Growth of moderately thermophilic iron-oxidizing bacterium strain TI-1 in synthetic medium

The moderately thermophilic iron-oxidizing bacterium strain TI-1, which lacks enzyme systems involved in CO₂ fixation, grows at 45°C in Fe²⁺ medium supplemented with yeast extract to give a maximum cell growth of 1.0 × 10⁸ cells per ml, but does not grow in Fe²⁺ medium without yeast extract. To elucidate the physiology of the strain, a synthetic medium was developed. It was found that the best synthetic medium was Fe²⁺-6AA, containing Fe²⁺, salts, and the following six l-amino acids: alanine, aspartic acid, glutamic acid, arginine, serine, and histidine. In this medium, strain TI-1 showed a maximum cell growth of 10 × 10⁸ cells/ml. The six amino acids in the Fe²⁺-6AA medium were used not only as a carbon source but also as a source of nitrogen. Inorganic nitrogen sources, such as ammonium ion, hydrazine, hydroxylamine, nitrite, and nitrate, were not used as a sole source of nitrogen, but rather strongly inhibited the utilization of the six amino acids at 1 mM. In the Fe²⁺ (10 mM)-6AA medium supplemented with 21 mM Fe³⁺, reduction of Fe³⁺ to Fe²⁺ that was dependent on the added amino acids was observed, suggesting another role of the amino acids in the growth of strain TI-1. Washed, intact cells of strain TI-1 had the activity to reduce Fe³⁺ to Fe²⁺.     

Effects of Nutrient Supply on Seed Growth of Field Bean (Vicia faba L.). 1. Effects of Different Sulphur and Methionine Regimes on Cotyledon Growth, Protein and Uncombined Amino Acids In vitro

The effects of differemt S and methionine regimes on growthof developing Vicia faba cotyledons in vitro were studied. Basalmedium (containing adequate S) supplemented with 05 mM methioninemarginally increased d. wt and uncombined amino acid accumulationbut adding 1–5 mM methionine inhibited both growth andprotein accumulation. Sulphur deficiency reduced both d. wtand protein accumulation but incresed accumulation of uncombinedamino acids. Adding 1 mM methionine to the S-deficient mediumrestored growth, normal protein and uncombined amino acid acnunulation.High sulphate medium (7.5 mM ) decreased d. wt, protein anduncombined amino acid accumulation. High sulphate medium or basal medium+methionine (05 mM) changedthe proportions of the seed proteins; legumin increased butvicilin decreased. Sulphur deficiency caused a relative increasein vicilin but a decrease in legumin. The different S and methionineregimes markedly changed the composition of the uncombined aminoacids, especially those derived from aspartic acid but not thecomposition of the protein fraction, except during S deficiency. The data presented indicates a flexibility in the storage proteincomposition of developing cotyledons grown in vitro, with theS and methionine status having a regulatory effect. Vicia faba L., field bean, cotyledon, growth, in vitro culture, uncombined amino acids, protein composition, legumin, vicilin, methionine, sulphur     

Metabolic activation and hepatotoxicity. Effect of cysteine, N-acetylcysteine, and methionine on glutathione biosynthesis and bromobenzene toxicity in isolated rat hepatocytes.

