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.     

Nitrate assimilation in the forage legume Lotus japonicus L.

Nitrate assimilation in the model legume, Lotus japonicus, has been investigated using a variety of approaches. A gene encoding a nitrate-inducible nitrate reductase (NR) has been cloned and appears to be the only NR gene present in the genome. Most of the nitrate reductase activity (NRA) is found in the roots and the plant assimilates the bulk of its nitrogen in that tissue. We calculate that the observed rates of nitrate reduction are compatible with the growth requirement for reduced nitrogen. The NR mRNA, NRA and the nitrate content do not show a strong diurnal rhythm in the roots and assimilation continues during the dark period although export of assimilated N to the shoot is lower during this time. In shoots, the previous low NR activity may be further inactivated during the dark either by a phosphorylation mechanism or due to reduced nitrate flux coincident with a decreased delivery through the transpiration stream. From nitrate-sufficient conditions, the removal of nitrate from the external medium causes a rapid drop in hydraulic conductivity and a decline in nitrate and reduced-N export. Root nitrate content, NR and nitrate transporter (NRT2) mRNA decline over a period of 2 days to barely detectable levels. On resupply, a coordinated increase of NR and NRT2 mRNA, and NRA is seen within hours.     

Ammonium and nitrate uptake in gap,generalist and understory species of the genus Piper

Summary We studied root net uptake of ammonium (NH ₄ ⁺ ) and nitrate (NO ₃ ^– ) in species of the genus Piper (Piperaceae) under high, intermediate and low photosynthetically active photon flux densities (PFD). Plants were grown hydroponically, and then transferred to temperature controlled (25° C) root cuvettes for nutrient uptake determinations. Uptake solutions provided NH ₄ ⁺ and NO ₃ ^– simultaneously (both) or separately (single). In the first experiment, seven species of Piper, from a broad range of rainforest light habitats ranging from gap to understory, were screened for mineral nitrogen preference (100 M NH ₄ ⁺ and/or 100 M NO ₃ ^– ) at intermediate PFD (100 mol m^–2 s^–1). Preference for NH ₄ ⁺ relative to NO ₃ ^– , defined as the ratio of NH ₄ ⁺ (both):NO ₃ ^– (both) net uptake, was higher in understory species than in gap species. Ammonium repression of NO ₃ ^– uptake, defined as the ratio of NO ₃ ^– (single): NO ₃ ^– (both) net uptake, was also higher in understory species as compared to gap species. In a second set of experiments, we examined the effect of nitrogen concentration (equimolar, 10 to 1000 M) on NH ₄ ⁺ preference and NH ₄ ⁺ repression of NO ₃ ^– net uptake at high (500 mol m^–2 s^–1) and low (50 mol m^–2 s^–1) PFD in a gap (P. auritum), generalist (P. hispidum) and understory species (P. aequale). All species exhibited negligible NH ₄ ⁺ repression of NO ₃ ^– net uptake at high PFD. At low PFD, NH ₄ ⁺ preference and repression of NO ₃ ^– net uptake occurred in all species (understory > generalist > gap), but only at intermediate nitrogen concentrations, i.e. between 10 and 200 M. Ammonium repression of net NO ₃ ^– uptake decreased or increased rapidly (in < 48 h) after transitions from low to high or from high to low PFD respectively. No significant diurnal patterns in NO ₃ ^– or NH ₄ ⁺ net uptake were observed.CIWDPB publication # 1130     

Nitrate reductase and glutamine synthetase activities,nitrate and ammonia assimilation,in the unicellular alga Cyanidium caldarium

