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.     

l-Lysine-2-monooxygenase production by strains of Pseudomonas fluorescens and P. putida

Twelve strains of Pseudomonas fluorescens and P. putida were grown in a synthetic medium that contained l-lysine as the only source of carbon and nitrogen, and screened for l-lysine-2-monooxygenase production. Best production was by P. putida BKM B-1458 at 30 IU/g wet wt biomass when grown in a shake-flask but 25 IU/g in a 250-l fermenter.     

Assimilation of ethylenediamine as nitrogen source by the cyanobiont Nostoc ANTH

Nostoc ANTH metabolizes ethylenediamine (EDA) as sole nitrogen source but not as a carbon source. EDA is assimilated by the glutamine synthetase-glutamate synthase pathway. EDA represses heterocyst formation and nitrogenase activity but this is reversed by l-methionine-dl-sulphoximine.The authors are with the Department of Microbiology, Barkatullah University, Bhopal 462 026, India     

Altered control of glutamate dehydrogenases in ornithine utilization mutants of Pseudomonas aeruginosa

Two classes of ornithine-nonutilizing (oru) mutants of Pseudomonas aeruginosa PAO were investigated. Strains carrying the oru-310 mutation were entirely unable to grow on l-ornithine as the only carbon and nitrogen source and were affected in the assimilation of a variety of nitrogen sources (e.g., amino acids, nitrate). The oru-310 mutation caused changes in the regulation of the catabolic NAD-dependent glutamate dehydrogenase; this enzyme was no longer inducible by glutamate but instead could be induced by ammonia. The oru-310 locus was cotransducible with car-9 and tolA in the 10 min region of the chromosome. An oru-314 mutant was severely handicapped in ornithine medium but could grow when a good carbon source was added; the mutant also showed pleiotropic growth effects related to nitrogen metabolism. The oru-314 mutation affected the regulation of the anabolic NADP-dependent glutamate dehydrogenase, which was no longer repressed by glutamate but showed normal derepression in the presence of ammonia. The oru-314 locus was mapped by transduction near met-9011 at 55 min. Both oru mutants could grow on l-glutamate, l-proline, or l-ornithine amended with 2-oxoglutarate, albeit slowly. We speculate that insufficient 2-oxoglutarate concentrations might account, at least in part, for the Oru^- phenotype of the mutants.     

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.     

Carbon and nitrogen utilization and acid production by mycelia of the ectomycorrhizal fungusTricholoma bakamatsutake in vitro

Mycelial growth of an isolate ofT. bakamatsutake was tested in media with C/N ratio ranging from 0 to 50 and with 32 carbon and 12 nitrogen sources. The isolate grew best at the C/N ratio of 30. It utilized the monosaccharidesd-glucose,d-mannose, andd-fructose, the disaccharide trehalose, and polysaccharide pectin among the carbon sources; and yeast extract,l-glutamic acid, and ammonium compounds among the nitrogen sources. The growth of ten isolates and secretion of gluconic and oxalic acids were compared ind-glucose, trehalose, and pectin media. The utilization ofd-glucose, trehalose, and pectin differed among the ten isolates, but all the isolates secreted gluconic acid in thed-glucose media and oxalic acid in the pectin media.     

Nutritional requirements ofXanthomonas phaseoli var.fuscans

As found by Starr (1946),l-glutamic acid is necessary for the growth ofXanthomonas phaseoli var.fuscans. According to our results, the growth is stimulated byl-asparagine in the presence ofl-glutamic acid;l-asparagine itself, however, does not serve as a source of carbon and nitrogen.Xanthomonas phaseoli var.fuscans grew well in a medium containing tryptone. Some peptides of the acidic fraction isolated from tryptone affected the growth as much as tryptone itself. Vitamins and plant growth substances did not affect the growth of the bacteria; proteins appeared to be a poor carbon and nitrogen source. On substituting glucose in a glutamic acid-containing medium with another saccharide, the growth of the bacteria was found equal or better in media containing mannose, sucrose, fructose, maltose or starch. The bacteria grew less satisfactorily in media containing galactose and cellobiose as compared with media containing glucose.     

Effect of nitrogen concentration on the activity of manganese peroxidase ofPhanerochœte chrysosporium

Manganese peroxidase as an extracellular enzyme is produced by the white rot fungusPhanerochœte chrysosporium under nutrient nitrogen or carbon limitation. The effect of nitrogen concentration on the activity of manganese peroxidase was studied using ammonium nitrate andl-asparagine as nitrogen sources. The highest activity of the enzyme was observed in cultures grown in a medium containing 75 mg/L ammonium nitrate and 0.15 g/Ll-asparagine. Manganese peroxidase was not detectable in cultures grown in the presence 0.5 g/L ammonium nitrate and 1 g/Ll-asparagine.     

