UPTAKE SPECIFICITY FOR ORGANIC SUBSTRATES BY THE MARINE DIATOM MELOSIRA NUMMULOIDES1

Melosira nummuloides, clone Mel-3, shows a very high specificity with regard to its ability to take up organic substrates. Amino acids supplied in the medium at 1 X 10^-4 M are taken up at initial rates of the same order of magnitude as that of photoassirnilation of COj. However, sugars, sugar alcohols, or organic acids supplied at the same concentration are not taken up. The mechanism for uptake of amino acids appears to require energy, since tlie uptake of the amino acid analog α-aminoisobutyric acid is strongly inhibited by 2 f-dinitrophenul. The uptake mechanism does not appear to be inducible. The ability of M. numinuloides to utilize amino acids as a nitrogen source is quite restricted. Arginine, ghttamine, asparagine, proline, and glutamic acid were good nitrogen sources. Seventeen other amino acids, including α-aminoisobutyric acid, were unsatisfactory for growth, although they were rapidly taken up from the medium.     

Analysis of the interactions of sulfur‐containing amino acids in membrane proteins

The interactions of Met and Cys with other amino acid side chains have received little attention, in contrast to aromatic–aromatic, aromatic–aliphatic or/and aliphatic–aliphatic interactions. Precisely, these are the only amino acids that contain a sulfur atom, which is highly polarizable and, thus, likely to participate in strong Van der Waals interactions. Analysis of the interactions present in membrane protein crystal structures, together with the characterization of their strength in small‐molecule model systems at the ab‐initio level, predicts that Met–Met interactions are stronger than Met–Cys ≈ Met–Phe ≈ Cys–Phe interactions, stronger than Phe–Phe ≈ Phe–Leu interactions, stronger than the Met–Leu interaction, and stronger than Leu–Leu ≈ Cys–Leu interactions. These results show that sulfur‐containing amino acids form stronger interactions than aromatic or aliphatic amino acids. Thus, these amino acids may provide additional driving forces for maintaining the 3D structure of membrane proteins and may provide functional specificity.     

Oligopeptide assimilation and transport by Oenococcus oeni

Aims: Oenococcus oeni is a slow‐growing wine bacterium with a low growth yield. It thrives better on complex nitrogen sources than on free amino‐acid medium. We aimed to characterize the oligopeptide use of this micro‐organism. Methods and Results: Several peptides of two to eight amino‐acid residues were able to provide essential amino acids. The disappearance of various peptides from extracellular medium was assessed with whole cells. Initial rates of utilization varied with the peptide, and free amino acids were released into the medium. Conclusions: Oenococcus oeni was able to transport the oligopeptides with two to five amino‐acid residues tested and to hydrolyse them further. Significance and Impact of the Study: This study has clear implications for the relationship between wine nitrogen composition and the ability of O. oeni to cope with its environment.     

AMINO ACID TRANSPORT IN NITZSCHIA OVALIS ARNOTT1,2

The marine diatom Nitzschia ovalis possesses at, least 3 amino acid uptake systems, specific for transport of acidic, polybasic, and, neutral amino acids. Maximum uptake velocity (V_max) for each, site is inversely related to the nitrogen content of the cell, and to the nitrogen available in the culture medium. Transport, of polybasic amino acids occurs throughout the course of growth in batch, culture, but the V_max increases dramatically as the culture ages and nitrogen/cell reaches a low value. K_s does not, change significantly. Acidic and neutral amino acids are taken up only by cells harvested from nitrogen-poor culture. It appears that amino acid transport is repressed by high concentrations of nitrogen in the medium. Under natural conditions, where nitrogen concentrations are low, the contribution of amino acid uptake to the nitrogen economy of Nitzschia populations may be significant.     