Freshly isolated rat hepatocytes contained a high level (30–40 nmol/10⁶ cells) of reduced glutathione (GSH) which decreased steadily upon incubation in an amino acid containing medium lacking cysteine and methionine. This decrease in GSH level was prevented, and turned into a slight increase, when either cysteine, N-acetylcysteine, or methionine was also present in the medium. The amino acid uptake into hepatocytes was more rapid with cysteine than with methionine. Cystine was not taken up, or taken up very slowly, by the cells and could not be used to prevent the decrease in GSH level which occurred in the absence of cysteine and methionine. The level of GSH in hepatocytes freshly isolated from rats pretreated with diethylmaleate was markedly decreased (to ~5 nmol/10⁶ cells) but increased rapidly upon incubation of the cells in a medium containing amino acids including either cysteine, N-acetylcysteine, or methionine. Again, cysteine was taken up into the cells more rapidly than methionine. The rate of uptake of cysteine was moderately enhanced in hepatocytes with a lowered level of intracellular GSH as compared to cells with normal GSH concentration. Exclusion of glutamate and/or glycine from the medium did not markedly affect the rate of resynthesis of GSH by hepatocytes incubated in the presence of exogenously added cysteine or methionine. Incubation of hepatocytes with bromobenzene in an amino acid-containing medium lacking cysteine and methionine resulted in accelerated cell damage. Addition of either cysteine, N-acetylcysteine, or methionine to the medium caused a decrease in bromobenzene toxicity. The protective effect was dependent, however, on the time of addition of the amino acid to the incubate; e.g., the effect on bromobenzene toxicity was greatly reduced when either cysteine or methionine was added after 1 h of preincubation of the hepatocytes with bromobenzene as compared to addition at zero time. This decrease in protective effect in bromobenzene-exposed cells was related to a similar decrease in the rate of uptake of cysteine and methionine into hepatocytes preincubated with bromobenzene. The rate of uptake, and incorporation into cellular protein, of leucine was also markedly inhibited in hepatocytes preincubated with bromobenzene. In contrast, there was no measurable change in the rate of release of leucine from cellular protein as a result of incubation of hepatocytes with bromobenzene. It is concluded that the presence of cysteine, N-acetylcysteine, or methionine in the medium protects hepatocytes from bromobenzene toxicity by providing intracellular cysteine for GSH biosynthesis and suggested that an inhibitory effect on amino acid uptake may contribute to the cytotoxicity of bromobenzene in hepatocytes.     

Loss of organic acids, amino acids, k, and cl from barley roots treated anaerobically and with metabolic inhibitors

Excised roots of barley (Hordeum vulgare, var. Campana) lost organic acids, amino acids, K⁺, and Cl⁻ within 15 minutes after initiation of anaerobic treatment or treatment with NaCN and 2,4-dinitrophenol. Initial loss of organic acids when roots were placed under N₂ is attributed to a decarboxylation reaction, possibly catalyzed by phosphoenolpyruvate carboxykinase. Organic and amino acids began to leak from the roots to the bathing medium after 1 to 2 hours under N₂, indicating injury to cell membranes. During the first hour of anaerobic treatment, K⁺ loss from low-salt roots was equivalent to organic acid loss. Potassium loss from roots containing high levels of KCl was approximately equal to organic acid plus amino acid loss; and Cl⁻ loss was approximately equal to amino acid loss. It is postulated that, within cells, organic acids may electrostatically bind an equivalent quantity of cations and that amino acids may bind an equivalent quantity of both cations and anions.     

Submerged culture conidial germination and conidiation of the bioherbicideColletotrichum truncatum are influenced by the amino acid composition of the medium

Summary Submerged culture experiments were conducted to determine the optimal nitrogen source for rapidly producing conidia of the bioherbicide,Colletotrichum truncatum. Germination ofC. truncatum conidial inocula in submerged culture occurred most rapidly (>95% in 6 h) in media provided with a complete complement of amino acids. When (NH₄)₂SO₄, urea, or individual amino acids were provided as the sole nitrogen source, conidial germination was less than 20% after 6 h incubation. Conidia production was delayed inC. truncatum cultures grown in media with urea or individual amino acids as nitrogen sources compared to cultures supplied with Casamino acids or complete synthetic amino acid nitrogen sources. The use of methionine, lysine, tryptophan, isoleucine, leucine or cysteine as a sole nitrogen source severely inhibitedC. truncatum conidia production. Media with synthetic amino acid mixtures less these inhibitory amino acids produced significantly higher conidia yields compared to media with amino acid mixtures containing these amino acids. When various amounts of each individual inhibitory amino acid were added to media which contained amino acid mixtures, cysteine and methionine were shown to be most effective in reducing conidiation. An optimal nitrogen source forC. truncatum conidiation in submerged culture should contain a complete mixture of amino acids with low levels of cysteine, methionine, leucine, isoleucine, lysine and tryptophan for rapid conidiation and optimal conidia yield.The mention of firm names or trade products does not imply that they are endorsed or recommended by the US Department of Agriculture over other firms or similar products not mentioned.     

