Nitrogen catabolite regulation of spiramycin production inStreptomyces ambofaciens

Spiramycin production byStreptomyces ambofaciens is controlled by the nitrogen source present in the culture medium. Thus, amino acids according to the mode of catabolism (transamination or deamination) influenced the spiramycin production differently. Arginine, whose catabolism led to an important excretion of ammonium, gave a slight spiramycin production of 5.3 mg. g^–1 dry cell weight; however, the introduction of an ammonium trapping agent [0.25% Mg₃(PO₄)₂] enhanced spiramycin production by 415%. The use of a neutral culture medium showed the existence of a critical phase during which the ammonium pulse had maximum negative effects on spiramycin production. Among these negative effects, the ammonium pulse provoked an increase in the growth rate, which was partially responsible for the decrease of the spiramycin production. The inhibitory effects of ammonium on spiramycin production were mitigated when the growth rate was controlled by the phosphate concentration. In addition, protease activities were limited on a culture medium in which ammonium was present and spiramycin production was null, whereas on lysine, where spiramycin production was favored, protease activities were higher.     

Lysine degradation in Candida maltosa: occurrence of a novel enzyme,acetyl-CoA: L-lysine N-acetyltransferase

The yeast Candida maltosa can utilize L-lysine as sole nitrogen and sole carbon source accompanied by accumulation of -N-acetyl-L-lysine, indicating that lysine is metabolized by way of N-acetylated intermediates. A novel lysine acetyltransferase catalyzing the first step in this pathway, the N-acetylation of the -amino group of L-lysine, was found in this yeast. The enzyme, acetyl-CoA:L-lysine N-acetyltransferase, is strongly induced in cells grown on L-lysine as sole carbon source. The enzyme is specific for both L-lysine and acetyl-CoA. The K _m values are 10 mM for L-lysine and 0.33 mM for acetyl-CoA. The enzyme has a maximum activity at pH 8.1.Dedicated to Prof. Dr. F. Böttcher in occasion of his 60th birthday     

Some physiological functions of the L-leucine dehydrogenase in Bacillus subtilis

Mutants of Bacillus subtilis constitutive for L-leucine dehydrogenase synthesis were selected. Using these mutants we could determine two functional roles for the L-leucine dehydrogenase. This enzyme liberates ammonium ions from branched chain amino acids when supplied as the sole nitrogen source. Another function is to synthesize from L-isoleucine, L-leucine, and L-valine the branched chain -keto acids which are precursors of branched chain fatty acid biosynthesis. These results together with the inducibility of the enzyme suggest that the L-leucine dehydrogenase has primarily a catabolic rather than an anabolic function in the metabolism of Bacillus subtilis.     

Effect of ammonium ions on glycolipid production byCandida (Torulopsis)apicola

The effect of ammonium on growth ofCandida apicola and on production of sophorose lipid was studied. Sophorose lipid production increased with increasing initial ammonium sulphate concentration. Both growth and product formation were strongly reduced at 73.6mm ammonium. With 58.9mm ammonium a microcrystalline sophorose lipid was formed. The ratio of the two isomers of the sophorose lipid, harbouring either - or -1 hydroxy fatty acid, was influenced by the initial concentration of ammonium. Both production kinetics, yields and profiles of the total cellular fatty acids express alterations with enlarged ammonium concentrations. These results suggest regulatory effects of ammonium onC. apicola and its sophorose lipid synthesis.Dedicated to Prof. Dr. Fritz Wagner on the occasion of his 65th birthday     

Isolation and characterization of a marine methanogenic bacterium from the biofilm of a shiphull in Los Angeles harbor

A marine mesophilic, irregular coccoid methanogen, which shows close resemblance toMethanococcus sp., was isolated from the biofilm of shiphulls docked in Los Angeles harbor. Hydrogen plus carbon dioxide or formate served as substrates for methanogenesis in a mineral salt medium. The isolate did not use acetate and methanol as sole source of carbon and energy. The organism had an optimal pH range of 6.8–7.0 and a temperature optimum of 37°C. Elevated levels of sodium chloride were required for optimum growth. Optimum levels of total sulfide and magnesium chloride for growth were 1.0mm and 10mm respectively. The isolate used ammonia as nitrogen source. The concentration of 30mm ammonium chloride supported maximum growth of the isolate.     

