In vitro amino acid requirements of Bacillus anthracis cells

In the process of batch cultivation in a synthetic medium B. anthracis cells actively consume free amino acids. The maximum amino acid consumption per unit of biomass occurs at the exponential phase of growth, but the consumption of serine is maintained at a high level also during the stationary phase. At the same time the consumption of proline by both vaccine and virulent strains is insignificant at the stationary phase of growth. In contrast to B. anthracis virulent strains, vaccine strains have been shown to consume no lysine, histidine, arginine and tryptophan.     

Cellular Changes Accompanying the Transition from Minimal to Maximal Rate of Extracellular Enzyme Secretion by Bacillus amyloliquefaciens

S ummary . During exponential growth of Bacillus amyloliquefaciens in a maltose–L-amino acids medium at 30°, cellular protein, RNA and DNA increased in parallel. After the exponential phase, extracellular protein, including α-amylase, was secreted into the medium at a quarter of the maximum rate of total cellular protein synthesis. The free amino acid pool for protein and the 'nucleotide'pool for RNA both increased fourfold during the transition to the post-exponential phase to 4.0 and 1.6%, respectively, of the cellular dry weight. Subsequently, the nucleotide pool did not change significantly whilst the free amino acid pool was reduced to 2/3 of its maximum size. When post-exponential phase, exoprotein-secreting bacteria were transferred to fresh culture medium, growth was re-established and there was a 4–5 fold reduction in nucleotide pool size accompanied by a loss of exoenzyme-forming ability.     

Growth and development kinetics of Bacillus thuringiensis in batch culture

The kinetics of Bacillus thuringiensis growth and its assimilation of nutrient substances were studied under the conditions of batch cultivation in a complex medium containing yeast extract and in a chemically defined medium with amino acids. The growth of B. thuringiensis can be divided into five phases: exponential growth; decelerated growth; stationary phase when protein crystals are formed; stationary phase when spores are formed; lysis of sporangia releasing spores. The first phase may in turn be subdivided into three stages according to changes in the specific growth rate and substrate assimilation: a high specific growth rate and no glucose assimilation; an abrupt drop in mu and the beginning of intensive glucose assimilation from the medium; a new rise in the specific growth rate. As follows from the results of studying the kinetics of B. thuringiensis growth in a chemically defined medium, the above changes in the exponential growth phase are due to the fact that the culture assimilates yeast extract components in the complex medium or amino acids in the chemically defined medium during this phase, and then starts to assimilate glucose and ammonium in the following phases of growth.     

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.     

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.     

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.     

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     

Changes in yeast amino acid pool with respiratory versus fermentative metabolism

The precursors of the amino acid yeast pool are intermediates of either the glycolytic or the tricarboxilic acid pathway (TCA). Accordingly, the influence of the metabolism (fermentative or respiratory) on the internal amino acid pool of the yeast Saccharmyces cerevisiae was established by measuring the intracellular amino acid concentration of the "grande" strain IF1256 and its "petite" mutant either in steady-state or when shifting from fermentative to respiratory conditions. Under steady-state conditions, when the cells only respire, there is a decrease in nearly all the amino acids whose precursors are intermediates of the glycolytic pathway. When the metabolism is exclusively fermentative, the opposite change takes place. This effect is not observed in those amino acids whose precursors come from the TCA cycle. However, in continuous culture and at dilution rates lower than 0.06 h(-1), there is an enormous increase in the concentration of all the amino acids in both strains, whatever their precursor, whereas, in batch cultures, both strains undergo variations in the concentration of most amino acids, when entering stationary growth phase. Results therefore indicate that, the fact that the cells ferment or respire effectively affect their amino acid pool according to their precursors coming from the glycolytic or the TCA pathway, but other parameters, such as growth rate or exponential versus stationary phase, have a much stronger effect on yeast amino acid concentration.     

Amino acids are general growth inhibitors of Nicotiana silvestris in tissue culture

