Regulation of amino acid transport in growing cells of Streptomyces hydrogenans

(1) The active uptake of different amino acids by growing cells of Streptomyces hydrogenans was shown to be correlated with the physiological age of the cells. During the lag phase of growth the transport capacity increased and attained its highest level when the growth rate was maximum. During further growth the transport capacity declined progressively. The lowest transport activity was observed when the culture shifted into the stationary growth phase. (2) Such modulation of transport capacity was independent on the presence or absence of amino acids in the growth medium of the cells. (3) The size and the composition of the pool of free intracellular amino acids was also undergoing substantial variations during the growth cycle of the culture. In the lag phase, the levels of all amino acids decreased markedly and attained their lowest values at the end of this phase. During further growth the pool size was slowly replenished. (4) Removal of the pool resulted in a considerable gain of transport capacity. Therefore, it was concluded that active amino acid transport in growing Streptomyces hydrogenans is under feedback control by intracellular amino acids. (5) Quantitatively, the modulation of the pool size could not fully account for the variation of the transport capacity. Since a pool-independent stimulation of transport was found to be correlated with the increase of the growth rate of the cells, the possibility is discussed that the stimulation of transport is either due to increased levels of distinct RNA species, which might provide positive feedback signals for transport, or by increased rates of de novo synthesis of transport limiting proteins.List of Abbreviations AIB 2-aminoisobutyric acid - CM complete medium - MM mineral medium     

Messenger ribonucleic acid content of Bacillus amyloliquefaciens throughout its growth cycle compared with Bacillus subtilis 168

The messenger RNA contents of Bacillus amyloliquefaciens and B. subtilis 168, grown in a 1% maltose-0.5% casein hydrolysate complex medium, were determined throughout their growth cycles by a hybridization technique. In both cases there was a level equal to about 3% of the total cellular RNA during the exponential phase. In B. subtilis this level was maintained into the stationary phase. By contrast, in B. amyloliquefaciens the proportion of messenger RNA increased after the end of exponential growth levelling off in the stationary phase at a value twice that observed in exponential growth. The total messenger RNA in each organism was resolved into two components, that involved in the formation of cell proteins and that concerned in extracellular protein production, by determining the relative rates of incorporation of l-[¹⁴C]valine into the two protein fractions. In both cases the cell protein component was the same and remained a relatively constant proportion of the total cellular material throughout the growth cycles. The exoprotein mRNA paralleled exoprotein secretion in each species, remaining at a constant low level in B. subtilis and undergoing a tenfold increase after the end of exponential growth in B. amyloliquefaciens. Applying a serial hybridization procedure to B. amyloliquefaciens, no evidence was obtained for the accumulation of a specific component of the messenger RNA in the exponential or post-exponential phase of growth, which was not detected by hybridization.     

Changes in the intracellular pool of free amino acids of Streptococcus lactis under different cultivation conditions]

The intracellular pool of free amino of Streptococcus lactis--lysine producer contains a good number of amino acids when cultivated on the corn medium. Glutamic acid, proline, alanine, lysine, leucine, histidine and arginine are in predominance. An almost complete amino acid pool develops at an early exponential phase of Str. lactis growth under stationary cultivation conditions. The content of free amino acids increases 4-fold during the transition from the early exponential phase to the stationary phases under submerged cultivation conditions. This can be attributed to a more intensive amino acid exchange during the medium stirring than during stationary cultivation.     

Effect of growth temperature and media composition on the fatty acid composition of Bacillus stearothermophilus AN 002

The influence of growth temperature, media composition and cell age on the chemical composition of Bacillus stearothermophilus strain AN 002 has been determined. The total cellular protein decreased and the free amino acid content increased with growth temperature, in both exponential and stationary growth phase. The protein and free amino acid contents of cells were higher in the stationary phase than in the exponential phase, irrespective of growth temperature and media composition. The RNA content was only reduced in cells grown at 55° C. No significant variations were observed in the DNA and carbohydrate contents with respect to growth temperature and cell age. The total lipid and fatty acid compositions on the other hand varied as a function of growth temperature, cell age and media composition. Differences in the relative concentrations of even, odd and branched chain fatty acids were noticed. Novariation was observed in the antiiso and unsaturated fatty acids with respect to growth temperature. The unique variations in the fatty acid composition and total lipids at the growth temperature of 50° C and their variations in the stationary growth phase seem to be characteristic for B. stearothermophilus AN 002.     