Nitrogen-limited continuous cultures of Cyanidium caldarium contained induced levels of glutamine synthetase and nitrate reductase when either nitrate or ammonia was the sole nitrogen source. Nitrate reductase occurred in a catalytically active form. In the presence of excess ammonia, glutamine synthetase and nitrate reductase were repressed, the latter enzyme completely. In the presence of excess nitrate, intermediate levels of glutamine synthetase activity occurred. Nitrate reductase was derepressed but occurred up to 60% in a catalytically inactive form.Cell suspensions of C. caldarium from nitrate- or ammonialimited cultures assimilated either ammonia or nitrate immediately when provided with these nutrients. In these types of cells, as well as in cells grown with excess nitrate, the rate of ammonia assimilation was 2.5-fold higher than the rate of nitrate assimilation. It is proposed that the reduced rate at which nitrate was assimilated as compared to ammonia might be due to regulatory mechanisms which operate at the level of nitrate reductase activity.     

Nitrate reductase activity in leaves and roots ofAlnus glutinosa (L) Gaertner

Summary Thein vivo nitrate reductase activity (NRA) was determined inAlnus glutinosa plants grown nonsymbiotically on ammonium, nitrate, a combination of both, or symbiotically with atmospheric nitrogen as the only nitrogen source. Root NRA was absent when ammonium or atmospheric nitrogen was the nitrogen source. With nitrate in the culture solution the roots showed a high NRA. However, the leaf NRA behaved quite differently: with negligible activities on all nitrogen sources except atmospheric nitrogen. The foliar NRA measured, however, is likely not due to the activity of the plant but of microbial origin. Methods commonly used to facilitate produced nitrite to leak out of the tissue, such as addition of propanol and cutting the plant material, did not increase the nitrite release from the leaves. A turbidity developed when testing the samples for nitrite which was positively correlated with the NRA. Populations of microorganisms in the phyllosphere did not differ between the nutritional treatments. Bacteria, able to grow on a low-nitrogen medium, were present on the leaves. Nitrifiers could not be detected. The bacteria on the leaves appear to produce nitrite when incubated with leaf material. Grassland Species Research Group, Publication no. 106     

Combined effects of endo- and exogenous trehalose on stress tolerance and biocontrol efficacy of two antagonistic yeasts

Effects of endo- and exogenous trehalose on viability of two antagonistic yeasts, Cryptococcus laurentii (Kuffer.) Skinner and Rhodotorula glutinis (Fresen.) Harrison, were investigated after being treated with rapid-freezing, slow-freezing and freeze-drying, respectively. The accumulation of intracellular trehalose in the two yeasts was induced by culturing the yeast cells in trehalose-containing medium, which significantly enhanced viabilities of both yeasts in the slow-freezing test. Trehalose, as an exogenous protectant, at the concentration of 5% or 10% could markedly increase survivals of the two yeasts when subjected to freeze-drying. When combined with exogenous trehalose as a protective substance, the yeasts containing high intracellular trehalose level showed higher viabilities as compared to those containing low levels under both freezing and freeze-drying stresses. The highest survival of C. laurentii and R. glutinis were 90% and 97% after freeze-drying, respectively, compared to 63% and 28% for the yeasts with lower intracellular trehalose levels. These results may be due to the fact that a combined effect occurred between endo- and exogenous trehalose of yeast cells. The combined effect on C. laurentii and R. glutinis also resulted in the highest level of biocontrol efficacy against blue mold in apple fruit caused by Penicillium expansum Link, and reduced the disease indexes to 45 and 56, respectively, compared to 94 and 81 in the untreated control. Meanwhile, the combination of endo- and exogenous trehalose significantly increased population of both yeasts in apple wounds, especially at the first 48 h after inoculation, which might explain the reason of the improvement in biocontrol effects of the two yeasts.     