Pattern of CO2 fixation during vegetative development and gametic differentiation in Chlamydomonas reinhardtii

The rate of C¹⁴O₂ incorporation into amino acids and organic acids in C. reinhardtii is a function of particular stages of development in the life cycle of the alga. Gametic differentiation in nitrogen free medium is accompanied by a reduced rate of amino acid synthesis and a higher synthesis of organic acids than that found for the cells undergoing vegetative development. The addition of ammonium to differentiating gametes results in an increased synthesis of amino acids, particularly the basic ones, and a concomitant reduction in organic acid synthesis.     

Significance of Extracellular Protease for Growth of a Heterotrophic Bacterium, Aeromonas salmonicida

The role of protease produced by a heterotrophic bacterium during growth was investigated with Aeromonas salmonicida, the pathogen of fish furunculosis, strain A-7301 and its protease-deficient mutant NTG-1 induced by mutagenesis. Strain A-7301 produced extracellular protease in a mixed amino acid medium (composed of Gly, Ala, Val, Ile, Leu, Thr, Ser, Cys, Met, Phe, Tyr, Lys, Arg, Pro, His, Try, Asp, Asn, Glu, and Gln at equal concentrations of 0.1 g/liter). Its multiplication rate was limited by the amounts of amino acids present, whereas strain NTG-1 showed no protease production despite considerable growth similar to that of A-7301. There was no difference between A-7301 and NTG-1 in amino acid requirements for growth, and seven amino acids (Gly, Ala, Val, Thr, Cys, Met, and His) were found to be indispensable. A defined level of the mixed amino acids (0.4 to 0.5 g/liter) was needed for A-7301 to initiate a large production of protease. Neither of the strains grew well in a casein medium, to which no amino acids were added. However, when a protease fraction obtained from extracellular products of A-7301 by DEAE-cellulose column chromatography was added, NTG-1 successfully reproduced in the casein medium. These results indicate that the extracellular protease plays an important role in supplying A. salmonicida cells with available amino acids as nutrients and that higher growth is closely associated with protease production which stimulates further reproduction.     

Participation of an extracellular deaminase in amino acid utilization by Neurospora crassa

A strain of Neurospora crassa defective in amino acid transport can utilize a variety of amino acids for growth when readily metabolizable nitrogen is limiting. Growth is accompanied by the production of an extracellular deaminase that converts the amino acid to its respective keto acid plus equimolar quantities of utilizable nitrogen in the ammonium ion form. Production of the deaminase is subject to ammonium repression. The relationship between the ability of an amino acid to trigger deaminase production and the presence of particular amino acid permease deficiencies is complex. Four classes of amino acids have been defined with respect to this relationship. The existence of multiple extracellular deaminases is discussed.     

Extracellular deamination of l-amino acids by Chlamydomonas reinhardtii cells

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

Improvement of pea biomass and seed productivity by simultaneous increase of phloem and embryo loading with amino acids

The development of sink organs such as fruits and seeds strongly depends on the amount of nitrogen that is moved within the phloem from photosynthetic‐active source leaves to the reproductive sinks. In many plant species nitrogen is transported as amino acids. In pea (Pisum sativum L.), source to sink partitioning of amino acids requires at least two active transport events mediated by plasma membrane‐localized proteins, and these are: (i) amino acid phloem loading; and (ii) import of amino acids into the seed cotyledons via epidermal transfer cells. As each of these transport steps might potentially be limiting to efficient nitrogen delivery to the pea embryo, we manipulated both simultaneously. Additional copies of the pea amino acid permease PsAAP1 were introduced into the pea genome and expression of the transporter was targeted to the sieve element‐companion cell complexes of the leaf phloem and to the epidermis of the seed cotyledons. The transgenic pea plants showed increased phloem loading and embryo loading of amino acids resulting in improved long distance transport of nitrogen, sink development and seed protein accumulation. Analyses of root and leaf tissues further revealed that genetic manipulation positively affected root nitrogen uptake, as well as primary source and sink metabolism. Overall, the results suggest that amino acid phloem loading exerts regulatory control over pea biomass production and seed yield, and that import of amino acids into the cotyledons limits seed protein levels.     