Development of a Synthetic Minimal Medium for Listeria monocytogenes

A defined solid and liquid minimal medium, HTM, which contained methionine and cysteine as the sole amino acids, was developed for Listeria monocytogenes. Complex broth-grown L. monocytogenes had to adapt to HTM by inducing amino acid biosyntheis. HTM is the simplest minimal medium available for growth of L. monocytogenes.     

Preparation of 15N-labeled l-alanine by immobilized l-alanine dehydrogenase: Differential incorporation of 15N in bacterial proteins

This paper describes a system for continuous synthesis of ¹⁵N-labeled l-alanine from lactic acid, ¹⁵NH₄Cl and NADH, which uses immobilized alanine dehydrogenase and soluble lactate dehydrogenase as enzyme sources. Lactic acid acts both as hydrogen donor for the regeneration of NADH and as pyruvate source, thus providing the carbon skeleton of l-alanine. Citrobacter freundi grown on synthetic media containing 17 unlabeled amino acids and l-(¹⁵N)alanine as nitrogen source, incorporated 66% of ¹⁵N into alanine found in bacterial proteins. When ¹⁵N-labeled glutamic acid, aspartic acid or glycocol were added to the synthetic growth media, their ¹⁵N was “diluted” among different amino acids of bacterial proteins. Isotope enrichment of l-(¹⁵N)lysine found in newly synthesized proteins of C. freundi was practically unchanged as compared to the isotope content of free amino acid in the growth medium.     

Amino Acid Metabolism of Lemna minor L. : I. Responses to Methionine Sulfoximine

When Lemna minor L. is supplied with the potent inhibitor of glutamine synthetase, methionine sulfoximine, rapid changes in free amino acid levels occur. Glutamine, glutamate, asparagine, aspartate, alanine, and serine levels decline concomitantly with ammonia accumulation. However, not all free amino acid pools deplete in response to this inhibitor. Several free amino acids including proline, valine, leucine, isoleucine, threonine, lysine, phenylalanine, tyrosine, histidine, and methionine exhibit severalfold accumulations within 24 hours of methionine sulfoximine treatment. To investigate whether these latter amino acid accumulations result from de novo synthesis via a methionine sulfoximine insensitive pathway of ammonia assimilation (e.g. glutamate dehydrogenase) or from protein turnover, fronds of Lemna minor were prelabeled with [¹⁵N]H₄⁺ prior to supplying the inhibitor. Analyses of the ¹⁵N abundance of free amino acids suggest that protein turnover is the major source of these methionine sulfoximine induced amino acid accumulations. Thus, the pools of valine, leucine, isoleucine, proline, and threonine accumulated in response to the inhibitor in the presence of [¹⁵N]H₄⁺, are ¹⁴N enriched and are not apparently derived from ¹⁵N-labeled precursors. To account for the selective accumulation of amino acids, such as valine, leucine, isoleucine, proline, and threonine, it is necessary to envisage that these free amino acids are relatively poorly catabolized in vivo. The amino acids which deplete in response to methionine sulfoximine (i.e. glutamate, glutamine, alanine, aspartate, asparagine, and serine) are all presumably rapidly catabolized to ammonia, either in the photorespiratory pathway or by alternative routes.     

Inhibition of yeast Δ1-pyrroline-5-carboxylate dehydrogenase by common amino acids and the regulation of proline catabolism

A yeast glutamate auxotroph (glt_{1 ? 1}), blocked in the tricarboxylic acid cycle at aconitase, is shown to possess catabolic pathways to glutamate from proline, arginine and glutamine, and grows on any of these amino acids in a minimal medium. This mutant does not, however, grow on these amino acids in a medium containing the full complement of common amino acids minus glutamate. The mechanism of this growth failure involves partial inhibition of the catabolic routes to glutamate by more than half the common amino acids. In the case of proline catabolism, this inhibition is localized principally at the enzyme Δ¹-pyrroline-5-carboxylate: NAD(P)⁺ oxidoreductase by in vitro studies. Similar results with this enzyme prepared both from yeast and from beef kidney mitochondria suggest that the inhibition observed may be the basis of a regulatory mechanism of general significance.     