S-formylglutathione: The substrate for formate dehydrogenase in methanol-utilizing yeasts

Formaldehyde dehydrogenase and formate dehydrogenase were purified 45- and 16-fold, respectively, from Hansenula polymorpha grown on methanol. Formaldehyde dehydrogenase was strictly dependent on NAD and glutathione for activity. The K _mvalues of the enzyme were found to be 0.18 mM for glutathione, 0.21 mM for formaldehyde and 0.15 mM for NAD. The enzyme catalyzed the glutathine-dependent oxidation of formaldehyde to S-formylglutathione. The reaction was shown to be reversible: at pH 8.0 a K _mof 1 mM for S-formylglutathione was estimated for the reduction of the thiol ester with NADH. The enzyme did not catalyze the reduction of formate with NADH. The NAD-dependent formate dehydrogenase of H. polymorpha showed a low affinity for formate (K _mof 40 mM) but a relatively high affinity for S-formylglutathione (K _mof 1.1 mM). The K _mvalues of formate dehydrogenase in cell-free extracts of methanol-grown Candida boidinii and Pichia pinus for S-formylglutathione were also an order of magnitude lower than those for formate. It is concluded that S-formylglutathione rather than free formate is an intermediate in the oxidation of methanol by yeasts.     

Nitrate induction in spruce: an approach using compartmental analysis

Using ¹³NO ₃ ^– -efflux analysis, the induction of nitrate uptake by externally supplied nitrate was monitored in roots of intact Picea glauca (Moench) Voss. seedlings over a 5-d period. In agreement with our earlier studies, efflux analysis revealed three compartments, which have been identified as surface adsorption, apparent free space, and cytoplasm. While induction of nitrate uptake was pronounced, NO ₃ ^– fluxes in induced plants were decidedly lower and the induction response was slower than in other species. Influx rose from 0.1 mol·g^–1·h^–1 (measured at 100 M [NO ₃ ^– _o) in uninduced plants to a maximum of 0.5 mol·g^–1h^–1 after 3 d of exposure to 100 M [NO ₃ ^– _o and declined to 0.3–0.4 mol·g^–1h^–1 at the end of the 5-d period. Efflux remained relatively constant around 0.02-0.04 mol·g^–1h^–1, but its percentage with respect to influx declined from initially high values (around 30%) to steady-state values of 4–7%. Cytoplasmic [NO ₃ ^– ] ranged from the low micromolar in uninduced plants to a maximum of 2 mM in plants fully induced at 100 M [NO ₃ ^– ]_o. In-vivo root nitrate reductase activity (NRA) was measured over the same time period, and was found to follow a similar pattern of induction as influx. The maximum response in NRA slightly preceded that of influx. It increased from 25 nmol·g^–1·h^–1 without prior exposure to NO ₃ ^– to peak values around 150 nmol· g^–1h^–1 after 2 d of exposure to 100 M [NO ₃ ^– ]_o. Subsequently, NRA declined by about 50%. The dynamics of flux partitioning to reduction, to the vacuole, the xylem, and to efflux during the induction process are discussed.The research was supported by an Natural Sciences and Engineering Research Council, Canada, grant to Dr. A.D.M. Glass and by a University of British Columbia Graduate Fellowship to Herbert J. Kronzucker. Our thanks go to Dr. M. Adam and Mr. P. Culbert at the particle accelerator facility TRIUMF on the University of British Columbia campus for providing ¹³N, to Drs. R.D. Guy and S. Silim for providing plant material, and to Dr. M.Y. Wang, Mr. J. Bailey, Mr. J. Mehroke and Mr. J. Vidmar for essential assistance in experiments.     

Nitrate reductase and its role in nitrate assimilation in plants

Nitrate reductase (EC 1.6.6.1) is an enzyme found in most higher plants and appears to be a key regulator of nitrate assimilation as a result of enzyme induction by nitrate. The biochemistry of nitrate reductase has been elucidated to a great extent and the role that nitrate reductase plays in regulation of nitrate assimilation is becoming understood.     

