Chemically defined media for the cultivation of Naegleria: pathogenic and high temperature tolerant species

Chemically defined minimal media for the cultivation of high temperature tolerant and pathogenic Naegleria spp. have been developed. A defined minimal medium, identical for N. fowleri and N. lovaniensis, consists of eleven amino acids (arginine, glycine, histidine, isoleucine, leucine, methionine, phenylalanine, proline, threonine, tryptophan, and valine), six vitamins (biotin, folic acid, hemin, pyridoxal, riboflavin, and thiamine), guanosine, glucose, salts, and metals. Three of the four strains of Naegleria fowleri tested (ATCC 30100, ATCC 30863, and ATCC 30896) and two strains of N. lovaniensis (ATCC 30467 and ATCC 30569) could be cultured beyond ten subcultures on this medium. For N. fowleri ATCC 30894 diaminopimelic acid, or lysine, or glutamic acid was also required. Mean generation time was reduced and population density increased for all strains with the introduction of glutamic acid. Glucose could be eliminated from the minimal medium only if glutamic acid was present. Without glucose, mean generation time increased and population density decreased. Diaminopimelic acid could substitute for lysin for ATCC 30894, indicating that Naegleria species may synthesize their lysine via the DAP pathway. Naegleria fowleri ATCC 30100 could be adapted to grow without serine or glycine in the minimal medium with glutamic acid added, but with mean generation time increased and population density decreased. The strain could be grown in the minimal medium in the absence of metals. For growth of N. australiensis ATCC 30958, modification of the medium by increasing metals ten-fold, substituting guanine for guanosine and adding lysine, glutamic acid, and six vitamins (p-aminobenzoic acid, choline chloride, inositol, vitamin B12, nicotinamide, and Ca pantothenate) was required.     

Study of amino acid utilization by isogenic strains of Escherichia coli in relation to the culture growth and biosynthesis of penicillin amidase

Dynamics of free amino acid utilization by isogenic strains of Escherichia coli differing in intensity of their growth and levels of penicillin acylase biosynthesis in media containing corn steep liquor or peptone was studied. It was shown that in both the media some amino acids such as serine, threonine, glutaminic and asparaginic acids were actively utilized by the strains mainly during the culture intensive growth while others such as glycine, alanine and tyrosine were actively utilized during the enzyme biosynthesis. Intensively utilized arginine and proline were probably used for the growth and biosynthesis. The other amino acids were not utilized completely from the media. The lowest levels of their utilization were observed when the strains were cultivated in the medium with peptone.     

Serine utilization by Klebsiella aerogenes.

Klebsiella aerogenes was found to contain a specific L-serine dehydrase that was induced by threonine, glycine or leucine, but not by its substrate. Cellular concentrations were sensitive to carbon rather than nitrogen sources in the growth medium. A nonspecific isoleucine-sensitive L-threonine dehydrase supplemented the specific L-serine dehydrase activity. K. aerogenes also contains a leucine-inducible L-threonine dehydrogenase which probably initiated a threonine-utilization pathway in which the serine-specific dehydrate participated. Strains that were altered in their ability to metabolize serine differed in either L-serine dehydrase or L-threonine dehydrase activity. Thus, K. aerogenes growing on L-serine as a sole nitrogen source relies upon two enzymes that metabolize the amino acid as subsidiary functions.     

Utilization of l-threonine by a species of Arthrobacter. A novel catabolic role for `aminoacetone synthase''