Biochemical properties of yeast l-asparaginase

Only a single l-asparaginase has been found in the yeast Saccharomyces cerevisiae. The enzyme is synthesized constitutively, and its functioning is not controlled by the products of its activity. The apparent K_m for the yeast l-asparaginase reaction is 2.5×10^–4 m. Activity is greatest at pH 8.5 and is unaffected by the ionic strength of reaction mixtures. l-Asparagine can serve as the sole nitrogen source for cell metabolism but cannot serve as the sole supply of carbon. Active l-asparaginase is necessary for the use of l-asparagine as a nitrogen donor for cell growth. This requirement suggests a possible way in which l-asparaginase-deficient strains of yeast or other organisms might easily be selected.G.E.J. was supported by U.S. Public Health Service Predoctoral Fellowship No. 5 F01 GM36,437.     

Studies on Aspergilli XVI. Effect of pH,temperature, and carbon and nitrogen interaction

Summary The effect of pH, temperature, and carbon and nitrogen interaction on the growth and sporulation ofAspergillus nidulans (Eidam)Wint.,A. rugulosus Thom &Raper,A. variecolor (Berk. &Br.)Thom &Raper andA. quadrilineatus was studied. All the moulds could grow on a wide range of pH (2.0 to 12.0) but the growth was poor on too acid and too alkaline media. Best growth ofA. rugulosus, A. quadrilineatus, andA. violaceus was seen at pH 6.5 and that ofA. nidulans andA. variecolor at pH 7.0. In general maximum production of perithecia was recorded between pH 6.0 and 8.0.All the above species ofAspergillus under study could grow between a temperature range of 10° C–48° C, but the growth was poor at 10° C and 48° C. The present moulds showed good growth at 20° C, 25°C, and 30° C. At 40° CA. nidulans andA. rugulosus showed moderate growth while the rest of the Aspergilli attained good growth. Temperatures between 20° C–30° C favoured excellent perithecial production.In general, little improvement in growth was noted on media containing good carbon and nitrogen sources. Malic acid was found to be useless when supplied singly. But, poor growth was recorded when supplied in combination with amino acids, amide, and peptone. This was due to the fact that these N sources also supplied carbon for their metabolism.     

An asparaginase of Aspergillus nidulans is subject to oxygen repression in addition to nitrogen metabolite repression

Summary Of five amidohydrolase activities subject to nitrogen metabolite repression in Aspergillus nidulans, l-asparaginase shows clearest evidence of also being subject to repression by atmospheric oxygen. Such oxygen repressibility is only evident under nitrogen metabolite derepressed conditions. Asparaginase levels are also considerably elevated by areA300, an altered function allele of the positive acting wide domain regulatory gene areA mediating nitrogen metabolite repression and are drastically reduced by loss of function mutations in areA. A. nidulans has two l-asparaginase enzymes and it has been shown by the use of appropriate mutants that these regulatory effects are exerted on the expression of that specified by the ahrA gene but probably not that specified by the apnA gene. Present address: (until 25 August, 1988) Department of Genetics, University of Georgia, Athens, GA 30602, USA     

Regulation of differentiation of the dimorphic fungusPaecilomyces viridis by nitrogen sources,antibiotics and metabolic inhibitors

The effect of nitrogen and carbon sources, vitamins, antibiotics and metabolic inhibitors on growth and differentiation ofPaecilomyces viridis was investigated. Sodium nitrate,l-asparagine,l-proline and peptone were found to be suitable nitrogen sources for mycelial growth (M) in a synthetic medium with glucose.Paecilomyces viridis could also grow slowly in a synthetic medium containing benzylpenicillin or bacitracin as the only nitrogen sources and very slowly even in a medium with polymyxin as the nitrogen source. Ammonium salts, area,l-arginine,d, l-aspartic acid andl,-serine were found to support intensive sporulation. Partially yeast-like growth (Y) was facilitated by NaNO₂, (NH₄)₂SO₄, NH₄NO₃, urea,d, l-alanine,l-arginine,d, l-aspartic acid,l-cysteine,l-glutamic acid andl-serine. Partially yeastlike growth could be observed in a medium with peptone and at an initial pH of 2. The following compounds appear as suitable carbon sources for mycelial growth:d-glucose,d-galactose,d-mannose, maltose, sucrose, chitin andd-mannitol. No changes in morphology could be detected on any of the 25 used carbon sources in a synthetic medium with NaNO₃. Yeast-like growth was induced by the antibiotics azalomycin F, cyanein (brefeldin A), griseofulvin and monorden (radicicol). After removal of the antibiotics, mycelial growth was restored. Sporulation was stimulated by chloramphenicol, 2-deoxy-d-glucose, furancarboxylic acid and stipitatic acid. Deformation of phialides was observed after treatment with actinomycin D, amphotericin B, boromycin, citrinin, cycloheximide, cytochalasin D, fungicidin and scopathricin. Microcyclic conidiation or growth of phialides directly from conidia were induced by cycloheximide, desertomycin, ethidium bromide and 5-fluorouracil.     

