The Role of Low-Molecular-Weight Nitrogen Compounds in the Osmotolerance of Rhodococcus erythropolis and Arthrobacter globiformis

Investigations showed that Rhodococcus erythropolis E-15 and Arthrobacter globiformis 2F cells respond to osmotic shock by increasing the synthesis of free amino acids, primarily glutamic acid (80% of the intracellular free amino acid pool). The osmoprotective role of glutamic acid follows from its beneficial effect on the growth of bacteria in high-salinity media. It was found that the addition of this amino acid to the growth medium at a concentration of 2 mM shortened the lag phase and increased the growth rate and biomass yield of either of the two bacteria. The addition of another osmoprotectant, trehalose, to the high-salinity growth medium of R. erythropolis E-15 at the same concentration (2 mM), restored the growth parameters of this bacterium to the control values.     

Growth conditions of and emetic toxin production by Bacillus cereus in a defined medium with amino acids

The growth and emetic toxin (cereulide) production of Bacillus cereus strains in defined culture media were studied. We found that a fully synthetic medium (CADM) allowed the production of emetic toxin and the addition of glucose enhanced it. By subtracting each amino acid from CADM, we found that only three amino acids, valine, leucine and threonine, were essential for growth and toxin production by B. cereus. The addition of high levels (50 mM) of leucine, isoleucine and glutamic acid decreased the toxin production. Other amino acids had no effect at this concentration.     

Analysis of glutathione-enhanced differentiation by microfilariae of Onchocerca lienalis (Filarioidea: Onchocercidae) in vitro

Reduced glutathione (GSH), but not its oxidized form (GSSG), stimulated development of Onchocerca lienalis microfilariae to the late first-larval stage in vitro. The degree and frequency of development was dose-related with a peak of activity at 15 mM, a concentration that is similar to known intracellular levels of GSH. To determine the mode(s) of action of this multifunctional compound, other reducing agents (L-cysteine, dithiothreitol), cysteine delivery agents (N-acetyl-L-cysteine, L-thiazolidine-4-carboxylic acid, L-2-oxothiazolidine-4-carboxylic acid), cysteine analogues (S-methyl-L-cysteine, D-glucose-L-cysteine, cysteine ethyl ester), free-component amino acids of GSH (glutamic acid, cysteine, and glycine), a specific metabolic inhibitor of gamma-glutamyl synthetase (buthionine sulfoximine), and an inhibitor of gamma-glutamyl transpeptidase (gamma-glutamyl glutamic acid) were also tested at concentrations of 0.01-50 mM in this system. N-acetyl-L-cysteine at 1-5 mM and D-glucose-L-cysteine at 2.5-10 mM significantly enhanced development. In contrast to those worms maintained in GSH-supplemented medium, microfilariae exposed to GSH for only the first 24 hr showed no enhancement by day 7 in culture. Neither buthionine sulfoximine nor gamma-glutamyl glutamic acid at 0.01-35 mM inhibited the effects of 15 mM GSH or 1 mM N-acetyl-L-cysteine. Results indicate that GSH or other cysteine analogues possessing a free sulfhydryl group must be present in the extranematodal environment to support microfilarial differentiation in vitro.     

Regulation of internal solute concentrations of marine Vibrio alginolyticus in response to external NaCl concentration.

Slightly halophilic marine Vibrio alginolyticus grown in the range of NaCl from 0.2 to 1.5 M maintained the total internal solute concentration always higher than the external medium by about 0.25 osM. The concentrations of macromolecules such as DNA, RNA, and protein were little affected by the increase in medium NaCl. The internal K+ concentration was kept to about 400 mM in the range of medium NaCl from 0.4 to 0.8 M; it rose to 510 mM when the bacterium was grown in 1.5 M NaCl, indicating that K+ increased only slightly in response to the large increase in medium NaCl. Thus, in contrast to the case of nonhalophilic and extremely halophilic bacteria, K+ was unlikely to act as a major component to regulate the internal solute concentration of marine V. alginolyticus. The internal Na+ and Cl- concentrations were maintained always lower than those in the growth medium, but they increased in response to the increase in medium NaCl. The concentration of internal Na+ was close to that of K+ at the concentration of medium NaCl that supports the optimal growth of this organism. The total amino acid content of V. alginolyticus increased from 76 to 413 mM by the increase in medium NaCl from 0.2 to 1.5 M. The concentrations of glutamic acid and prolined were 254 and 72 mM, respectively, when grown in 1.5 M NaCl. These results indicated that Na+, Cl- and amino acids, especially glutamic acid and proline, contributed to the regulation of internal solute concentration of V. alginolyticus in response to the increased external NaCl.     