The Nutrition of Parauronema acutum*

SYNOPSIS. A symbiote-free strain of Parauronema acutum, 110–3, a small marine hymenostome ciliate has been cultured in a synthetic medium consisting of amino acids, purine derivatives, vitamins, lipids and artificial sea water. Populations of ～ 1.3 × 10⁶ per ml were obtained in 5 to 6 days at 27 C in the dark in medium prepared in sea water, density = 1.015 g/cc at a surface to volume ratio of 5 cm²/ml. The pH optimum was 7.2. The following amino acids were determined to be essential for the growth of this strain: arginine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine (or glycine), threonine, tryptophan and valine: guanine, guanosine or guanylic acid, but not adenine, adenosine, adenylic acid, hypoxanthine, inosine, inosinic acid, xanthine, xanthosine, or xanthylic acid, satisfied the need for a purine for growth of this organism. Pyrimidines were not required for growth. Of the vitamins tested, folic acid, nicotinamide, d-pantothenic acid, pyridoxal HCl, riboflavin, thiamine HCl and thioctic acid were essential for growth; biotin was not. Growth in the absence of lipids was transplantable, but amounted to ～ 3% that obtained in medium containing a mixture of asolectin, animal cephalin and Tween 80. Asolectin alone at high concentrations was almost as effective as the lipid mixture in supporting growth. Purified phospholipids such as phosphatidyl serine, phosphatidyl choline and phosphatidyl inositol were less effective on an individual basis. In minimal medium containing only the “essential” amino acids, growth was less than 5% that obtained in the complete medium, but could be restored to maximal by the addition of either glutamic acid or aspartic acid. A number of substances, including sugars, amino acids and Krebs cycle intermediates, partially restored growth under these conditions. Only glycogen, starch and glucose-1-phosphate, tested individually, were as effective as glutamic acid or aspartic acid in restoring growth to optimal levels.     

Influence of amino acids,mammalian serum,and osmotic pressure on the proliferation of Drosophila cell lines

In the D22 medium of ECHALIER and OHANESSIAN for the culture of Drosophila cell lines lactalbumin hydrolysate could be replaced by a synthetic amino acids mixture. In spite of the presence of yeast extract and fetal calf serum the omission of any one of arginine, asparagine, cysteine, histidine, methionine, proline, serine, or threonine prevented cell proliferation. Of these eight amino acids cysteine had to be added in concentrations higher than 0.1 mM. Without much effect on cell proliferation foetal calf serum could be reduced from 10% to 2% or be replaced by 1% horse serum or 1% porcine serum. Cells could grow in media of osmolarities from 225 mOsm up to 400 mOsm depending on the osmotic agent used. Chloride concentrations up to 80 mM were compatible with proliferation as was a wide range of sodium/potassium ratios.     

Insights into Actinobacillus succinogenes Fermentative Metabolism in a Chemically Defined Growth Medium

Chemically defined media allow for a variety of metabolic studies that are not possible with undefined media. A defined medium, AM3, was created to expand the experimental opportunities for investigating the fermentative metabolism of succinate-producing Actinobacillus succinogenes. AM3 is a phosphate-buffered medium containing vitamins, minerals, NH₄Cl as the main nitrogen source, and glutamate, cysteine, and methionine as required amino acids. A. succinogenes growth trends and end product distributions in AM3 and rich medium fermentations were compared. The effects of NaHCO₃ concentration in AM3 on end product distribution, growth rate, and metabolic rates were also examined. The A. succinogenes growth rate was 1.3 to 1.4 times higher at an NaHCO₃ concentration of 25 mM than at any other NaHCO₃ concentration, likely because both energy-producing metabolic branches (i.e., the succinate-producing branch and the formate-, acetate-, and ethanol-producing branch) were functioning at relatively high rates in the presence of 25 mM bicarbonate. To improve the accuracy of the A. succinogenes metabolic map, the reasons for A. succinogenes glutamate auxotrophy were examined by enzyme assays and by testing the ability of glutamate precursors to support growth. Enzyme activities were detected for glutamate synthesis that required glutamine or α-ketoglutarate. The inability to synthesize α-ketoglutarate from glucose indicates that at least two tricarboxylic acid cycle-associated enzyme activities are absent in A. succinogenes.     

