Evidence for cooperation between cells during sporulation of the yeast Saccharomyces cerevisiae.

Diploid Saccharomyces cerevisiae cells heterozygous for the mating type locus (MATa/MAT alpha) undergo meiosis and sporulation when starved for nitrogen in the presence of a poor carbon source such as potassium acetate. Diploid yeast adenine auxotrophs sporulated well at high cell density (10(7) cells per ml) under these conditions but failed to differentiate at low cell density (10(5) cells per ml). The conditional sporulation-deficient phenotype of adenine auxotrophs could be complemented by wild-type yeast cells, by medium from cultures that sporulate at high cell density, or by exogenously added adenine (or hypoxanthine with some mutants). Adenine and hypoxanthine in addition to guanine, adenosine, and numerous nucleotides were secreted into the medium, each in its unique temporal pattern, by sporulating auxotrophic and prototrophic yeast strains. The major source of these compounds was degradation of RNA. The data indicated that differentiating yeast cells cooperate during sporulation in maintaining sufficiently high concentrations of extracellular purines which are absolutely required for sporulation of adenine auxotrophs. Yeast prototrophs, which also sporulated less efficiently at low cell density (10(3) cells per ml), reutilized secreted purines in preference to de novo-made purine nucleotides whose synthesis was in fact inhibited during sporulation at high cell density. Adenine enhanced sporulation of yeast prototrophs at low cell density. The behavior of adenine auxotrophs bearing additional mutations in purine salvage pathway genes (ade apt1, ade aah1 apt1, ade hpt1) supports a model in which secretion of degradation products, uptake, and reutilization of these products is a signal between cells synchronizing the sporulation process.     

Initiation of Bacillus subtilis sporulation by the stringent response to partial amino acid deprivation

We have controlled the rates at which three different amino acids were available to auxotrophs of Bacillus subtilis by avoiding active transport of the respective substrate. The active transport of oxomethylvalerate, a precursor of isoleucine, was prevented by a kauA mutation, the uptake of L-aspartate was competed by 20 mM L-glutamate, and D-methionine was used instead of L-methionine. When in this way conditions of partial amino acid deprivation were achieved, a partial "stringent response" occurred which included the increase of ppGpp and pppGpp, and the decrease of GTP; such conditions initiated sporulation. In the corresponding relaxed (relA) mutants, the changes of guanine nucleotides were greatly reduced and no sporulation was observed at any substrate concentration; but addition of decoyinine produced a further decrease of GTP and caused sporulation.     

The decrease of guanine nucleotides initiates sporulation of Bacillus subtilis

Massive sporulation of Bacillus subtilis normally begins when carbon, nitrogen or phosphorus sources able to support rapid growth are no longer available. Sporulation can also be induced in exponentially growing cultures, in the presence of rapidly utilizable ammonia, glucose and phosphate if growth is partially but not completely inhibited either by inhibitors of nucleotide synthesis (hadacidin, decoyinine or 6-azauracil) or by purine deprivation in purine and especially in guanine auxotrophs. All these conditions allowing sporulation result in a decrease in the intracellular concentration of guanosine di- and triphosphates and usually uridine di- and triphosphates while other nucleotides decrease in some but increase in other cases. A decrease of uracil nucleotides alone, in a uracil auxotroph, does not produce massive sporulation. Our results demonstrate that the partial reduction of a guanine nucleotide, probably relative to some other compound, suffices to initiate sporulation. This reduction may always play a decisive role in the initiation of sporulation, as we have observed it under all conditions so far known to produce massive sporulation.     

The decrease of guanine nucleotides initiates sporulation of Bacillus subtilis.