Utilization of amino acids to enhance glutathione production in Saccharomyces cerevisiae

The effects of amino acids on glutathione (GSH) production by Saccharomyces cerevisiae T65 were investigated in this paper. Cysteine was the most important amino acids, which increased intracellular GSH content greatly but inhibited cell growth at the same time. The suitable amino acids addition strategy was two-step addition: in the first step, cysteine was added after two hours culture to 2 mM and then, the three amino acids (glutamic acid, glycine, and serine) were added after seven hours culture. The optimum concentration of those three key amino acids (10 mM glutamic acid, 10 mM glycine, and 10 mM serine) was obtained by orthogonal matrix method. With the optimum amino acids addition strategy a 1.63% intracellular GSH content was obtained in shake flask culture. Intracellular GSH content was 55.2% higher than the experiments without three amino acids addition. The cell biomass and GSH yield were 9.4 g/L and 153.2 mg/L, respectively. Using this amino acids addition strategy in the fed-batch culture of S. cerevisiae T65, GSH content, the biomass, and GSH yield reached 1.41%, 133 g/L, and 1875 mg/L, respectively, after 44 hours fermentation. GSH yield was about 2.67 times as that of amino acids free.     

Development of Defined, Minimal, and Complete Media for the Growth of Hyphomicrobium neptunium

A complete synthetic medium containing 15 amino acids, a minimal synthetic medium (GAMS) containing 4 amino acids, and a supplemented minimal medium (GAMS + calcium pantothenate) have been developed for the cultivation of Hyphomicrobium neptunium ATCC 15444. Depending on the complexity of the synthetic media, generation times were approximately 2 to 3 times longer, and maximum cell densities were 0.3 to 0.9 log₁₀ lower than in ZoBell marine broth 2216. The fates of ¹⁴C-labeled amino acids in GAMS were monitored. Results suggested that H. neptunium was auxotrophic for methionine, utilized glutamic acid as a primary energy source, and readily anabolized and catabolized serine and aspartic acid. Individual amino acid concentrations above 125 mM induced prolonged lag periods, whereas only methionine was not growth limiting at a concentration as low as 2 mM.     

Synthesis of imidazol-2-yl amino acids by using cells from alkane-oxidizing bacteria

Sixty-one strains of alkane-oxidizing bacteria were tested for their ability to oxidize N-(2-hexylamino-4-phenylimidazol-1-yl)-acetamide to imidazol-2-yl amino acids applicable for pharmaceutical purposes. After growth with n-alkane, 15 strains formed different imidazol-2-yl amino acids identified by chemical structure analysis (mass and nuclear magnetic resonance spectrometry). High yields of imidazol-2-yl amino acids were produced by the strains Gordonia rubropertincta SBUG 105, Gordonia terrae SBUG 253, Nocardia asteroides SBUG 175, Rhodococcus erythropolis SBUG 251, and Rhodococcus erythropolis SBUG 254. Biotransformation occurred via oxidation of the alkyl side chain and produced 1-acetylamino-4-phenylimidazol-2-yl-6-aminohexanoic acid and the butanoic acid derivative. In addition, the acetylamino group of these products and of the substrate was transformed to an amino group. The product pattern as well as the transformation pathway of N-(2-hexylamino-4-phenylimidazol-1-yl)-acetamide differed in the various strains used.     

Nitrogen Starvation-Induced Changes in Amino Acid and Free Ammonium Pools in <Emphasis Type="Italic">Schizophyllum commune</Emphasis> Colonies

Wood-decay fungi depend upon recycling of nitrogen-containing molecules to maintain growth in nitrogen-deficient environments. One of the pools that can support growth in these organisms is the pool of free amino acids. The free amino acid (AA) composition of Schizophyllum commune mycelium grown on the surface of nitrogen-rich (M = 6.6 mM L-asparagine) and nitrogen-poor medium (M01 = 0.06 mM L-asparagine) has been examined: When mycelium is grown on M, alanine, glutamate, and asparagine account for almost 2/3 of the amino acid pool. The free amino acid concentration is reduced by 75% for mycelium grown on the M01 medium, with alanine and glutamate predominating. In addition, free NH₄⁺ increases by 60% in nitrogen-deprived mycelia. Except for asparagine, which is absorbed by the apices, the concentration of all free amino acids is higher in the centers of M-grown, 4-day-old mycelia than in the apices. Hyphae grown to exponential growth on M and transferred to M01 for 12 h show greater free amino acid and NH₄⁺ concentrations in the apices, most likely indicating increased translocation to the apices.     