1. A species of Arthrobacter (designated Arthrobacter 9759) was isolated from soil by its ability to grow aerobically on l-threonine as sole source of carbon atoms, nitrogen atoms and energy; the organism also grew well on other sources of carbon atoms including glycine, but no growth was obtainable on aminoacetone or dl-1-aminopropan-2-ol. 2. During growth on threonine, (14)C from l-[U-(14)C]threonine was rapidly incorporated into glycine and citrate, and thereafter into serine, alanine, aspartate and glutamate. 3. With extracts of threonine-grown cells supplied with l-[U-(14)C]threonine, evidence was obtained of the NAD and CoA-dependent catabolism of l-threonine to produce acetyl-CoA plus glycine. Short-term incorporation studies in which [2-(14)C]acetate and [2-(14)C]glycine were supplied (a) to cultures growing on threonine, and (b) to extracts of threonine-grown cells, showed that the acetyl-CoA was metabolized via the tricarboxylic acid cycle and glyoxylate cycle whereas the glycine was converted into pyruvate via the folate-dependent ;serine pathway'. 4. The threonine-grown organism contained ;biosynthetic' threonine dehydratase and a potent NAD-linked l-threonine dehydrogenase but possessed no l-threonine aldolase activity. 5. Evidence was obtained that the acetyl-CoA and glycine produced from l-threonine had their immediate origin in the alpha-amino-beta-oxobutyrate formed by the threonine dehydrogenase; the CoA-dependent cleavage of this compound was catalysed by an alpha-amino-beta-oxobutyrate CoA-ligase, which was identified with ;aminoacetone synthase'. A continuous spectrophotometric assay of this enzyme was developed, and it was found to be inducibly synthesized only during growth on threonine and not during growth on acetate plus glycine. 6. By using a reconstituted mixture of separately purified l-threonine dehydrogenase and alpha-amino-beta-oxobutyrate CoA-ligase (i.e. ;aminoacetone synthase'), l-[U-(14)C]threonine was broken down to [(14)C]glycine plus [(14)C]acetyl-CoA (trapped as [(14)C]citrate). 7. There was no evidence of aminoacetone metabolism by Arthrobacter 9759 even though a small amount of this amino ketone appeared in the culture medium during growth on threonine.     

Chemically Defined Media for the Cultivation of Naegleria: Pathogenic and High Temperature Tolerant Species

Chemically defined minimal media for the cultivation of high temperature tolerant and pathogenic Naegleria spp. have been developed. A defined minimal medium, identical for N. fowleri and N. lovaniensis, consists of eleven amino acids (arginine, glycine, histidine, isoleucine, leucine, methionine, phenylalanine, proline, threonine, tryptophan, and valine), six vitamins (biotin, folic acid, hemin, pyridoxal, riboflavin, and thiamine), guanosine, glucose, salts, and metals. Three of the four strains of Naegleria fowleri tested (ATCCr?30100, ATCCr?30863, and ATCCr?30896) and two strains of N. lovaniensis (ATCCr?30467 and ATCCr?30569) could be cultured beyond ten subcultures on this medium. For N. fowleri ATCCr?30894 diaminopimelic acid, or lysine, or glutamic acid was also required. Mean generation time was reduced and population density increased for all strains with the introduction of glutamic acid. Glucose could be eliminated from the minimal medium only if glutamic acid was present. Without glucose, mean generation time increased and population density decreased. Diaminopimelic acid could substitute for lysine for ATCCr?30894, indicating that Naegleria species may synthesize their lysine via the DAP pathway. Naegleria fowleri ATCCr?30100 could be adapted to grow without serine or glycine in the minimal medium with glutamic acid added, but with mean generation time increased and population density decreased. The strain could be grown in the minimal medium in the absence of metals. For growth of N. australiensis ATCCr?30958, modification of the medium by increasing metals ten-fold, substituting guanine for guanosine and adding lysine, glutamic acid, and six vitamins (p-aminobenzoic acid, choline chloride, inositol, vitamin B₁₂, nicotinamide, and Ca pantothenate) was required.     

Characterization of interaction of active-site serine mutants of tissue-type plasminogen activator with plasminogen activator inhibitor-1

To define determinants of interactions of tissue-type plasminogen activator (t-PA) with plasminogen activator inhibitor type-1 (PAI-1), we utilized site-directed mutagenesis to substitute either threonine or glycine for the active-site serine of tissue-type plasminogen activator. Assays of conditioned media of transfected cells demonstrated that the threonine substitution markedly decreased but did not entirely abolish plasminogen activating activity. In contrast, the glycine substitution yielded a mutant with absolutely no detectable plasminogen activating activity. Wild-type t-PA formed stable complexes with PAI-1. However, even when exogenous inhibitor was present in the medium or purified mutant was added to plasma that had been rendered PAI-1-rich in vivo, the mutants were present in the free form exclusively judging from results of fibrin autography and Western blot analysis. Thus, despite maintenance of some residual plasminogen-activating activity associated with preservation of the hydroxyl group at the active site, the threonine mutant did not form stable complexes with inhibitor. The glycine mutant, developed so that steric hindrance or other unfavorable interactions at the modified active site would be minimal, was similarly incapable of forming complexes with PAI-1. These results show that the presence of an active site serine residue is necessary for formation of stable complexes between t-PA and PAI-1.     