Changes in Free Amino Acid Production and Intracellular Amino Acid Pools of Bacillus licheniformis as a Function of Culture Age and Growth Media

Changes in the endogenous intracellular amino acid pool and total free amino acid production in Bacillus licheniformis grown in minimal media were investigated. The total intracellular pool increased during exponential growth and then decreased rapidly after the end of growth. Most of the amino acids were present at low concentrations, but glutamate and alanine comprised 60 to 90% of the total intracellular free amino acid at most times during the growth cycle. It was concluded that, in addition to providing monomers for protein synthesis, the intracellular amino acid pool may be maintained for the storage of energy-providing metabolic intermediates and possibly as a balance to the ionic strength of the medium. The total free amino acid production by the cell was found to be dependent upon the composition of the salts medium as well as the culture age under conditions in which the carbon and nitrogen sources were the same. A 10-fold increase in extracellular amino acid was observed as the cells changed from vegetative to sporulation metabolism, mostly due to the extrusion of intracellular amino acid. The impact of this increase upon amino acid uptake and pulse-labeling studies using unwashed cells is discussed.     

Soy Peptides Enhance Heterologous Membrane Protein Productivity during the Exponential Growth Phase of Saccharomyces cerevisiae

In this study, the production of eight G protein-coupled receptors by Saccharomyces cerevisiae was compared using two types of media, one of which contained soy peptides and the other free amino acids. Yeast cell growth improved in the medium with soy peptides, and the expression levels of six of the receptors increased during the exponential phase by an average of 2.3-fold as against the free amino acid-based medium. The enhancement of protein expression by soy peptides can be explained by alleviation of metabolite stress due to amino acid source depletion caused by heterologous protein expression.     

Why do rotifer populations present a typical sigmoid growth curve?

To determine the underlying processes to population growth in the rotifer Brachionus plicatilis, we conducted an experiment using 1.5 ml cultures for 70 days. All individuals were transferred daily to culture media containing algae, and the number of individuals, clutch sizes and number of deaths were counted. The population dynamics showed a typical sigmoid curve. The population density increased exponentially from 10 to 682 individuals during the first 7 days (exponential growth phase), and gradually up to about 1500 individuals during the next 30 days (post-exponential growth phase). The population density then remained at a constant level with small fluctuations during the rest of the experimental period (stationary phase). Mortalities appeared from the post-exponential growth phase and were almost constant at about 2% throughout the experimental period. The clutch size decreased from 5 to 1 during the first 5 days, and afterwards females laid only one egg each. The proportion of non-reproductive females increased from 30% (exponential growth phase) to 80% (post-exponential growth phase) to 90% (stationary phase). These results suggest that the exponential growth phase resulted from the imbalance between a high birth rate and a low death rate, while the stationary phase was maintained by the compensation between low birth and death rates.     

Soy peptides enhance heterologous membrane protein productivity during the exponential growth phase of Saccharomyces cerevisiae

In this study, the production of eight G protein-coupled receptors by Saccharomyces cerevisiae was compared using two types of media, one of which contained soy peptides and the other free amino acids. Yeast cell growth improved in the medium with soy peptides, and the expression levels of six of the receptors increased during the exponential phase by an average of 2.3-fold as against the free amino acid-based medium. The enhancement of protein expression by soy peptides can be explained by alleviation of metabolite stress due to amino acid source depletion caused by heterologous protein expression.     

Stability of rapidly labelled messenger ribonucleic acid in Bacillus amyloliquefaciens during the phases of minimum and maximum extracellular enzyme formation.