Glutamine synthetase of Chlamydomonas: its role in the control of nitrate assimilation

Work is described which suggests that glutamine synthetase (GS) could play an important and direct regulatory role in the control of NO₃ assimilation by the alga. In both steady-state cells and ones disturbed physiologically by changes in light or nitrogen supply the assimilation of NO₃ appears to be limited by the activity of GS. Moreover although in normal cells NH₃ can completely inhibit NO₃ uptake, promote the deactivation of nitrate reductase (NR) and repress the synthesis of NR and nitrite reductase (NIR), these controls are relaxed in cells in which GS is deactivated by treatment with L-methionine-DL-sulfoximine (MSO). It is proposed that the reversible deactivation of GS may play an important part in the regulation of NO₃ assimilation although it is still not clear whether the enzyme itself or products of its metabolism are responsible.Abbreviations GS glutamine synthetase - GS_s glutamine synthetase, synthetase activity - GS_t glutamine synthetase, transferase activity - NR nitrate reductase - NIR nitrite reductase - GDH glutamate dehydrogenase - CHX cycloheximide - MSO L-methionine-DL-sulfoximine - FAD flavine adenine dinucleotide     

The roles of nitrate and ammonium in the regulation of the development of nitrate reductase in Chlamydomonas reinhardii

The regulation of the development of nitrate reductase (NR) activity in Chlamydomonas reinhardii has been compared in a wild-type strain and in a mutant (nit-A) which possesses a modified nitrate reductase enzyme that is non-functional in vivo. The modified enzyme cannot use NAD(P)H as an electron donor for nitrate reduction and it differs from wild-type enzyme in that NR activity is not inactivated in vitro by incubation with NAD(P)H and small quantities of cyanide; it is inactivated when reduced benzyl viologen or flavin mononucleotide is present. After short periods of nitrogen starvation mutant organisms contain much higher levels of terminal-NR activity than do similarly treated wild-type ones. Despite the inability of the mutant to utilize nitrate, no nitrate or nitrite was found in nitrogen-starved cultures; it is therefore concluded that the appearance of NR activity is not a consequence of nitrification. After prolonged nitrogen starvation (22 h) the NR level in the mutant is low. It increases rapidly if nitrate is then added and this increase in activity does not occur in the presence of ammonium, tungstate or cycloheximide. Disappearance of preformed NR activity is stimulated by addition of tungstate and even more by addition of ammonium. The results are interpreted as evidence for a continuous turnover of NR in cells of the mutant with ammonium both stimulating NR breakdown and stopping NR synthesis. Nitrate protects the enzyme from breakdown. Reversible inactivation of NR activity is thought to play an insignificant rôle in the mutant.Abbreviations NR nitrate reductase - BV benzyl viologen     

Nitrate or ammonium nutrition in french bean

Summary Bean Plants were grown in a greenhouse in sand irrigated with nutrient solutions containing either 2 mM NO ₃ ^– or 2 mM NH ₄ ⁺ . After 45 days fresh weight of NH ₄ ⁺ plants was half that of NO ₃ ^– plants. Cation concentration in NH ₄ ⁺ plants was 30% less than in NO ₃ ^– plants. Amino acids (SER, ASN, GLN) accummulated 3 to 10 times more in NH ₄ ⁺ plants. The concentration of organic acids (malic, malonic, citric) was 10 to 30 times higher in NO ₃ ^– plants. The ATP-costings for the synthesis of amino acids and organic acids in NH ₄ ⁺ plants was half that of NO ₃ ^– ones: therefore it could not account for the reduction of growth in the ammonium-fed plants.     