Characterization of nitrogen-fixing bacteria from a temperate saltmarsh lagoon,including isolates that produce ethane from acetylene

Nitrogen-fixing bacteria were isolated from sediments and water of a saltmarsh lagoon on the west coast of South Africa, and characterized according to factors that regulate nitrogen fixation in the marine environment. The majority of isolates were assigned to the Photobacterium or Vibrio genera on the basis of physiological and biochemical characteristics. One isolate was further assigned to the species Vibrio diazotrophicus. Carbohydrate utilization by each diazotrophic isolate was examined. Abilities of the isolates to utilize a range of mono-, di-, and polysaccharides largely reflected the predicted availability of organic carbon and energy in the lagoon, except that chitin was not utilized. Biochemical tests on the utilization of combined nitrogen showed that one isolate could utilize nitrate, and that this strain was susceptible to full repression of nitrogenase activity by 10mm nitrate. Urease activity was not detected in any of the isolates. In the absence of molybdenum two of the isolates, a Photobacterium spp. and V. diazotrophicus, reduced acetylene to ethylene and ethane, a property frequently associated with the activity of alternative nitrogenases. Addition of 25µM molybdenum inhibited ethane production by V. diazotrophicus, but stimulated ethylene and ethane production by the Photobacterium isolate. Addition of 28µM vanadium did not appear to regulate ethane production by either strain. Assays of nitrogenase activity in sediments from which some isolates were obtained indicated that molybdenum was not limiting nitrogenase activity at naturally-occurring concentrations. Southern hybridizations of the chromosomes of these strains with the anfH and vnfH genes of Azotobacter vinelandii and the nifH gene of Klebsiella pneumoniae indicated the presence of only one nitrogenase in these isolates.Correspondence to: B.J. Tibbles.     

Effect of methionine-sulfoximine on nitrate uptake and photosynthesis in the cyanobacteriumAnabaena doliolum Bharadwaja

l-Methionine-dl-sulfoximine (MSX) stimulated nitrate uptake but inhibited¹⁴CO₂ fixation and O₂ evolution inAnabaena doliolum. Nitrate uptake was inhibited by ammonium (NH ₄ ⁺ ) in the absence of MSX, but not in the presence of MSX. Glutamine or a derivative of it appears to be the actual negative effector of nitrate utilization. In presence of nitrate, MSX-treated cells ofA. doliolum evolve more O₂ than do untreated cells. Our results suggest a close relation between photoassimilation of carbon and utilization of nitrogen.     

d-Pentose metabolism byBacteroides vulgatus strain 8482

Bacteroides vulgatus strain 8482 metabolizedd-arabinose by a mechanism involving a 32 (top to bottom) cleavage of the arabinose carbon skeleton. During growth in the presence of 1-¹⁴C-d-arabinose, acetate, propionate, and succinate were labeled, but during growth in the presence of 5-labeledd-arabinose, only labeled acetate and succinate were formed. The metabolism ofd-ribose by strain 8482 differed from that ford-arabinose. Strain 8482 converted glycolic acid and glycine to acetate and succinate, but not propionate, by a mechanism involving cleavage of the glycine and glycolic acid carbon skeletons and equilibration of carbons 1 and 2 of glycolic acid and glycine with nonequivalent metabolic pools. The metabolism ofd-arabinose,d-ribose,d-glycine, andd-glycolic acid by strain 8482 was similar, in some respects, to that ofBacteroides fragilis strain 2044, but differed substantially from the metabolism of the same substances byBacteroides ruminicola strain B₁4.     

