Effect of chloramphenicol and actinomycin D on extracellular alkaline RNAse biosynthesis by Bacillus intermedius

By means of chloramphenicol it was found that biosynthesis of alkaline exocellular RNAase was repressed in Bacillus intermedius by inorganic phosphate. Actinomycin D at a low concentration stimulates RNAase biosynthesis in a medium with a minimal phosphorus concentration in model experiments with washed cells and in the batch culture. As a result, the activity of RNAase rises 2-4 times. The stimulating effect of actinomycin D decreases when phosphorus concentration in the medium is increased The effect of actinomycin D is maximal if the antibiotic is added to the medium when the specific growth rate of the bacterium falls down and the rate of RNAase biosynthesis rises.     

Regulation of Nitrate Reductase in the Basidiomycete Ustilago maydis

Nitrate reductase was induced in Ustilago maydis by growth in medium containing only nitrate as the nitrogen source. Ammonium ions repressed the enzyme and led to a rapid loss of activity. Ammonium did not inhibit the enzyme in vitro; although amino acids partially did so, this cannot account for the rapid loss of in vivo activity which occurred when the ammonium was added. Experiments with cycloheximide and actinomycin D, together with measurements of protein turnover, suggested that nitrate reductase is actively broken down when cells with fully induced activity are transferred to medium containing ammonium ions.     

Control of actinomycin D biosynthesis in Streptomyces parvullus: regulation of tryptophan oxygenase activity

Tryptophan oxygenase (tryptophan 2,3-dioxygenase) activity increases immediately before the initiation of actinomycin D production by Streptomyces parvullus. We have attempted to discern whether this increase is due to a release from catabolite repression or to the synthesis of an inducer substance. The standard culture medium (glutamic acid-histidine-fructose medium) used in antibiotic production studies with S. parvullus contains l-glutamate as a major constituent. l-Glutamate is almost totally consumed before the onset of actinomycin D synthesis. The addition of 10 mM l-glutamate at this stage completely abolished actinomycin D production as well as tryptophan oxygenase synthesis. Fourteen amino acids were tested for a similar effect. Of these, l-glutamate and l-aspartate had the most dramatic effect on tryptophan oxygenase and beta-galactosidase (beta-d-galactosidase), another inducible enzyme. Standard glutamic acid-histidine-fructose medium, preincubated for 23 h to remove l-glutamate, allowed the synthesis of actinomycin D and tryptophan oxygenase by cells at a stage of growth normally considered too early for antibiotic production. A chemically defined medium lacking l-glutamate and adjusted to pH 8.0 was designed to simulate the preincubation medium. The transfer of cells to this artificial preincubation medium resulted in the appearance of tryptophan oxygenase as early as 19 h before normal synthesis occurred, eliminating the possibility that an inducer molecule is synthesized and excreted during the preincubation period. The results of these studies suggest that the increase in tryptophan oxygenase activity before the onset of actinomycin D synthesis, as well as the synthesis of actinomycin D itself, is due to a release from l-glutamate catabolite repression.     

Metabolic regulation in Streptomyces parvulus during actinomycin D synthesis, studied with 13C- and 15N-labeled precursors by 13C and 15N nuclear magnetic resonance spectroscopy and by gas chromatography-mass spectrometry.

Recent studies have suggested that the onset of synthesis of actinomycin D in Streptomyces parvulus is due to a release from L-glutamate catabolic repression. In the present investigation we showed that S. parvulus has the capacity to maintain high levels of intracellular glutamate during the synthesis of actinomycin D. The results seem contradictory, since actinomycin D synthesis cannot start before a release from L-glutamate catabolic repression, but a relatively high intracellular pool of glutamate is needed for the synthesis of actinomycin D. Utilizing different labeled precursors, D-[U-13C]fructose and 13C- and 15N-labeled L-glutamate, and nuclear magnetic resonance techniques, we showed that carbon atoms of an intracellular glutamate pool of S. parvulus were not derived biosynthetically from the culture medium glutamate source but rather from D-fructose catabolism. A new intracellular pyrimidine derivative whose nitrogen and carbon skeletons were derived from exogenous L-glutamate was obtained as the main glutamate metabolite. Another new pyrimidine derivative that had a significantly reduced intracellular mobility and that was derived from D-fructose catabolism was identified in the cell extracts of S. parvulus during actinomycin D synthesis. These pyrimidine derivatives may serve as a nitrogen store for actinomycin D synthesis. In the present study, the N-trimethyl group of a choline derivative was observed by 13C nuclear magnetic resonance spectroscopy in growing S. parvulus cells. The choline group, as well as the N-methyl groups of sarcosine, N-methyl-valine, and the methyl groups of an actinomycin D chromophore, arose from D-fructose catabolism. The 13C enrichments found in the peptide moieties of actinomycin D were in accordance with a mechanism of actinomycin D synthesis from L-glutamate and D-fructose.     

