Occurrence of the stringent response in Streptomyces sp. and its significance for the initiation of morphological and physiological differentiation

Streptomyces sp. MA406-A-1 produced formycin (a nucleoside antibiotic) in parallel with cell growth in a synthetic medium. When the synthetic medium was supplemented with 1% (w/v) Casamino acids, however, formycin was produced only after the end of exponential growth. The intracellular ppGpp pool increased gradually towards the end of exponential growth and was maximal at the beginning of formycin production. After shift down from Casamino acids medium to synthetic medium, the ppGpp pool increased immediately, while the GTP pool decreased; under such conditions, the ability to produce formycin increased eightfold. Relaxed (rel) mutants, the first isolated for a Streptomyces species, were found at high incidence (10%) among spontaneous thiopeptin-resistant isolates and had severely reduced abilities to accumulate ppGpp. These rel mutants also failed to produce formycin under the usual culture conditions and exhibited numerous pleiotropic effects such as an inability to produce melanin and an extended delay of aerial mycelium formation. Thus Streptomyces sp. exhibited a typical stringent response, and the response initiated (or was needed for) the induction of secondary metabolism. The response may have also participated in the initiation of aerial mycelium formation by decreasing the intracellular GTP pool.     

The role of histidine in regulating the synthesis of purine nucleotides in Escherichia coli cells]

Coordination of GTP and 5-aminoimidazole-4-carboxamide riboside 5'-phosphate pools changes was studied. The CTP pool is an important component of Escherichia coli metabolism, while AICAR 5'-phosphate being one of alarmones controls the synthesis of GTP. Main attention was paid to histidine, the biosynthesis of which is connected with formation of purine nucleotides. The expression of the histidine operon and biosynthesis of histidine are shown to change the AICAR pool and help the formation of the GTP pool. The ribosomal antibiotics streptomycin and chloramphenicol may cause the temporary deficiency of GTP eliminated by the increase of alarmone AICAR pool. The latter event is concluded to cause the increase in GTP pool independent of the means of AICAR accumulation (C1-pholatedependent restriction of metabolization or, vice versa, the stimulation in the histidine biosynthesis pathway).     

The size of the guanosine triphosphate pool active in the synthesis of stable RNA by stage 6 oocytes of Xenopus laevis

The size of the kinetic pool of GTP used in the synthesis of RNA by stage 6 Xenopus laevis oocytes was calculated by expanding the endogenous pool by measured amounts and analysing the incorporation of [³H]GTP into RNA. In experiments on 5 animals, measurements of kinetic pools of GTP ranged from 190.3 to 314.6 pmoles/oocyte and are similar to previously obtained estimates of the chemically extractable GTP pool. Calculations of the rate of stable RNA synthesis ranged from 0.99 to 1.95 pmoles of GTP incorporated per hour per oocyte. These values were also consistent with previous measurements which were based on the assumption that the entire GTP pool provided precursors for stable RNA synthesis. Consequently, it has been concluded that the GTP pool active in RNA synthesis is indistinguishable from the total chemical pool of GTP.     

Endogenous purine metabolism in the conidia of wild type and certain adenine mutants of Neurospora crassa. I. The nature of the reserve pools and pool utilization during adenine starvation.

Conidia of four adenine auxotrophs (ad 9, ad 3B, ad 8 and ad 4) of Neurospora crassa differ in their ability to germinate on adenine-deficient medium. A large percentage of the ad 9 and ad 3B mutant conidia germinate while those of ad 8 and ad 4 mutant do not. No correlation was found between the size of the conidial purine reserves and the conidial ability to germinate. In all the strains the major fraction of the conidial purine reserve pools was inosine. The ad 8 and ad 4 mutants are blocked after IMP formation in the adenine biosynthetic pathway and therefore cannot use the stored inosine for germination. Pool-utilization studies indicated that in all strains investigated some of the purine reserves were lost from the conidia during incubation. In the most readily germinating strain, ad 9, only small amounts of the purine pool were lost from the conidia and a large portion of the reserve pool was used for nucleic acid synthesis. The nature of the purine reserves present in the conidia, and the ability of the strains to prevent loss of the stored purines from the conidia appear to be among the factors influencing the conidial germination of the adenine mutants of N. crassa.     

