Associative cellulolysis and N2 fixation by co-cultures of Trichoderma harzianum and Clostridium butyricum: the effects of ammonium-N on these processes

Mixed cultures of the cellulolytic fungus Trichoderma harzianum with the anaerobic diazotroph Clostridium butyricum were shown to co-operatively degrade cellulose and utilize the degradation products for N₂ fixation. Cellulose degradation and N₂ fixation were stimulated by small (0.1 mg/ml) additions of (NH₄)₂SO₄. The (NH₄₂SO₄ stimulates cellulolysis thereby increasing the supply of cellulose degradation products to the diazotroph. In aerobic environments the anaerobe depends on the respiration of the aerobe to create anaerobic microsites. The N source increased O₂ uptake by the fungus increasing the number of sites suitable for the development of the anaerobe. Stimulation in the growth of T. harzianum by (NH₄₂SO₄ resulted in increased growth and N₂ fixation by Cl. butyricum.     

Serum Proteins Promoting [3H]Thymidine Uptake by Trypanosoma (Schizotrypanum) cruzi (Chagas) in vitro

SYNOPSIS. Five proteins capable of stimulating [³H]thymidine uptake by Trypanosoma cruzi in vitro were isolated from fetal calf serum by (NH₄)₂SO₄ precipitation and ion exchange column chromatography. The proteins were partially characterized by immunodiffusion, immunoelectrophoresis, polyacrylamide gel disc electrophoresis, and SDS electrophoresis. As estimated by SDS electrophoresis, using 4 standards, the molecular weight of protein 1 was 100,000, that of protein 2 was 76,000. and that of proteins 3–5 was 68,000 daltons.     

Variations in the amino acid composition of xylem sap of Betula pendula Roth. trees due to remobilization of stored N in the spring

Seasonal patterns of N translocation in the xylem sap of Betula pendula were studied, to determine whether specific amino acids were recovered in spring as a consequence of N remobilization. Seedlings were grown in sand culture and provided with ¹⁵NH₄¹⁵NO₃ (at 2·2 atom percent excess) for one growing season. The following winter dormant trees were transplanted into fresh sand and given N at natural abundance thereafter. Destructive harvests were taken during bud burst and leaf growth to determine the pattern of ¹⁵N remobilization and N uptake, along with isolation of xylem sap for analysis of their amino acid profiles and ¹⁵N enrichment by GC-MS. ¹⁵N remobilization occurred immediately following bud burst, while N derived from root uptake did not appear in the leaves until 12 d after bud burst. During N remobilization there was a 10-fold increase in the concentration of N in the xylem sap, due predominantly to increases in citrulline and glutamine. The ¹⁵N enrichment of these two amino acids demonstrated the increase in their concentration in the xylem sap following bud burst was due to N remobilization. These results are discussed in relation to measuring N remobilization and storage capacity of trees in the field.     

Time-course of inorganic nitrogen uptake and incorporation by natural populations of freshwater phytoplankton

SUMMARY. 1. Time-course measurements of NH₄⁺ and NO₃⁻uptake were made on the natural phytoplankton populations in a eutrophic lake at a time when these nutrients were at their lowest annual concentration.
2. Both NH₄⁺ and NO₃⁻ uptake was increased at least five-fold during the first 5 min of incubation following near saturating pulses of these nutrients.
3. Elevated uptake was also observed following low level (∼2μg N 1⁻¹) pulses of NH₄⁺ and NO₃⁻, but substrate depletion during the first hour of incubation may have been partially responsible for this apparent enhancement.
4. Incorporation of ^I5N into TCA-insoluble material (protein) following the saturating NH₄⁺ pulse was increased less than total cellular ¹⁵N uptake, whereas no elevation of ¹⁵N incorporation into protein was observed following a saturating NO₃⁻pulse.
5. The percentage of ^I5N incorporated into protein, with respect to total cellular uptake, was ∼32% and ∼12% for NH₄⁺ and NO₃⁻, respectively, following 5 h of incubation.     

Nitrogen regulation of erythromycin formation in Streptomyces erythreus

Abstract Erythromycin formation decreased in Streptomyces erythreus as a function of the ammonium concentration present in the medium. Total inhibition of synthesis was obtained with 100 mM NH₄Cl but medium pH and culture growth were not significantly affected. A similar effect was obtained with NH₄NO₃ or (NH₄)₂SO₄ indicating that ammonium ion probably repressed formation of antibiotic.     