Massive sporulation of Bacillus subtilis normally begins when carbon, nitrogen or phosphorus sources able to support rapid growth are no longer available. Sporulation can also be induced in exponentially growing cultures, in the presence of rapidly utilizable ammonia, glucose and phosphate if growth is partially but not completely inhibited either by inhibitors of nucleotide synthesis (hadacidin, decoyinine or 6-azauracil) or by purine deprivation in purine and especially in guanine auxotrophs. All these conditions allowing sporulation result in a decrease in the intracellular concentration of guanosine di- and tri-phosphates and usually uridine di- and triphosphates while other nucleotides decrease in some but increase in other cases. A decrease of uracil nucleotides alone, in a uracil auxotroph, does not produce massive sporulation. Our results demonstrate that the partial reduction of a guanine nucleotide, probably relative to some other compound, suffices to initiate sporulation. This reduction may always play a decisive role in the initiation of sporulation, as we have observed it under all conditions so far known to produce massive sporulation.     

Effect of long-term phosphate starvation on the levels and metabolism of purine nucleotides in suspension-cultured Catharanthus roseus cells

The effect of long-term phosphate (Pi) starvation of up to 3 weeks on the levels of purine nucleotides and related compounds was examined using suspension-cultured Catharanthus roseus cells. Levels of adenine and guanine nucleotides, especially ATP and GTP, were markedly reduced during Pi-starvation. There was an increase in the activity of RNase, DNase, 5'- and 3'-nucleotidases and acid phosphatase, which may participate in the hydrolysis of nucleic acids and nucleotides. Accumulation of adenosine, adenine, guanosine and guanine was observed during the long-term Pi starvation. Long-term Pi starvation markedly depressed the flux of transport of exogenously supplied [8-(14)C]adenosine and [8-(14)C]adenine, but these labelled compounds which were taken up by the cells were readily converted to adenine nucleotides even in Pi-starved cells, in which RNA synthesis from these precursors was significantly reduced. The activities of adenosine kinase, adenine phosphoribosyltransferase and adenosine nucleosidase were maintained at a high level in long-term Pi starved cells.     

Regulatory Role of Adenine Nucleotides in the Biosynthesis of Guanosine 5′-Monophosphate

Derepression of the synthesis of inosine 5′-monophosphate (IMP) dehydrogenase and of xanthosine 5′-monophosphate (XMP) aminase in pur mutants of Escherichia coli which are blocked in the biosynthesis of adenine nucleotides and guanine nucleotides differs in two ways from derepression in pur mutants blocked exclusively in the biosynthesis of guanine nucleotides. (i) The maximal derepression is lower, and (ii) a sharp decrease in the specific activities of AMP dehydrogenase and XMP aminase occurs, following maximal derepression. From the in vivo and in vitro experiments described, it is shown that the lack of adenine nucleotides in derepressed pur mutants blocked in the biosynthesis of adenine and guanine nucleotides is responsible for these two phenomena. The adenine nucleotides are shown to play an important regulatory role in the biosynthesis of guanosine 5′-monophosphate (GMP). (i) They induce the syntheses of IMP dehydrogenase and XMP aminase. (The mechanism of induction may involve the expression of the gua operon.) (ii) They appear to have an activating function in IMP dehydrogenase and XMP aminase activity. The physiological importance of these regulatory characteristics of adenine nucleotides in the biosynthesis of GMP is discussed.     

Effect of methionine on chemotaxis by Bacillus subtilis.

Bacillus subtilis, like Escherichia coli and Salmonella typhimurium, carries out chemotaxis by modulating the relative frequency of smooth swimming and tumbling. Like these enteric bacteria, methionine auxotrophs starved for methionine show an abnormally long-period of smooth swimming after addition of attractant. This "hypersensitive" state requires an hour of starvation for its genesis, which can be hastened by including alanine, a strong attractant, in starvation medium. Susceptibility to repellent, which causes transient tumbling when added, if anything, increases slightly by starvation for methionine. The results are interpreted by postulating the existence of a methionine-derived structure that hastens recovery of attractant-stimulated bacteria back to normal.     