Hydroxy amino acid metabolism in Pseudomonas cepacia: role of L-serine deaminase in dissimilation of serine, glycine, and threonine.

Growth of Pseudomonas cepacia (P. multivorans) on serine depended upon induction of a previously undescribed L-serine deaminase distinct from threonine deaminase. Formation of the enzyme was induced during growth on serine, glycine, or threonine. The induction pattern reflected a role of the enzyme in catabolism of these three amino acids. Both threonine and glycine supported growth of serine auxotrophs and were presumably converted to serine and pyruvate in the course of their degradation. Mutant strains deficient in serine deaminase, or unable to use pyruvate as a carbon source, failed to utilize serine or glycine and grew poorly with threonine, whereas strains deficient in threonine dehydrogenase or alpha-amino beta-ketobutyrate:coenzyme A ligase (which together convert threonine to glycine and acetyl coenzyme A) failed to utilize threonine or derepress serine deaminase in the presence of this amino acid. The results confirm for the first time the role of alpha-amin beta-ketobutyrate:coenzyme A ligase in threonine degradation and indicate that threonine does not mimic serine as an inducer of serine deaminase.     

The growth and nutrition of Clostridium sporogenes NCIB 8053 in defined media

Various defined and minimal media are described for the growth of Clostridium sporogenes NCIB 8053. The organism requires 10 amino acids and one vitamin for growth, whilst three other vitamins are growth stimulatory. L-alpha-hydroxy acid analogues can replace eight, and fatty acid analogues four, of these amino acids. The organism may generate free energy by a variety of Stickland reactions. Most Stickland acceptors, but not glycine, stimulate the growth of this organism on glucose. Nonetheless, cells grown in the presence of glycine will reductively deaminate it. The media described support the growth of several other strains of this species. The simplified growth media which we have developed permit quantitative studies of the physiology of this organism.     

The growth and nutrition of Clostridium sporogenes NCIB 8053 in defined media

Various defined and minimal media are described for the growth of Clostridium sporogenes NCIB 8053. The organism requires 10 amino acids and one vitamin for growth, whilst three other vitamins are growth stimulatory. L-α-hydroxy acid analogues can replace eight, and fatty acid analogues four, of these amino acids. The organism may generate free energy by a variety of Stickland reactions. Most Stickland acceptors, but not glycine, stimulate the growth of this organism on glucose. Nonetheless, cells grown in the presence of glycine will reductively deaminate it. The media described support the growth of several other strains of this species. The simplified growth media which we have developed permit quantitative studies of the physiology of this organism.     

The Amino Acid Requirements of Acanthamoeba sp. Neff

SYNOPSIS. Acanthamoeba sp. Neff can synthesize 4 of the 10 essential amino acids, namely, histidine, lysine, threonine and tryptophan. However, in this minimal medium, the non-essential amino acid glycine was also required for growth. Serine or threonine could replace glycine, but the growth rate was reduced.     

Regulatory role for l-arginine in the utilization of amino acids by pig small-intestinal bacteria

We recently reported that bacteria from the pig small intestine rapidly utilize and metabolize amino acids (AA). This study investigated the effect of L-arginine on the utilization of AA by pure bacterial strains (Streptococcus sp., Escherichia coli and Klebsiella sp.) and mixed bacterial cultures derived from the pig small intestine. Bacteria were incubated at 37°C for 3 h in anaerobic AA media containing 0-5 mmol/L of arginine to determine the effect of arginine on the bacterial utilization of AA. Amino acids in the medium plus cell extracts were analyzed by high-performance liquid chromatography. Results indicated concentration-dependent increases in the bacterial utilization of arginine and altered fluxes of arginine into ornithine and citrulline in the bacteria. Net glutamine utilization increased in pure bacterial strains with increased concentrations of arginine. With the addition of arginine, net utilization of threonine, glycine, phenylalanine and branched-chain AA increased (P<0.05) in Streptococcus sp. and Klebsiella sp., but decreased in E. coli. Net utilization of lysine, threonine, isoleucine, leucine, glycine and alanine by jejunal or ileal mixed bacteria decreased (P<0.05) with the addition of arginine. Complete utilization of asparagine, aspartate and serine were observed in pig small-intestinal bacteria after 3 h of incubation. Overall, the addition of arginine affected the metabolism of the arginine-family of AA and the serine- and aspartate-family of AA in small-intestinal bacteria and reduced the utilization of most AA in ileal mixed bacteria. These novel findings indicate that arginine exerts its beneficial effects on swine nutrition partially by regulating AA utilization and metabolism in the small-intestinal microbiota.     