The growth of Nicotiana silvestris in suspension culture is inhibited by all of the common protein amino acids at the millimolar level, except for L-glutamine. A defined experimental system for growth/inhibition studies has been established, and growth studies were carried out with cells that had been maintained in the exponential growth phase for at least 10 generations (EE cells). The following results were obtained after particularly detailed studies with aromatic amino acids. The onset of inhibition was preceded by a duration of normal growth rate which varied within a range of 12 to 48 h. The degree of inhibition was directly proportional to amino acid concentration and inversely related to the initial cell density of the inoculum. A slowed, but still exponential rate of growth persisted during an early phase of inhibition. Under sufficiently severe conditions, this was followed by progressive diminution of growth rate and eventual lysis. The most drastic inhibitory effects caused by aromatic amino acids were in the order: phenylalanine, tryptophan and tyrosine. When EE cells cultivated under conditions of growth inhibition were diluted into fresh medium, immediate resumption of growth at the uninhibited rate occurred and persisted. On the other hand, when growth-inhibited EE cells were diluted into medium containing the same concentration of amino acid used in the first round of growth, an initial burst of uninhibited growth lasting about 24 h was followed by a drastic, progressively declining growth rate which deteriorated to cell death and lysis. When cells in stationary phase were used as an inoculum, as is done in typical growth characterizations with suspension cultures, the sensitivity to inhibition during the subsequent exponential growth phase was several-fold greater than was the case with EE cells. Hypotheses that growth inhibition might be caused by ammonia toxicity, keto-acid toxicity, or by inhibition of nitrate utilization were ruled out. Observations that provide new insight are: (i)growth-inhibited cells undergo drastic plasmolysis, (ii) L-glutamine is an effective antagonist of amino-acid inhibitors, and (iii) growth-inhibited cells exhibit a transient restoration of normal growth rate upon dilution into fresh growth medium. These results implicate a linkage of amino acids with osmotic regulation and nitrogen metabolism.     

Conditions of the biosynthesis of an extracellular subtilisin-like proteinase by Bacillus pumilus KMM 62

The influence of the cultivation conditions on Bacillus pumilus KMM 62 growth and effectiveness of the production of a subtilisin-like serine proteinase were investigated. Enzyme accumulation in the culture fluid reached the maximum value after 32 and 46-48 h of growth; it depends on the composition of the nutrient medium. The ratio of the concentrations of two main components of the medium, peptone and inorganic phosphate, which was optimal for enzyme biosynthesis was determined by multifactor experiments. Ammonium salts, when introduced as an additional nitrogen source, had different effects on the proteinase biosynthesis at different growth stages: they suppress enzyme production at the early stationary growth phase and stimulate the biosynthesis of the enzyme after 46-48 h of growth. Complex organic substrates (albumin, casein, hemoglobin, and gelatin) have a repressive effect on the biosynthesis of the enzyme. The effect of amino acids on culture growth and enzyme biosynthesis during the early and late stationary growth phase is different. Hydrophilic amino acids, glutamine, and glutamic acid exhibit the most pronounced repressive action on biosynthesis. The activity of different regulatory mechanisms for the synthesis of this proteinase is assumed at the early and late stationary stages of growth.     

Determination of metabolic flux changes during fed-batch cultivation from measurements of intracellular amino acids by LC-MS/MS

Metabolic flux analysis using (13)C-labeled substrates is a well-developed method for investigating cellular behavior in steady-state culture condition. To extend its application, in particular to typical industrial conditions, such as batch and fed-batch cultivations, a novel method of (13)C metabolic flux analysis is proposed. An isotopomer balancing model was developed to elucidate flux distributions in the central metabolism and all amino acids synthetic pathways. A lysine-producing strain of Escherichia coli was cultivated by fed-batch mode in a growth medium containing yeast extract. Mass distribution data was derived from both intracellular free amino acids and proteinogenic amino acids measured by LC-MS/MS, and a correction parameter for the protein turnover effect on the mass distributions of intracellular amino acids was introduced. Metabolic flux distributions were determined in both exponential and stationary phases. Using this new approach, a culture phase-dependent metabolic shift was detected in the fed-batch culture. The approach presented here has great potential for investigating cellular behavior in industrial processes, independent of cultivation modes, metabolic phase and growth medium.     

Derepression of asparaginase II during exponential growth of Saccharomyces cerevisiae on ammonium ion

The biosynthesis of asparaginase II in Saccharomyces cerevisiae is sensitive to nitrogen catabolite repression. In cell cultures growing in complete ammonia medium, asparaginase II synthesis is repressed in the early exponential phase but becomes derepressed in the midexponential phase. When amino acids such as glutamine or asparagine replace ammonium ion in the growth medium, the enzyme remains repressed into the late exponential phase. The three nitrogen compounds permit a similar rate of cell growth and are assimilated at nearly the same rate. In the early exponential phase the internal amino acid pool is larger in cells growing with glutamine or asparagine than in cells growing with ammonium sulfate as the sole source of nitrogen.     