Endogenous purine metabolism in the conidia of wild type and certain adenine mutants of neurospora crassa I. The nature of the reserve pools and pool utilization during adenine starvation

Conidia of four adenine auxotrophs (ad 9, ad 3B, ad 8 and ad 4 of Neurospora crassa differ in their ability to germinate on adenine-deficient medium. A large percentage of the ad 9 and ad 3B mutant conidia germinate while those of ad 8 and ad 4 mutant do not. No correlation was found between the size of the conidial purine reserves and the conidial ability to germinate. In all the strains the major fraction of the conidial purine reserved pools was inosine. The ad 8 and ad 4 mutants are blocked after IMP formation in the adenine biosynthetic pathway and therefore cannot use the stored inosine for germination. Pool-utilization studies indicated that in all strains investigated some of the purine reserved were lost from the conidia during incubation. In the most readily germinating strain, ad 9, only small amounts of the purine pool were lost from the conidia and a large portion of the reserve pool was used for nucleic acid synthesis. The nature of the purine reserves present in the conidia, and the ability of the strains to prevent loss of the stored purines from the conidia appear to be among the factors influencing the conidial germination of the adenine mutants of N. crassa.     

GTP-sensitivity of the energy-dependent Ca2+ storage pool in permeabilized pancreatic acinar cells

Isolated rabbit pancreatic acinar cells, permeabilized by saponin treatment and incubated in the presence of 0.1 microM free Ca2+, accumulated 3.3 nmol of Ca2+/mg of acinar protein in an energy-dependent pool. Part of this energy-dependent pool could be released by GTP in a polyethylene glycol-dependent manner. The kinetics of GTP-induced release of Ca2+ showed a biphasic pattern with an initial rapid phase followed by a sustained slower phase. In contrast, IP3-induced release of Ca2+ was completed within 30 s following addition of IP3. No reuptake of Ca2+ was observed following GTP- or IP3-induced release of Ca2+. The GTP effect was independent of IP3 and not inhibited by Ca2+, indicating that the IP3-operated Ca2+ channel is not involved in GTP-induced release of Ca2+. The size of the IP3-releasable pool was not affected by GTP, indicating that GTP, when added to permeabilized acinar cells, does not promote the coupling between IP3-insensitive and IP3-sensitive Ca2+ accumulating organelles. Thus, in permeabilized acinar cells, GTP and IP3 act on different Ca2+ sequestering pools. Interestingly, however, comparison of the size of the GTP-releasable pool with that of the IP3-releasable pool for the cell preparations used in the present study, revealed an inversed relationship, indicating that at the time of permeabilization the GTP-releasable pool can be coupled to a greater or lesser extent to the IP3-releasable pool. This suggests that, in the intact cell, a GTP-dependent mechanism may exist that controls the size of the IP3-releasable pool by coupling IP3-insensitive to IP3-sensitive organelles. Moreover, this suggests that the extent of coupling is preserved during permeabilization.     

Neurospora crassa conidial germination: role of endogenous amino acid pools.

The levels of the endogenous amino acid pools in conidia, germinating conidia, and mycelia of wild-type Neurospora crassa were measured. Three different chromatographic procedures employing the amino acid analyzer were used to identify and quantitatively measure 28 different ninhydrin-positive compounds. All of the common amino acids were detected in conidial extracts except proline, methionine, and cystine. The levels of these three amino acid pools were also very low in mycelia. During the first hour of germination in minimal medium, the levels of most of the free amino acid pools decreased. The pool of glutamic acid, the predominant free amino acid in conidia, decreased 70% during the first hour. Very little glutamic acid or any other amino acid was excreted into the medium. During the first 20 min of germination, the decrease in the glutamic acid pool was nearly equivalent to the increase in the aspartic acid pool. The aspartic acid and lambda-aminobutyric acid pools were the only amino acid pools that increased to maximum levels within the first 20 min of germination and then decreased. It is proposed that an important metabolic event that occurs during the early stages of conidial germination is the production of reduced pyridine nucleotides. The degradation of the large glutamic acid pool existing in the conidia (2.5% of the conidial dry weight) could produce these reduced coenzymes.     