Effective extracellular trehalose production by<Emphasis Type="Italic"> Cellulosimicrobium cellulans</Emphasis>

A bacterium isolated from a petal of Casa Blanca Lily (ST26 strain) produced a marked amount of extracellular trehalose (-d-glucopyranosyl-[1,1]--d-glucopyranose) in culture medium containing glucose. 16S rDNA-based phylogeny showed that ST26 belongs to, or is related to, Cellulosimicrobium cellulans, a close relative of Cellulomonas spp. Various Cellulomonas strains obtained from culture collections also showed extracellular trehalose productivity, suggesting that trehalose production is a common property of this bacterial genus. ST26 accumulated trehalose in medium supplied with glucose but not with sucrose, glycerol or maltose. Effective extracellular trehalose production by ST26 was achieved by supplying 0.5–1% ammonium sulfate and 0.5–1% CaCO₃. The addition of CaCO₃ adjusted the pH of the culture to around 5.0. The optimized culture conditions yielded trehalose from glucose at a conversion rate of 61%. The addition of ammonium sulfate greatly reduced the dry cell weight of ST26 and intracellular content of trehalose, which suggests that the addition of ammonium sulfate makes ST26 cells leak trehalose into the medium. ST26 effectively propagated in minimal medium containing trehalose as a sole carbon source, which suggests that trehalose serves as a carbohydrate reserve of this organism.The nucleotide sequence of 16S rDNA of ST26 has been submitted to the DDBJ databank under accession number AB109293     

Regulation of α-aminoadipyl-cysteinyl-valine,isopenicillin N synthetase,isopenicillin N isomerase and deacetoxycephalosporin C synthetase by nitrogen sources in Streptomyces lactamdurans

Summary Ammonium and asparagine produced a concentration-dependent reduction of cephamycin C biosynthesis by Streptomyces lactamdurans. Addition of ammonium salts at 1 mM concentration reduced cephamycin biosynthesis by resting cells of S. lactamdurans, whereas concentrations of asparagine above 10 mM were required to get the same effect. High ammonium concentrations decreased glutamine synthetase activity in cell extracts of S. lactamdurans in parallel to the reduction of antibiotic biosynthesis. Ammonium supplementation decreased the pool of glutamic acid and glutamine whereas the intracellular content of ammonium, alanine, and phosphoserine increased significantly. The pool of the tripeptide (l--aminoadipyl)-l-cysteinyl-d-valine, an intermediate in cephamycin biosynthesis, was greatly reduced in ammonium-supplemented cultures. Isopenicillin N synthetase, that converts the tripeptide (l--aminoadipyl)-l-cysteinyl-d-valine to isopenicillin N, isopenicillin N isomerase (that isomerises isopenicillin N to penicillin N) and deacetoxycephalosporin C synthetase (converting penicillin N into deacetoxycephalosporin C) were also reduced in ammonium-supplemented cultures. However, the activities of these enzymes were not inhibited in vitro by 40 mM ammonium, suggesting that the enzymes were repressed but not inhibited by ammonium in vivo.     

Organic or mineral nitrogen source during Penicillium camembertii growth on a glucose limited medium

Penicillium camembertii was cultivated on a carbon-limited medium (glucose). Two nitrogen sources were compared, a mineral, ammonium, and an organic nitrogen source, lysine. Among the amino acids convenient nitrogen sources for P. camembertii, lysine was chosen since it cannot be assimilated as a carbon source for cell biosynthesis. During culture on glucose and ammonium, a decline phase immediately followed growth after glucose depletion, since no energy source remained in the medium. On the contrary, on glucose and lysine, a stationary state was recorded after glucose depletion, since lysine was used as the energy supply for cell maintenance, leading to the release of the corresponding carbon as CO₂, while nitrogen from lysine was released as ammonium.     

