Sporulation Events Research Articles

The induction of sporulation in the aquatic fungus Blastocladiella emersonii is dependent on extracellular calcium

Induction of sporulation in Blastocladiella emersonii is absolutely dependent on extracellular calcium. Vegetative cells grown in media with or without calcium do not sporulate in media devoid of calcium or in CaCl 2 with EGTA. Calcium channel blockers, CoCl 2 and nifedipine, and ionophore A23187 inhibited the induction of sporulation. The calmodulin antagonists trifluoperazine and chlorpromazine inhibited the sporulation when present in the cultures at least 60 min after induction. So, calcium that is accumulated during growth is not sufficient or is not mobilized to initiate sporulation, and a calcium influx is likely to occur by type II calcium channel functions, essential for the response to nutritional starvation. A calmodulin-like protein has been suggested to mediate calcium events in sporulation.

The induction of sporulation in the aquatic fungus blastocladiella emersonii is dependent on extracellular calcium

Induction of sporulation in Blastocladiella emersonii is absolutely dependent on extracellular calcium. Vegetative cells grown in media with or without calcium do not sporulate in media devoid of calcium or in CaCl(2) with EGTA. Calcium channel blockers, CoCl(2) and nifedipine, and ionophore A23187 inhibited the induction of sporulation. The calmodulin antagonists trifluoperazine and chlorpromazine inhibited the sporulation when present in the cultures at least 60 min after induction. So, calcium that is accumulated during growth is not sufficient or is not mobilized to initiate sporulation, and a calcium influx is likely to occur by type II calcium channel functions, essential for the response to nutritional starvation. A calmodulin-like protein has been suggested to mediate calcium events in sporulation.

Cyclical systemic colonization inVerticillium-infected tomato

It is generally believed thatVerticilliummoves slowly up a plant through spore trapping sites in the vascular tissue in a stepwise fashion. In a resistant plant, rapid defence responses around these sites slow pathogen growth and often restrict the fungus to the root and stem base. In a susceptible plant, the fungus escapes and eventually proliferates logarithmically in the upper stem and leaves. In the present study Craigella tomatoes were infected withVerticillium albo-atrum. Amounts of pathogen were monitored in the upper stems by a PCR-based quantitative assay and S1nuclease protection was used to study expression of a fungal induced phenylalanine ammonia lyase gene (tPAL5) during a 2–21 day time course. Even in resistant plants fungal colonization and PAL gene expression were found to be fully systemic from the earliest sporulation events (i.e. 2–4 days post-inoculation) and to occur in cycles, the PAL gene cycle following the colonization cycle. Peaks of pathogen growth occurred at 2–4 days and 12–15 days, succeeded by intermittent periods of fungal elimination. The results indicated that wilt is more systemic and also more dynamic in terms of fungal population fluctuations than previously thought.

Restricted transcription from sigma H or phosphorylated spo0A dependent promoters in the temperature-sensitive secA341 mutant of Bacillus subtilis.

The temperature-sensitive secA341 mutation of Bacillus subtilis affects sporulation and sporulation-associated events as well as protein secretion and cell septation. With lacZ or bgaB fusion genes, we examined the expression of the early sporulation genes in the mutant strain. Transcriptional expression of delta H dependent kinA, spo0A (Ps), phrC, spoVG, and citG (p2) genes was blocked by the secA341 mutation at 37 degrees C. On the other hand, neither repression of the abrB gene nor induction of the spoH (delta H) gene was affected. Active RNA polymerase containing delta H was, however, found to be produced in the mutant cells. Expression of the phosphorylated Spo0A dependent spoIIG operon was also blocked. Thus the secA341 mutation blocks some step(s) or factor(s) required for delta H-dependent transcription in vivo.

The CDK-activating kinase CAK1 can dosage suppress sporulation defects of smk1 MAP kinase mutants and is required for spore wall morphogenesis in Saccharomyces cerevisiae.

Mitogen-activated protein (MAP) kinase pathways are evolutionarily conserved kinase cascades that are required for the response of eukaryotic cells to a wide variety of environmental stimuli. MAP kinase pathways are also required for the execution of developmental and differentiative programs in a variety of cell and tissue types. SMK1 encodes a developmentally regulated MAP kinase in yeast that is required for spore wall morphogenesis. Cyclin-dependent kinase-activating kinases (CAKs) phosphorylate a conserved threonine residue in the activating loop of cyclin-dependent kinases. CAK1 encodes the major CAK activity in yeast and is required for cell cycle progression. The work presented here demonstrates that CAK1 functions positively in the spore wall morphogenesis pathway. First, CAK1 has been isolated as a dosage suppressor of a conditional smk1 mutant that is defective for spore wall morphogenesis. Second, CAK1 mRNA accumulates during spore development contemporaneously with SMK1 mRNA. Third, cak1 mutant strains have been isolated that are able to complete meiosis I and II but are specifically defective in assembly of the spore wall. These results show that cell cycle progression and morphogenetic pathways can be regulated by a single gene product and suggest mechanisms for coordinating these processes during development.