Formation of secreted acid phosphatase during the growth of Saccharomyces cerevisiae yeasts on different sources of carbon and nitrogen nutrition

The effect of certain components in the growth medium on the secretion of acid phosphatase was studied with Saccharomyces cerevisiae. The presence of phosphate at a concentration of 10 mM in the medium inhibited the formation of repressible forms of this enzyme. The synthesis of the secreted enzyme depended on the sources of carbon and nitrogen nutrition. The enzyme yield was highest in a medium with sucrose as a carbon source and ammonium chloride as a nitrogen source. The secretion of acid phosphatase is stimulated by an increase in the sugar content and a deficiency of the nitrogen source in the medium.     

The nature of the increase in ribonuclease activity in germinating seeds of cowpea treated with gibberellic acid or cyclic-amp

There was a two- to three-fold stimulation of RNAase activity by the application of GA₃ and cAMP to cowpea seedlings. The increase in activity was inhibited by the administration of actinomycin D, cycloheximide, cordycepin, 5-fluorouracil and amino acid analogues. Purification of labelled RNAase revealed that GA₃ and cAMP enhanced the RNAase activity predominantly by its fresh synthesis.     

Biosynthesis of the Bacillus intermedius subtilisin-like serine proteinase by the recombinant Bacillus subtilis strain

The effect of certain nutrients on the growth and production of the Bacillus intermedius subtilisin-like serine proteinase by the recombinant strain Bacillus subtilis AJ73(pCS9) was studied. Glucose was found to inhibit the synthesis of proteinase in the early (28 h of growth) but not in the late stationary phase (48 h of growth). The inhibitory effect of the other mono- and disaccharides studied was less pronounced. Casamino acids added to the medium at concentrations of 0.1-1% as an additional carbon and nitrogen source stimulated enzyme biosynthesis. Individual amino acids (cysteine, asparagine, glutamine, tryptophan, histidine, and glutamate) also stimulated enzyme biosynthesis in the early stationary phase by 25-30%, whereas other amino acids (valine, leucine, alanine, and aspartate) were ineffective or even slightly inhibitory to enzyme production. The stimulatory effect of the first group of amino acids on the synthesis of proteinase in the late stationary phase was negligible. In contrast, the bivalent ions Ca2+, Mg2+, and Mn2+ stimulated biosynthesis of proteinase in the late stationary phase (by 20-60%) and not in the early stationary phase. The data indicate that there are differences in the biosyntheses of proteinase by the recombinant B. subtilis strain during the early and late periods of the stationary phases.     

Isolation and characterization of an actinomycin D-sensitive mutant of Saccharomyces cerevisiae.

A single mutation in Saccharomyces cerevisiae conferred sensitivity to low concentrations of actinomycin D. Treatment with actinomycin D preferentially inhibited synthesis of rRNA's. Residual rRNA synthesized was processed normally. Total protein synthesis and inducibility of the enzyme maltase were relatively unaffected at concentrations of actinomycin D which severely inhibited rRNA synthesis.     

Biosynthesis of the Bacillus intermedius subtilisin-like serine proteinase by the recombinant Bacillus subtilis strain

The effect of certain nutrients on the growth and production of the Bacillus intermedius subtilisin-like serine proteinase by the recombinant strain Bacillus subtilis AJ73(pCS9) was studied. Glucose was found to inhibit the synthesis of proteinase in the early (28 h of growth) but not in the late stationary phase (48 h of growth). The inhibitory effect of the other mono-and disaccharides studied was less pronounced. Casamino acids added to the medium at concentrations of 0.1–1% as an additional carbon and nitrogen source stimulated enzyme biosynthesis. Individual amino acids (cysteine, asparagine, glutamine, tryptophan, histidine, and glutamate) also stimulated enzyme biosynthesis in the early stationary phase by 25–30%, whereas other amino acids (valine, leucine, alanine, and aspartate) were ineffective or even slightly inhibitory to enzyme production. The stimulatory effect of the first group of amino acids on the synthesis of proteinase in the late stationary phase was negligible. In contrast, the bivalent ions Ca²⁺, Mg²⁺, and Mn²⁺ stimulated biosynthesis of proteinase in the late stationary phase (by 20–60%) and not in the early stationary phase. The data indicate that there are differences in the biosyntheses of proteinase by the recombinant B. subtilis strain during the early and late periods of the stationary phases.     