Analysis of free amino acids during fermentation by Bacillus subtilis using capillary electrophoresis

A high performance capillary electrophoresis (HPCE) method was presented to identify and quantitate free amino acids during fermentation by Bacillus subtilis. Amino acids, pre-column derivatized with phenylisothicyanate, were separated and characterized by HPCE. In order to optimize separation conditions, the assay was developed by varying the β-cyclodextrin concentration and pH of the background electrolyte. A buffer system comprising 30 mM phosphate and 3 mM β-cyclodextrin at pH 7.0, voltage of 20 kV and detection wavelength of 254 nm showed the best results, with 17 out of 20 phenylthioncarbamyl amino acids in a solution adequately separated. For quantification, p-aminobenzoic acid was added as an internal standard. Analysis of free amino acids in Bacillus subtilis culture medium using this method revealed good consistency with the values obtained using conventional ninhydrin-based amino acid analyzer. Four free amino acids (aspartic acid, glutamic acid, proline, and tyrosine) concentration in an extracellular matrix during fermentation by Bacillus subtilis were mainly monitored using this method.     

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.     

The effect of citric acid on growth of proteolytic strains of Clostridium botulinum

In strictly anaerobic conditions in a culture medium adjusted to pH 5.2 with HCl and incubated at 30 degrees C, inocula containing less than 10 vegetative bacteria of Clostridium botulinum ZK3 (type A) multiplied to give greater than 10(8) bacteria per ml in 3 d. Growth from an inoculum of between 10 and 100 spores occurred after a delay of 10-20 weeks. Citric acid concentrations of 10-50 mmol/l at pH 5.2 inhibited growth from both vegetative bacteria and spore inocula, a concentration of 50 mmol/l increasing the number of vegetative bacteria or of spores required to produce growth by a factor of approximately 10(6). The citric acid also reduced the concentration of free Ca2+ in the medium. The inhibitory effect of citric acid on vegetative bacteria at pH 5.2 could be prevented by the addition of Ca2+ or Mg2+ and greatly reduced by Fe2+ and Mn2+. The addition of Ca2+, but not of the remaining divalent metal ions, restored the concentration of free Ca2+ in the medium to that in the citrate-free medium. The inhibitory effect of citric acid on growth from a spore inoculum was only partially prevented by Ca2+. Citric acid (50 mmol/l) did not inhibit growth of strain ZK3 at pH 6 despite the greater chelating activity of citrate at pH 6 than at pH 5.2. The effect of citric acid and Ca2+ at pH 5.2 on vegetative bacteria of strains VL1 (type A) and 2346 and B6 (proteolytic type B) was similar to that on strain ZK3.     

The Uptake and Utilization of Bacteria, Amino Acids and Nucleic Acid Components by the Rumen Ciliate Eudiplodinium maggii

Washed suspensions of the rumen ciliate protozoon Eudiplodinium maggii grown in vitro and incubated anaerobically engulfed all the bacteria tested except for Bacteroides ruminicola and Klebsiella aerogenes. There was considerable variation (160–9100 bacteria/h/protozoon at an external concentration of 10¹⁰ bacteria/ml) in the rate at which the bacteria were engulfed, but Eu. maggii showed some preference for bacteria of rumen origin. Some of the bacteria were digested with the release of soluble materials into the medium. Free amino acids were incorporated from an 0.1 mM solution at rates of 0.13 to 0.84 pmol/h/protozoon. Evidence is presented that Eu. maggii could obtain half the amino acids required for growth by the engulfment and digestion of bacteria and half by the uptake of free amino acids. Eudiplodinium maggii incorporated uridine 5' monophosphate and also hydrolysed this to uridine and then to uracil which was reduced to dihydrouracil. These products all appeared in the medium. Ribose was incorporated by the protozoon and appeared as glucose in protozoal and bacterial polysaccharide; none was incorporated as such into protozoal nucleic acid.     