Chemical and cytological changes during the autolysis of yeasts

Summary Cell suspensions ofSacharomyces cerevisiae, Kloeckera apiculata andCandida stellata were autolyzed in phosphate buffer, pH 4.5, for up to 10 days. Cell dry weights decreased by 25–35% after 10 days. Based on initial cell dry weight, the soluble autolysate consisted of: carbohydrate (principally polysaccharide) 3–7%; organic acids 3–6%; protein 12–13%; free amino acids 8–12%; nucleic acid products 3–5%; and lipids 1–12%. The main organic acids in autolysates were propionic, succinic and acetic and the main amino acids were phenylalanine, glutamic acid, leucine, alanine and arginine. Approximately 85–90% of cellular RNA and 25–40% of cellular DNA were degraded during autolysis. Both neutral lipid and phospholipid components were degraded, with neutral lipids but not phospholipids being found in autolysates. Scanning and transmission electron micrographs showed retention of cell wall structure and shape during autolysis, but there was extensive intracellular disorganization withinS. cerevisiae andC. stellata. There were differences in the autolytic behavior ofK. apiculata compared withS. cerevisiae andC. stellata.This paper is dedicated to Professor Herman Jan Phaff in honor of his 50 years of active research which still continues.     

Integrin-like proteins in Candida spp. and other microorganisms

The vertebrate integrins provide a paradigm for cell surface proteins involved in adhesion and morphogenesis. However, homologs of integrins have been found in more primitive organisms. This review will discuss the evidence for surface proteins in Candida albicans and Candida tropicalis that contain motifs reminiscent of integrins and will analyze the contributions of one of these proteins, Int1p, to adhesion, morphogenesis, and virulence. Other microorganisms thought to express integrin-like proteins will also be addressed.     

Nitrate deposition in northern hardwood forests and the nitrogen metabolism of Acer saccharum marsh

It is generally assumed that plant assimilation constitutes the major sink for anthropogenic Nitrate NO ₃ ^– deposited in temperate forests because plant growth is usually limited by nitrogen (N) availability. Nevertheless, plants are known to vary widely in their capacity for NO ₃ ^– uptake and assimilation, and few studies have directly measured these parameters for overstory trees. Using a combination of field and greenhouse experiments, we studied the N nutrition of Acer saccharum Marsh. in four northern hardwood forests receiving experimental NO ₃ ^– additions equivalent to 30 kg N ha^–1 year^–1. We measured leaf and fine-root nitrate reductase activity (NRA) of overstory trees using an in vivo assay and used ¹⁵N to determine the kinetic parameters of NO ₃ ^– uptake by excised fine roots. In two greenhouse experiments, we measured leaf and root NRA in A. saccharum seedlings fertilized with 0–3.5 g NO ₃ ^– –N m^–2 and determined the kinetic parameters of NO ₃ ^– and NH ₄ ⁺ uptake in excised roots of seedlings. In both overstory trees and seedlings, rates of leaf and fine root NRA were substantially lower than previously reported rates for most woody plants and showed no response to NO ₃ ^– fertilization (range = non-detectable to 33 nmol NO ₂ ^– g^–1 h^–1). Maximal rates of NO ₃ ^– uptake in overstory trees also were low, ranging from 0.2 to 1.0 mol g^–1 h^–1. In seedlings, the mean V _max for NO ₃ ^– uptake in fine roots (1 mol g^–1 h^–1) was approximately 30 times lower than the V _max for NH ₄ ⁺ uptake (33 mol g^–1 h^–1). Our results suggest that A. saccharum satisfies its N demand through rapid NH ₄ ⁺ uptake and may have a limited capacity to serve as a direct sink for atmospheric additions of NO ₃ ^– .     

Nitrate nutrition ofDeschampsia flexuosa (L.) Trin. in relation to nitrogen deposition in Sweden

Summary Current and maximally induced nitrate reductase activity (NRA), total-N, nitrate, K, P, Ca, Mg, Mo and sucrose in leaves ofDeschampsia flexuosa was measured three times during the vegetation period in forests along a deposition gradient (150 km) in south Sweden, in north Sweden where the nitrogen deposition is considerably lower, and at heavily N-fertilized plots. In addition, the interaction between nitrogen nutrition and light was studied along transects from clearings into forest in both south and north Sweden. Plants from sites with high nitrogen deposition had elevated current NRA compared to plants from less polluted sites, indicating high levels of available soil nitrate at the former. Current NRA and total N concentration in grass from sites with high deposition resembled those found at heavily N-fertilized plots. Under such circumstances, the ratio current NRA: maximally induced NRA as well as the concentration of nitrate was high, while the concentration of sucrose was low. This suggests that the grass at these sites was already utilizing a large portion of its capacity to assimilate nitrate. Light was found to play an important role in the assimilation of nitrate; leaf concentration of sucrose was found to be negatively correlated with both nitrate and total N. Consequently, grass growing under dense canopies in south Sweden is not able to dilute N by increasing growth. The diminished capacity of the grass to assimilate nitrate will increase leaching losses of N from forests approaching N saturation.     