N-15 in vivo NMR spectroscopic investigation of nitrogen deprived cell suspensions of the green alga Chlorella fusca

The possibility to apply N-15 in vivo NMR spectroscopy to study algal N-metabolism has been investigated. N-15 labelled cells of the green alga Chlorella fusca, subjected to nitrogen starvation and N-14 labelled cells supplied with K¹⁵NO₃ after prolonged nitrogen starvation were monitored by N-15 in vivo NMR spectroscopy at different times after the change in their nitrogen supply. During 20–40 min, necessary for the acquisition of 1 spectrum, the cells were under dark anaerobic conditions, but the relative amounts of the metabolites detected did not change. Signals from 2 acid amides, from the side chain nitrogens of arginine and lysine, from prolin as well as 4 signals from α amino groups of amino acids were detected. Besides two signals not yet reported in the literature were found. They may be due to amino compounds, but not to amino acids. The amount of free amino acids in the cells increases not only upon resupply of nitrogen starved cells with nitrate but also during the first hours after nitrate depletion. The spectra obtained from N-15 labelled autospores show that N-15 in vivo NMR spectroscopy can be applied to the investigation of N metabolism of the cells.     

Olfactory discrimination of complex mixtures of amino acids by the black bullhead Ameiurus melas

On the basis of previous findings of behavioural discrimination of amino acids and on the knowledge of electrophysiology of the catfish (genera Ictalurus and Ameiurus) olfactory organs, behavioural experiments that investigated olfactory discrimination of amino acid mixtures were carried out on the black bullhead Ameiurus melas. Repeated presentations of food‐rewarded mixtures released increased swimming activity measured by counting the number of turns >90° within 90 s of stimulus addition. Non‐rewarded amino acids and their mixtures released little swimming activity, indicating that A. melas discriminated between the conditioned and the non‐conditioned stimuli. Two questions of mixture discrimination were addressed: (1) Are A. melas able to detect components within simple and complex amino acid mixtures? (2) What are the smallest differences between two complex mixtures that A. melas can detect? Three and 13 component mixtures tested were composed primarily of equipotent amino acids [determined by equal electroolfactogram (EOG) amplitude] that contained L‐Cys at ×100 the equipotent concentration. Ameiurus melas initially perceived the ternary amino acid mixture as its more stimulatory component alone [i.e. cysteine (Cys)], whereas the conditioned 13 component mixture containing the more stimulatory L‐Cys was perceived immediately as different from L‐Cys alone. The results indicate that components of ternary mixtures are detectable by A. melas but not those of more complex mixtures. To test for the smallest detectable differences in composition between similar multimixtures, all mixture components were equipotent. Initially, A. melas were unable to discriminate the mixtures of six amino acids from the conditioned mixtures of seven amino acids, whereas they discriminated immediately the mixtures of four and five amino acids from the conditioned mixture. Experience with dissimilar mixtures enabled the A. melas to start discriminating the seven‐component conditioned mixture from its six‐component counterparts. After fewer than five training trials, A. melas discriminated the mixtures of nine and 10 amino acids from a conditioned mixture of 12 equipotent amino acids; however, irrespective of the number of training trials, A. melas were unable to discriminate the 12 component mixture from its 11 component counterparts.     

Multivalent Induction of Biodegradative Threonine Deaminase

To determine the inducer(s) of the biodegradative threonine deaminase in Escherichia coli, the effects of various amino acids on the synthesis of this enzyme were investigated. The complex medium used hitherto for the enzyme induction can be completely replaced by a synthetic medium composed of 18 natural amino acids. In this synthetic medium, the omission of each of the seven amino acids threonine, serine, aspartic acid, methionine, valine, leucine, and arginine resulted in the greatest loss of enzyme formation. These seven amino acids did not significantly influence the uptake of other amino acids into the cells. Furthermore, they did not stimulate the conversion of inactive enzyme into an active form, since they did not affect the enzyme level in cells in which protein synthesis was inhibited by chloramphenicol. Threonine, serine, aspartic acid, and methionine failed to stimulate enzyme production in cells in which messenger ribonucleic acid synthesis was arrested by rifampin, whereas valine, leucine, and arginine stimulated enzyme synthesis under the same conditions. Therefore, the first four amino acids appear to act as inducers of the biodegradative threonine deaminase in E. coli and the last three amino acids appear to be amplifiers of enzyme production. The term "multivalent induction" has been proposed for this type of induction, i.e., enzyme induction only by the simultaneous presence of several amino acids.     