H+ uptake increases GTP-induced connection of inositol 1,4,5-trisphosphate- and caffeine-sensitive calcium pools in pancreatic microsomal vesicles.

Evidence suggests that GTP but not GTP gamma S activates Ca2+ movement between myo-inositol 1,4,5-trisphosphate (IP3)-sensitive and -insensitive Ca2+ pools (1). Measuring 45Ca2+ uptake into pancreatic microsomal vesicles we have determined the sizes of three different Ca2+ pools which release Ca2+ in response 1) to IP3, 2) to caffeine, and 3) to both IP3 and caffeine ("common" Ca2+ pool). In the presence of GTP the size of the IP3-sensitive Ca2+ pool is decreased whereas the "common" Ca2+ pool is increased as compared to control Ca2+ pool sizes in the presence of GTP gamma S. This effect of GTP is inhibited by bafilomycin B1, a specific inhibitor of vacuolar type H+ ATPases (2). We conclude that GTP induced connection between IP3- and caffeine-sensitive Ca2+ pools is triggered by intravesicular acidification and involves function of small GTP-binding proteins, known to mediate interorganelle transfer.     

Response of Guanosine 5′-Triphosphate Concentration to Nutritional Changes and Its Significance for Bacillus subtilis Sporulation

We have investigated the changes in the guanosine 5'-triphosphate (GTP) and P-ribosyl-PP pools in stringent and relaxed strains of Bacillus subtilis under conditions frequently used to initiate sporulation. After a shift-down from a Casamino Acids-glutamate to a glutamate medium (Sterlini-Mandelstam shift-down), the pools of adenosine 5'-triphosphate and P-ribosyl-PP increased in both strains; in the stringent strain, ppGpp and pppGpp increased and GTP decreased rapidly, whereas in the relaxed strain, ppGpp and pppGpp increased only slightly and GTP decreased only slowly and less extensively. The stringent strain sporulated well, whereas the relaxed strain sporulated late and poorly. Addition of decoyinine, an inhibitor of guanosine 5'-monophosphate synthetase, caused a further decrease of GTP and initiated good sporulation of the relaxed strain. After a shift-down from a glucose-lactate to a lactate medium (Ramaley-Burden shift-down) the pool of P-ribosyl-PP (and GTP) decreased in both strains, indicating a shortage of purine precursors. This shift-down also caused a stringent response which prevented the consumption of nucleotides, as shown by the maintenance of adenosine 5'-triphosphate at a high concentration in the stringent strain but not in the relaxed strain. After a delay, the relaxed strain, in which GTP decreased as fast as in the stringent strain, sporulated also as efficiently. In nutrient sporulation medium the stringent strain and, less effectively, the relaxed strain accumulated ppGpp and pppGpp transiently towards the end of exponential growth. Eventually, the P-ribosyl-PP pool decreased drastically in both strains. In all cases the initiation of sporulation was correlated with a significant decrease of GTP. Granaticin, an antibiotic which prevents the charging of leucyl-transfer ribonucleic acid, was used to show that the stringent response inhibited the formation of xanthosine monophosphate from inosine monophosphate. It prevented the accumulation of xanthosine monophosphate in decoyinine-treated cultures of the stringent strain but not in those of the relaxed strain.     

Formvar membrane laid on artificial medium induces haustorium-like structure formation in powdery mildew fungi

We aimed to develop an artificial membrane system to observe the infection process of the obligate biotrophic powdery mildew fungi without the use of living plant cells. The conidia of Blumeria graminis and Erysiphe pisi conidia were inoculated on a formvar membrane laid on an artificial medium. Germinated conidia frequently formed appressoria and then penetrated the membrane to form haustorium-like structures in the artificial medium. Secondary hyphae elongation was also observed after the formation of haustorium-like structures. These results suggested that the formvar membrane laid on artificial medium induced the formation of haustorium-like structures that have roles in the formation of secondary hyphae.     

Molecular cloning and characterization of the obg gene of Streptomyces griseus in relation to the onset of morphological differentiation.