Reduced Development of Grey Mould (Botrytis cinerea) in Bean and Tomato Plants by Calcium Nutrition

Fertilization of bean plants grown in perlite with 1 and 3 mM CaCl₂ or Ca(NO₃)₂ reduced severity of grey mould as compared with control plants or plants fertilized with 5 mM of the compounds. Fertilization with Ca(NO₃)₂ reduced severity leaf grey mould and fruit ghost spots of tomato plants grown in perlite by 70 and 45%, respectively. The rate of decrease varied with the position of the fruits on the plants. Leaves from plants treated with calcium or otherwise [KNO₃, (NH₄)₂SO₄] produced less ethylene than leaves of nontreated plants. Rate of growth of B. cinerea was lower on growth medium prepared from washings from leaves of calcium fertilized plants than from leaves from other treatments. The fertilizer combination Ca(H₂PO₄)₂+ CaSO₄ (1 and 3 g/kg soil) applied once to tomato plants grown in soil reduced severity of leaf grey mould by 80 % (significant at P = 0.05) but 1–3 g CaSO₄/kg soil only tended to reduce disease severity (30–40 %, not significant) as compared with the control. The compounds CaCl₂ and Ca(NO₃)₂ increased significantly ( P = 0.05) the growth of B. cinerea on synthetic medium when applied at rates of 1 0–10.0 mM whereas reduction of growth was observed with 0.1 mM of the compounds and of CaSO₄.     

Characterization of cyclic nucleotide phosphodiesterase activity in Phaseolus vulgaris

Cyclic nucleotide phosphodiesterase (3',5'-cyclic nucleotide nucleotidohydrolase, EC 3.1.4.17) activity isolated from Phaseolus vulgaris L. cv. Limberg seedlings was partially purified and characterized by fractional (NH₄)₂SO₄ precipitation, DEAE-cellulose chromatography, chromatography on 3',5'-cAMP-agarose, gel permeation chromatography and chromatofocusing. A crude enzyme preparation, a 30–65% (NH₄)₂SO₄ pellet, showed an acidic pH optimum. The enzyme activity was stimulated by imidazole and divalent cations such as Ca²⁺, Mg²⁺ and Mn²⁺, whereas NaF, PP_i and Fe³⁺ were inhibitory. Isobutylmethylxanthine had no significant effect on the plant enzyme. An M_I of 42 000 was estimated by gel permeation high performance liquid chromatography. By chromatography on 3',5'-cAMP-agarose a phosphodiesterase was resolved that produced 5'-AMP as sole reaction product.     

Culture conditions affect the chemical composition of the exopolysaccharide synthesized by the fungus Aureobasidium pullulans

Aims:  To identify if culture conditions affect the chemical composition of exopolysaccharide (EPS) produced by Aureobasidium pullulans .
Methods and Results:  In batch airlift and continuously stirred tank (CSTR) reactors the EPS produced with low (0·13 g l⁻¹ N) initial NaNO₃ or (NH₄)₂SO₄ levels contained pullulan, with maltotriose as its major component, similar to that synthesized in the airlift reactor with high (0·78 g l⁻¹ N) initial NaNO₃ levels. EPS produced by CSTR grown cultures with high (NH₄)₂SO₄ levels contained little pullulan, possibly because of a population shift from unicells to mycelium. This chemical difference may explain why total EPS yields did not fall as they did with cultures grown under identical conditions with high NaNO₃ levels, where the pullulan component of the EPS disappeared. EPS synthesized in N-limiting chemostat cultures of A. pullulans changed little with growth rate or N source, being predominantly pullulan consisting of maltotriose units.
Conclusions:  While the EPS chemical composition changed little under N-limiting conditions, high initial medium N levels determined maltotriose content and/or pullulan content possibly by dictating culture morphology.
Significance and Impact of the Study:  These results emphasize the requirement of all studies to determine EPS chemical composition when examining the influence of culture conditions on EPS yields.     

Purification and N-terminal amino acid sequence of Enterocin CRL 35, a 'pediocin-like' bacteriocin produced by Enterococcus faecium CRL 35

M.E. FARÍAS, R.N. FARÍAS, A.P. DE RUIZ HOLGADO AND F. SESMA. 1996. Enterocin CRL 35, a bacteriocin produced by Enterococcus faecium CRL 35 that inhibits food-borne pathogens, was purified by precipitation with (NH₄)₂SO₄, gel filtration, ion exchange and reverse phase chromatography. The partial N-terminal amino acid sequence indicated a strong homology with other 'pediocin-like bacteriocins' previously described.     