The nitrogen requirements ofGluconobacter,Acetobacter andFrateuria

The nitrogen requirements of 96Gluconobacter, 55Acetobacter and 7Frateuria strains were examined. Only someFrateuria strains were able to grow on 0.5% yeast extract broth or 0.5% peptone broth. In the presence ofd-glucose ord-mannitol as a carbon source, ammonium was used as the sole source of nitrogen by all three genera. With ethanol, only a fewAcetobacter strains grew on ammonium as a sole nitrogen source. Singlel-amino acids cannot serve as a sole source of carbon and nitrogen for growth ofGluconobacter, Acetobacter orFrateuria. The singlel-amino acids which were used by most strains as a sole nitrogen source for growth are: asparagine, aspartic acid, glutamine, glutamic acid, proline and alanine. SomeAcetobacter andGluconobacter strains deaminated alanine, asparagine, glutamic acid, threonine, serine and proline. NoFrateuria strain was able to develop on cysteine, glycine, threonine or tryptophan as a sole source of nitrogen for growth. An inhibitory effect of valine may explain the absence of growth on this amino acid. No amino acid is “essential” forGluconobacter, Acetobacter orFrateuria.     

Evaluation of alternative nitrogen and carbon sources for sugarbeet suspension culture platings in development of cell selection schemes

Summary Low molecular weight nitrogenous impurity compounds as well as raffinose are negative quality factors that interfere with efficient processing of sugarbeet (Beta vulgaris L.) for sucrose. In order to identify nutrient media for cell selection of biochemical mutants or transgenics that might have reduced levels of these processing impurities, the ability of 10 endogenous compounds to serve as sole nitrogen or carbon source for suspension plating and subculture callus growth was evaluated. The most productive concentrations of nitrate, ammonium, l-glutamine, l-glutamate, urea, and l-proline as sole nitrogen sources supported plating callus growth at 106, 159, 233, 167, 80, and 52%, respectively, as well as the historical 60 mM mix of nitrate and ammonium in Murashige-Skoog medium. Glycine betaine and choline did not support growth. d(+) Raffinose and d(+) galactose supported plating callus growth only 67 and 25%, respectively, as well as sucrose as sole carbohydrate source. No callus growth occurred on glutamine, glutamate, or glycine betaine as the sole carbon or carbon plus nitrogen source. Platings on either nitrate or ammonium as sole nitrogen source did not differ in sensitivity to the nitrate uptake inhibitor phenylglyoxal, suggesting that phenylglyoxal lacks the specificity for use in selection for mutants of nitrate uptake. The ability of raffinose to be used as the carbon source, and glutamine or glutamate as the nitrogen source, may preclude their use for selection of genetic variants accumulating less of these processing impurities. However, mutants or transgenics able to utilize either glutamine, glutamate, or glycine betaine might be selectable on media containing any one of these as carbon, nitrogen, or carbon plus nitrogen source, respectively, that is incapable of supporting wild-type cell growth.     

Purification and characterization of glutamine synthetase from the commerical mushroom Agaricus bisporus

Agaricus bisporus glutamine synthetase, a key enzyme in nitrogen metabolism, was purified to apparent homogeneity. The native enzyme appeared to be a GS-II type enzyme. It has a molecular weight of 325 kDa and consists of eight 46-kDa subunits. Its pI was found at 4.9. Optimal activity was found at 30°C. The enzyme had low thermostability. Stability declined rapidly at temperatures above 20°C. The enzyme exhibits a K _m for glutamate, ammonium, and ATP of 22mm, 0.16mm and 1.25mm respectively in the biosynthetic reaction, with optimal activity at pH 7. The enzyme is slightly inhibited by 10mm concentrations of l-alanine, l-histidine, l-tryptophan, anthranilic acid, and 5-AMP and was strongly inhibited by methionine sulfoximine and phosphinothricine. For the transferase reaction K _i-values were 890 m and 240 m for methionine sulfoximine and phosphinothricine respectively. For the biosynthetic reaction K _i was 17 m for both methionine sulfoximine and phosphinothricine.     

A chemically defined medium for cephalosporin C production by Paecilomyces persicinus

A chemically defined medium was developed for the biosynthesis of cephalosporin C by Paecilomyces persicinus Nicot strain P-10. Glucose served as the major carbon source and nitrogen was supplied by five amino acids, l-arginine, l-aspartic acid, l-glutamic acid, glycine and dl-methionine. Omission of any of the first four diminished or prevented production of cephalosporin C; omission of methionine did not. Methionine is not critical for the production of cephalosporin C in this defined medium. Production of the antibiotic was affected by the concentrations of inorganic salts employed. Biotin was required for growth and cephalosporin C synthesis. The addition of l-lysine precursors to the medium did not influence cephalosporin C levels and l-lysine itself inhibited antibiotic production. Known precursors of -lactam antibiotics as well as oleic acid did not affect biosynthesis of cephalosporin C. Chemical changes occurring in the defined medium revealed that glucose was efficiently utilized after 96 hours incubation whereas total soluble nitrogen levels increased following an initial sharp decrease. Mycelial weight and cephalosporin C production were both maximal after 96 hours incubation. Mycelial nitrogen was highest after 48 hours incubation whereas mycelial lipid levels were greatest after 72 hours.     

