Ammonium uptake by Chlorella

The preincubation of Chlorella cells with glucose caused a tenfold increase of the maximal uptake rate of ammonium without change in the K _m (2 M). A similar stimulation of ammonium uptake was found when the cells were transferred to nitrogen-free growth medium. The time-course of uptake stimulation by glucose revealed a lag period of 10–20 min. The turnover of the ammonium transport system is characterized by a half-life time of 5–10 h, but in the presence of light 30% of uptake activity stayed even after 50 h. 6-Deoxyglucose was not able to increase the ammonium uptake rate. These data together were interpreted as evidence for induction of an ammonium transport system by a metabolite of glucose. Mechanistic studies of the ammonium transport system provided evidence for the electrogenic uptake of the ammonium ion. The charge compensation for NH ₄ ⁺ entry was achieved by immediate K⁺ efflux from the cells, and this was followed after 1 min by H⁺ extrusion. Ammonium accumulated in the cells; the rate of uptake was sensitive to p-trifluoromethoxy-carbonylcyanide-phenylhydrazon and insensitive to methionine-sulfoxime. Uptake studies with methylamine revealed that methylamine transport is obviously catalyzed by the ammonium transport system and, therefore, also increased in glucose-treated Chlorella cells.Abbreviation p.c. packed cells     

Catabolite inactivation of the glucose transport system in Saccharomyces cerevisiae

The sugar transport systems of Saccharomyces cerevisiae are irreversibly inactivated when protein synthesis is inhibited. This inactivation is responsible for the drastic decrease in fermentation observed in ammonium-starved yeast and is related to the occurrence of the Pasteur effect in these cells. Our study of the inactivation of the glucose transport system indicates that both the high-affinity and the low-affinity components of this system are inactivated. Inactivation of the high-affinity component evidently requires the utilization of a fermentable substrate by the cells, since inactivation did not occur during carbon starvation, when a fermentable sugar was added to starved cells, inactivation began, when the fermentation inhibitors iodoacetate or arsenate were added in addition to sugars, the inactivation was prevented, when a non-fermentable substrate was added instead of sugars, inactivation was also prevented. The inactivation of the low-affinity component appeared to show similar requirements. It is concluded that the glucose transport system in S. cerevisiae is regulated by a catabolite-inactivation process.     

Expression of a heterologous xylose transporter in a <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> strain engineered to utilize xylose improves aerobic xylose consumption

The goal of this investigation was to determine the effect of a xylose transport system on glucose and xylose co-consumption as well as total xylose consumption in Saccharomyces cerevisiae. We expressed two heterologous transporters from Arabidopsis thaliana in recombinant xylose-utilizing S. cerevisiae cells. Strains expressing the heterologous transporters were grown on glucose and xylose mixtures. Sugar consumption rates and ethanol concentrations were determined and compared to an isogenic control strain lacking the A. thaliana transporters. Expression of the transporters increased xylose uptake and xylose consumption up to 46% and 40%, respectively. Xylose co-consumption rates (prior to glucose depletion) were also increased by up to 2.5-fold compared to the control strain. Increased xylose consumption correlated with increased ethanol concentration and productivity. During the xylose/glucose co-consumption phase, strains expressing the transporters had up to a 70% increase in ethanol production rate. It was concluded that in these strains, xylose transport was a limiting factor for xylose utilization and that increasing xylose/glucose co-consumption is a viable strategy for improving xylose fermentation.     

Control of cell morphology of the yeast Saccharomyces cerevisiae by nutrient limitation in continuous culture

Saccharomyces cerevisiae IFO 0203, a polyploid yeast used in ethanol production in Japan, grows as ovoid cells in unstirred batch culture and on fully nutritive agar plates (2% w/v glucose; 0.67% w/v Difco yeast nitrogen base).
Extensively branched pseudohyphae formed on 0.01% w/v ammonium sulphate plates within a few days. In continuous culture with high oxygen supply and limiting glucose, cells were elongated but growth was vigorous and the daughter cells separated well after budding.
Limitation of growth by either nitrogen source or oxygen during continuous culture resulted in formation of truncated, occasionally branched, pseudohyphae up to five cells in length.     

