined under the conditoin of various concentration of glucose. As the glucose concentratin was increased, the amounts of biotin required for maximum growth also increased proportionally to the glucose concentrations. The optimal amounts of biotin for maximum accumulation of glutamic acid were smaller than those for maximum growth of cells at any glucose concentration. It is suggested that the process of glutamic acid accumulation is inevitably associated with the process of cell multiplication by both experiments of successive culture of cells grown under the dose of biotin sufficient and deficient for maximum growth.     

Organic and inorganic nitrogen source effects on the metabolism of Clostridium acetobytulicum

Summary The effects of organic and inorganic nitrogen combinations on cell growth, solvent production and nitrogen utilization by Clostridium acetobutylicum ATCC 824 was studied in batch fermentations. Fermentations in media with 10 mM glutamic acid, as the organic nitrogen source, and 0 mM to 10 mM ammonium chloride, as the inorganic nitrogen source had a solvent yield of 0.8 to 1.08 mmol solvent/mmol glucose used, with a slow fermentation rate (2 mmol solvent/l h^-1). When media contained 20 mM or 30 mM glutamic acid as well as 2.5 to 7.5 mM ammonium chloride the fermentation rate increased (5.5 mmol/l h^-1) while the solvent yield remained constant (0.86 to 0.96 mmol solvent/mmol glucose used). Total solvent production was higher in media containing 20 mM or 30 mM glutamic acid than with 10 mM glutamic acid.     

End products of glucose and glutamine metabolism by L929 cells

Products of glucose and glutamine metabolism by L929 cells were detected and quantitated by gas chromatography and mass spectrometry of the oxime-trimethylsilyl derivatives. This method allowed detection and identification of all major carboxylic and amino acids produced in the system. Although lactic acid was expected to be the major product, alanine, citric, glutamic, aspartic, and pyruvic acids were also released into the culture medium at significant rates. Incorporation of labeled carbon from D-[U-13C]glucose showed that the alanine, lactic, and pyruvic acids were derived from glucose as was one-third of the citric acid carbon. The rate of glucose utilization for production of these end products was 29-fold greater than the rate of glucose oxidation to CO2, and calculated ATP production from alanine and pyruvate synthesis exceeded that from lactate synthesis by nearly 2-fold. Utilization of glutamine for synthesis of aspartic, glutamic, and citric acids also exceeded the rate of glutamine oxidation, thereby making end-product synthesis from glucose and glutamine the dominant cellular metabolic activity. In the absence of glucose, synthesis and intracellular levels of aspartic and glutamic acids increased, whereas synthesis and cell content of the other acids decreased markedly. This response is consistent with the metabolic pattern proposed by Moreadith and Lehninger (Moreadith, R.W., and Lehninger, A.L. (1984) J. Biol. Chem. 259, 6215-6221) in which much of the glutamine used by these cells is converted to aspartate in the absence of a pyruvate source and to aspartate or citrate in the presence of pyruvate.     

Nutritional Requirements of Staphylococcus aureus S-6

A synthetic medium was devised for growth of Staphylococcus aureus strain S-6. The growth yield in synthetic medium was compared to that in complex medium containing an equivalent amount of protein hydrolysate. Enterotoxin B formation in the two media was also compared. The defined medium was composed of inorganic salts, 11 amino acids (glycine, valine, leucine, threonine, phenylalanine, tyrosine, cysteine, methionine, proline, arginine, and histidine), and three vitamins (thiamine, nicotinic acid, and biotin). Biotin was a growth factor requirement of S-6 when glutamic acid but not glucose was used as a carbon source. The quantity of enterotoxin B produced in the defined medium was about one-seventh of that produced in complex medium, even though the growth yields were similar.     

Amino acid production in isolated rat liver mitochondria

Amino acid analyses of mitochondrial membranes are compared with the amino acid composition of whole mitochondria (Alberti, 1964) and found to be very similar except in the cystine content. The composition of the endogenous amino acids found in freshly prepared mitochondria has been established and shown to differ considerably from the amino acid composition of membranes or whole mitochondria. The amino acids produced during anaerobic incubation of mitochondria at pH7.4, on the other hand, resemble the membrane in composition, supporting the view that neutral proteinase activity is responsible for their appearance. Aerobic incubation produces a similar pattern of amino acids except that amino acids such as proline, serine, asparagine, glutamic acid and glutamine, which can be metabolically utilized under aerobic conditions, are present to a smaller extent. The presence of large relative concentrations of endogenous taurine, cysteic acid and oxidized glutathione and the accumulation of taurine during incubation is found. The selective retention of taurine and cysteic acid within the mitochondria is established. It is proposed that the first step in the degeneration of isolated mitochondria results from lipid hydroperoxide accumulation caused by the lack of glutathione reductase in isolated mitochondria.     