Biosynthesis of amino acids in Clostridium pasteurianum

1. Clostridium pasteurianum was grown on a synthetic medium with the following carbon sources: (a) (14)C-labelled glucose, alone or with unlabelled aspartate or glutamate, or (b) unlabelled glucose plus (14)C-labelled aspartate, glutamate, threonine, serine or glycine. The incorporation of (14)C into the amino acids of the cell protein was examined. 2. In both series of experiments carbon from exogenous glutamate was incorporated into proline and arginine; carbon from aspartate was incorporated into glutamate, proline, arginine, lysine, methionine, threonine, isoleucine, glycine and serine. Incorporations from the other exogenous amino acids indicated the metabolic sequence: aspartate --> threonine --> glycine right harpoon over left harpoon serine. 3. The following activities were demonstrated in cell-free extracts of the organism: (a) the formation of aspartate by carboxylation of phosphoenolpyruvate or pyruvate, followed by transamination; (b) the individual reactions of the tricarboxylic acid route to 2-oxoglutarate from oxaloacetate; glutamate dehydrogenase was not detected; (c) the conversion of aspartate into threonine via homoserine; (d) the conversion of threonine into glycine by a constitutive threonine aldolase; (e) serine transaminase, phosphoserine transaminase, glycerate dehydrogenase and phosphoglycerate dehydrogenase. This last activity was abnormally high. 4. The combined evidence indicates that in C. pasteurianum the biosynthetic role of aspartate and glutamate is generally similar to that in aerobic and facultatively aerobic organisms, but that glycine is synthesized from glucose via aspartate and threonine.     

Threonine formation via the coupled activity of 2-amino-3-ketobutyrate coenzyme A lyase and threonine dehydrogenase.

The enzymes L-threonine dehydrogenase and 2-amino-3-ketobutyrate coenzyme A (CoA) lyase are known to catalyze the net conversion of L-threonine plus NAD+ plus CoA to NADH plus glycine plus acetyl-CoA. When homogeneous preparations of these two enzymes from Escherichia coli were incubated together for 40 min at 25 degrees C with glycine, acetyl-CoA, and NADH, a 36% decrease in the level of glycine (with concomitant NADH oxidation) was matched by formation of an equivalent amount of threonine, indicating that this coupled sequence of enzyme-catalyzed reactions is reversible in vitro. Several experimental factors that affect the efficiency of this conversion in vitro were examined. A constructed strain of E. coli, MD901 (glyA thrB/C tdh), was unable to grow unless both glycine and threonine were added to defined rich medium. Introduction of the plasmid pDR121 (tdh+kbl+) into this strain enabled the cells to grow in the presence of either added glycine or threonine, indicating that interconversion of these two amino acids occurred. Threonine that was isolated from the total pool of cellular protein of MD901/pDR121 had the same specific radioactivity as the [14C]glycine added to the medium, establishing that threonine was formed exclusively from glycine in this strain. Comparative growth rate studies with several strains of E. coli containing plasmid pDR121, together with the finding that kcat values of pure E. coli 2-amino-3-ketobutyrate CoA lyase favor the cleavage of 2-amino-3-ketobutyrate over its formation by a factor of 50, indicate that the biosynthesis of threonine is less efficient than glycine formation via the coupled threonine dehydrogenase-2-amino-3-ketobutyrate lyase reactions.     