Ability of casamino acids to support gellan production by Sphingomonas paucimobilis ATCC 31461

The ability of casamino acids and vitamin-assay casamino acids to support gellan production by Sphingomonas paucimobilis ATCC 31461 was examined in a medium containing glucose or corn syrup as the carbon source relative to yeast extract supplementation. When glucose or corn syrup served as the carbon source, the presence of yeast extract in the growth medium stimulated gellan production by strain ATCC 31461 on casamino acids. Using vitamin-assay casamino acids as the nitrogen source, the addition of vitamins lowered gellan synthesis by glucose-grown cells regardless of yeast extract supplementation while gellan elaboration by corn syrup-grown strain ATCC 31461 cells could only be increased by supplementing vitamins into medium lacking yeast extract. Independent of carbon source, the absence of yeast extract in the medium reduced biomass production. Biomass production by the strain grown on either carbon source was increased by supplementing vitamins in the medium containing yeast extract.     

Extracellular ribonuclease formation in Bacillus subtilis and its stimulation by actinomycin D

1. Extracellular ribonuclease is produced linearly for at least 3hr. by washed post-logarithmic-phase cells of Bacillus subtilis suspended in a medium containing maltose (1%) and casein hydrolysate (0·5%). 2. Low concentrations of actinomycin D (less than 2μg./ml.) stimulate ribonuclease formation, the maximum effect being observed with a concentration of 1μg./ml. Concentrations greater than 2μg./ml. are inhibitory. There is no parallel stimulation of α-amylase formed under the same conditions, and [¹⁴C]uracil incorporation into a perchloric acid-insoluble form is inhibited. 3. The actinomycin D-induced stimulation is not due to the presence of an activator, nor is the inhibition due to the release of an inhibitor by the cells. The effect is on the amount of ribonuclease produced in the medium. 4. Extracellular ribonuclease formation is partially inhibited by anaerobiosis, 2,4-dinitrophenol, sodium azide and by chloramphenicol and puromycin. 5. High concentrations of antibiotic do not completely inhibit ribonuclease formation, but a basal amount of enzyme representing 20min. synthesis in an uninhibited system is always produced. This `antibiotic-insensitive' enzyme could possibly represent preformed enzyme `in the pipe-line' en route to secretion. 6. The stimulated appearance of ribonuclease in the presence of 1μg. of actinomycin D/ml. is shown to be dependent on enzyme synthesis. The mechanism of this effect is discussed.     

Mediation of growth hormone-enhanced expression of the cartilage phenotype in vitro by the availability of the essential amino acid valine

Without increasing cell number, ovine growth hormone was shown to stimulate the incorporation of ²⁵SO₄ by cultured chick embryo chondrocytes into chondroitin sulfate. Since the stimulation of sulfation by growth hormone was abolished when the amino acid concentrations in the medium were doubled, the relationship between amino acids and growth hormone in promoting the synthesis of acid mucopolysaccharides was investigated. Comparison of the incorporation of various labeled amino acids into trichloroacetic acid-soluble and insoluble material revealed that growth hormone promoted the incorporation of only valine into trichloroacetic acid-insoluble material. Furthermore, growth hormone stimulated valine incorporation into both extracellular and intracellular protein, rather than preferentially into extracellular chondromucoprotein. Growth hormone gave a 4-fold stimulation of valine incorporation into collagen without stimulating collagen synthesis. That growth hormone enhances sulfation by stimulating valine availability was further supported by the observations: (a) doubling only the valine concentration in the medium enhanced sulfation; (b) in medium with twice the normal valine concentration, sulfation failed to be further stimulated with the addition of growth hormone; and (c) in medium with all the other amino acids except valine at twice normal concentrations, growth hormone enhanced sulfation. In addition the temporal relationships and synthetic events occurring between growth hormonealtered valine availability and enhanced chondromucoprotein synthesis were studied. It was found that growth hormone-promoted valine incorporation into acid-insoluble material is a rapid effect that can be detected by 10 min after hormone addition and does not require RNA synthesis. Increased valine availability is rapidly reversed after growth hormone removal ( ). On the other hand, growth hormone- and valine-enhanced chondromucoprotein synthesis are slower responses, taking over 24 hr of treatment for a maximal stimulation, and are mediated by RNA synthesis, as indicated by actinomycin D sensitivity. Enhanced chondromucoprotein synthesis is also relatively stable after removal of growth hormone or valine ( ).The evidence suggests that the availability of a single amino acid, valine, plays a regulatory role in the synthesis of a specialized cellular product and that growth hormone acts at some level to alter the availability of this essential amino acid.     

CRF-stimulated biosynthesis of ACTH in a cell-free system from rat anterior pituitaries

A cell-free system consisting of ribosomes, pH 5 enzymes and supernatant prepared from rat anterior pituitaries was found to be active in the incorporation of 3H-serine into ACTH. The rate of biosyntesis of ACTH, in a cell-free system as, measured by the incorporation of radioactive amino acid, and the rate of biological activity were markedly increased by the addition of CRF. The synthesis of ACTH was significantly inhibited by puromycin and RNAase but was not significantly inhibited by actinomycin D and DNAase.     