Methionineless Death in Escherichia coli

Methionine auxotrophs of strains derived from Escherichia coli 15 lose their colony-forming ability when deprived of this amino acid. Late addition of methionine to liquid cultures did not restore plating efficiency but permitted growth of surviving cells. This phenomenon, termed methionineless death (mld), was not observed with methionine auxotrophs of E. coli strains B, W, or K(12), nor was a similar amino acidless death observed with corresponding auxotrophs of E. coli 15 for arginine, tryptophan, proline, isoleucine, and leucine. Mld was not dependent upon the genetic site determining methionine auxotrophy, nor did it affect the decarboxylation of methionine or the stability of methionyl-transfer ribonucleic acid synthetase activity of starved cells. Death was not altered by the presence of spermine or spermidine but was abolished by the methionine analogue, alpha-methylmethionine. Simultaneous starvation of another amino acid in a multiple auxotroph also significantly reduced mld, suggesting a possible role of protein synthesis. The onset of mld is correlated with a lower net increase of deoxyribonucleic acid.     

Evidence that Bacillus subtilis sporulation induced by the stringent response is caused by the decrease in GTP or GDP.

Partial amino acid deprivation of Bacillus subtilis, which evokes the stringent response, initiates sporulation not because the highly phosphorylated guanine nucleotides guanosine-5'-diphosphate-3'-diphosphate (ppGpp) and guanosine-5'-triphosphate-3'-diphosphate (pppGpp) increase but because GTP decreases. This was shown with a mutant (Myc) partially resistant to mycophenolate, an inhibitor of IMP dehydrogenase. Upon amino acid deprivation, the Myc mutant (62032) showed the usual increase in ppGpp and pppGpp but a reduced decrease in GTP, and only few cells sporulated. Extensive sporulation was restored by the addition of mycophenolate or decoyinine, and inhibitor of GMP synthetase, which caused a further decrease in GTP.     

INTERACTIONS BETWEEN UV‐B EXPOSURE AND PHOSPHORUS NUTRITION. II. EFFECTS ON RATES OF DAMAGE AND REPAIR1

The interactive effects of P starvation and exposure to UV radiation (UVR) on rates of damage ( k ) and repair ( r ), modeled from exposure response curves (ERCs), in the chlorophyte microalga Dunaliella tertiolecta Butcher were investigated. When nutrient‐replete cells were exposed to the UVR during growth, k and r both increased by approximately 62% and 100%, respectively. However, when cells were starved of phosphorus, k increased by a similar amount as observed in replete cells, but r decreased by about 70%, explaining the increased susceptibility of cells to UVR‐induced inhibition of photosynthesis under P starvation. Although not specifically investigated in this study, it is argued that the decreased repair capacity under P starvation is due to a decline in nucleotides such as ATP and GTP, which are necessary for protein repair.     

Novel (p)ppGpp0 suppressor mutations reveal an unexpected link between methionine catabolism and GTP synthesis in Bacillus subtilis

In bacteria, guanosine (penta)tetra-phosphate ([p]ppGpp) is essential for controlling intracellular metabolism that is needed to adapt to environmental changes, such as amino acid starvation. The (p)ppGpp⁰ strain of Bacillus subtilis, which lacks (p)ppGpp synthetase, is unable to form colonies on minimal medium. Here, we found suppressor mutations in the (p)ppGpp⁰ strain, in the purine nucleotide biosynthesis genes, prs, purF and rpoB/C, which encode RNA polymerase core enzymes. In comparing our work with prior studies of ppGpp⁰ suppressors, we discovered that methionine addition masks the suppression on minimal medium, especially of rpoB/C mutations. Furthermore, methionine addition increases intracellular GTP in rpoB suppressor and this effect is decreased by inhibiting GTP biosynthesis, indicating that methionine addition activated GTP biosynthesis and inhibited growth under amino acid starvation conditions in (p)ppGpp⁰ backgrounds. Furthermore, we propose that the increase in intracellular GTP levels induced by methionine is due to methionine derivatives that increase the activity of the de novo GTP biosynthesis enzyme, GuaB. Our study sheds light on the potential relationship between GTP homeostasis and methionine metabolism, which may be the key to adapting to environmental changes.     