Permeability properties of peroxisomal membranes from yeasts

We have studied the permeability properties of intact peroxisomes and purified peroxisomal membranes from two methylotrophic yeasts. After incorporation of sucrose and dextran in proteoliposomes composed of asolectin and peroxisomal membranes isolated from the yeasts Hansenula polymorpha and Candida boidinii a selective leakage of sucrose occurred indicating that the peroxisomal membranes were permeable to small molecules. Since the permeability of yeast peroxisomal membranes in vitro may be due to the isolation procedure employed, the osmotic stability of peroxisomes was tested during incubations of intact protoplasts in hypotonic media. Mild osmotic swelling of the protoplasts also resulted in swelling of the peroxisomes present in these cells but not in a release of their matrix proteins. The latter was only observed when the integrity of the cells was disturbed due to disruption of the cell membrane during further lowering of the concentration of the osmotic stabilizer. Stability tests with purified peroxisomes indicated that this leak of matrix proteins was not associated with the permeability to sucrose. Various attempts to mimic the in vivo situation and generate a proton motive force across the peroxisomal membranes in order to influence the permeability properties failed. Two different proton pumps were used for this purpose namely bacteriorhodopsin (BR) and reaction center-light-harvesting complex I (RCLH_I complex). After introduction of BR into the membrane of intact peroxisomes generation of a pH-gradient was not or barely detectable. Since this pump readily generated a pH-gradient in pure liposomes, these results strengthened the initial observations on the leakiness of the peroxisomal membrane fragments. Generation of a membrane potential () was also not observed when RCLH_I complex was introduced into vesicles of purified peroxisomal membranes. The significance of the observed permeability of isolated yeast peroxisomal membranes to small molecules with respect to current and future in vitro import studies is discussed.Abbreviations CL cardiolinin - PE phosphatidylethanolamine - PC phosphatidylcholine - MES 2-(N-Morpholino)ethanesulfonic acid - R₁₈ octadecyl Rhodamine B Chloride - SUVs small unilamellar vesicles - RCLH_I-complex reaction center-light-harvesting complex I - BR bacteriorhodopsin - DCCD N,N-dicyclohexylcarbodiimide     

Virulence of Candida parapsilosis, Candida orthopsilosis, and Candida metapsilosis in reconstituted human tissue models

Candida parapsilosis is an increasingly important human pathogen. To study the interactions of C. parapsilosis with human tissues, we evaluated the effects of the CBS 604 type strain and three different clinical isolates on reconstituted human oral epithelial and epidermal tissues. The newly described species Candida orthopsilosis and Candida metapsilosis were also examined in these models. Microscopy of reconstituted tissues infected with yeast cells revealed severe attenuation, morphological changes and cellular damage. C. orthopsilosis caused damage similar to C. parapsilosis isolates, whereas C. metapsilosis was less virulent. To further quantitate tissue damage, we measured lactate dehydrogenase (LDH) in the culture supernatant. The relative LDH measurements correlated with our histopathological observations. We also examined the effect of the lipase inhibitor Ebelactone B and proteinase inhibitor Pepstatin A, to establish the utility of this model for studying factors of C. parapsilosis virulence. Both Ebelactone B and Pepstatin A reduced the destruction of epidermal and epithelial tissues. Our data show that reconstituted human tissues are extremely useful for modeling host interactions with C. parapsilosis and for studying fungal virulence factors.