Response of a wild type and a non-nitrogen-fixing mutant of Anabaena doliolum towards different amino acids

The effects of various amino acids on growth and heterocyst differentiation have been studied on wild type and a heterocystous, non-nitrogen-fixing (het+ nif-) mutant of Anabaena doliolum. Glutamine, arginine and asparagine showed maximum stimulation of growth. Serine, proline and alanine elicited slight stimulation of growth of wild type but failed to show any stimulatory effect on mutant strain. Valine, glutamic acid, iso-leucine and leucine at a concentration of as low as 0.1 mM were inhibitory to growth of parent type. Methionine, aspartic acid, threonine, cysteine, and tryptophan did not affect growth at concentrations lower than 0.5 mM. But at 1 mM, these amino acids were inhibitory. In addition to the stimulatory effects of glutamine, arginine and asparagine, the heterocyst frequency was also repressed by these amino acids. Glutamine and arginine at 2 mM completely repressed heterocyst differentiation in the mutant strain; however, other amino acids failed to repress the differentiation of heterocysts. Our results suggest that glutamine and arginine are utilized as nitrogen sources. This is strongly supported from the data of growth and heterocyst differentiation of mutant strain, where at least with glutamine there is good growth without heterocyst formation. Studies with glutamine and arginine on other N2-fixing blue-green algae may reveal the regulation of the heterocyst-nitrogenase sub-system.     

Nutrient modifications for improved growth of Brassica nigra cell suspension cultures

Cell pellet yield of two Brassica nigra suspension cultures was stimulated by amino acid supplements in the growth medium. This could confound the interpretation of amino acid feeding studies involved in characterizing amino acid metabolism mutants. The nutritional requirements of one of the Brassica nigra suspension cultures growing in modified Murashige & Skoog medium were therefore reviewed. Sucrose at 2% w/v was growth limiting and amino or organic acid supplements stimulated growth rate and yield. Increasing sucrose to 6% and supplementing with 15 mM sodium succinate increased maximum cell pellet volume by 2.7 times and maximum dry weight by 2.8 times, stimulated cell enlargement and produced similar maximum numbers of cells per culture. The further addition of an amino acid supplement of 4 mM alanine, 4 mM glutamine and 1 mM glutamate produced no further improvement. The revised medium was more strongly buffered, supported cell growth for a longer period and permitted a 30-fold reduction in the minimum cell inoculum. Cells grown in the revised medium are 10-fold more resistant to growth inhibition by the tryptophan analogue 5MT. These advantages recommend the revised medium for amino acid feeding, mutant isolation and similar studies.     

Glutamic acid potentiates hepatocyte response to mitogens in primary culture

Adult rat hepatocytes in primary culture responded to epidermal growth factor (EGF) by increased DNA synthesis. When hepatocytes were cultured in Leibovitz L-15 medium, their response to EGF was low compared with that in Williams' medium E or Koga's medium L. Furthermore, female rat hepatocytes showed almost no response to the mitogenic action of EGF compared with male rat hepatocytes in L-15 medium. Addition of glutamic acid (1–20 μM) to EGF-containing L-15 medium not only enhanced DNA synthesis > tenfold in both male and female hepatocytes, but eliminated the sex differences in DNA synthesis. Aspartic acid, glutamine, or ornithine at 20 mM did not replace the glutamic acid effect on DNA synthesis. Proline also enhanced EGF-induced DNA synthesis, although it was less effective than glutamic acid. Therefore, this effect may be specific to a high concentrations of glutamic acid. Glutamic acid by itself did not stimulate DNA synthesis at any concentrations tested. In the presence of glutamic acid, EGF showed a dose-dependent (0.5–20 ng/ml) stimulation of DNA synthesis with a maximal effect at 10 ng/ml. Almost the same effect was obtained with transforming growth factor alpha (0.5–20 ng/ml). Glutamic acid also induced an expansion of the mitogenic action of angiotensin II. Since glutamic acid did not affect [¹²⁵I]EGF binding to hepatocytes or its processing, the effect may occur internal to the receptor. These results suggest that glutamic acid modulates the sensitivity of the hepatocyte response to mitogens © 1994 Wiley-Liss, Inc.     

Amino acid uptake and protein synthesis in rat testes: stimulation by dissociable factors

Low concentration (25 mM) of sodium in the incubation medium produced a decrease in the amino acid uptake by the testis tissue as well as a reduction in the response to FSH. In this experimental condition, the basal protein synthesis and the stimulatory effect of FSH was not modified. The subcutaneous administration of testosterone to 15 day old rats increased the protein synthesis in the testis without any modification in the amino acid uptake. The addition of DBcAMP (1 mM) or glucose (14 mM) to the incubation medium increased the protein synthesis in the testes of immature (12 day-old) or prepubertal (32 day-old) rats respectively. The amino acid uptake was not modified. In immature rat testes, with protein synthesis completely inhibited by cycloheximide, the restoration of the sodium concentration in the incubation medium to normal levels produced an increase in amino acid uptake. The results above seem to indicate that protein synthesis and amino acid uptake in rat testes tissue can be regulated, at least partially, by different factors.     