Bald mutants of Streptomyces griseus that prematurely undergo key events of sporulation.

To identify the structural defects of nonsporulating mutants of Streptomyces griseus, the wild-type strain and class III bald mutants were examined by using transmission electron microscopy, ultrasonic treatment, and fluorescence microscopy after the induction of submerged sporulation by phosphate starvation. In the wild-type strain, submerged sporulation was marked by the relatively synchronous formation of sporogenic hyphae, nucleoid segregation, deposition of sporulation septa, and subsequent thickening of the spore walls during maturation. All of the class III mutants prematurely synthesized sporulation septa and thick spore walls. The class IIIA and C mutants formed sporogenic hyphae earlier than the wild-type strain and underwent nucleoid segregation in parallel with sporulation septum formation. In the class IIIB (bld4) mutant, DNA segregation appeared to be uncoupled from septum formation. The results indicate that the class III mutants are defective in loci that are involved in the regulation of key events of Streptomyces morphogenesis.

Characterization of cell cycle events during the onset of sporulation in Bacillus subtilis

To elucidate the process of asymmetric division during sporulation of Bacillus subtilis, we have measured changes in cell cycle parameters during the transition from vegetative growth to sporulation. Because the propensity of B. subtilis to grow in chains of cells precludes the use of automated cell-scanning devices, we have developed a fluorescence microscopic method for analyzing cell cycle parameters in individual cells. From the results obtained, and measurements of DNA replication fork elongation rates and the escape time of sporulation from the inhibition of DNA replication, we have derived a detailed time scale for the early morphological events of sporulation which is mainly consistent with the cell cycle changes expected following nutritional downshift. The previously postulated sensitive stage in the DNA replication cycle, beyond which the cell is unable to sporulate without a new cell cycle, could represent a point in the division cycle at which the starved cell cannot avoid attaining the initiation mass for DNA replication and thus embarking on another round of the cell cycle. The final cell cycle event, formation of the asymmetric spore septum, occurs at about the time in the cell cycle at which the uninduced cell would have divided centrally, in keeping with the view that spore septation is a modified version of vegetative division.

The importance of morphological events and intercellular interactions in the regulation of prespore‐specific gene expression during sporulation in Bacillus subtilis

We have established a time course for the early morphological events of sporulation in Bacillus subtilis and related this to changes in gene expression, particularly those occurring in the prespore compartment. We have also systematically studied the effects of mutations in various regulatory (spo) genes on prespore-specific gene expression. On the basis of these results, and those of other laboratories, at least four distinct temporal classes of prespore-specific gene expression can now be distinguished. The first class begins within 15 min of the formation of the spore septum, and requires the sigma F form of RNA polymerase. The second class, also directed by RNA polymerase containing sigma F, begins soon after the completion of prespore engulfment, and depends on an intercellular signal from the mother cell. This transcription results in synthesis of sigma G. However, sigma G activity, directing the third class of gene expression, appears only about 30 min later and is dependent on the completion of prespore engulfment and on further interactions with the mother cell. The fourth class of gene expression has been described. The results demonstrate that the prespore programme of gene expression incorporates a series of control points modulated by information from the mother cell and on progress through the morphogenetic process.

Transition-state regulators: sentinels of Bacillus subtilis post-exponential gene expression.

When Bacillus subtilis encounters a nutrient-depleted environment, it expresses a wide variety of genes that encode functions in alternative pathways of metabolism and energy production. Expression of these genes first occurs during the transition from active growth into stationary phase and is controlled by a class of proteins termed transition-state regulators. In several instances, a given gene is redundantly controlled by two or more of these regulators and many of these regulators control genes in numerous different pathways. The AbrB, Hpr and Sin proteins are the best-studied examples of these regulatory molecules. Their role is to prevent inappropriate and possibly detrimental functions from being expressed during exponential growth when they are not needed. They serve as elements integrating sporulation with ancillary stationary-phase phenomena and appear to participate in the timing of early sporulation events and in fine-tuning the magnitude of gene expression in response to specific environmental conditions.