Growth, pectate lyase production and solute accumulation by Erwinia chrysanthemi under osmotic stress: effect of osmoprotectants

Glycine betaine strongly stimulated the growth rate of five strains of Erwinia chrysanthemi when grown in a synthetic medium at 0·986, 0·983 and 0·980 a _w (NaCl) whereas in four strains, little effect was observed compared with the control. Proline, dimethyl glycine, carnitine and pipecolic acid also actedas osmoprotectants. Glutamate and trehalose, commonly accumulated by enteric bacteria in response to osmotic stress, failed to act as osmoprotectants when supplied exogenously. Glycine betaine and pipecolic acid partially overcame the inhibition of pectate lyase release by NaCl in strain ECC. ¹³C NMR spectroscopy of two osmotically-stressed strains showed that glycine betaine was accumulated intracellularly from synthetic media containing the exogenous osmoprotectant. However, both strains also synthesized and accumulated trehalose in addition to glycine betaine in response to osmotic stress in complex media containing glycine betaine.     

Glycine fermentation by Clostridium histolyticum

Clostridium histolyticum grew on glycine, arginine, or threonine as sole substrate. Arginine degradation preceded that of glycine and partially inhibited that of threonine when two amino acids were present. Each amino acid seemed to be individually catabolized, not by a Stickland type of reaction. Glycine fermentation required the presence of complex ingredients. Therefore, an effect of selenite on glycine catabolism could only be demonstrated after scavenging selenium contamination by preculturing Peptostreptococcus glycinophilus in that medium. C. acidiurici was not suited as selenium accumulating organism as C. histolyticum was inhibited by the residual uric acid. Arginine catabolism was unaffected by seleniuum depriviation. The labelling pattern obtained in acetate after incubation of C. histolyticum with [1-¹⁴C]- or [2-¹⁴C]glycine strongly indicated the metabolism of glycine via the glycine reductase pathway.     

Effects of gibberellic acid and L-methionine on C1 metabolism in aerated carrot disk

Aeration of carrot storage tissue disks in water was accompanied by net folate synthesis and by changes in the specific activities of key folate-dependent enzymes. Disks aerated in 0.1 mM gibberellic acid (GA₃) for 48 hr contained higher concentrations of methyltetrahydrofolates but aeration in 5 mM L-methionine reduced net folate synthesis. Gibberellic acid also increased the specific activities of 5,10-methylenetetrahydrofolate reductase (E.C. 1.1.1.68), serine hydroxymethyltransferase (E.C. 2.1.2.1) and 5-methyltetrahydrofolate: homocysteine transmethylase. The levels of these enzymes in disks aerated in L-methionine (5 mM) were comparable or slightly higher than those of disks aerated in water. Activity of the reductase and 10-formyltetrahydrofolate synthetase (E.C. 6.3.4.3) was inhibited by L-methionine in vitro. Aeration increased ability to incorporate formate [¹⁴C] into serine, glycine and methionine. Disks aerated for 36 hr in 0.1 mM GA₃ incorporated greater amounts of ¹⁴C into free methionine but those aerated in L-methionine (5 mM) had less ability to metabolize formate and the specific radioactivities of free glycine, serine and methionine were low.     

Effects of amino acid additions on ammonium stressed CHO cells

Ammonium is a toxic and inhibitory byproduct of mammalian cell metabolism. At the end of a typical recombinant protein production campaign, the ammonium concentration can be as high as 10 mM, mainly due to glutamine metabolism. Intracellular pH (pH(i)) levels are sensitive to ammonium, which negatively impacts both cell growth and recombinant protein productivity. Ammonium also negatively affects the recombinant protein glycosylation profile, thus altering quality. Many strategies have been adopted to reduce ammonium accumulation, with limited results. This study investigated the addition of amino acids to the growth media for Chinese hamster ovary (CHO) cell cultures as a means of mitigating the negative effects of ammonium. Threonine, proline, and glycine additions improved CHO cell growth and recombinant protein levels. Further, the threonine, proline, and glycine additions positively impacted important metabolic parameters, including glucose consumption, lactate production, glutamine utilization, and final ammonium levels. Additionally, threonine, proline, and glycine increased the level of alpha(2,3)-linked sialic acid, galactose-beta(1,4)-N-acetylglucosamine, and alpha(2,6)-linked sialic acid residues on the recombinant tissue plasminogen activator (t-PA). Thus, threonine, proline, and glycine can be used to mitigate some of the toxic effects of ammonium on cell growth, recombinant protein productivity, and protein quality.     