The stability of rapidly labelled hybridizable messenger RNA in both exponential and post-exponential phase cells of Bacillus amyloliquefaciens was measured in terms of the rate of loss of its radioactivity. In the exponential phase, where 96% of the mRNA was specific for cell proteins and only 4% was exoprotein mRNA, the label was lost exponentially from the rapidly labelled hybridizable mRNA fraction with a half-life of six minutes at 30 °C. The antibiotic rifampicin, at a concentration of 10 μg/ml, had no effect on the characteristics of decay of this exponential-phase mRNA. In the post-exponential phase, where there were equal amounts of cell protein and exoprotein-specific mRNA, rapidly labelled hybridizable mRNA decayed exponentially in the presence of rifampicin (10 μg/ml), with a half-life of six minutes at 30 °C. In the absence of rifampicin the characteristics of decay were more complex. The evidence available suggested that this was due to the superimposition of a component attributable to reincorporation of degradation products of radioactive RNA on the characteristic exponential decay pattern of the post-exponential mRNA.Measurement of the stability of active mRNA, by studying the loss of ability to incorporate l-[¹⁴C]leucine into protein in the presence of rifampicin (10 μg/ml), gave half-lives of 4.5 minutes and six minutes, respectively, for exponential and post-exponential material.     

INTRACELLULAR AND EXTRACELLULAR AMINO ACID POOLS OF THE MARINE DIATOM PHAEODACTYLUM TRICORNUTUM (BACILLARIOPHYCEAE) GROWN ON UNENRICHED SEAWATER IN HIGH-CELL-DENSITY DIALYSIS CULTURE1

The consumption of inorganic macronutrients (NO₃^?+ NO₂^?, NH₄⁺, and PO₄^?3) and the composition of intra- and extracellular dissolved free amino acid pools (IDFAA and EDFAA, respectively) were determined in continuous-reservoir batch dialysis cultures of the marine diatom Phaeodactylum tricornutum Bohlin maintained on unenriched natural seawater as a growth medium. Nutrient diffusion (Nd), which equals the nutrient uptake of the culture, increased with the cell density and the age of the culture. A concentration of 6.77 × 10⁷ cells · mL^?1 was obtained in stationary phase, which coincided with the NO₃^?+ NO₂^? diffusion limit (Nd_max) of the dialysis apparatus. The Nd_max for NH₄⁺ occurred much earlier, at the end of exponential growth, whereas Nd_max for PO₄^?3 was not attained during the growth cycle of the culture, even in early stationary phase. A significant depletion (77%) of the IDFAA pool during exponential phase was followed by a reestablishment–to approximately 60% of the initial level–of internal pools during linear and stationary growth phases. This recovery occurred during the illuminated portion of the photoperiod (12:12 h LD) and involved principally the amino acids GLN, GLU, β-GLU, and ASN. The recovery of GLN and ASN levels was particularly significant, because the intracellular concentrations of these amino acids were higher at the end of the growth cycle than before. The EDFAA pool was generally dominated by the amino acids SER and GLY+THR; however, during active growth, ORN and LYS often constituted an important fraction. The EDFAA concentration increased until linear growth phase was reached, during which a higher concentration of total free amino acids was attained in darkness than under illumination. The EDFAA component diminished afterward, and in stationary phase this fraction returned to concentrations equivalent to those observed at the beginning of the growth cycle. The variations in EDFAA concentrations were expressed by a pronounced decrease in the cellular excretion of amino acids with increasing cell density. These cellular responses of Phaeodactylum tricornutum in dense culture, specifically the regulation of amino acid excretion and intracellular pool size, may affect the N-conversion coefficient (Y_N). Consequently, by prolonging the linear phase of growth and reducing the concentration of autoinhibitory metabolites by diffusion, a markedly enhanced final cell density can be achieved in cultures grown on natural unenriched seawater.     