Nutritional Features of Bacteroides fragilis subsp. fragilis

Studies of three reference strains of Bacteroides fragilis subsp. fragilis showed that they grow well in a minimal defined medium containing glucose, hemin, vitamin B₁₂, minerals, bicarbonate-carbon dioxide buffer, NH₄Cl, and sulfide. The vitamin B₁₂ requirement of 0.1 ng/ml was replaced with 7.5 μg of methionine. Cysteine or sulfide was an excellent source of sulfur, thioglycolate was a poor source, and thiosulfate, methionine, β-mercaptoethanol, dithiothreitol, sulfate, or sulfite did not serve as sole sources of sulfur. Neither single amino acids, nitrate, urea, nor a complex mixture of L-amino acids or peptides effectively replaced ammonia as the nitrogen source. Comparative studies with a few strains of other subspecies of B. fragilis including B. fragilis subsp. vulgatus, B. fragilis subsp. thetaiotaomicron, and B. fragilis subsp. distasonis indicate that they exhibit similar growth responses in the minimal medium. A single strain of B. fragilis subsp. ovatus required other materials. The results indicate the great biosynthetic ability of these organisms and suggest that, in their ecological niche within the large intestine, many nutrients such as amino acids are in very low supply, whereas materials such as ammonia, heme, and vitamin B₁₂, or related compounds, must be available during much of the time.     

De Novo Synthesis of Amino Acids by the Ruminal Bacteria Prevotella bryantii B14, Selenomonas ruminantium HD4, and Streptococcus bovis ES1

The influence of peptides and amino acids on ammonia assimilation and de novo synthesis of amino acids by three predominant noncellulolytic species of ruminal bacteria, Prevotella bryantii B₁4, Selenomonas ruminantium HD4, and Streptococcus bovis ES1, was determined by growing these bacteria in media containing ¹⁵NH₄Cl and various additions of pancreatic hydrolysates of casein (peptides) or amino acids. The proportion of cell N and amino acids formed de novo decreased as the concentration of peptides increased. At high concentrations of peptides (10 and 30 g/liter), the incorporation of ammonia accounted for less than 0.16 of bacterial amino acid N and less than 0.30 of total N. At 1 g/liter, which is more similar to peptide concentrations found in the rumen, 0.68, 0.87, and 0.46 of bacterial amino acid N and 0.83, 0.89, and 0.64 of total N were derived from ammonia by P. bryantii, S. ruminantium, and S. bovis, respectively. Concentration-dependent responses were also obtained with amino acids. No individual amino acid was exhausted in any incubation medium. For cultures of P. bryantii, peptides were incorporated and stimulated growth more effectively than amino acids, while cultures of the other species showed no preference for peptides or amino acids. Apparent growth yields increased by between 8 and 57%, depending on the species, when 1 g of peptides or amino acids per liter was added to the medium. Proline synthesis was greatly decreased when peptides or amino acids were added to the medium, while glutamate and aspartate were enriched to a greater extent than other amino acids under all conditions. Thus, the proportion of bacterial protein formed de novo in noncellulolytic ruminal bacteria varies according to species and the form and identity of the amino acid and in a concentration-dependent manner.     