Effect of changes in nitrogen nutrition on the polyamine content of Alnus glutinosa

Abstract. The principal polyamines in Alnus glutinosa roots, nodules and root pressure sap, putrescine, spermidine and spermine, were quantified by reversed-phase, high-performance liquid chromatography with fluorescence detection following precolumn derivatization with 9-fluorenylmethyl chloroformate and 1-ada-mantanamine. Putrescine was the major component of all tissues and sap. It comprised 70% or more of the polyamine pool except in roots of KNO₃-fed plants, in which similar amounts of putrescine and spermidine were present at levels five-fold lower than plants fed (NH₄)₂SO₄. Polyamine levels in nodules were 50% greater than in roots. The polyamine content of roots and nodules was not altered significantly when the nitrogen nutrition was changed from sole reliance on nitrogen fixation to partial or complete utilization of (NH₄)₂SO₄. However, the polyamine content of root pressure sap from nodulated plants increased almost four-fold when they were fed with increasing concentrations of NH₄NO₃, although the total polyamine content remained low (5mmol m⁻³ sap). The polyamine content of the Alnus root system changed with plant age. In particular, the spermidine content of both roots and nodules was higher in 10- as compared to 16-week-old plants.     

Changes in leaf water status associated with salinity in mature, field grown Prunus salicina

Seasonal and diurnal measurements of leaf water potential (ψ₁), relative water content (RWC) and stomatal conductance (g_s) were made in the field on 19-year old Prunus salicina (L.) cv. Santa Rosa, a deciduous fruit tree species, irrigated with 3 different concentrations of saline water over a 3 year period (1985-1987). With the exception of stage III of fruit growth, little or no treatment difference in Φ₁, leaf turgor potential (Φ_p), or RWC was noted during the day. Seasonal averages of morning (0700-0900) and afternoon (1500-1700) Φ_p did not decline with increasing salinity, indicating long-term osmotic adjustment in this species. Maintenance of leaf water status under saline conditions was in part a consequence of increased stomatal closure, with a subsequent reduction in leaf transpiration rate. However, during stage III of fruit growth, an increase in mean afternoon (1200-1700) stomatal conductance of 26-117%, independent of salinity treatment, was observed in 1985 and again in 1987. Higher conductance values during this period may be associated with rapid fruit expansion and greater assimilate demand. The observed increase in conductance resulted in greater leaf water loss and larger measured differences in midday ψ₁ between salinity treatments. This research indicates that for Prunus salicina in the field, salinity stress resulted in leaf water deficits only during the final period of fruit expansion and ripening.     

Possible evidence for novel metabolism of nitrogen gas by a green alga

We report the isolation of a cukaryotic green alga ( Chlorella , strain WPI-2) which accumulates large stores of nitrogen (N) during growth in N-free medium and seems to incorporate¹⁴N₂, yet does not reduce acetylene to ethylene. Total N accumulation during growth on N-free medium and in gases free of combined N was measured by three methods: Kjeldahl, oxidative pyrolysis via chemiluminescence (Antek N analyzer), and Dumas (Coleman N analyzer). Increases in N ranging from 22–64%± 1% were observed. Isotope dilution studies using cells labelled with ¹⁵NO ₃- and then shifted to ¹⁴N₂ in N-free medium showed dilution of the ¹⁵N isotope by ¹⁴N from 5.67 to 5.32%± 0.05%. Using a variety of conditions, we were unable to demonstrate the reduction of acctylene to ethylene by WPI-2, although diazotrophic cyanobacteria gave positive results. Although the data on WPI-2 are not conclusive in establishing this alga as a diazotroph, the data do suggest that within the Chlorophyceae there may exist a novel form of nitrogen gas metabolism.     

Effect of pH, temperature and culture medium composition on the production of an extracellular cysteine proteinase by Micrococcus sp. INIA 528

Growth and proteinase production by Micrococcus sp. INIA 528 in a batch-operated laboratory fermentor were investigated, with trypticase soy broth as the basal medium for studies on optimum temperature, pH and medium composition. Maximum growth was recorded at 34°C and pH 715, whereas optimum temperature and pH for proteinase production were 31°C and pH 6.25. Maximum rate of enzyme production occurred during the late log and early stationary phases of growth. Addition of 5.0 g 1^-1 yeast extract, 1.0 g 1^-1 glucose, 1.0 g 1^-1 MgSO₄ or 1.0 g 1^-1 K₂HPO₄ to basal medium resulted in a lower enzyme yield, but supplementation of basal medium with 2.5 g 1^-1 (NH₄)₂SO₄ increased enzyme production by 45%. A high initial biomass added to fresh broth supplemented with 2.5 g 1^-1 (NH₄)₂SO₄ only increased enzyme activity by 19%, compared to the maximum enzyme activity achieved with the standard inoculum.     

