Effect of culture media on the composition of free amino acids in Saccharomyces cerevisiae yeast

The quantitative and qualitative compositions of free amino acids of the yeast Saccharomyces cerevisiae Y-503 cultivated in different nutrient media were studied by liquid chromatography. The yeast grown in the medium containing geothermal water was shown to accumulate more amino acids. During lyophilization, the stabilization of the physiological activity of the yeast in this nutrient medium was observed. The increased biological value of dry yeast was shown to depend on the content of free amino acids, including essential amino acids: arginine, histidine, leucine, isoleucine, lysine, threonine, serine and phenylalanine.     

Effects of Nutrient Media on the Composition of Free Amino Acids in Saccharomyces cerevisiae

The quantitative and qualitative compositions of free amino acids of the yeast Saccharomyces cerevisiaeY-503 cultivated in different nutrient media were studied by liquid chromatography. The yeast grown in a medium containing geothermal water was shown to accumulate more amino acids. During lyophilization, the stabilization of the physiological activity of the yeast in this nutrient medium was observed. The increased biological value of dry yeast was shown to depend on the content of free amino acids, including essential amino acids: arginine, histidine, leucine, isoleucine, lysine, threonine, serine, and phenylalanine.     

A possible role of histidine in a nickel resistant mechanism of Saccharomyces cerevisiae

When a nickel resistant strain N08 of S. cerevisiae was grown in a Ni-supplemented medium, approximately 70% of the nickel is distributed in the soluble fraction. The soluble fraction was chromatographed on Sephadex G-10 and the fraction contained both nickel and large amounts of histidine. When cells were grown in medium containing various combinations of nickel and magnesium and which exhibited approximately 50% growth inhibition, a molar ratio of intracellular histidine and nickel contents remained constant at 1.2-1.4, indicating that the increase in histidine content is correlated with nickel accumulation. The wild type strain 0605-S6, however, exhibits no increase in histidine content when grown in a Ni-supplemented medium, and, therefore, a nickel-resistant mechanism of yeast appears to be the formation of histidine-nickel complexes.     

The effect of yeast autolysates on the level and distribution of free amino acids in yeast cells of the Candida genus

The work was aimed at studying the effect of yeast autolysate on the content and subcellular distribution of free amino acids in yeast cells. The overall pool of free amino acids decreased 1.5-2 times when mineral nitrogen in the growth medium was substituted either completely or partly by yeast autolysate. As was shown using the technique of differential extraction, the vacuolar pool of the cell is mainly responsible for the decrease in the content of free amino acids.     

The production of extracellular and intracellular free amino acids during aerated fermentation of glucose by Baker’s yeast (Saccharomyces cerevisiae)

During a study of the effects of a high level of NaCl on the content of free intracellular amino acids in baker’s yeast grown in aerated fermentation of glucose it was found (Malaneyet al. 1988, 1989; Malaney and Tanner 1988) that 0.6 mol/L exogenous NaCl significantly increased the content of free intracellular citrulline, glutamine, ornithine, arginine and lysine (all basic amino acids) over that observed at zero mol/L exogenous NaCl. (Exogenous is defined as salt added beyond that present in the mineral salts in the culture medium.) This paper describes the production and relative relationships of both extracellular and free intracellular amino acids byS. cerevisiae under conditions of high NaCl content in the growth medium at pH 5 and 32 °C. For early culture times (6 h), the production of glutamine, citrulline, valine, isoleucine, ornithine, lysine and histidine were all enhanced by the addition of NaCl. For late times (24 h), except for ornithine, the early-time-enhanced amino acids continued to be enhanced by the addition of NaCl. In addition, the yields of several other amino acids also were increased by exogenous salt at this late time. These include aspartic acid, threonine, glutamic acid, cystine, methionine, tyrosine, phenylalanine and arginine. Deceased, August 12, 1988. This study was supported by theNational Science Foundation (Grant No. CPE8209945) in conjunction with the NSF United States — Taiwan Cooperative Science Program.     