Stringent control of RNA synthesis inLactobacillus acidophilus

The amino acid regulation of RNA synthesis inLactobacillus acidophilus was studied and found to be of stringent character. The synthesis of RNA was inhibited in the absence of essential amino acids in the medium, this inhibition being released by chloramphenicol or chlortetracycline. The RNA synthesized in the presence of the above inhibitors was not stable. The results do not support the hypothesis that the release of RNA synthesis by chloramphenicol is due to an increased pool of free amino acids, in consequence to the inhibition of protein synthesis. Chloramphenicol removed the inhibition of RNA synthesis at the same rate as the amino acids themselves. The pool of free leucine or histidine decreased to 60% in the absence of exogenous amino acids and it was not raised on adding chloramphenicol. The results are in agreement with the assumption that the synthesis of ribosomal RNA in bacteria is controlled by the equilibrium between polysomes and free ribosomes. Further, the results point to a possible limiting role of proteins in the regulation of ribosomal RNA synthesis.     

Nitrogen Starvation-Induced Changes in Amino Acid and Free Ammonium Pools in <Emphasis Type="Italic">Schizophyllum commune</Emphasis> Colonies

Wood-decay fungi depend upon recycling of nitrogen-containing molecules to maintain growth in nitrogen-deficient environments. One of the pools that can support growth in these organisms is the pool of free amino acids. The free amino acid (AA) composition of Schizophyllum commune mycelium grown on the surface of nitrogen-rich (M = 6.6 mM L-asparagine) and nitrogen-poor medium (M01 = 0.06 mM L-asparagine) has been examined: When mycelium is grown on M, alanine, glutamate, and asparagine account for almost 2/3 of the amino acid pool. The free amino acid concentration is reduced by 75% for mycelium grown on the M01 medium, with alanine and glutamate predominating. In addition, free NH₄⁺ increases by 60% in nitrogen-deprived mycelia. Except for asparagine, which is absorbed by the apices, the concentration of all free amino acids is higher in the centers of M-grown, 4-day-old mycelia than in the apices. Hyphae grown to exponential growth on M and transferred to M01 for 12 h show greater free amino acid and NH₄⁺ concentrations in the apices, most likely indicating increased translocation to the apices.     

BIOCHEMICAL CHANGES DURING GROWTH AND ENCYSTMENT OF THE CELLULAR SLIME MOLD POLYSPHONDYLIUM PALLIDUM

The growth of the cellular slime mold, Polysphondylium pallidum, was studied on a semidefined medium in shaken suspension. When the medium contained large quantities of particulate material, growth was more rapid and the cellular size and protein content were smaller than when growth occurred on a medium containing less particulate material. The cellular levels of DNA, RNA, and protein; of lysosomal enzymes (acid phosphatase, acid proteinase); and of peroxisomal enzymes (catalase) were assayed during growth and the subsequent stationary phase that led eventually to encystment. Only DNA remained at a constant cellular level. Encystment of exponentially growing cells could also be initiated by washing them and introducing them into a soluble peptone medium. The rate of encystment was proportional to the osmolarity of this medium. The encystment process was followed with respect to the cellular levels of DNA, RNA, protein, carbohydrates, acid phosphatase, acid β-N-Ac-glucosaminidase, and catalase. The most dramatic change occurred in the cellular cellulose content, which increased by at least an order of magnitude by the time encystment was morphologically complete. It was concluded that the encystment of this slime mold in suspension exhibits a number of biochemical similarities to the development of this and other cellular slime molds on a surface.     

Studies on the Nitrogen Metabolism in Ectomycorrhizae

Concentrations of free and bound amino acids were determined in 1) the mycorrhizal fungus Boletus variegatus Fr. 2) nonmycorrhizal root systems of aseptically grown Pinus sylvestris L. seedlings, and 3) mycorrhizal root systems of seedlings developed aseptically using the two symbionts. Arginine (total) was the major amino acid constituent in the mycelium of B. variegatus (18%–22%) during the exponential phase of growth. While 59%–86% of the available arginine was bound during the acceleration phase of growth, in the logarithmic phase 63%–75% was in the free pool. There were differences in the proportions between the individual amino acids in the bound fraction at different stages of growth suggesting production of diverse proteins. Twenty per cent of the amino acid content of uninfected P. sylvestris root systems was arginine. Infection of the root systems by the fungal symbiont did not result in an increase but a slight decrease in the free arginine content of the composite structure. Almost all other amino acids in the free pool were found in higher concentrations in the mycorrhizal root system. It is suggested that arginine synthesis in B. variegatus is repressed by the arginine available in the host. The mycorrhizal fungus possibly metabolizes the host arginine pool ultimately resulting in more efficient protein synthesis in both the partners.     