The effects of amino acids and ammonium on the growth of plant cells in suspension culture

A suspension culture of soybean (Glycine max L.) was grown on a defined medium in which the nitrogen sources were nitrate (25 mM) and ammonium (2 mM). The cells did not grow on nitrate unless the medium was supplemented with ammonium or glutamine. The l- and d-isomers of 12 amino acids tested singly could not replace ammonium. Most amino acids (4 mM) inhibited growth when the cells were cultured on nitrate and ammonium. Cells from five other plants (Reseda luteoli L.; Triticum monococcum L.; flax, Linum usitatissimum L.; horseradish, Amoracia lapathifolia Gilib; Haplopappus gracilis L.) grew on the defined medium with nitrate (25 mM) as the sole nitrogen source. Higher cell yields were obtained when ammonium (2 mM) or glutamine also was present. Supplementing the defined medium with high concentrations of ammonium (20 mM) inhibited growth of soybean, Haplopappus, and wheat cells. Addition of citrate (5 mM) relieved the inhibitory effects of ammonium in soybean and wheat cells but not in the Haplopappus cells.     

Purification and properties of glutamine synthetase from Nocardia asteroides

Glutamine synthetase (GS, EC 6.3.1.2) from Nocardia asteroides was purified to homogeneity by ammonium sulfate precipitation, Sephadex G-150, and DEAE-Sepharose chromatography. The native molecular weight of the purified enzyme was determined to be 720 kDa. SDS-PAGE analysis of the purified preparation revealed a single band corresponding to 59 kDa, indicating the possible presence of 12 identical subunits. The divalent cations Mn^2- and Mg²⁺ were found to be essential for optimal transferase and biosynthetic activity, respectively. The optimal pH and temperature for both activities of the enzyme were found to be 7.2 and 50°C. Amino acids such as l-alanine, glycine, and aspartate inhibited the GS activity. The K _m values for the substrates of the biosynthetic reaction ATP, glutamate, and ammonium chloride were found to be 400 m, 7.7mm, and 200 m, respectively. Addition of ammonium chloride to the nitrogen-limited culture resulted in a decrease of GS transferase and biosynthetic activities. Phosphodiesterase treatment of the extract from ammonia-shocked cultures showed an increase in GS transferase activity. The results indicate the possible regulation of GS by covalent modification.     

A mutant of Saccharomyces cerevisiae lacking catabolic NAD-specific glutamate dehydrogenase Growth characteristics of the mutant and regulation of enzyme synthesis in the wild-type strain

NAD-specific glutamate dehydrogenase (GDH-B)¹ was induced in a wild-type strain derived of - 1278b by -amino acids, the nitrogen of which according to known degradative pathways is transferred to 2-oxoglutarate. A recessive mutant (gdhB) devoid of GDH-B activity grew more slowly than the wild type if one of these amino acids was the sole source of nitrogen. Addition of ammonium chloride, glutamine, asparagine or serine to growth media with inducing -amino acids as the main nitrogen source increased the growth rate of the gdhB mutant to the wild-type level and repressed GDH-B synthesis in the wild type. Arginine, urea and allantoin similarly increased the growth rate of the gdhB mutant and repressed GDH-B synthesis in the presence of glutamate, but not in the presence of aspartate, alanine or proline as the main nitrogen source. These observations are consistent with the view that GDH-B in vivo deaminates glutamate. Ammonium ions are required for the biosynthesis of glutamine, asparagine, arginine, histidine and purine and pyrimidine bases. Aspartate and alanine apparently are more potent inducers of GDH-B than glutamate.Anabolic NADP-specific glutamate dehydrogenase (GDH-A) can not fulfil the function of GDH-B in the gdhB mutant. This is concluded from the equal growth rates in glutamate, aspartate and proline media as observed with a gdhB mutant and with a gdhA, gdhB double mutant in which both glutamate dehydrogenases are lacking. The double mutant showed an anomalous growth behaviour, growth rates on several nitrogen sources being unexpectedly low.The following abbreviations and symbols are used GDH-A NADP-specific glutamate dehydrogenase [l-glutamate - NADP⁺ oxido-reductase (deaminating), EC 1.4.1.4] - gdhA genotype associated with GDH-A deficiency - GDH-B NAD-specific glutamate dehydrogenase, [L-glutamate NAD⁺ oxido-reductase (deaminating), EC 1.4.1.2] - gdhB genotype associated with GDH-B deficiency - gdhCR genotype associated with derepressed GDH-B synthesis - specific growth rate (h^-1) - x cell density - t time (h)     