Experimental Studies on the Formation of Volatile Nitrogen Compounds Induced by Pseudomonas Fragi in A Synthetic Medium with Amino Acids as Source of Nitrogen

The influence of the growth of Pseudomonas fragi strain F 111 was studied in a synthetic medium. It was shown that volatile nitrogen compounds were rapidly liberated as a result of the decomposition at 5°C of arginine and histidine. A later and slower increase of TVN was observed in media, which contained lysine or urea. From the other 7 amino acids which were included in the test, no increase of TVN was observed to be caused by the strain F 11. It was shown that within the range of 0.05 to 0.8 % histidine added to the basic salt solution the amount of TVN liberated was correlated to the amount of histidine included in the media. At the TVN maximum approx. 50 % of the amino acid nitrogen of the medium was recovered as TVN. It was further shown that the liberation of TVN was correlated to the added amount of arginine and histidine included in the growth medium. The presence of lysine also influenced on the TVN maximum which was reached. In the synthetic medium to which arginine or histidine was added the growth of strain F 111 was stimulated by the presence of glucose. The acids produced by the oxidative breakdown of glucose were neutralized partly by the volatile bases produced in the decomposition of amino acids. The ability of the Pseudomonas fragi to grow in anaerobic conditions and to produce enzymes which could decompose histidine was studied. It was concluded that the anaerobic breakdown of histidine was similar to the anaerobic breakdown of arginine reported by other authors.     

Copper extrusion after accumulation during growth of copper-tolerant yeast Yarrowia lipolytica

The Cu2+-tolerant yeast Yarrowia lipolytica accumulated Cu2+ until the late logarithmic phase. Thereafter, Cu2+ was temperature-dependently extruded into phosphate-limited culture medium containing high concentrations of heavy metal ions but not into 10 mM 2-(N-morpholino)ethane sulfonic acid (MES) buffer (pH 6.0). Peptone in the culture medium played an important role in the extrusion, which proceeded even when peptone was substituted with cysteine or histidine, but not with any other amino acid tested.     

Alteration in the Amino Acid Content of Yeast During Growth Under Various Nutritional Conditions

Yeast cells grown under optimal and suboptimal concentrations of biotin were analyzed for the amino acid content of their soluble pool and cellular protein. Optimally grown yeast cells exhibited a maximum amino acid content after 18 hr of growth. Biotin-deficient cells were depleted of all amino acids at 26 and 43 hr, with alanine, arginine, aspartate, cysteine, glutamate, isoleucine, leucine, lysine, methionine, serine, threonine, and valine being present in less than half the concentration observed in biotin-optimal cells. At early time intervals, the amino acid pool of biotin-deficient yeast contained lower concentrations of all amino acids except alanine. After more prolonged incubation, several amino acids accumulated in the pool of biotin-deficient yeast, but citrulline and ornithine accumulated to appreciable levels. The addition of aspartate to the growth medium resulted in a decrease in the amino acid content of biotin-optimal cells but caused a marked increase in the concentration of amino acids in biotin-deficient cells. The pools of biotin-deficient yeast grown in the presence of aspartate displayed a marked reduction in every amino acid with the exception of aspartate itself. These data provide evidence that the amino acid content of yeast cells and their free amino acid pools are markedly affected by biotin deficiency as well as by supplementation with aspartate, indicating that aspartate plays a major role in the nitrogen economy of yeast under both normal as well as abnormal nutritional conditions.     

Characterization of Avt1p as a vacuolar proton/amino acid antiporter in Saccharomyces cerevisiae

Several genes for vacuolar amino acid transport were reported in Saccharomyces cerevisiae, but have not well been investigated. We characterized AVT1, a member of the AVT vacuolar transporter family, which is reported to be involved in lifespan of yeast. ATP-dependent uptake of isoleucine and histidine by the vacuolar vesicles of an AVT exporter mutant was lost by introducing avt1? mutation. Uptake activity was inhibited by the V-ATPase inhibitor: concanamycin A and a protonophore. Isoleucine uptake was inhibited by various neutral amino acids and histidine, but not by γ-aminobutyric acid, glutamate, and aspartate. V-ATPase-dependent acidification of the vesicles was declined by the addition of isoleucine or histidine, depending upon Avt1p. Taken together with the data of the amino acid contents of vacuolar fractions in cells, the results suggested that Avt1p is a proton/amino acid antiporter important for vacuolar compartmentalization of various amino acids.     

Vba2p, a vacuolar membrane protein involved in basic amino acid transport in Schizosaccharomyces pombe

A recent study filling the gap in the genome sequence in the left arm of chromosome 2 of Schizosaccharomyces pombe revealed a homolog of budding yeast Vba2p, a vacuolar transporter of basic amino acids. GFP-tagged Vba2p in fission yeast was localized to the vacuolar membrane. Upon disruption of vba2, the uptake of several amino acids, including lysine, histidine, and arginine, was impaired. A transient increase in lysine uptake under nitrogen starvation was lowered by this mutation. These findings suggest that Vba2p is involved in basic amino acid transport in S. pombe under diverse conditions.     