Carrier-mediated Uptake of Arginine and Urea by Chlamydomonas reinhardtii

Chlamydomonas reinhardtii possesses a high affinity, highly specific carrier involved in uptake of exogenous arginine. Carrier-mediated uptake of other amino acids cannot be detected, even in cultures maintained on amino acids as a nitrogen source or starved for nitrogen. This fact may contribute to the difficulty of isolating strains auxotrophic for amino acids other than arginine; conventional selection media may not supply adequate quantities of amino acids to permit growth of auxotrophs. A urea carrier is also present in C. reinhardtii but is readily distinguished from the arginine carrier on the basis of kinetic properties and sensitivity to a range of structural analogs. Ammonia appears to play a major role in regulating (depressing) activity of the arginine uptake system. Activity of the urea uptake system is elevated in nitrogen-starved cultures and elevated even further in the presence of urea or arginine. Extensive, independent fluctuations in the two uptake systems observed in semisynchronous cultures suggest that both are subject to modulation by a complex set of interacting endogenous and exogenous factors.     

Oryza sativa Lysine‐Histidine‐type Transporter 1 functions in root uptake and root‐to‐shoot allocation of amino acids in rice

In agricultural soils, amino acids can represent vital nitrogen (N) sources for crop growth and yield. However, the molecular mechanisms underlying amino acid uptake and allocation are poorly understood in crop plants. This study shows that rice (Oryza sativa L.) roots can acquire aspartate at soil concentration, and that japonica subspecies take up this acidic amino acid 1.5‐fold more efficiently than indica subspecies. Genetic association analyses with 68 representative japonica or indica germplasms identified rice Lysine‐Histidine‐type Transporter 1 (OsLHT1) as a candidate gene associated with the aspartate uptake trait. When expressed in yeast, OsLHT1 supported cell growth on a broad spectrum of amino acids, and effectively transported aspartate, asparagine and glutamate. OsLHT1 is localized throughout the rice root, including root hairs, epidermis, cortex and stele, and to the leaf vasculature. Knockout of OsLHT1 in japonica resulted in reduced root uptake of amino acids. Furthermore, in ¹⁵N‐amino acid‐fed mutants versus wild‐type, a higher percentage of ¹⁵N remained in roots instead of being allocated to the shoot. ¹⁵N‐ammonium uptake and subsequently the delivery of root‐synthesized amino acids to Oslht1 shoots were also significantly decreased, which was accompanied by reduced shoot growth. These results together provide evidence that OsLHT1 functions in both root uptake and root to shoot allocation of a broad spectrum of amino acids in rice.     

Studies on utilization of 2-ketoglutarate,glutamate and other amino acids by the unicellular alga Cyanidium caldarium

Two strains of Cyanidium caldarium which possess different biochemical and nutritional characteristics were examined with respect to their ability to utilize amino acids or 2-ketoglutarate as substrates.One strain utilizes alanine, glutamate or aspartate as nitrogen sources, and glutamate, alanine or 2-ketoglutarate as carbon and energy sources for growth in the dark. The growth rate in the dark on 2-ketoglutarate is almost twice as high or higher than that on glutamate or alanine. During growth or incubation of this alga on amino acids, large amounts of ammonia are formed; however, ammonia formation is strongly inhibited by 2-ketoglutarate. The capacity of the alga to form ammonia from amino acids is inducible and develops fully only when the cells are grown or incubated in the presence of glutamate.By contrast, the other strain of Cyanidium caldarium cannot utilize alanine or aspartate as nitrogen sources. It utilizes glutamate only very poorly and does not excrete ammonia into the external medium. This strain is unable to utilize amino acids or 2-ketoglutarate as carbon and energy sources for heterotrophic growth.Cell-free extracts were tested for the occurrence of enzymes which could account for amino acid metabolism and ammonia formation.     