Morphological differentiation in microorganisms is usually accompanied by a decrease in intracellular GTP pool size, as has been demonstrated in bacillaceae, streptomycetaceae, and yeasts. The obg gene, which codes for a GTP-binding protein belonging to the GTPase superfamily of proteins, was cloned from Streptomyces griseus IFO13189. The gene is located just downstream of the genes for ribosomal proteins L21 and L27, encoded a protein of 478 amino acids (51 kDa), and possessed three consensus motifs which confer GTP-binding ability; Obg protein expressed in Escherichia coli bound GTP, as demonstrated using a UV cross-linking method. Introduction of multiple copies of obg into wild-type S. griseus suppressed aerial mycelium development in cells on solid media. However, no effect on streptomycin production was detected, indicating that Obg is involved in the regulation of the onset of morphological but not physiological differentiation. Multiple copies of obg also suppressed submerged spore formation in liquid culture. Southern hybridization studies indicated that genes homologous to obg exist widely in streptomycetes, and an obg homolog was successfully cloned from S. coelicolor A3(2). We propose that by monitoring the intracellular GTP pool size, the Obg protein is involved in sensing changes in the nutritional environment leading ultimately to morphological differentiation.     

A decrease in GTP content is associated with aerial mycelium formation in Streptomyces MA406-A-1

Aerial mycelium formation by Streptomyces sp. MA406-A-1, a formycin-producing strain, was suppressed by the presence of excess nutrient. In such suppressed cultures, decoyinine, which specifically inhibits GMP synthetase, initiated the formation of aerial mycelium at concentrations which only partially inhibited growth. The intracellular GTP pool of organisms growing in liquid culture markedly decreased on the addition of decoyinine. Decoyinine was also effective in initiating aerial mycelium formation of three other Streptomyces spp. examined. Regardless of the successful initiation of aerial mycelium formation, the ability of the cells to produce antibiotics (formycin or actinomycin D) did not increase, but decreased, on the addition of decoyinine. It is concluded that aerial mycelium formation by Streptomyces results from a decrease in the pool of GTP (or GDP), whereas antibiotic synthesis results from a different signal(s).     

An inhibitor of eIF2 activity in the sRNA pool of eukaryotic cells

Eukaryotic protein synthesis is a multi-step and highly controlled process that includes an early initiation complex containing eukaryotic initiation factor 2 (eIF2), GTP, and methionine-charged initiator methionyl-tRNA (met-tRNAi). During studies to reconstruct formation of the ternary complex containing these molecules, we detected a potent inhibitor in low molecular mass RNA (sRNA) preparations of eukaryotic tRNA. The ternary complex inhibitor (TCI) was retained in the total sRNA pool after met-tRNAi was charged by aminoacyl tRNA synthetase, co-eluted with sRNA by size exclusion chromatography, but resolved from met-tRNAi by ion exchange chromatography. The adverse effect of TCI was not overcome by high GTP or magnesium omission and was independent of GTP regeneration. Rather, TCI suppressed the rate of ternary complex formation, and disrupted protein synthesis and the accumulation of heavy polymeric ribosomes in reticulocyte lysates in vitro. Lastly, a component or components in ribosome depleted cell lysate significantly reversed TCI activity. Since assembly of the met-tRNAi/eIF2/GTP ternary complex is integral to protein synthesis, awareness of TCI is important to avoid confusion in studies of translation initiation. A clear definition of TCI may also allow a better appreciation of physiologic or pathologic situations, factors, and events that control protein synthesis in vivo.     

Effects of temperature,light, carbon and nitrogen nutrition on reproduction in Phoma medicaginis