The presence of two types of β-cyanoalanine synthase in germinating seeds and their responses to ethylene

Germinating seeds of many species contain two types of β-cyanoalanine synthase (CAS, EC 4.4.1.9) that convert HCN to β-cyanoalanine. One is cytoplasmic CAS (cyt-CAS), which is precipitated by 50 to 60% (NH₄)₂SO₄ and has a pH optimum of 10.5. Cytoplasmic CAS is present at high levels in dry seed and its activity does not increase during imbibition. The activity of cyt-CAS is not affected by exogenously applied ethylene (C₂H₄), except in rice ( Oryza sativa cv. Sasanishiki). The second type of CAS found in seed is mitochondrial CAS (mit-CAS), which is precipitated by 60 to 70% (NH₄)₂SO₄ and has a pH optimum of 9.5. Mitochondrial CAS is present at low levels in dry seed, and its activity increases greatly during imbibition in the seeds of all species tested. Exposure to C₂H₄ stimulated mit-CAS activity in seeds of rice, barley ( Hordeum vulgare cv. Hadakamugi). cucumber ( Cucumis sativus cv. Kagafushinari) and cocklebur ( Xanthium pennsylvanicum ). The increase in the mit-CAS activity in cocklebur in response to C₂H₄ commenced alter a lag period of 2 to 3 h when the duration of soaking was short (16 h), but commenced without a lag period when the seeds were soaked for three months. Application of both chloramphenicol and cycloheximide to the axial and cotyledonary tissues of cocklebur seeds strongly inhibited growth as well as the increase in mit-CAS activity. It is postulated that the mit-CAS is synthesized de novo during imbibition and that its activity is regulated by C₂H₄, CO₂ which also promotes seed germination in some species, was ineffective m stimulating mit-CAS activity in cocklebur seeds.     

Synthesis of glutamate by mitochondria – An anaplerotic function for glutamate dehydrogenase

The photorespiratory nitrogen cycle was initially thought to be a closed cyclic process. If this were true the loss of glutamate, glutamine, serine or glycine to other processes, such as protein synthesis or export from the leaves, would not be possible in a stoichiometric sense. However, recent studies with [¹⁵N]-labeled amino acids show that there are alternative sources of nitrogen for photorespiration, indicating that the nitrogen cycle is not a closed cyclic system. In addition recent work with ¹⁵NH₄Cl and [¹⁵N]-glycine and a metabolically competent mitochondria system has shown that glutamate is synthesized in the mitochondria. Hence the glutamate dehydrogenase (GDH, EC 1.4.1.2) in mitochondria could also be active in the reassimilation of NH₄. We would like to propose that one function of mitochondrial GDH is to synthesize glutamate from some of the NH₄ released by photorespiration and that this glutamate represents a reserve for use in biosynthetic reactions.     

Amylase from Lactobacillus cellobiosus D-39 isolated from vegetable wastes: Purification and characterization

An organism producing α-amylase and identified as Lactobacillus cellobiosus D-39 was recently isolated from vegetable wastes. Its amylase was purified by (NH₄)₂SO₄ precipitation and DEAE cellulose column chromatography and was obtained in crystalline form. It was fairly stable at a broadly neutral pH range and maximally active at 50°C. The molecular weight was 22 500 daltons as determined by SDS gel electrophoresis.     

Nitrogen concentration, ammonium/nitrate ratio and NaCl interaction in vegetative and reproductive growth of peanuts

Peanuts ( Arachis hypogaea L. cv. Shulamit) grown with NO₃⁻ and saline water in hydroponics responded positively to addition of nitrogen (N) in their vegetative growth, but not in desert dune sand. In order to clarify these conflicting results, peanut plants were grown in a greenhouse pot experiment with fine calcareous sand. The nutrient solution contained 0 or 50 m M NaCl and 2 or 6 m M N in the form of Ca(NO₃)₂, NH₄NO₃ or (NH₄)₂SO₄. Three replicates were harvested after 48 days (beginning of reproductive stage) and three after 109 days (pod filling). In addition, gynophores were treated with 0, 50, 100, 150 or 200 m M NaCl outside the growth pot to check their sensitivity to salt. Shoot dry weight became greater with increasing NH₄⁺/NO₃⁻ ratio. Increasing the N concentration from 2 to 6 m M did not change shoot dry weight of the NH₄NO₃ or NH₄⁺-fed plants, but caused a reduction in shoot dry weight of NO₃⁻-fed plants. Shoot dry weight was not affected by increasing the NaCl concentration to 50 m M . Salt caused an increase in the number of gynophores per plant and a reduction of the mean pod weight. A NaCl concentration of 100 m M and above reduced gynophore vitality. It is concluded that the salt sensitivity of peanut plants resides mainly in the sensitivity of the reproductive organs.     