Proline transport in Saccharomyces cerevisiae.

The yeast Saccharomyces cerevisiae is capable of utilizing proline as the sole source of nitrogen. Mutants of S. cerevisiae with defective proline transport were isolated by selecting for resistance to either of the toxic proline analogs L-azetidine-2-carboxylate or 3,4-dehydro-DL-proline. Strains carrying the put4 mutation are defective in the high-affinity proline transport system. These mutants could still grow when given high concentrations of proline, due to the operation of low-affinity systems whose existence as confirmed by kinetic studies. Both systems were repressed by ammonium ions, and either was induce by proline. Low-affinity transport was inhibited by histidine, so put4 mutants were unable to grow on a medium containing high concentrations of proline to which histidine has been added.     

The importance of amino acids as carbon sources for Micromonospora echinospora (ATCC 15837)

AIMS: This study set out to investigate the effect of amino acids on the uptake of glucose by Micromonospora eichinospora (ATCC 15837). METHODS AND RESULTS: The specific rate of glucose uptake was found to be reduced when organic nitrogen components were present in the medium. Radioactive uptake studies revealed that the Km for glucose in this organism was 53 mm, indicating a low affinity for uptake compared with other actinomycete sugar transport systems. Individual amino acids negatively influenced the rate of glucose transport, suggesting a relationship between amino acid metabolism and glucose uptake in this organism. The sugar transport system was found to be an active process being inhibited by ionophores and KCN. CONCLUSIONS: The data suggest a direct link between amino acid metabolism and glucose uptake at the level of sugar transport. SIGNIFICANCE AND IMPACT OF THE STUDY: This study shows that the uptake of glucose, a major carbon source for many antibiotic fermentations, is significantly reduced in the presence of amino acids. This fact should inform the medium design and feeding regimes of fermentations involving similar actinomycetes.     

Transport of fructose by a proton symport in a brewing yeast

Abstract Saccharomyces cerevisiae IGC4261, a brewing strain, transported fructose and sorbose but not glucose by a high-affinity, low-capacity proton symport. The symport was not subject to glucose repression and coexisted with the facilitated diffusion system for glucose, fructose, sorbose and other sugars. Transport by the symport was accumulative. The stoichiometry was one proton per molecule of fructose. Maltose acted as a non-competitive inhibitor.     

Ammonium permease-based sensing mechanism for rapid ammonium activation of the protein kinase A pathway in yeast

In the yeast Saccharomyces cerevisiae starvation for nitrogen on a glucose-containing medium causes entrance into G0 and downregulation of all targets of the PKA pathway. Re-addition of a nitrogen source in the presence of glucose causes rapid activation of trehalase and other PKA targets. Trehalase activation upon ammonium re-supplementation is dependent on PKA activity, but not on its regulatory subunit nor is it associated with an increase in cAMP. In nitrogen-starved cells, ammonium transport and activation of trehalase are most active in strains expressing either the Mep2 or Mep1 ammonium permease, as opposed to Mep3. The non-metabolizable ammonium analogue, methylamine, also triggers activation of trehalase when transported by Mep2 but not when taken up by diffusion. Inhibition of ammonium incorporation into metabolism did not prevent signalling. Extensive site-directed mutagenesis of Mep2 showed that transport and signalling were generally affected in a similar way, although they could be separated partially by specific mutations. Our results suggest an ammonium permease-based sensing mechanism for rapid activation of the PKA pathway. Mutagenesis of Asn246 to Ala in Mep2 abolished transport and signalling with methylamine but had no effect with ammonium. The plant AtAmt1;1, AtAmt1;2, AtAmt1;3 and AtAmt2 ammonium transporters sustained transport and trehalase activation to different extents. Specific mutations in Mep2 affected the activation of trehalase differently from induction of pseudohyphal differentiation. We also show that Mep permease involvement in PKA control is different from their role in haploid invasive growth, in which Mep1 sustains and Mep2 inhibits, in a way independent of the ammonium level in the medium.     