Seed and Embryo Germination in Syringa vulgaris and S. reflexa as Affected by Temperature during Seed Development

A nitrogen source was needed for the flowering of Lemna gibba L., a long-day plant, and L. perpusilla Torr., a shortday plant. The level of endogenous amino acids analyzed by an Amino Acid Analyzer, rose during the first few inductive cycles, but was reduced during later stages of the flowering process. Serine and threonine levels increased during the light period and decreased during the dark period in L. perpusilla. Exogenous serine and threonine added to the culture medium at 10^?6M increased the rate of flowering by more than 35% over the controls. Cysteine inhibited flowering, while other amino acids had little or no promotive effect on flowering. Serine and threonine increased flowering rate in L. perpusilla only when added during a dark period of the inductive cycle. The addition of amino acids during a light period not followed by a dark period had no effect on flowering.     

Amino acid uptake in Xenopus laevis oocytes.

The isolated oocytes from Xenopus laevis are able to take up radioactive amino acids from the exogenous medium. Most amino acids tested are taken up to reach concentrations higher than the extracellular medium. The initial uptake velocities vary with the external amino acid concentration in a Michaelis-Menten fashion. Aspartic acid requires concentrations an order of magnitude higher than the five other amino acids tested to reach half the maximal uptake velocity. The uptake mechanism seems to be specific for groups of analogous amino acids, as can be determined by competition studies. The amino acid groups for which there is some evidence of uptake specificity would be aromatic, aliphatic, acidic and basic. Amino acid pools of oocytes show that these cells can concentrate amino acids from Xenopus blood, as well as from artificial media.     

Xanthine accumulation and vacuolization inChlamydomonas reinhardtii cells

Summary Utilization of xanthine as the sole nitrogen source for growth byChlamydomonas reinhardtii cells involved the formation of a transient, intracellular pool of xanthine. Up to 20% of the total xanthine supplied to the medium was not assimilated after uptake but stored in the cells at concentrations that exceeded xanthine solubility in water. At the subcellular level, a massive accumulation of starch grains in the chloroplast and the appearance of many vacuoles in the cytoplasm distinguished xanthine-grown from ammonium-grown cells. Starch accumulation, but not development of vacuoles, was also observed in N-starved cells. Uptake experiments with radio-labelled xanthine showed that this accumulates only in the cytoplasm, most probably inside vacuoles. The electron-dense material observed in vacuoles of xanthine-grown cells suggests that the intracellular xanthine is in part solid xanthine.     

The amino acid transport systems of the autotrophically grown green alga Chlorella

The kinetic analysis of l-amino acid uptake by the green alga Chlorella revealed at least seven different uptake systems to be present in cells grown autotrophically with nitrate as nitrogen source. There is a ‘general system’ which transports most neutral and acidic amino acids, a system for short-chain neutral amino acids including proline, a system for basic amino acids including histidine, a special system for acidic amino acids, and specific systems for methionine, glutamine and threonine. The ‘general system’ is possibly the same as that which can be stimulated by incubation of cells in glucose plus ammonium (Sauer, N. (1984) Planta 161, 425–431). The incubation of Chlorella in glucose induces the increased synthesis of six amino acid uptake systems, namely the above-mentioned system for short-chain neutral amino acids, a threonine system, a methionine system, and a glutamine system. These results indicate that the uptake of l-amino acids by the green alga Chlorella is as complex as in other free-living organisms such as bacteria or yeast. The small number of amino acid uptake systems found in cells of higher plants, i.e. two or three, seems therefore to be a consequence of integration of the cells in a tissue supplying a relatively constant environment, and not a consequence of autotrophic growth on mineral carbon and mineral nitrogen.