Polyhydric alcohol production and intracellular amino acid pool in relation to halotolerance of the yeast Debaryomyces hansenii

Changes in polyol production and the intracellular amino acid pool were followed during the growth cycle of Debaryomyces hansenii in 4 mM and 2.7 M NaCl media. The intracellular levels of polyols were markedly enhanced by high salinity, the dominant solutes being glycerol in log phase cells and arabinitol in stationary phase cells. At low salinity arabinitol was the most prominent intracellular solute throughout the growth cycle. There were no major changes in the composition of the total amino acid pool with changes in cultural salinity. The amount of total free amino acids related to cell dry weight was 15–50% lower in cells cultured in 2.7 M NaCl as compared to 4 mM NaCl media.After subtraction of contributions from intracellular polyols the calculated cellular C/N ratio was found to be unaffected by cultural age and salinity during the late log and early stationary phase. On prolonged incubation of stationary phase cells, this ratio decreased, particularly at high salinity. The sensitivity of cells towards exposure to high salinity was measured in terms of the length of the lag phase after transference to 2.7 M NaCl media. This lag phase decreased with increasing intracellular polyol concentrations. At a given polyol content, stationary phase cells were considerably less sensitive than were log phase cells.When cultured at high salinity the mutant strain, 26-2b, grew more slowly and retained less of the total polyol produced during the early growth stages than did the wildtype. Exogenously supplied mannitol, arabinitol, and glycerol stimulated the growth of the mutant in saline media. Erythritol was without effect.Abbreviations GLC gas-liquid chromatography - TCA trichloroacetic acid     

Conditions of the biosynthesis of an extracellular subtilisin-like proteinase by <Emphasis Type="Italic">Bacillus pumilus</Emphasis> KMM 62

The influence of the cultivation conditions on Bacillus pumilus KMM 62 growth and effectiveness of the production of a subtilisin-like serine proteinase were investigated. Enzyme accumulation in the culture fluid reached the maximum value after 32 and 46–48 h of growth; it depends on the composition of the nutrient medium. The ratio of the concentrations of two main components of the medium, peptone and inorganic phosphate, which was optimal for enzyme biosynthesis was determined by multifactor experiments. Ammonium salts, when introduced as an additional nitrogen source, had different effects on the proteinase biosynthesis at different growth stages: they suppress enzyme production at the early stationary growth phase and stimulate the biosynthesis of the enzyme after 46–48 h of growth. Complex organic substrates (albumin, casein, hemoglobin, and gelatin) have a repressive effect on the biosynthesis of the enzyme. The effect of amino acids on culture growth and enzyme biosynthesis during the early and late stationary growth phase is different. Hydrophilic amino acids, glutamine, and glutamic acid exhibit the most pronounced repressive action on biosynthesis. The involvement of different regulatory mechanisms of the synthesis of this proteinase is assumed in the early and late stationary phases of growth.     

Utilization of 15N-labelled yeast hydrolysate in Lactococcus lactis IL1403 culture indicates co-consumption of peptide-bound and free amino acids with simultaneous efflux of free amino acids

Lactococcus lactis subsp. lactis IL1403 was grown in medium containing unlabelled free amino acids and ¹⁵N-labelled yeast hydrolysate to gain insight into the role of peptides as a source of amino acids under conditions where free amino acids are abundant. A mathematical model was composed to estimate the fluxes of free and peptide-derived amino acids into and out of the intracellular amino acid pool. We observed co-consumption of peptides and free amino acids and a considerable efflux of most free amino acids during growth. We did not observe significant differences between the peptide consumption patterns of essential and non-essential amino acids, which suggests that the incorporation of a particular amino acid is more dependent on its availability in a readily assimilated form than the organism’s auxotrophy for it. For most amino acids the contribution of peptide-bound forms to the formation of biomass was initially between 30 and 60 % with the remainder originating from free amino acids. During the later stages of fermentation we observed a decrease in the utilization of peptide-bound amino acids, thus indicating that the more readily assimilated peptides are gradually exhausted from the medium during growth.     

Free amino acids in milk containing different amounts of dry substances after its fermentation by lactic acid Streptococci]

The content of free amino acids in milk containing different amounts of dry substances (11.2, 16, 18%) was measured. An increase in the content of dry substances reduced the capacity of lactic acid streptococci to accumulate threonine, proline, alanine, tyrosine and elevated their capacity to accumulate serine, cystine, norleucine, phenylalanine. The content of free amino acids was much higher during continuous cultivation than at the end of the stationary phase.     