Involvement of endocytosis in catabolite inactivation of the K+ and glucose transport systems in Saccharomyces cerevisiae

Abstract The possible relationship between endocytosis and catabolite inactivation of plasma membrane proteins in Saccharomyces cerevisiae has been investigated. Using mutants with an increased rate of endocytosis we have shown that there is a positive correlation between the rate of endocytosis and the rate of inactivation of the K⁺ and glucose transport systems. It is concluded that endocytosis is involved in catabolite inactivation of these two transport systems.     

Effects of ethanol and acetic acid on the transport of malic acid and glucose in the yeast Schizosaccharomyces pombe: implications in wine deacidification

Abstract Ethanol and acetic acid, at concentrations which may occur during wine-making, inhibited the transport of l-malic acid in Schizosaccharomyces pombe . The inhibition was non-competitive, the decrease of the maximum initial velocity following exponential kinetics. Glucose transport was not significantly affected either by ethanol (up to 13%, w/v) or by acetic acid (up to 1.5%, w/v). The uptake of labelled acetic acid followed simple diffusion kinetics, indicating that a carrier was not involved in its transport. Therefore, the undissociated acid appears to be the only form that enters the cells and is probably responsible for the toxic effects. Accordingly, deacidification by Ss. pombe during wine fermentation should take place before, rather than after, the main alcoholic fermentation by Saccharomyces cerevisiae .     

High capacity xylose transport in Candida intermedia PYCC 4715

Xylose-utilising yeasts were screened to identify strains with high xylose transport capacity. Among the fastest-growing strains in xylose medium, Candida intermedia PYCC 4715 showed the highest xylose transport capacity. Maximal specific growth rate was the same in glucose and xylose media (mu(max)=0.5 h-1, 30 degrees C). Xylose transport showed biphasic kinetics when cells were grown in either xylose- or glucose-limited culture. The high-affinity xylose/proton symport system (Km = 0.2 mM, Vmax = 7.5 mmol h-1 g-1) was more repressed by glucose than by xylose. The less specific low-affinity transport system (K = 50 mM, Vmax = 11 mmol h-1 g-1) appeared to operate through a facilitated-diffusion mechanism and was expressed constitutively. Inhibition experiments showed that glucose is a substrate of both xylose transport systems.     

Some observations on oscillatory changes in the growth rate of Saccharomyces cerevisiae in aerobic continuous undisturbed culture.

Oscillatory changes in the growth rate were observed in undisturbed continuous culture of Saccharomyces cerevisiae on sugar-cane molasses media when nitrogen sources (2.56 to 6.17 g/liter of ammonium sulfate or 1.22 g/liter of urea) were added to the feeding mash and when the air rate was 1.3 to 1.6 v/v/m. The oscillations were not affected by the addition of yeast extract. The suppression of the nitrogen source during the continous test leads to a nonoscillatory transient state. No oscillations occured at all when no nitrogen source was added to the medium and/or the air rate was equal to zero or equal to about 3.3 v/v/m. The oscillatory responses of the system were affected by a previous anaerobic continuous cultivation of the yeast.     

Inhibition of amino acid transport by ammonium ion in Saccharomyces cerevisiae.

The rate of transport of L-amino acids by Saccharomyces cerevisiae epsilon 1278b increased with time in response to nitrogen starvation. This increase could be prevented by the addition of ammonium sulfate or cycloheximide. A slow time-dependent loss of transport activity was observed when ammonium sulfate (or ammonium sulfate plus cycloheximide) was added to cells after 3 h of nitrogen starvation. This loss of activity was not observed in the presence of cycloheximide alone. In a mutant yeast strain which lacks the nicotinamide adenine dinucleotide phosphate-dependent (anabolic) glutamate dehydrogenase, no significant decrease in amino acid transport was observed when ammonium sulfate was added to nitrogen-starved cells. A double mutant, which lacks the nicotinamide adenine dinucleotide phosphate-dependent enzyme and in addition has a depressed level of the nicotinamide adenine dinucleotide-dependent (catabolic) glutamate dehydrogenase, shows the same sensitivity to ammonium ion as the wild-type strain. These data suggest that the inhibition of amino acid transport by ammonium ion results from the uptake of this metabolite into the cell and its subsequent incorporation into the alpha-amino groups of glutamate and other amino acids.     