Aerobiology ofClaviceps purpureain Kentucky Bluegrass

Airborne ascospores of Claviceps purpurea were monitored with volumetric spore traps in two fields of Kentucky bluegrass in Oregon during 1989 and 1990. A diurnal periodicity in ascospore release was observed, with peak catches between 1:00 and 6:00 a.m. Sporulation events (one or more consecutive days on which ascospores were trapped) generally began 2-3 days after rain and continued for 1-16 days. Cumulative number of ascospores trapped paralleled cumulative hours per day with rain. Ascospore release in the field was not correlated with temperature or relative humidity. Under controlled temperature conditions, however, acospore release increased with rise in temperature from 5 to 20 C

A structured model for vegetative growth and sporulation inBacillus thuringiensis

A mathematical model has been developed for the delta-endotoxin producing Bacillus thuringiensis. The structure of the model involves the processes taking place during vegetative growth, those leading to the initiation of sporulation under conditions of carbon and/or nitrogen limitation, and the sporulation events. The key features in the model are the pools of compounds, such as PRPP, IMP, ADP/ATP, GDP/GTP, pyrimidine nucleotides, NAD/NADH2, amino acids, nucleic acids, cell wall, and vegetative and sporulation proteins. These, along with sigma-factors that control the nature of RNA-polymerase during the different phases, effectively stimulate the vegetative growth and sporulation. The initiation of sporulation is controlled by the intracellular concentration of GTP. Results of simulation of vegetative growth, initiation of sporulation, spore protein formation, and production of delta-endotoxin under C- or N-limitation are presented.

The cdc30 mutation in Saccharomyces cerevisiae affects phosphoglucose isomerase, the cell cycle and sporulation.

Spontaneous revertants of the cdc30 mutation in Saccharomyces cerevisiae simultaneously regained the ability to grow and divide at 36.5 degrees C on glucose-containing media along with a more thermostable phosphoglucose isomerase (PGI). An independently isolated allele of cdc30 gave a similar phenotype to that previously described including temperature-sensitivity of PGI. Isoelectric focussing allowed the separation of two isoenzymes of PGI. These results all support the idea that two genes--PGI1 and CDC30--are responsible for PGI activity in yeast. Diploid strains homozygous for the cdc30 mutation sporulated poorly in potassium acetate irrespective of whether the cells had previously been cultured at a temperature that was permissive or restrictive for cell cycle progression. This was not surprising because a strain defective in PGI would not be expected to be able to complete the gluconeogenic events of sporulation.

Ammonium ions and glutamine inhibit sporulation of Coprinus cinereus basidia assayed in vitro

Basidia of Coprinus cinereus (Schaeff.: Fr) S. F. Gray are committed to their developmental pathway, continuing through meiosis and sporulation even when excised from their parental fruit body. A technique is described which permits this in vitro differentiation to be used as a rapid, small-scale bioassay for chemicals which interfere with these morphogenetic processes. Of a range of compounds tested, only ammonium and glutamine, and some structural analogues, were able to inhibit basidium differentiation. Growth was not inhibited; instead the differentiation inhibitors caused vegetative hyphal tips to grow out from regions of the basidial apparatus expected to be in active growth during sporulation. Depending on the stage reached at the time of exposure to the inhibitors, vegetative hyphal tips emerged from the four apical sites for sterigmata, from the tips of sterigmata, from partially formed or abnormal spores, and from the basal regions of the basidium from which paraphyses would be expected to arise. The experiments show that ammonium ions and glutamine halt meiocyte differentiation. Reports of similar effects in other organisms, animals and plants as well as fungi, may imply that sporulation events are generally sensitive to ammonium inhibition.

Fed-batch with biomass recycle and batch production of 2,3-butanediol from glucose by Bacillus polymyxa

The production of 2,3-butanediol from glucose by Bacillus polymyxa in batch was sensitive to both protein concentration and aeration rate. Two fed-batch experiments which were resupplied from a reservoir containing urea as the sole source of nitrogen, and incorporated total biomass recycle resulted in yields of 65.84 mM and 69.66 mM of 2,3-butanediol per 100 mM of glucose utilized. No massive sporulation events were detected. In addition, fed-batch with recycle yielded more butanediol and less acetate than any batch run.

Subcellular differentiation in sporulating yeast cells

We have previously described the induction of two sets of sporulation-specific mRNAs in Saccharomyces cerevisiae. Herein we correlate the appearance of these RNAs with the major morphogenic events of sporulation, and we analyze the spatial distribution of the RNAs within the ascus. Several observations suggest that the first set of messages is involved in spore wall synthesis. In fractionation experiments, these mRNAs are detected in the ascal cytoplasm but not in developing spores, indicating that the proteinaceous component of the spore wall is synthesized from the external compartment. The second set of messages is induced later in the course of spore maturation. These mRNAs accumulate within the spores and, unlike the first set of mRNAs, are retained in mature asci until the early stages of germination. We conclude that the development of ascospores proceeds through the differentiation of functionally distinct subcellular compartments.