Nitrogen Metabolism of Vicia faba

Vicia faba plants were grown for four and six weeks without externally supplied nitrogen. Some nitrogen was transported to the plant axis from the cotyledons throughout this period, but the amount available was insufficient to support maximum shoot growth. During this period the protein content of the shoot declined whilst the free amino acids, especially aspartic acid, glutamic acid, histamine and the combined pool for threonine, serine, asparagine and glutamine and ammonia, increased in amount. In contrast to the shoot the protein content of the root increased as did their free amino acid content, but the increase in the latter was less than in the shoot and only the combined value for threonine, serine, asparagines and glutamine increased significantly. During tbe last two weeks growth, some soluble non-amino acid compound appeared to donate nitrogen to the pool of free amino acids in the root and shoot.     

The Role of Low-Molecular-Weight Nitrogen Compounds in the Osmotolerance of Rhodococcus erythropolis and Arthrobacter globiformis

Investigations showed that Rhodococcus erythropolis E-15 and Arthrobacter globiformis 2F cells respond to osmotic shock by increasing the synthesis of free amino acids, primarily glutamic acid (80% of the intracellular free amino acid pool). The osmoprotective role of glutamic acid follows from its beneficial effect on the growth of bacteria in high-salinity media. It was found that the addition of this amino acid to the growth medium at a concentration of 2 mM shortened the lag phase and increased the growth rate and biomass yield of either of the two bacteria. The addition of another osmoprotectant, trehalose, to the high-salinity growth medium of R. erythropolis E-15 at the same concentration (2 mM), restored the growth parameters of this bacterium to the control values.     

Changes in fatty acids,amino acids and carbon/nitrogen biomass during nitrogen starvation of ammonium- and nitrate-grownIsochrysis galbana

Growth of cells ofIsochrysis galbana with either nitrate or ammonium as the N-source, and the effects of subsequent N-starvation of these cells, were compared. During exponential N-sufficient growth nitrate-grown cells had double the fatty acid content of the ammonium-grown cells but lower concentrations of a few amino acids. Following resuspension in N-free medium the fatty acid content of the ammonium-grown cells increased to that of the nitrate-grown cells, but there was no further increase in fatty acid content on a C-biomass or cellular basis during the following 4 days for either culture. Fatty acid synthesis was continuous during N-starvation, while it occurred during the light-phase only in exponential growth. The proportion of 18:1n9 fatty acid increased from 10 to 25% total fatty acids during N-starvation. Intracellular free amino acid content decreased in a similar manner in both cultures on N-starvation, the ratio of intracellular free amino-N/cell-C falling more rapidly than overall cellular N/C. It was concluded that optimal amino acid and fatty acid content would be attained by growth in the presence of excess nitrate. Measurements of chlorophyll and carotenoid content and ofin vivo fluorescence indicated that these parameters had potential for monitoring the C and N biomass in cultures grown under relatively constant (not necessarily continuous) illumination.     

Distribution of amino acids in the cellular fractions ofStaphylococcus aureus

WhenStaphylococcus aureus cells were labeled with a single radioactive amino acid for 20 minutes, the highest activity, except for alanine, leucine, and glycine, was found in the free pool. Significant amounts of the above amino acids and also valine and methionine were incorporated into the protein — cell wall fraction.Cells previously labeled with a single amino acid underwent a net loss of radioactivity when transferred to buffer, glucose, or complete medium. An exception was glycine. The greatest loss in activity occurred in the free pool.While some amino acids (alanine, cystine) were transferred from the free pool to the protein — cell wall fraction under all conditions tested, others (glutamic acid, proline) were transferred only under conditions of growth.Cells labeled with certain single amino acids and then transferred to a complete medium lost a significant portion of the label. The most extreme case noted was proline, but other amino acids also effluxed from the cell under these conditions.