Kinetic studies on glucose and xylose transport in Saccharomyces cerevisiae

Zero trans-influx assays of glucose and xylose were performed using Saccharomyces cerevisiae to investigate transport characteristics under high and low glucose conditions. Under high glucose conditions, most glucose was transported by the low-affinity transporter. The high-affinity transporter was expressed under low glucose conditions, transporting over 50% glucose. Inhibition kinetics revealed that xylose was transported by both high- and low-affinity glucose transporters. Affinities of both glucose transporters for xylose were very low under high glucose condition but increased to a similar level to glucose under low glucose condition. The maximum rate of xylose transport increased by 85%, while an overall maximum glucose transport rate decreased by 42% under low glucose condition, indicating the presence of other transport system for sugars except for glucose. It was suggested that expression of the high-affinity transporter and increased affinity of glucose transporters for xylose under low glucose condition would provide a fermentation strategy for enhancing the productivity of xylitol by recombinant S. cerevisiae harboring the xylose reductase gene.     

植物吸收转运无机氮的生理及分子机制

氮是植物生长必需的营养元素。植物从土壤中吸收的氮素主要是NO3-和NH4 等无机氮源。植物吸收NO3-和NH4 的系统均有高亲和转运系统(high-affinity transport system,HATS)和低亲和转运系统(low-affinity transport system,LATS)之分。近10多年的研究已对这些转运系统的分子基础有了较好的理解,本文着重对近年来植物吸收无机氮分子机制的研究进展进行了综述。     

General properties and regulation of arginine transporting systems in Saccharomyces cerevisiae

The transport of arginine-¹⁴C by exponentially growing cellsof Saccharomyces cerevisiae (ATCC 9763) was studied in the presenceof various amino acids, ammonium and urea. Arginine transportwas inhibited when the cells were preincubated with these compoundsfor 1 hr. Little or no inhibition of transport occurred whenthe preincubation period was omitted. Kinetics studies revealedthat arginine was transported by two distinct systems havinghigh and low affinities for this amino acid. At given arginineconcentrations the high affinity system was capable of transportingarginine molecules at approximately seventy times the rate ofthe low affinity system. The general requirements for arginine transport revealed energyand temperature dependencies in addition to sensitivity to anumber of metabolic inhibitors. Transfer of cells to N-freemedium was accompanied by increased rates of transport. Thisincrease was shown for the uptake of ten different amino acids.For L-arginine, this increase was prevented by addition of cycloheximide. Analyses of amino acid pools, after various experimental treatments,failed to reveal any consistent correlation between transportrates and the concentrations of individual amino acids or ammonium. It is concluded that arginine transport of S. cerevisiae isregulated by inhibition and repression. In this respect theavailability of ammonium would appear to be of prime importancein the development of transport activity. (Received December 5, 1975; )     

Nitrate transport in the cyanobacterium Anacystis nidulans

A significant progress in the knowledge of different aspects of nitrate transport in the unicellular cyanobacterium Anacystis (Synechococcus ) has been achieved in the last few years. The main contributions of our group are summarized in this article and discussed in relation to other information available. Endergonic accumulation of nitrate into the cells, indicative of the operation of an active nitrate transport system, has been experimentally substantiated and methods established to evaluate and analyze the activity of the system. Nitrate transport activity is sensitive to regulation exerted by products of both ammonium and CO₂ assimilation, thus providing evidence that photosynthetic nitrate assimilation in cyanobacteria is primarily controlled at the level of substrate supply to the cell. The expression of nitrate transport was also shown to be under nitrogen control, being repressed when ammonium is used as the nitrogen source. A 47-kDa polypeptide, which is a major plasma membrane component in nitrate-grown cells but is virtually absent in ammonium-grown cells, was identified as an essential component of the nitrate transporter. More recently, evidence of a strict Na'-dependence of active nitrate transport has been obtained, Δμ(Na⁺) appearing as the driving force of a sodium-nitrate symport system. Kinetic studies indicate also that the nitrate transporter may transport nitrite into the cell.     