The immunosuppressant FK506 inhibits amino acid import in Saccharomyces cerevisiae.

The immunosuppressants cyclosporin A, FK506, and rapamycin inhibit growth of unicellular eukaryotic microorganisms and also block activation of T lymphocytes from multicellular eukaryotes. In vitro, these compounds bind and inhibit two different types of peptidyl-prolyl cis-trans isomerases. Cyclosporin A binds cyclophilins, whereas FK506 and rapamycin bind FK506-binding proteins (FKBPs). Cyclophilins and FKBPs are ubiquitous, abundant, and targeted to multiple cellular compartments, and they may fold proteins in vivo. Previously, a 12-kDa cytoplasmic FKBP was shown to be only one of at least two FK506-sensitive targets in the yeast Saccharomyces cerevisiae. We find that a second FK506-sensitive target is required for amino acid import. Amino acid-auxotrophic yeast strains (trp1 his4 leu2) are FK506 sensitive, whereas prototrophic strains (TRP1 his4 leu2, trp1 HIS4 leu2, and trp1 his4 LEU2) are FK506 resistant. Amino acids added exogenously to the growth medium mitigate FK506 toxicity. FK506 induces GCN4 expression, which is normally induced by amino acid starvation. FK506 inhibits transport of tryptophan, histidine, and leucine into yeast cells. Lastly, several genes encoding proteins involved in amino acid import or biosynthesis confer FK506 resistance. These findings demonstrate that FK506 inhibits amino acid import in yeast cells, most likely by inhibiting amino acid transporters. Amino acid transporters are integral membrane proteins which import extracellular amino acids and constitute a protein family sharing 30 to 35% identity, including eight invariant prolines. Thus, the second FK506-sensitive target in yeast cells may be a proline isomerase that plays a role in folding amino acid transporters during transit through the secretory pathway.     

Toxicity of Copper, Cobalt, and Nickel Salts Is Dependent on Histidine Metabolism in the Yeast Saccharomyces cerevisiae

The pH-dependent inhibition of 22 metal salts have been systematically investigated for the yeast Saccharomyces cerevisiae. We have established that the inhibition of growth by Cu, Co, or Ni salts is markedly enhanced by histidine auxotrophy and by increasing the pH of the medium. Each of the his1-his7 mutant strains were unable to grow in the presence of elevated levels of Cu, Co, or Ni at nearly neutral pHs, in contrast to His(+) strains, which grew under these conditions. The Cu, Co, or Ni inhibition was reversed by the addition of histidine to the medium. Deletion of the high-affinity histidine permease Hip1p in His(-) strains resulted in even greater sensitivity to Cu, Co, and Ni and the requirement of an even higher level of histidine to reverse the inhibition. These results suggest that intracellular histidine, most likely in the vacuole, diminishes the pH-dependent toxicity of Cu, Co, and Ni. Furthermore, the toxicity of many salts is exacerbated in strains with a defective vacuolar H(+)-ATPase, which abolishes the ability of yeast to maintain an acidic vacuole, a compartment known to sequester metal compounds. We suggest that the accumulation of histidine in the vacuole is a normal process used to detoxify Cu, Co, and Ni.     

Stimulation of amino acid accumulation in neuroblastoma and astrocytoma cells byl-histidine

Uptake of amino acids by cultured neuroblastoma and astrocytoma cells was studied in the presence and absence ofl-histidine. Intracellularly accumulated histidine was assumed to induce accumulation of radioactively labeled amino acids from medium by means of exchange transport. Neuroblastoma cells accumulated more histidine than astrocytoma cells and were more sensitive to the enhancement of the uptake of other large neutral amino acids by histidine. Histidine also increased glutamic acid uptake in astrocytoma cells, but reduced it in neuroblastoma cells. The greatest differences between the cell lines in amino acid uptake without histidine were found with acidic amino acids (astrocytoma cells accumulated them more than neuroblastoma cells) and with taurine (the reverse was found). The uptake and exchange mechanisms for some neutral and acidic amino acids may thus be dissimilar in the plasma membranes of cultured cells of neuronal and glial origin.     