Methionine catabolism and production of volatile sulphur compounds by OEnococcus oeni

AIMS: During malolactic fermentation (MLF), the secondary metabolisms of lactic acid bacteria (LAB) contribute to the organoleptic modification of wine. To understand the contribution of MLF, we evaluated the capacity of various wine LAB to metabolize methionine. METHODS AND RESULTS: Using gas chromatography (GC) coupled either with mass spectrometry (MS) or a flame photometry detector in sulphur mode (FPD), we studied this metabolism in laboratory media and wine. In laboratory media, several LAB isolated from wine were able to metabolize methionine. They formed methanethiol, dimethyl disulphide, 3-(methylsulphanyl)propan-1-ol and 3-(methylsulphanyl)propionic acid. These are known to have powerful characteristic odours and play a role in the aromatic complexity of wine. In various red wines, after MLF only the 3-(methylsulphanyl)propionic acid concentration increased significantly, as verified with several commercial starter cultures. This compound, which is characterized by chocolate and roasted odours, could contribute to the aromatic complexity produced by MLF. CONCLUSIONS: This study shows that LAB isolated from wine, especially OEnococcus oeni strains, the major species in MLF, are able to metabolize methionine to form volatile sulphur compounds. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first study to demonstrate the capacity of wine LAB to metabolize methionine.     

One-carbon metabolism in Neurospora crassa wild-type and in mutants partially deficient in serine hydroxymethyltransferase.

1. The concentrations of folate-dependent enzymes in Neurospora crassa Lindegren A wild type (FGSC no. 853), Ser-l mutant, strain H605a (FGSC no. 118), and for mutant, strain C-24 (FGSC no. 9), were compared during exponential growth on defined minimal media. Both mutants were partially lacking in serine hydroxymethyltransferase, but contained higher concentrations of 10-formyltetrahydrofolate synthetase than did the wild type. Mycelia of the mutants contained higher concentrations of these enzymes when growth media were supplemented with 1mM-glycine. In the wild-type, this glycine supplement also increased the specific activities of 5,10-methylenetetrahydrofolate dehydrogenase and 5,10-methylenetetrahydrofolate reductase. 5. During growth, total folate and polyglutamyl folate concentrations were greatest in the wild-type. Methylfolates were not detected in mutant Ser-l, and were only present in the for mutant after growth in glycine-supplemented media. Exogenous glycine increased folate concentration threefold in the wild type, mainly owing to increases in unsubstituted polyglutamyl derivatives. 3. Feeding experiments using 14C-labelled substrates showed that C1 units were generated from formate, glycine and serine in the wild type. Greater incorporation of 14C occurred when mycelia were cultured in glycine-supplemented media. Formate and serine were precursors of C1 units in the mutants, but the ability to cleave glycine was slight or lacking.     

Membrane potential of rat hepatoma cells in culture: Influence of factors affecting amino acid transport

The effect has been studied of various media, hormones and of amino acids on the membrane potential of rat hepatoma cells in culture measured by microelectrode impalement. Cells in Eagle's minimal essential medium plus 5% serum had a value which varied daily from about 5–8 mV, inside negative. The membrane potential of rat hepatocytes was measured to be 8.7 ± 0.2mV, inside negative. The membrane potential of the hepatoma cells was decreased by insulin and increased by glucagon. Membrane potential was unaffected by change of medium to Hanks' or Earle's balanced salt solutions or deprivation of serum. It was, however, reduced in cells in phosphate-buffered saline and by reduction of pH. The former effect was shown to be due to the higher [Na⁺] of phosphat-buffered saline as opposed to the other media. Addition of alanine, glycine, serine, proline and methylaminoisobutyrate all reduced membrane potential by 2–3 mV. Smaller decreases were seen with methionine, leucine and phenylalanine, but none with glutamine, threonine, BCH (2-aminonorborane-2-carboxylic acid) and D-alanine. The results are compared with the effects of similar conditions on aminoisobutyrate uptake. Whilst there was a correlation under some conditions there was not under others. It is concluded that for the hepatoma cells factors additional to the membrane potential must exert some influence on the capacity for amino acid transport.