Translocation of amino acids from stem nodules of Sesbania rostrata demonstrated by GC-MS in planta 15N isotope dilution

We report a novel use of the ¹⁵N dilution technique to detail the translocation of amino compounds in the legume Sesbania rostrata . The conventional ¹⁵N dilution technique follows the dilution of ¹⁵N within a labelled plant, as ¹⁴N₂ is fixed by symbiotic bacteria. In our experiments, stem-nodulated Sesbania rostrata were enriched by feeding with ¹⁵N ammonium nitrate for 2 weeks, followed by a 1 week period where the only N available to the plants was via nitrogen fixation of atmospheric N₂. We measured the composition, concentration and ¹⁵N enrichment of amino compounds in various plant tissues, both above and below the stem nodules, using GC-MS and isotopic abundance mass spectrometry techniques. Approximately 28% of the total N in the stem nodules was derived from internal plant sources. The ureides allantoic acid and allantoin were not abundant in xylem, leaf or nodule tissues. The amides asparagine and glutamine were the major export products from stem nodules although a wide range of other amino compounds are also synthesized. Amino acids within the nodules had a low level of enrichment, demonstrating that a small fraction (≈ 11%) was derived from outside the nodules, and significant cycling of N (28% of xylem N) through the root system was revealed by measurements of ¹⁵N distribution and amino acid concentrations.     

Assimilation of nitrate and ammonium by the Scots pine (Pinus sylvestris) seedling under conditions of high nitrogen supply

Seedlings of Scots pine ( Pinus sylvestris L.) were grown on perlite for 21 days under controlled conditions. Apart from the water control, KNO₃ (15 m M ), (NH₄)₂SO₄ (7.5 m M ), and NH₄NO₃ (15 m M ) were offered to study the effects of a high nitrogen supply on nitrogen assimilation. In some experiments 1.3 m M potassium was added to the basic ammonium solutions. In labelling studies nitrate and ammonium were 2.3 atom%¹⁵N-enriched. It was found that over the 21-day period approximately three times more ammonium-N was taken up than nitrate-N. However, nitrate and ammonium, applied simultaneously, were taken up to the same extent as if they were applied separately (additivity). The presence of K⁺ in the medium did not affect N-uptake. Among the soluble N-containing compounds nitrate, ammonium and 8 amino acids were quantified. It was found that assimilation of nitrate can cope with the uptake of NO⁻₃ under all circumstances. Neither free nitrate nor ammonium or amino acids accumulated to an extent exceeding the values of water-grown seedlings. On the other hand, in case of high ammonium supply considerably more nitrogen was taken up than could be incorporated into nonsoluble N-containing substance ('protein'). The remaining nitrogen was found to accumulate in intermediary storage pools (free NH₄⁺, glutamine, asparagine, arginine). Part of this accumulated N could be incorporated into protein when potassium was offered in the nutrient solution. It is concluded that potassium is a requirement for a high rate of protein synthesis not only in crop plants but also in conifers.     

Two tertiary branched tetraamines, N4-aminopropylnorspermidine and N4-aminopropylsperimidine, are the major polyamines in thermophilic eubacteria, Thermoleophilum

Abstract Two novel polyamines were found as major polyamines of Thermoleophilum album and Thermoleophilum minutum , which are Gram-negative eubacteria obligate for thermophily and n -alkane substrates. They were identified as tertiary branched tetraamines, N⁴-aminopropylnorspermidine ( tris (3-aminopropyl)amine) [NH₂(CH₂)₃N-((CH₂)₃NH₂)(CH₂)₃NH₂ or N(CH₂CH₂CH₂NH₂)₃] and N⁴-aminopropylspermidine [NH₂(CH₂)₃N((CH₂)₃NH₂)(CH₂)₄NH₂] by high-performance liquid chromatography and gas chromatography-mass spectrometry.