Biosynthesis of a trypanocide by Chromobacterium violaceum

Radio-isotope studies indicated not only that l-tryptophan can serve as carbon source for synthesis of the trypanocide, violacein by Chromobacterium violaceum (BB-78 strain) but also that isatin and indole 3-acetic acid are both important metabolic intermediates. Using 3-indolyl [2-¹⁴C] and [1-¹⁴C] acetic acid, it was found that the carboxylic carbon was not eliminated and that indole-3-acetic acid was incorporated intact into the pigment structure. N-Ethyl(5-hydroxy-indol-3-yl)-2-indolylethylamide is also an important metabolic intermediate in the violacein biosynthesis. This is the first report of a metabolic scheme for violacein synthesis which includes an intermediate other than l-tryptophan.     

Utilization of benzylpenicillin as carbon,nitrogen and energy source by a Pseudomonas fluorescens strain

A bacterium which utilizes benzylpenicillin as carbon, nitrogen and energy source was isolated from a lake sediment. The organism was identified as a strain of Pseudomonas fluorescens with a GC content of 59.71 Mol %. After growth of the organism on a mineral salts medium containing benzylpenicillin, the derivatives benzylpenicilloic acid, benzylpenilloic acid and benzylpenicillenic acid were found in culture media. There was no indication that the phenylacetate side chain of benzylpenicillin is decomposed. In uninoculated culture media benzylpenicillin, benzylpenicilloic acid and benzylpenicillenic acid were demonstrable. The following compounds were found to be absent from inoculated or uninoculated culture fluids: d-penicillamine, l-valine, l-cysteine, benzylpenillic acid and 6-aminopenicillanic acid. The organism possesses penicillinase. Penicillin acylase was not demonstrable. The reaction product of penicillinase, benzylpenicilloic acid, supports only little growth. There is no growth on 6-aminopenicillanic acid with or without NH₄Cl. Relatively little growth occurs on 6-aminopenicillanic acid in the presence of phenylacetic acid.The data indicate that the nucleus of the benzylpenicillin molecule is utilized as carbon, nitrogen and energy source. During growth a part of the substrate is destroyed into scarcely usable benzylpenicilloic acid; hereby the antibiotic is detoxified.Abbreviations TLC thin-layer chromatography - DNPH 2,4-dinitrophenylhydrazine     

Differential uptake of fumarate by Candida utilis and Schizosaccharomyces pombe

The dicarboxylic acid fumarate is an important intermediate in cellular processes and also serves as a precursor for the commercial production of fine chemicals such as l-malate. Yeast species differ remarkably in their ability to degrade extracellular dicarboxylic acids and to utilise them as their only source of carbon. In this study we have shown that the yeast Candida utilis effectively degraded extracellular fumarate and l-malate, but glucose or other assimilable carbon sources repressed the transport and degradation of these dicarboxylic acids. The transport of both dicarboxylic acids was shown to be strongly inducible by either fumarate or l-malate while kinetic studies suggest that the two dicarboxylic acids are transported by the same transporter protein. In contrast, Schizosaccharomyces pombe effectively degraded extracellular l-malate, but not fumarate, in the presence of glucose or other assimilable carbon sources. The Sch. pombe malate transporter was unable to transport fumarate, although fumarate inhibited the uptake of l-malate. Received: 15 March 2000 / Received revision: 4 July 2000 / Accepted: 9 July 2000     

Conversion of L-Hydroxyproline to glutamate by extracts of strains of Pseudomonas convexa and Pseudomonas fluorescens

Summary Three strains of Pseudomonas convexa and three strains of Pseudomonas fluorescens were found able to utilize L-hydroxyproline as sole source of carbon and nitrogen. Sonic extracts of these organisms converted L-hydroxyproline to glutamic acid.     

Molecular analysis of inorganic nitrogen assimilation in yeasts

Assimilation of nitrate and various other inorganic nitrogen compounds by different yeasts was investigated. Nitrate, nitrite, hydroxylamine, hydrazine, ammonium sulphate, urea and L-asparagine were tested as sole sources of nitrogen for the growth of Candida albicans, C. pelliculosa, Debaryomyces hansenii, Saccharomyces cerevisiae, C. tropicalis, and C. utilis. Ammonium sulphate and L-asparagine supported the growth of all the yeasts tested except D. hansenii while hydroxylamine and hydrazine failed to support the growth of any. Nitrate and nitrite were assimilated only by C. utilis. Nitrate utilization by C. utilis was also accompanied by the enzymatic activities of NAD(P)H: nitrate oxidoreductase (EC 1.6.6.2) and NAD(P)H: nitrite oxidoreductase (EC 1.6.6.4), but not reduced methyl viologen-or FAD-nitrate oxidoreductases (EC 1.7.99.4). It is demonstrated here that nitrate and nitrite reductase activities are responsible for the ability of C. utilis to assimilate primary nitrogen.