Ca2+-Guanine Nucleotide Interactions in Brain Membranes. II. Characteristics of [3H]Guanosine Triphosphate and [3H]β, γ-Imidoguanosine 5'-Triphosphate Binding and Catabolism in the Rat Hippocampus and Striatum

Abstract: With [³H]guanosine triphosphate ([³H]GTP) and [³H]β, γ -imidoguanosine 5′-triphosphate ([³H]GppNHp) as the labelled substrates, both the binding and the catabolism of guanine nucleotides have been studied in various brain membrane preparations. Both labelled nucleotides bound to a single class of noninteracting sites (K_D= 0.1-0.5 μm ) in membranes from various brain regions (hippocampus, striatum, cerebral cortex). Unlabelled GTP, GppNHp, and guanosine diphosphate (GDP) but not guanosine monophosphate (GMP) and guanosine competitively inhibited the specific binding of [³H]guanine nucleotides. Calcium (0.1–5 mm ) partially prevented the binding of [³H]GTP and [³H]GppNHp to hippocampal and striatal membranes. This resulted from both an increased catabolism of [³H]GTP (into [³H]guanosine) and the likely formation of Ca-guanine nucleotide^2- complexes. The blockade of guanine nucleotide catabolism was responsible for the enhanced binding of [³H]GTP to hippocampal membranes in the presence of 0.1 mm -ATP or 0.1 mm -GMP. Striatal lesions with kainic acid produced both a 50% reduction of the number of specific guanine nucleotide binding sites and an acceleration of [³H]GTP and [³H]GppNHp catabolism (into [³H]guanosine) in membranes from the lesioned striatum. This suggests that guanine nucleotide binding sites were associated (at least in part) with intrinsic neurones whereas the catabolising enzyme(s) would be (mainly) located to glial cells (which proliferate after kainic acid lesion). The characteristics of the [³H]guanine nucleotide binding sites strongly suggest that they may correspond to the GTP subunits regulating neurotransmitter receptors including those labelled with [³H]5-hydroxytryptamine ([³H]5-HT) in the rat brain.     

Induction of Bacterial Differentiation by Adenine- and Adenosine-Analogs and Inhibitors of Nucleic Acid Synthesis

Abstract

Several adenine- or adenosine-analogs, which inhibited growth and decreased the intracellular GTP pool, induced sporulation of Bacillus subtilis. The inducers were added to cultures growing in a medium containing excess ammonium ions, glucose, and phosphate in which cells normally cannot differentiate. They included compounds that are modified in the ribose unit (decoyinine, psicofuranine, cordycepin) or are substituted within the purine ring or at the 6-N position of adenosine (6-methylaminopurine, zeatin, 6-anilinopurine, formycin). Their effects on the cellular concentration of nucleotides were also measured. All sporulation inducers except formycin-A caused a decrease of GMP, GDP and GTP, some by inhibiting IMP dehydrogenase and others by inhibiting GMP synthetase. In contrast, formycin-A caused an increase of GMP while GDP and GTP decreased. Therefore, the compound (signal) controlling sporulation is GDP or GTP but not GMP. Antibiotics inhibiting growth by direct inhibition of nucleic acid synthesis did not induce sporulation.     

The synthesis of S-adenosylmethionine by mutants with defects in S-adenosylmethionine synthetase

Summary Some metK mutants of Salmonella typhimurium with constitutive methionine biosynthesis have no detectable S-adenosylmethionine (SAM) synthetase, the enzyme which converts methionine to SAM, the postulated corepressor of the methionine pathway. However these mutants are not auxotrophic for SAM, an essential compound for many reactions. Here it is shown that these mutants have normal functioning of pathways involving SAM and do in fact produce SAM at as high levels as wild-type. Also, SAM synthetase is shown to be dispensible for growth but not for methionine regulation. These results indicate that there is another route of SAM synthesis independent of SAM synthetase. This route probably also uses methionine as substrate as metK mutants are shown to convert methionine to SAM as efficiently as analogous non-metK strains. The existence of a second route of SAM synthesis makes it necessary to postulate a compartmentalization of SAM made via the SAM synthetase reaction from SAM made in any other way to explain the reduced ability of metK mutants to repress methionine biosynthesis.     