Reproduction by three isolates ofPhoma medicaginis growing on potato dextrose agar was studied. The formation of pycnidia was optimum at 30°C whereas the optimum for the formation of conidia was 20°C. Light consistently increased the numbers of pycnidia and conidia over those formed in darkness and more conidia were produced in light at 23°C than at 30°C. The effects of carbon and nitrogen sources on reproduction were studied using modified Richard's medium as the basal medium. Fourteen carbohydrates, 11 amino acids, 9 inorganic nitrogen sources and urea were evaluated by replacing the carbohydrate or nitrogen in Richard's medium with the test substance. Generally, the monosaccharides and disaccharides were about alike and superior to polysaccharides for the production of pycnidia. The carbon sources were about equally useful for production of conidia, but the polysaccharides were superior to the other two classes of carbon sources when the number of conidia/pycnidium was calculated. Generally, the formation of pycnidia and conidia was favored by nitrate more than by ammonium nitrogen sources. The average number of conidia/pycnidium was greatest, however, when the nitrogen source was NH₄NO₃. All amino acids tested appeared to be useful nitrogen sources for production of pycnidia but none were especially good for conidia production. L-isoleucine was superior to the other amino acids tested. Of three isolates used, Illinois 23 consistently produced more pycnidia and conidia that did isolates Minnesota 2 and Missouri 5. Usually the significant interactions between isolates and other treatments were due to a greater response by isolate Ill. 23. It was concluded that the reproduction ofP. medicaginis varies significantly with the isolates of the fungus, the environment, and nutrients as well as with interactions among these factors, and conclusions about the influence of a particular factor will depend on whether the formation of pycnidia, conidia or conidia/pycnidium is being studied.Paper No. 7425, Scientific Journal Series, Minnesota Agricultural Experiment Station.     

Hereditary variation and genetic recombination in Koji-molds (Aspergillus oryzae and Asp. sojae). I. Natural variation

Natural variation in monospore lines of Koji-molds (Asp. oryzae and Asp. sojae), isolated from commercial Koji material or soil and from laboratory stock cultures, has been observed. We can divide the 58 strains of Koji-molds investigated into two groups; one group consists of inconstant strains which are very liable to produce natural variants, and the other consists of strains which remain constant through successive single spore culture. The inconstant strains develop colonies bearing various proportions of conidia and aerial mycelium (X-type). They generally form large conidia (Asp. oryzae var. magnasporus) but sometimes medium sized conidia (Asp. oryzae s. str.), which produce large conidia occasionally. The colonies of the constant strains show abundant conidial formation and smooth surfaces (C-type). The conidia are mostly small (Asp. oryzae var. microsporus) but sometimes medium in size (Asp. oryzae s. str.). The colony types of the variants are as follows: C (Conidial type, whole colony covered with conidia), M (mycelial type), R (restricted in growth rate), St (sterile type, little sporulation on all media tested), Nit (requiring reduced nitrate, very faint growth on Czapek's agar), and LS (semi lethal, growth cease immediately after germination). Pedigree cultures of the 8 inconstant strains have been made, but no definite segregation ratios for each variant type have been recognized through successive generations. The LS and N types commonly occur spontaneously from the M-type.     

Effect of histidine on purine nucleotide synthesis and utilization in Neurospora crassa.

Histidine affects de novo purine nucleotide synthesis and purine nucleotide pool utilization in Neurospora crassa. The former effect was assessed qualitatively by the presence or absence of purple pigment production in ad3B and ad3A mutants. Tryptophan also affected the de novo purine nucleotide synthesis. The effect of histidine on purine nucleotide pool utilization resulted in stimulated germination of ad8 and ad4 mutant conidia in adenine-deficient medium. Increased germination was correlated with increased net levels of nucleic acids in these strains. Possible mechanisms for the dual action of histidine are discussed.     

纯培养下拟茎点霉分生孢子的形成及意义

以18种共23个拟茎点霉菌株为材料,比较了在25℃、12h光照(40w日光灯)/d条件下不同培养基上甲、乙型分生孢子的形成情况。结果表明,苜蓿煎汁+Czapek培养基能促使多数拟茎点霉菌株形成甲、乙两型分生孢子,其形态及大小也与自然寄主上的相一致。根据纯培养获得的形态特征对原始描述缺乙型分生孢子特征记载的Phomopsis durionis, homopsis sterculicola, Phomopsis macadamii, Phomopsis lucumicola和Phomopsis tinea进行了补充描述。     