Methylamine and ammonia transport in Saccharomyces cerevisiae.

Methylamine (methylammonium ion) entered Saccharomyces cerevisiae X2180-A by means of a specific active transport system. Methylamine uptake was pH dependent (maximum rate between pH 6.0 and 6.5) and temperature dependent (increasing up to 35 C) and required the presence of a fermentable or oxidizable energy source in the growth medium. At 23 C the vmax for methylamine transport was similar 17 nmol/min per mg of cells (dry weight) and the apparent Km was 220 muM. The transport system exhibited maximal activity in ammonia-grown cells and was repressed 60 to 70 percent when glutamine or asparagine was added to the growth medium. There was no significant derepression of the transport system during nitrogen starvation. Ammonia (ammonium ion) was a strong competitive inhibitor of methylamine uptake, whereas other amines inhibited to a much lesser extent. Mutants selected on the basis of their reduced ability to transport methylamine (Mea-R) simultaneously exhibited a decreased ability to transport ammonia.     

Role of the Npr1 kinase in ammonium transport and signaling by the ammonium permease Mep2 in Candida albicans

The ammonium permease Mep2 induces a switch from unicellular yeast to filamentous growth in response to nitrogen limitation in Saccharomyces cerevisiae and Candida albicans. In S. cerevisiae, the function of Mep2 and other ammonium permeases depends on the protein kinase Npr1. Mutants lacking NPR1 cannot grow on low concentrations of ammonium and do not filament under limiting nitrogen conditions. A G349C mutation in Mep2 renders the protein independent of Npr1 and results in increased ammonium transport and hyperfilamentous growth, suggesting that the signaling activity of Mep2 directly correlates with its ammonium transport activity. In this study, we investigated the role of Npr1 in ammonium transport and Mep2-mediated filamentation in C. albicans. We found that the two ammonium permeases Mep1 and Mep2 of C. albicans differ in their dependency on Npr1. While Mep1 could function well in the absence of the Npr1 kinase, ammonium transport by Mep2 was virtually abolished in npr1Δ mutants. However, the dependence of Mep2 activity on Npr1 was relieved at higher temperatures (37°C), and Mep2 could efficiently induce filamentous growth under limiting nitrogen conditions in npr1Δ mutants. Like in S. cerevisiae, mutation of the conserved glycine at position 343 in Mep2 of C. albicans to cysteine resulted in Npr1-independent ammonium uptake. In striking contrast, however, the mutation abolished the ability of Mep2 to induce filamentous growth both in the wild type and in npr1Δ mutants. Therefore, a mutation that improves ammonium transport by Mep2 under nonpermissible conditions eliminates its signaling activity in C. albicans.     

Characteristics of glucose uptake by glucose- and NH4-limited grown Penicillium ochrochloron at low, medium and high glucose concentration

Glucose uptake by Penicillium ochrochloron (formerly Penicillium simplicissimum) was studied from 0.01 to 400 mM glucose using chemostat culture and bioreactor batch culture. The characteristics of glucose uptake varied considerably with the conditions of growth, harvest and uptake assay. Glucose-limited grown mycelium showed one saturable transport system [K(S) below 0.01 mM; v(max) 1.1-1.2 mmol (g dry weight)(-1)h(-1)] plus a first order process (permeability P=1.2x10(-7)cm s(-1)). Ammonium-limited grown mycelium showed only one saturable transport system [K(S) 0.3-0.7 mM; v(max) 0.5-0.8 mmol (g dry weight)(-1)h(-1)]. During exponential growth at high glucose concentration (300-400 mM) a first order process was found with a P value of 5.6-9.3x10(-7)cm s(-1). After ammonium exhaustion a second first order phase showed a lower P value (6.1-9.3x10(-8)cm s(-1)). A similar change in permeability was also found after a re-evaluation of published data for Gibberella fujikuroi, Aspergillus niger, Aspergillus awamori and Saccharomycopsis lipolytica. For the first order processes simple diffusion was ruled out as a mechanism for glucose uptake. Glucose uptake by P. ochrochloron was controlled more strongly by metabolism than by transport and was not rate limiting for overflow metabolism.