Development and evaluation of a boronate inhibitor of gamma-glutamyl transpeptidase

Gamma-glutamyl transpeptidase (gamma-GT) plays a central role in the metabolism of glutathione and is also a marker for neoplasia and cell transformation. We have investigated the compound L-2-amino-4-boronobutanoic acid (ABBA) as a structural analog of the putative ternary complex formed by the enzyme, L-serine, and borate, proposed to function as a transition state analog inhibitor. ABBA was found to be a potent inhibitor of the enzyme, with Ki = 17 nM using typical assay conditions (pH 8, gamma-glutamyl-p-nitroanilide substrate, 20 mM glycyl-glycine acceptor). ABBA is a stable amino acid analog with pK values determined from 13C and 11B NMR to be 2.3, 11.0 (amino titration), and 7.9 (boronate titration). The structural similarity to glutamate suggested that it might function as a glutamate analog for some glutamate-dependent enzymes or receptors. Transamination of pyruvate by ABBA to yield alanine in the presence of glutamic pyruvic transaminase was demonstrated by 13C NMR. The 2-keto-4-boronobutanoic acid transamination product is apparently fairly labile to hydrolysis, leading to formation of 2-ketobutanoic acid plus borate. The latter is also subsequently transaminated to yield 2-aminobutanoic acid. Both of these metabolites were observed in the 13C NMR spectrum. However, the corresponding transamination of oxaloacetate by ABBA in the presence of glutamic oxaloacetic transaminase was not observed. Effects of ABBA on the growth of cultured rat liver cell lines ARL-15C1 (nontumorigenic, low gamma-GT activity) and ARL-16T2 (tumorigenic, high gamma-GT activity) were also investigated, both in standard Williams Media as well as in a low cysteine growth medium. A high concentration (1 mM) of ABBA inhibited the growth of both cell lines in both media, with the degree of inhibition greater in the low cysteine medium. Alternatively, growth inhibition by 10 microM ABBA could be observed only in the low cysteine media. In general, there were no significant differences between the two cell lines in terms of sensitivity to ABBA.     

Relationship between Na+-dependent cytoplasmic pH homeostasis and Na+-dependent flagellar rotation and amino acid transport in alkalophilic Bacillus

The cytoplasmic pH homeostasis of alkalophilic Bacillus strains required the presence of Na+ in the medium, and Li+ was found to be equivalently substitutable for Na+. Flagellar rotation and amino acid transport of these bacteria also required Na+ but Li+ was not substitutable for Na+. Na+ concentration of about 1 mM was enough for the cytoplasmic pH homeostasis, while more than 10 mM Na+ was required for the full activities of flagellar rotation and amino acid transport. The addition of 150 mM ethanolamine to the cells at pH 9.6 disrupted the pH homeostasis and increased the cytoplasmic pH close to the external pH. Under this condition, however, flagellar rotation and amino acid transport were not so much affected. Thus, it is clear that flagellar rotation and amino acid transport themselves require the presence of Na+ in the medium, independent of the Na+-dependent cytoplasmic pH homeostasis.     

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.     

Decrease of phosphate concentration in the medium by Brevibacterium casei cells

Brevibacteria able to decrease phosphate concentration in the medium are of interest for the study of the role of bacteria in the phosphorus cycle and for development of biotechnology of phosphate removal from waste. Brevibacterium casei, Brevibacterium linens, and Brevibacterium epidermidis grown in media with initial phosphorus concentrations of 1-11 mM were shown to decrease its concentration by 90%. The composition of the incubation medium required for B. casei to carry out this process was established. This process occurs in the absence of glucose but requires the presence of Mg2+, NH4+, and alpha-ketoglutarate. The latter two components may be replaced by amino acids metabolized to NH4+ and alpha-ketoglutarate: histidine, arginine, glutamine, proline, or glutamic acid. No formation of insoluble phosphate salts was observed when the media were incubated under the same conditions with heat-inactivated cells or without cells at pH 7-8.5.