A novel link between the biosynthesis of aromatic amino acids and transfer RNA modification in Escherichia coli

A transposon-induced mutation in Escherichia coli resulted in a lack of two modified nucleosides in the transfer ribonucleic acid. These nucleosides were identified as uridine-5-oxyacetic acid (cmo⁵U)² and its methylester, mcmo⁵U. Both became radioactively labelled using [methyl-¹⁴C]methionine as methyl donor when wild-type cells were grown in a defined rich medium. We believe that both nucleosides have hydroxyuridine as a common precursor, which should be methylated in the first modification step. However, in our system in vitro the tRNA from the mutant was not a methyl group acceptor, indicating that the step affected in the mutant occurs before the methylation step. Thus, the most likely biosynthetic pathway is: formation of (1) hydroxyuridine, (2) methoxyuridine. (3) cmo⁵U and, in some cases, (4) mcmo⁵U. The mutant had also become Aro^?, i.e. it required aromatic amino acids for growth. Genetic analysis revealed that the transposon Tn5 had been integrated close to or within the aroD gene, the gene product of which participates in the synthesis of shikimic acid. The common pathway of the biosynthesis of aromatic amino acids includes the genes aroB, D, E, A and C in that order, and any mutant defective in any of these genes lacked cmo⁵U and mcmo⁵U in their tRNA. When shikimic acid was included in the defined rich medium used, the Tn5-induced mutant regained the normal level of cmo⁵U and mcmo⁵U while an aroC mutant (distal to shikimic acid but prior to chorismic acid) did not. The rich medium used contained, besides the aromatic amino acids, all the precursors for the synthesis of folate, ubiquinone and enterochelin. Thus, chorismic acid itself or a metabolite of it in the synthetic pathway to vitamin K₂ or in an unknown pathway must play a pivotal role in this specific modification of the tRNA. These results reveal a novel link between the biosynthesis of amino acids and modification of tRNA.     

Growth and metabolism of the obligate photolithotroph Chlorobium thiosulfatophilum in the presence of added organic nutrients

Growth of Chlorobium vibrioforme f. thiosulfatophilum NCIB 8327 could be monitored by measurement of turbidity (E₆₀₀); absorbance at 745 and 665 nm; increase in methanol-extractable pigment (E₆₆₀); fixation of ¹⁴CO₂; and titration of thiosulphate and sulphide in the medium. Growth could be inhibited by formate, methionine, tryptophan, tyrosine, threonine, serine and glycine, but not by 14 other amino acids, shikimic acid, some alcohols, sugars or acetate. Inhibition could some-times be relieved by the presence of other amino acids. This was probably partly due to restoration of normal internal amino acid requirements by “feeding”, and partly because uptake of amino acids appeared to show some competition for two or more low specificity uptake systems. Numerous ¹⁴C-labelled amino acids, formate and glucose were shown to be photoassimilated by Chlorobium, and the labelling patterns obtained provided information on its pathways of intermediary biosynthesis. Growth inhibition by threonine could be related to the probable presence of a normal branched pathway for the synthesis of the aspartate family of amino acids, with an aspartokinase enzyme subject to strong inhibition by threonine and lysine, separately and in combination.     

Utilization of Ammonia Nitrogen by Intestinal Bacteria Isolated from Pigs

In a medium containing ammonia, proteose peptone, and cysteine as nitrogen sources, 17 of 24 Bacteroidaceae strains, 3 of Selenomonas strains, 1 of 7 curved rods, 3 of 7 Spirochaetaceae strains, 8 of 20 Eubacterium strains, 8 of 13 Peptococcaceae strains, 3 of 4 Clostridium strains, 19 of 20 Enterobacteriaceae strains, and 1 of 8 Streptococcus strains utilized ammonia nitrogen preferentially to proteose peptone nitrogen. To determine the ability of intestinal microbes to synthesize amino acids from ammonia, ammonia utilization by Bacteroides ruminicola strain 9 was studied in defined media containing ammonia and other nitrogen sources. In another medium containing ammonia, proteose peptone, and cysteine as nitrogen sources, ammonia was preferentially utilized even when the proteose peptone nitrogen content was eight times greater than that of ammonia nitrogen. In a medium containing ammonia, an amino acid, and cysteine, the lowest uptake of ammonia nitrogen was observed when the medium contained aspartic acid, glutamic acid, threonine, or alanine; but ammonia was utilized more effectively than any of the amino acids. Incorporation of ¹⁵N from [¹⁵N]ammonia into bacterial amino acids was studied. ¹⁵N was incorporated into every amino acid of B. ruminicola strain 9, and the highest uptake was observed in aspartic acid and alanine.     