GTP stimulates pregnenolone generation in isolated rat adrenal mitochondria

Pregnenolone synthesis from cholesterol by adrenal mitochondria isolated from ether-stressed rats exhibits a biphasic time course: upon the addition of a reducing substrate (e.g. malate), a rapid phase of pregnenolone formation occurs during the first 5 min, which has been interpreted as the metabolism of a steroidogenic pool of cholesterol, probably in the inner membrane. A slower rate follows, which is interpreted as translocation of cholesterol into the steroidogenic pool. While a 30-min preincubation of mitochondria with cholesterol alone did not affect the extent of the rapid phase, preincubation with GTP plus cholesterol extended the first phase, resulting in an up to 2-fold increase in pregnenolone synthesis by 20-30 min. The apparent Km for GTP was 0.1-0.4 mM, and stimulation was maximal with preincubation times of 10-30 min, depending upon incubation conditions. Exogenous cholesterol was not required to observe a stimulatory effect, indicating that GTP reorganizes the endogenous mitochondrial cholesterol pools. Nevertheless, stimulation was greater when exogenous cholesterol was provided, consistent with enhanced utilization of both endogenous and exogenous cholesterol. Stimulation by GTP was also seen in mitochondria isolated from cycloheximide-injected/ether-stressed rats, although the activity in these preparations was always lower than that in mitochondria from ether-stressed rats. The stimulation was specific for GTP, since many other nucleotides (e.g. ATP, GDP, and ITP) and GTP analogues (guanosine 5'-O-(3-thiotriphosphate and guanosine 5'-(beta,gamma-imino)triphosphate) had no effect. The GTP-activated state was reversible: after GTP hydrolysis by a mitochondrial GTPase, pregnenolone synthesis returned to the basal level. Sonic disruption of mitochondria abolished the stimulatory effect of GTP. These results suggest that GTP enhances pregnenolone synthesis by promoting the movement of cholesterol to the steroidogenic pool, consistent with a recently proposed general role for GTP in some vectorial transport processes (Bourne, H. R. (1988) Cell 53, 669-671).     

Influence of solute, pH, and incubation temperature on recovery of heat-stressed Wallemia sebi conidia

The influences of glucose, sorbitol, and NaCl in a basal enumeration medium at water activities (aw) from 0.82 to 0.97 on colony formation by sublethally heat-stressed Wallemia sebi conidia were determined. Over this aw range, glucose and sorbitol had similar effects on recovery, whereas at an aw of 0.82 to 0.92, NaCl had a detrimental effect. Colony diameters were generally largest on media containing sorbitol and smallest on media containing NaCl. Maximum colony size and viable population of heat-stressed conidia were observed on media at an aw of ca. 0.92. When the recovery incubation temperature was 20 degrees C, the number of uninjured conidia detected at an aw of 0.82 was reduced compared with the number detected at 25 degrees C, while at 30 degrees C, the number recovered at an aw of 0.97 was reduced. The effect on heat-stressed conidia was magnified. This suggests that W. sebi conidia may be more tolerant of aw values higher than the optimum 0.92 when the incubation temperature is decreased from the near optimum of 25 degrees C and less tolerant of aw values greater than 0.92 when the incubation temperature is higher than 25 degrees C. The sensitivity of heat-stressed conidia increased as the pH of the recovery medium was decreased from 6.55 to 3.71. W. sebi conidia dispersed in wheat flour at aw values of 0.43 and 0.71 and stored for up to 65 days at both 1 and 25 degrees C neither lost viability nor underwent sublethal desiccation or temperature injury.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of inhibitors of enzymatic DNA methylation on the formation of reproductive structures and carotenoid production in Neurospora crassa]

The effect of inhibitors of DNA methylation on light-sensitive developmental stages of the filamentous fungus Neurospora crassa was studied. Under conditions of nitrogen starvation, when blue light induced protoperithecia development and inhibited conidia formation, 5-azacytidine (3-300 microM) inhibited protoperithecia formation and stimulated conidia formation (a 700-fold increase after light induction). After treatment of the mycelium with 5-azacytidine, the protoperithecia formation was accompanied by inversely proportional changes in the formation of conidia, both in the dark and after illumination. In the mycelium cultivated on the Vogel's medium, 5-azacytidine (up to 30 microM) and methotrexate (up to 3 microM) stimulated the light-induced carotenoid synthesis by 30%, whereas higher concentrations of these agents were toxic to carotenoid synthesis and growth.