Cell-cell interactions in developmental lysis of Myxococcus xanthus

The developmental events of sporulation and fruiting body formation in the prokaryote Myxococcus xanthus are preceded by a stage of massive cell death. Two phenotypically complementable strains of M. xanthus defective in developmental lysis were identified from a group of conditional sporulation mutants. Mixture of the two lysis groups resulted in full complementation of lysis, sporulation, and fruiting body formation; efficient sporulation was observed only in strain mixtures where lysis was complemented. We have identified a cell-free extract from developing cells that phenotypically complemented lysis, sporulation, and fruiting body formation in one group of mutants; the active component of this extract appeared to be tightly cell associated. The effect of the cell-free extract could be replaced by exogenously supplied glucosamine or mannosamine.

Synthesis of sigma 29, an RNA polymerase specificity determinant, is a developmentally regulated event in Bacillus subtilis.

Using an immunological probe, we have determined that the synthesis of the Bacillus subtilis RNA polymerase promoter specificity determinant sigma 29 is a developmentally regulated event. sigma 29 is absent from vegetatively growing cells but is abundant in sporulating cells for a restricted (2-h) period during differentiation (hour 2 to hour 4 into the sporeforming process). The narrowness of this period suggests that sigma 29 is a regulatory factor that directs the transcription of a subpopulation of genes at a precise, intermediate stage of spore formation. This view predicts that sigma 29 should be dispensable for early sporulation events. We verified this prediction by an analysis of sigma 29 accumulation in mutants that are blocked at different stages of sporulation in which we show that cells can advance to at least an intermediate point in development (stage III) in the absence of detectable sigma 29. Lastly, our anti-sigma 29 antibody probe detected a second, previously unrecognized protein in Bacillus cell extracts that may be a precursor to sigma 29. This protein, P31 (molecular weight, 31,000) is synthesized earlier in sporulation than is sigma 29. It has a peptide profile that is similar to sigma 29 and is present in all Bacillus subtilis Spo- mutants that were tested and found to still be able to accumulate sigma 29.

Medium Promoting Sporulation of Bacillus larvae and Metabolism of Medium Components

A new medium, designated TMYGP broth, was developed that allowed the honeybee pathogen Bacillus larvae NRRL B-3650 to produce up to 5 x 10 spores per ml of culture (microscopic count). This species normally sporulates poorly, if at all, in artificial broth media. An aeration rate lower than that normally used to cultivate other Bacillus species was required for sporulation. During the exponential growth phase, acids were produced by catabolism of yeast extract components, causing a decrease in pH of the medium. Thereafter, the pH began to increase, probably because of derepression of the citric acid cycle and consumption of the acids. Only after this time did usage of glucose from the medium occur. Thus, glucose usage seems to be regulated by catabolite repression. The presence of glucose was needed for one or more of the later events of sporulation. Of many substances tested, only gluconic acid and glucosamine partially substituted for glucose as a requirement for sporulation. Pyruvate was also required for good sporulation. It was metabolized during the late-exponential phase of growth.

Elevated Riboflavin Requirement for Postphotoinductive Events in Sporulation of a Trichoderma Auxotroph

A riboflavin auxotroph of Trichoderma required more riboflavin for blue-light-induced conidiation than for growth. Colonies transferred after illumination from 0.2 micromolar (limiting conidiation, not growth) to 3.5 micromolar riboflavin (nonlimiting), responded the same as those grown at 3.5 micromolar. The additional riboflavin is therefore not required as a photoreceptor. The data do not rule out the hypothesis that cryptochrome(s) is/are flavin(s).

Synthesis and Order of Assembly of Spore Coat Proteins in Bacillus subtilis

Analysis of mature spores or their integuments by extraction with sodium dodecyl sulphate/dithioerythritol followed by electrophoresis shows that the coat contains four major proteins and about ten others. Nine of the 14 proteins begin to be synthesized in stage II or stage III and their synthesis must be controlled by stage II or stage III operons. Some of these proteins are incorporated into the spore structure from about t 4 onwards (i.e. 4 h after induction of sporulation). Their deposition in the coat between t 4 and t 7 is not stopped by chloramphenicol, with the exception of one protein (mol. wt 36000) which begins to be synthesized only at t 6. Spores were isolated at various stages from about t 5 onwards, and the surface proteins were labelled with 125I. The labelling patterns show that proteins which are exposed on the surface at t 5·3 are successively overlaid as the spores mature. It appears that the coat of the mature spore contains at least three layers. The outermost layer is mainly composed of an alkali-soluble protein (mol. wt 12 000), which is synthesized early (t 2), and the 36000 mol. wt protein, which is synthesized very late. Deposition of the former seems to require processing by proteolytic action, and both proteins are apparently necessary for the acquisition of resistance to lysozyme, though not for resistance to heat or organic solvents. The results are discussed in relation to the classification of sporulation events and the nomenclature of the genetic loci controlling sporulation.