Extracellular deamination of l-amino acids by Chlamydomonas reinhardtii cells

When grown in the light and in a Tris-acetate phosphate medium, cells of Chlamydomonas reinhardtii Dang. can use the following l-amino acids as a sole nitrogen source: asparagine, glutamine, arginine, lysine, alanine, valine, leucine, isoleucine, serine, methionine, histidine, and phenylalanine, whereas, in the absence of acetate, the cells only used l-arginine. The utilization system in the acetate medium consisted of an extracellular deaminating activity induced by l-amino acids; it took between 10 to 30 h before the system appeared in cells previously grown with ammonium. This deaminase activity was nonspecific, required an organic carbon source for its de-novo synthesis, and was sensitive to high ammonium concentration and light deprivation.Abbreviations HPLC high-performance liquid chromatography - TAP Tris-acetate-phosphate This work was supported by a grant of the CAICYT, Spain. The secretarial assistance of C. Santos and I. Molina is gratefully acknowledged.To whom correspondence should be addressed.     

Studies on Metabolism of Pyrrolidone Carboxylic Acid in Bacteria

The present investigation is concerned with l-glutamic acid production in the presence of pyrrolidone carboxylic acid and glucose in Bacillus megaterium st. 6126. This strain does not grow on dl-pyrrolidone carboxylic acid (dl-PCA)¹⁾ as the sole source of carbon and nitrogen. The optimal concentration of yeast extract required for the maximal production of l-glutamic acid was 0.005% under the conditions used. As the yeast extract concentration was increased, growth increased proportionally; but the l-glutamic acid production did not exceed the control’s to which glucose and ammonium chloride had been added. l-Glutamic acid produced by both growing cultures and resting cells was derived from glucose and ammonium salt of dl-PCA. Isotope experiments suggested that the l-glutamic acid produced was partially derived from ammonium salt of dl-PCA in the growing culture which had been supplemented with d-glucose-U-¹⁴C or dl-PCA-1-¹⁴C and that ammonium salt of dl-PCA was consumed as the source of nitrogen and carbon for l-glutamic acid.     

Controlled biosynthesis of Val7- and Leu7-surfactins

Summary Surfactin was found to consist of a mixture of two groups of homologous lipopeptides differing by their peptide sequence; Val⁷-surfactin was recently characterized as a minor companion of the previously described Leu⁷-surfactin. The addition of various -amino acids to the culture media led to variations in the production ratios of the two congeners. The supplementation of l-valine or l-isoleucine to the culture medium resulted in a selective enhancement of the production of the Val⁷-surfactin whereas this production was very low when l-leucine was the nitrogen source in the culture medium. Offprint requests to: F. Peypoux     

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.     

An organic solvent-tolerant protease from Pseudomonas aeruginosa strain K: Nutritional factors affecting protease production

An organic solvent-tolerant bacterium producing an organic solvent-stable protease was isolated from soil and identified as Pseudomonas aeruginosa strain K. Nutritional requirements for optimized protease production by this strain were investigated. Maximum protease activity was achieved with sorbitol as the sole carbon source, followed by starch and lactose at pH 7.0 and 37 °C. Dextrose, sucrose and glycerol greatly reduced the protease production. The best organic nitrogen source was casamino acid. Tryptone, soytone and yeast extract supported protease production while corn steep liquor and beef extract inhibited the protease activity. Significant protease production was observed with sodium nitrate as a sole nitrogen source however, ammonium nitrate completely inhibit it. More than 62% drop in production occurred in the presence of amino acids. Addition of metal ions such as K⁺, Mg²⁺ and Ca²⁺ maximized the enzyme production.