A family of basic amino acid transporters of the vacuolar membrane from Saccharomyces cerevisiae

Among the members of the major facilitator superfamily of Saccharomyces cerevisiae, we identified genes involved in the transport into vacuoles of the basic amino acids histidine, lysine, and arginine. ATP-dependent uptake of histidine and lysine by isolated vacuolar membrane vesicles was impaired in YMR088c, a vacuolar basic amino acid transporter 1 (VBA1)-deleted strain, whereas uptake of tyrosine or calcium was little affected. This defect in histidine and lysine uptake was complemented fully by introducing the VBA1 gene and partially by a gene encoding Vba1p fused with green fluorescent protein, which was determined to localize exclusively to the vacuolar membrane. A defect in the uptake of histidine, lysine, or arginine was also observed in the vacuolar membrane vesicles of mutants YBR293w (VBA2) and YCL069w (VBA3). These three VBA genes are closely related phylogenetically and constitute a new family of basic amino acid transporters in the yeast vacuole.     

Changes in the intracellular pool of free amino acids of Streptococcus lactis under different cultivation conditions]

The intracellular pool of free amino of Streptococcus lactis--lysine producer contains a good number of amino acids when cultivated on the corn medium. Glutamic acid, proline, alanine, lysine, leucine, histidine and arginine are in predominance. An almost complete amino acid pool develops at an early exponential phase of Str. lactis growth under stationary cultivation conditions. The content of free amino acids increases 4-fold during the transition from the early exponential phase to the stationary phases under submerged cultivation conditions. This can be attributed to a more intensive amino acid exchange during the medium stirring than during stationary cultivation.     

Basic amino acid inhibition of cell division and macromolecular synthesis in Saccharomyces cerevisiae.

Growth of Saccharomyces cerevisiae on poor nitrogen sources such as allantoin or proline was totally inhibited by addition of a non-degradable basic amino acid to the medium. Cells treated with lysine contained greatly reduced quantities of histidine and arginine. Conversely, lysine and histidine were severely reduced in arginase-deficient cells treated with arginine. When all three basic amino acids were present in the culture medium, growth was normal suggesting that synthesis of all three basic amino acids was decreased by an excess of any one of them. Inhibition of growth was accompanied by a fivefold increase in the observed ratio of budded to unbudded cells. These morphological changes suggested that DNA synthesis was inhibited. Consistent with this suggestion, addition of a basic amino acid to the culture medium substantially reduced the ability of the cells to incorporate [14C]uracil into alkali-resistant, trichloroacetic acid-precipitable material. RNA and protein synthesis, although decreased, were less sensitive to the effects of such additions.     

Effect of variations in salinity and alkalinity of applied irrigation waters on the amino acid make-up of Phaseolus aureus

Summary A marked decrease in the content of various amino acids was observed when the boron concentration was increased from 0.5 to 2.0 ppm particularly at low value of SAR and low level of conductivity. At medium level of conductivity alongwith low level of boron increase in value of SAR definitely suppressed the content of lysine, arginine plus histidine, aspartic acid plus glutamine, threonine plus alanine, proline and cystine in plants. Increase in levels of conductivity specially at low value of SAR and low level of boron substantially affected the content of lysine, arginine plus histidine, aspartic acid plus glutamine, threonine plus alanine, proline and cystine.     

Development of a minimal medium for Clostridium perfringens by using an anaerobic chemostat.

A minimal medium was developed for the cultivation of Clostridium perfringens in an anaerobic chemostat. Cultures of C. perfringens ATCC 3624 and NCTC 10240 were grown at 46 and 43 degrees C, respectively, in a glucose-limited, chemically defined medium at pH 7.2. The concentrations of amino acids, minerals, nucleotides, and vitamins, initially present in excess, were varied independently. The minimum concentration of each nutrient which would support 3 X 10(8) CFU/ml with a generation time of less than 40 min was determined and used to develop a reformulated defined medium. Atomic absorption spectroscopy and amino acid analyses of the reformulated medium indicated additional adjustments in nutrient content which led to the development of a minimal medium for each strain. The nutritional profile for each strain was similar. A decrease in the concentration of arginine, histidine, and tyrosine for strain 3624 and of arginine, histidine, and isoleucine for strain 10240 resulted in an increase in the optical density of each culture.     

Vba2p,A Vacuolar Membrane Protein Involved in Basic Amino Acid Transport in Schizosaccharomyces pombe

A recent study filling the gap in the genome sequence in the left arm of chromosome 2 of Schizosaccharomyces pombe revealed a homolog of budding yeast Vba2p, a vacuolar transporter of basic amino acids. GFP-tagged Vba2p in fission yeast was localized to the vacuolar membrane. Upon disruption of vba2, the uptake of several amino acids, including lysine, histidine, and arginine, was impaired. A transient increase in lysine uptake under nitrogen starvation was lowered by this mutation. These findings suggest that Vba2p is involved in basic amino acid transport in S. pombe under diverse conditions.