The role of amino acid catabolism in the formation of the tricarboxylic acid cycle intermediates and ammonia in anoxic rat heart

Amino acid catabolism, the tricarboxylic acid cycle intermediates and ammonia formation were studied in isolated perfused rat heart under anoxia. The total net anaplerosis due to amino acid degradation in anoxia was equal to that in oxygenation (6.29 and 6.09 mumol/g dry weight per h, respectively) as a result of the increased transamination of glutamic and aspartic acids. During anoxic perfusion, the rate of catabolism of glutamic and aspartic acids was 1.5-times higher than in normoxia, while depletion of branched-chain amino acids, lysine, proline, arginine and methionine, was inhibited. Alanine was the product of excessive degradation of glutamic and aspartic acids. Under anaerobic conditions, in spite of inhibition of amino acid deamination, ammonia formation was increased 2.7-fold as compared to oxygenation. The principal amount of ammonia (96%) was produced at degradation of adenine nucleotides. A 2.5-fold increase in the pool of the tricarboxylic acid cycle intermediates under anoxia was associated mainly with accumulation of succinate. The data suggest that the coupling of alanine- and aspartate amino transferases is a mechanism controlling the tricarboxylic acid cycle pool size in anoxic heart.     

Glutamic acid production byArthrobacter globiformis

Two hundred and fiftyArthrobacter strains were tested in a basal salts-glucose medium for their ability to produce glutamic acid; 50 strains produced small amounts of glutamic acid and alanine, as well as traces of other amino acids. Five biotin-dependent strains produced extraordinarily large amounts of glutamic acid. One of these, which was identified asA. globiformis, was selected for further study. Glutamic acid was only produced by this organism at biotin levels suboptimal for growth; maximal production (0.45 moles of glutamic acid per mole of glucose consumed) occurred at a biotin level of 10^–5 µg/ml. Other factors which markedly influenced glutamic acid production were temperature, (NH₄)₂SO₄ concentration, and pH of the growth medium.The taxonomy of glutamic acid-producing bacteria and the correlation between biotin deficiency and glutamic acid production are discussed.     

Treponema succinifaciens sp. nov., an anaerobic spirochete from the swine intestine

The morphology, the general physiological characteristics, and the energy-yielding metabolism of an obligately anaerobic spirochete isolated from the colon of a swine were studied. Electron microscopy showed that the helical spirochetal cells possessed an outer sheath, a protoplasmic cylinder, and 4 periplasmic fibrils in a 2-4-2 arrangement. The spirochete grew in an atmosphere of N₂ in prereduced media containing a carbohydrate, NaHCO₃, rumen fluid, yeast extract, peptone, l-cysteine, and inorganic salts. The spirochete fermented carbohydrates and required substrate amounts of CO₂ (HCO ₃ ^- ) for growth. Amino acids were not fermented. Major fermentation products of cells growing with glucose as the substrate and in the presence of CO₂ were acetate, formate, succinate, and lactate. Small amounts of 2,3-butanediol, pyruvate, and acetoin were also formed. Determinations of enzymatic activities in cell extracts, and of radioactivity in products formed by growing cells from [1-¹⁴C]glucose, indicated that this sugar was dissimilated to pyruvate via the Embden-Meyerhof pathway. The spirochetes used a coliform-type clastic reaction to metabolize pyruvate. Determinations of radioactivity in products formed from [¹⁴C]NaHCO₃ indicated that CO₂ was assimilated and used in succinate production. The guanine+cytosine content of the DNA was 36 mol%. This study indicates that this intestinal spirochete represents a new species of Treponema. It is proposed that the new species be named Treponema succinifaciens.Abbreviations cpm counts per minute - DTT dithiothreitol - EM Embden-Meyerhof - GC guanine plus cytosine - IgG immunoglobulin G - PC protoplasmic cylinder - PF periplasmic fibrils (axial fibrils) - OS outer sheath     

Function of Growth Factors for Rumen Microorganisms I. Nutritional Characteristics of Selenomonas ruminantium

Nutritional characteristics of Selenomonas ruminantium var. lactilytica isolated from a sheep rumen were studied. The organism required for growth the addition of a clarified rumen fluid to a Trypticase-yeast extract medium with either lactate or glucose as an energy source. The requirement for rumen fluid was found to be satisfied by volatile fatty acids in glucose media and by biotin in lactate media. Straight-chain saturated fatty acids with C(3) to C(10) carbon skeleton had been found to be effective. Among them, n-valerate was most effective at the lowest concentration. An abnormal morphology was observed with n-valerate-deficient glucose media. n-Valerate was essential in glucose media, and it was stimulatory in lactate media. Fermentation products from glucose were lactate, propionate, and acetate, and fermentation products from lactate were propionate and acetate. When cells were grown in a glucose medium containing n-valerate-C(14), the label was present in cell fractions. Almost all of the activity was found in lipid materials.