The effect of amino acids on the motile behavior of Bacillus subtilis

Constant levels of amino acids enhanced the velocity of Bacillus subtilis 60015 cells about 2-fold and stimulated the response in motility assays. The stimulation of velocity did not occur via the receptors for chemotaxis. Cysteine and methionine, general inhibitors of chemotaxis, both completely inhibited the smooth response in a temporal gradient of attractant. After methionine starvation B. subtilis 60015 showed no measurable response in a temporal gradient of attractant, this in contrast to the effect observed with some other bacteria. Addition of methionine to starved cells restored the response toward attractant. Revertants of B. subtilis 60015 for methionine requirement could not be starved and showed a normal behavior toward temporal gradients of attractant.Abbreviation O.D.₆₀₀ optical density measured at 600 nm     

Accumulation of 5′ guanine nucleotides by mutants of Brevibacterium ammoniagenes

5′Xanthylic acid was efficiently converted to 5′guanine nucleotides (5′GMP, 5′GDP, and 5′GTP) without being degraded to guanine via 5′GMP by decoyinine resistant mutants of strain KY 13315 which had been isolated from Brevibacterium ammoniagenes and was practically devoid of 5′nucleotide degrading activity. The concentration of phosphate in the medium showed a profound effect on the ratio of the accumulated 5′guanine nucleotides, making it possible to direct the fermentation towards 5′GMP or 5′GTP. A direct accumulation of 5′guanine nucleotides from carbohydrate was possible by mixed cultivation of a 5′XMP accumulating strain and a 5′XMP converting mutant. A maximum concentration of 9.67 mg of 5′guanine nucleotides per ml was obtained directly from glucose in such a mixed culture.     

Mutations affecting the biosynthesis of S-adenosylmethionine cause reduction of DNA methylation in Neurospora crassa.

A temperature-sensitive methionine auxotroph of Neurospora crassa was found in a collection of conditional mutants and shown to be deficient in DNA methylation when grown under semipermissive conditions. The defective gene was identified as met-3, which encodes cystathionine-gamma-synthase. We explored the possibility that the methylation defect results from deficiency of S-adenosylmethionine (SAM), the presumptive methyl group donor. Methionine starvation of mutants from each of nine complementation groups in the methionine (met) pathway (met-1, met-2, met-3, met-5, met-6, met-8, met-9, met-10 and for) resulted in decreased DNA methylation while amino acid starvation, per se, did not. In most of the strains, including wild-type, intracellular SAM peaked during rapid growth (12-18 h after inoculation), whereas DNA methylation continued to increase. In met mutants starved for methionine, SAM levels were most reduced (3-11-fold) during rapid growth while the greatest reduction in DNA methylation levels occurred later. Addition of 3 mM methionine to cultures of met or cysteine-requiring (cys) mutants resulted in 5-28-fold increases in SAM, compared with wild-type, at a time when DNA methylation was reduced approximately 40%, suggesting that the decreased methylation during rapid growth in Neurospora is not due to limiting SAM. DNA methylation continued to increase in a cys-3 mutant that had stopped growing due to methionine starvation, suggesting that methylation is not obligatorily coupled to DNA replication in Neurospora.     

The Mode of Action of a Carboxypeptidase from Malt

Certain methionine auxotrophs of Arthrobacter paraffineus and Bacillus species produce large amounts of O-acetylhomoserine (OAH). The methionine requirement of these auxotrophs could be satisfied by either cystathionine or homocysteine but not by homoserine. The cell-free extacts from the auxotrophs were found to be deficient in cystathionine ?-synthase activity. OAH and O-succinylhomoserine (OSH) could replace methionine in the auxotrophs which are deficient in homoserine-O-transacetylase. A methionine auxotroph of Corynebacterium glutamicum also produced OAH, and the blocked step in the auxotroph appeared to be between cystathionine and homocysteine.