Extracellular fibrinogenolytic enzyme of Aspergillus fumigatus: substrate-dependent variations in the proteinase synthesis and characterization of the enzyme

Abstract To get a better understanding of the role of the previously reported fibringenolytic enzyme of Aspergillus fumigatus , we investigated the in vitro conditions of enzyme synthesis and attempted to characterize it. Modification of the nitrogen source did not influence the extracellular serine-proteinase profile, but resulted in important quantitative differences in the yields in batch cultures. The enzyme synthesis appeared to be an inducible phenomenon in A. fumigatus since it was initiated exclusively in the presence of proteins or protein hydrolysate. Free amino acids or inorganic nitrogen compounds could not promote significant enzyme production. Moreover, peptone at a concentration of 0.1% appeared to be the best inducer of enzyme synthesis. Conversely, modification of the carbon source did not affect fungal growth or enzyme synthesis. However, the production of chymotrypsin was highly sensitive to the carbohydrate level in the culture medium and, with peptone as nitrogen source, highest yields were obtained in the presence of 0.3 or 0.5% glucose. Culture filtrates of A. fumigatus CBS 113.26 grown with peptone or nitrate as nitrogen source were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Comparison of the protein patterns suggested for the proteinase a molecular mass of 33 kDa which was confirmed by chromatographic purification of the enzyme through (Nα-CBZ)- d -phenylalanine agarose.     

Effect of actinomycin D on RNA synthesis in a stationary culture of Chinese hamster cells stimulated to proliferate]

The study of the effects of actinomycin D on stationary cultures of Chinese hamster cells and those stimulated by medium changing has revealed that RNA synthesis is more sensitive in the latter, the difference in the first place being attributed to the rate of labeled uridine incorporation into cell nucleoli. There is no significant difference in H3-actinomycin D uptake between stationary and stimulated cells, but the latter incorporate more labeled actinomycin D into their nuclei. A pronounced variation in sensitivity to actinomycin D is observed during the prereplicative period of stimulated cells. The first part of this period is more sensitive, which may be due to the necessity for stimulated cells to synthesize a great number of ribosomes to enter the mitotic cycle and to proceed through it.     

Glutathione formation in Penicillium chrysogenum: stimulatory effect of ammonium

Penicillium chrysogenum produced glutathione after growth in a defined medium containing 10 mM-NH4Cl as the sole source of nitrogen. The use of higher ammonium concentrations (100 mM) resulted in stimulation of growth and glutathione formation. In addition, increases in the intracellular pools of glutamate, alanine and glutamine, proportional to the amount of ammonium present in the medium were observed. Resting cell systems, prepared from cells previously grown with ammonium, were able to produce glutathione when incubated with ammonium or the amino acids glutamate, alanine and glutamine. A mutant lacking NADP-dependent glutamate dehydrogenase activity (which has a leaky phenotype on ammonium as sole nitrogen source) required glutamate to synthesize glutathione. Resting cell systems of this mutant, prepared from cells previously grown with ammonium, did not produce glutathione even when incubated with glutamate or glutamine. On the other hand, resting cell systems of this mutant produced glutathione if prepared from cells previously grown with glutamate. The addition of glutamate to resting cell systems of the wild-type strain stimulated the synthesis of gamma-glutamylcysteine synthetase, the first enzyme of glutathione biosynthesis.     

Kinetics of Penicillinase Induction and Variation of Penicillinase Translation in Staphylococcus aureus

At neutral pH, the rate of penicillinase synthesis by staphylococci declines gradually after removal of free inducer, while at pH 5.4 enzyme formation is generally linear for an extended period. Linear synthesis of penicillinase was observed at neutral pH in nonsaturating concentrations (1 μg/ml) of actinomycin D. The rate of enzyme synthesis, corrected for inhibition of growth caused by the antibiotic, was relatively independent of the time of actinomycin addition. The lag preceding linear enzyme formation increased with the interval between induction and the addition of actinomycin. The findings are consistent with the concept that, at neutral pH, “operons” activated by induction are rapidly repressed, while at pH 5.4, this process is delayed.

At a concentration of 4 μg/ml, actinomycin D blocked penicillinase messenger synthesis and also elicited a short-lived acceleration of the increase of penicillinase activity in uninduced and, late after induction, in induced cultures. This effect did not require a functional genomic repressor mechanism since it occurred also in a penicillinase-constitutive strain. It required protein synthesis and could not be attributed to a greater enzyme stability in the presence of actinomycin. The results suggest enhanced penicillinase translation after addition of actinomycin D.