Feedback-Resistant Acetohydroxy Acid Synthase Increases Valine Production in Corynebacterium glutamicum

Acetohydroxy acid synthase (AHAS), which catalyzes the key reactions in the biosynthesis pathways of branched-chain amino acids (valine, isoleucine, and leucine), is regulated by the end products of these pathways. The whole Corynebacterium glutamicum ilvBNC operon, coding for acetohydroxy acid synthase (ilvBN) and aceto hydroxy acid isomeroreductase (ilvC), was cloned in the newly constructed Escherichia coli-C. glutamicum shuttle vector pECKA (5.4 kb, Km^r). By using site-directed mutagenesis, one to three amino acid alterations (mutations M8, M11, and M13) were introduced into the small (regulatory) AHAS subunit encoded by ilvN. The activity of AHAS and its inhibition by valine, isoleucine, and leucine were measured in strains carrying the ilvBNC operon with mutations on the plasmid or the ilvNM13 mutation within the chromosome. The enzyme containing the M13 mutation was feedback resistant to all three amino acids. Different combinations of branched-chain amino acids did not inhibit wild-type AHAS to a greater extent than was measured in the presence of 5 mM valine alone (about 57%). We infer from these results that there is a single binding (allosteric) site for all three amino acids in the enzyme molecule. The strains carrying the ilvNM13 mutation in the chromosome produced more valine than their wild-type counterparts. The plasmid-free C. glutamicum ΔilvA ΔpanB ilvNM13 strain formed 90 mM valine within 48 h of cultivation in minimal medium. The same strain harboring the plasmid pECKAilvBNC produced as much as 130 mM valine under the same conditions.     

14CO2 dark fixation in the halophytic species mesembryanthemum crystallinum

The time course of ¹⁴CO₂ dark fixation was studied in leaves of the facultatively halophytic plant species Mesembryanthemum crystallinum cultivated with and without 400 mM NaCl in the nutrient medium. It is generally known from the literature that plants grown under saline conditions incorporate ¹⁴C predominately into amino acids. By contrast in leaves of M. crystallinum grown on NaCl and exposed to ¹⁴CO₂ in the dark, relatively more radioactivity is incorporated in the organic acids (especially malate) than in amino acids. The data obtained are discussed in relation to the NaCl induced Crassulacean acid metabolism in M. crystallinum reported earlier.     

Selection and characterization of ethionine-resistant alfalfa (Medicago sativa L.) cell lines

Summary Diploid alfalfa (HG2), capable of plant regeneration from tissue culture, was used to select variant cell lines resistant to growth inhibition due to ethionine (an analog of methionine). Approximately 10⁷ suspension-cultured cells were mutagenized with methane sulfonic acid ethylester and then plated in solid media containing ethionine. Callus colonies formed on media with 0.02 mM ethionine. Of the 124 cell lines recovered, 91 regenerated plants. After six months growth on media without ethionine, 15 of 110 cell lines of callus grew significantly better than HG2 on 1 mM ethionine. Several ethionine-resistant callus cultures were also resistant to growth inhibition due to the addition of lysine + threonine to the media. High concentrations, relative to unselected HG2 callus, of methionine, cysteine, cystathionine, and glutathione were found in some, but not all, ethionine-resistant callus cultures. Cell line R32, which had a ca. tenfold increase in soluble methionine, had a 43% increase in total free amino acids and a 40% increase in amino acids in protein as compared to unselected HG2 callus. Relative amounts of each amino acid in protein were the same in both.Abbreviation LT lysine + threonine in equimolar concentration