Cell-free extracts of A. paraffineus, C. glutamicum and Bacillus species catalyzed the formation of OAH from acetyl-CoA and homoserine, while a corresponding reaction with succinyl-CoA was not detected. Cystathionine γ-synthases in extracts of C. glutamicum and Bacillus species were specific for OAH, while the enzyme in extract of A. paraffineus was rather specific for OSH though it reacted with OAH to a certain extent.

These results indicate that the biosynthesis of l-methionine in these bacteria involves OAH.     

Effects of food deprivation on expression of growth hormone receptor and proximate composition in liver of black seabream Acanthopagrus schlegeli

The effects of food deprivation on the hepatic level growth hormone receptor (GHR) were investigated in black seabream (Acanthopagrus schlegeli) both at the protein level (by radioreceptor assay) and at the mRNA level (by ribonuclease protection assay). Serum levels of growth hormone (GH) and triiodothyronine (T₃) were also measured. Condition factor and hepatic proximate composition of the fish were also assessed. Significant decrease in hepatic GHR binding was recorded as early as on day 2 of starvation. On day 30 this decrease was even more pronounced, with the level in the starved fish reaching less than 20% the fed control level. A concomitant decrease in the hepatic GHR mRNA content was also noted during this period, with a progressive decrease from day 2 to day 30 of starvation. The extent of decrease in the mRNA content was less pronounced than the decrease in receptor binding, with the hepatic GHR mRNA content in the day 30 starved fish representing approximately 30% of the level in the fed control. In large contrast, serum GH level increased progressively during starvation. After 30 days of starvation, serum GH levels in the starved fish were more than three times the concentration found in the fed control. Serum T₃ levels, on the other hand, decreased during starvation, with the difference reaching significance on day 15 and day 30. After 30 days of starvation, serum T₃ levels in the starved fish were only approximately 40% the concentration found in the fed control. The hepatic lipid content exhibited an increasing trend during starvation. On day 30 the hepatic lipid content of the starved fish had doubled the level found in the fed control. However, the hepatic protein content did not exhibit much change during starvation. There was also a minor decrease in the moisture content of the liver during starvation, but the condition factor of the fish as a whole registered a gradual decrease during the course of food deprivation.     

Initiation of meiosis and sporulation in Saccharomyces cerevisiae does not require a decrease in cyclic AMP.

Meiosis and sporulation of Saccharomyces cerevisiae are initiated in a guanine auxotroph by guanine deprivation (E. Bautz Freese, Z. Olempska-Beer, A. Hartig, and E. Freese, Dev. Biol. 102:438-451, 1984). We used this condition to examine a hypothesis (K. Matsumoto, I. Uno, and T. Ishikawa, Cell 32:417-423, 1983) that initiation of meiosis requires a low level of cAMP. We found that, after guanine deprivation, the intracellular concentration of cAMP transiently decreased not more than 20% and not at all if the cAMP phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX) was added to the medium. Under these conditions, at least 76% of the cells sporulated in the absence of IBMX, and almost 100% sporulated in its presence. The sporulating cells continually excreted cAMP and utilized the gluconeogenic carbon source. The cells failed to sporulate efficiently and to form four-spored asci if simultaneously deprived of guanine and carbon. After guanine deprivation in glucose medium, sporulation remained suppressed and intracellular cAMP was unchanged. We conclude that, under conditions of guanine starvation, cAMP deficiency is not required for initiation of meiosis and sporulation, cAMP is produced in excess and excreted to the medium, the cells sporulate better if the cAMP concentration is increased by addition of IBMX, the cells require a gluconeogenic carbon source for complete and efficient sporulation, and suppression of sporulation by glucose is not mediated by cAMP.