State of adenosine phosphates during dehydration of yeast

Summary The effect of cultivation and dehydration conditions on the adenosine phosphate content of yeast cells has been studied. Irrespective of the cultivation conditions the total pool of adenosine phosphates was found to increase, mainly due to accumulation of ATP, during the exponential phase of cell growth and to decrease during transition of the culture into the stationary phase. Changes in the intracellular content of adenosine phosphates were parallel with changes in the respiratory activity of yeast cells cultivated under batch conditions. Yeast cells harvested at the exponential growth phase were sensitive to dehydration, losing a notable amount of adenosine phosphates as well as respiratory capacity during drying, leading to a massive dying-off of the cells. Yeast at the stationary phase was resistant to drying, and, during this process, accumulated ATP by mitochondrial oxidation of endogenous carbohydrates. The accumulated ATP was used by the dried yeast cells as an energy source in the first minutes of reactivation. On the basis of our results we recommend that the ATP content of dried yeast cells should be used as an indicator of their capacity to recover their viability by reactivation.     

Pools and protein synthesis in mammalian cells.

From the kinetics of incorporation into protein shown by four amino acids and one amino acid analogue in suspension cultured HeLa S-3 cells, two distinctly different patterns were observed under the same experimental conditions. An initial slow exponential incorporation followed by linear kinetics was characteristic of the two non-essential amino acids, glycine and proline, whereas the two essential amino acids studied, phenylalanine and leucine, showed linear kinetics of incorporation with no detectable delay. The analogue amino acid, p-fluorophenylalanine also showed immediate linear kinetics of incorporation. There was a poor correlation between the rate of formation of acid-soluble pools and incorporation kinetics. However, the rate of formation of the freely diffusible pool of amino acids correlated more closely with incorporation kinetics. The lack of direct involvement of the acid-soluble pool in protein synthesis was also demonstrated by pre-loading of pools before treatment of cells with labelled amino acids. The results partially support the hypothesis that precursor amino acids for protein synthesis come from the external medium rather than the acid-soluble pool, but suggest that the amino acid which freely diffuses into the cell from the external medium could also be the source.     

Influence of glutamate on growth, sporulation, and spore properties of Bacillus cereus ATCC 14579 in defined medium

A chemically defined medium in combination with an airlift fermentor system was used to study the growth and sporulation of Bacillus cereus ATCC 14579. The medium contained six amino acids and lactate as the main carbon sources. The amino acids were depleted during exponential growth, while lactate was metabolized mainly during stationary phase. Two concentrations of glutamate were used: high (20 mM; YLHG) and low (2.5 mM; YLLG). Under both conditions, sporulation was complete and synchronous. Sporulation started and was completed while significant amounts of carbon and nitrogen sources were still present in the medium, indicating that starvation was not the trigger for sporulation. Analysis of amino acids and NH4+ in the culture supernatant showed that most of the nitrogen assimilated by the bacteria was taken up during sporulation. The consumption of glutamate depended on the initial concentration; in YLLG, all of the glutamate was used early during exponential growth, while in YLHG, almost all of the glutamate was used during sporulation. In YLLG, but not in YLHG, NH4+ was taken up by the cells during sporulation. The total amount of nitrogen used by the bacteria in YLLG was less than that used by the bacteria in YLHG, although a significant amount of NH4+ was present in the medium throughout sporulation. Despite these differences, growth and temporal expression of key sigma factors involved in sporulation were parallel, indicating that the genetic time frames of sporulation were similar under both conditions. Nevertheless, in YLHG, dipicolinic acid production started later and the spores were released from the mother cells much later than in YLLG. Notably, spores had a higher heat resistance when obtained after growth in YLHG than when obtained after growth in YLLG, and the spores germinated more rapidly and completely in response to inosine, l-alanine, and a combination of these two germinants.     

The effect of nitrogen and carbon sources on proteinase production by Pseudomonas fluorescens

Some factors influencing the production of an extracellular proteinase by Pseudomonas fluorescens NCDO 2085 were studied. Proteinase production was optimal at 20C and pH 69 in static culture when calcium was included in the medium. Proteinase was not detectable in basal medium but could be induced by organic nitrogen compounds. The proteinase was produced in the exponential phase of growth on protein substrates but not until early stationary phase during growth on amino acids. The organism did not utilize lactose, the most abundant carbohydrate in milk. Citrate was readily utilized as an energy source but had a strong repressive effect on proteinase production. A medium containing sodium caseinate and pyruvate supported good growth and enzyme production. All the amino acids utilized as a sole carbon source, with the exception of serine, could induce proteinase production. Asparagine was the most effective amino acid inducer. Particular combinations of amino acids could induce or repress proteinase production. The regulation of proteinase production by Ps. fluorescens NCDO 2085 appears to be based on a balance between induction by low concentrations of low molecular weight degradation products and sensitivity to end product catabolite repression. The results suggest that the function of the proteinase is to ensure a supply of carbon rather than amino acids for protein synthesis.