Induction Of Sporulation Research Articles

Isolation and identification of Pyrenochaeta lycopersici, causal agent of tomato brown root rot

Reliable methods for the isolation of Pyrenochaeta lycopersici and induction of sporulation are described. The fungus was isolated from infested soil by a baiting technique, and from diseased root systems using a semi-selective medium. Sporulation was induced by culture on V8 Juice agar at 22 °C with a 16 h black light photoperiod.

Induction of sporulation in Pestalotiopsis palmarum by sodium chloride

The fungus Pestalotiopsis palmarum , which needs light for sporulation, was found to sporulate in the dark on a medium containing 0.34–1.02 m -NaCl. An incubation for at least 8 h was required for induction of sporulation. Non-ionic osmotic compounds were ineffective. KC1 also induced dark-sporulation. The results suggested that the monovalent cation, Na + , in NaCl was responsible for sporulation. In the presence of KC1, MgCl 2 , CaCl 2 and BaCl 2 , the effect of NaCl was reduced or abolished.

Induction of sporulation in Bacillus brevis. 1. Biochemical events and modulation of RNA synthesis during induction by tyrocidine.

Under conditions of severe nitrogen starvation, brought about by nutritional shift-down, Bacillus brevis ATCC 8185 was unable to sporulate unless supplemented with the peptide antibiotic tyrocidine. The induction of sporulation was highly specific for tyrocidine and required only very low concentrations of the peptide (5 microM). Tyrocidine-induced sporulation was accompanied by the typical sporulation-specific events (e.g. extracellular protease production and dipicolinate synthesis) as well as the formation of linear gramicidin. The addition of tyrocidine produced acute inhibition of RNA synthesis that was followed by a limited activation of transcription near the time of onset of linear gramicidin synthesis, when the first sporulation-specific changes were observed. These results provide direct evidence for a role of tyrocidine in sporulation of B. brevis and suggest that the action of the peptide antibiotic may involve the control of transcription. Such a notion is supported by earlier studies on the effects of tyrocidine and linear gramicidin on purified RNA polymerase.

Induction of sporulation in Bacillus brevis. 2. Dependence on the presence of the peptide antibiotics tyrocidine and linear gramicidin.

This paper presents evidence that the two peptide antibiotics tyrocidine and linear gramicidin, produced by Bacillus brevis ATCC 8185, are required for the induction of sporulation in the producer organism. When tyrocidine synthesis was specifically blocked with 2-amino-3-hydroxy-3-phenylpropanoic acid [Mach, B., Reich, E., and Tatum, E. L. (1963) Proc. Natl Acad. Sci. USA, 50, 175-181], sporulation and gramicidin synthesis were inhibited, but both processes could be restored by the addition of tyrocidine. Certain other amino acids such as L-tyrosine inhibited both sporulation and peptide antibiotic synthesis in nitrogen-limited cultures. When either tyrocidine or linear gramicidin was added together with L-tyrosine, neither sporulation nor peptide antibiotic synthesis was restored. On the other hand, the addition of both tyrocidine and linear gramicidin effectively reversed the inhibition of sporulation by L-tyrosine. These experiments demonstrate that sporulation of B. brevis depends on either the endogenous synthesis or the addition of both tyrocidine and linear gramicidin. The fact that endogenous as well as exogenous peptides could effect sporulation argues against the involvement of artifacts, such as the depletion of intracellular nucleotide pools caused by the surfactant properties of added peptide antibiotics.

Semi-continuous and Continuous Production of Aspergillus niger Spores in Submerged Liquid Culture

SUMMARY: Sporulation of Aspergillus niger was induced in continuous tower fermenters by restricting growth with nitrate limitation. Semi-continuous production of spores was achieved in a single-stage fermentation by alternating full-nutrient and nitrogen-deficient media to the culture. Continuous spore production was obtained in a two-stage fermentation system using the first stage for the growth of vegetative mycelium under optimum conditions and the second, larger, stage for sporulation induction in a constant nitrogen-deficient environment. Spores were produced from much simplified sporulation structures. Using a new sporulation index (Ω), which relates spore numbers produced to the quantity of substrate utilized, the average production efficiency of the two-stage continuous system was shown to be more than twice that of the semi-continuous production system. The tower fermenter was shown to be ideal for controlling organism morphology, thus providing conditions which allowed the development of continuous fungal sporulation.

Induction and Differentiation of Heterocysts in the Filamentous Cyanobacterium Cylindrospermum licheniforme

The fragmentation of Cylindrospermum licheniforme filaments resulted in the induction of a synchronous round of heterocyst differentiation. Young heterocysts (proheterocysts) appeared 12 to 15 h after fragmentation, and 12 to 15% of the cells became heterocysts within 24 h. NH4Cl or NaNO3 prevented the formation of new heterocysts, while differentiation was stimulated twofold by the absence of atmospheric N2. The sequence of appearance of heterocyst-specific characteristics was observed following filament fragmentation. Nitrogenase activity increased rapidly between 13 and 26 h, paralleling the increase in heterocyst frequency. Glycolipids unique to the heterocyst envelope were also synthesized between 13 and 26 h, and activities of 6-phosphogluconate dehydrogenase and glucose-6-phosphate dehydrogenase increased after 10 h and 90 h respectively. Aged medium induced sporulation in intact filaments within 1 d. but sporulation could occur no earlier than 2 d after filament fragmentation, indicating that the induction of sporulation requires the presence of mature (but not necessarily functional) heterocysts.

Modification of histones during the mitotic and meiotic cycle of yeast

Core histones (H2a ,H2b ,H3 ,H4) of yeast have been shown to be similar to histones from higher organisms [ 1 ] whereas a protein corresponding to histone Hl seems to be absent from this simple unicellular eukaryote [2]. Also the organization of yeast chromatin into nucleosomes closely resembles that of higher eukaryotes [ 11. Histones of other organisms have been found to be subject to various modifications. Phosphorylation, especially that of histones Hl and H3, occurs predominantly during chromosome condensation for mitosis in higher organisms [3] as well as in the slime mold, Physarum polycephalum [4]. Phosphorylation of histone H2a was suggested to be involved in interphase heterochromatin structure in a cell cycle-independent mamrer [5-71. Highly acetylated histone H4 was discovered specifically in transcriptionally active, DNase I-sensitive, chromatin [8-lo], its concentration is inversely correlated with the initiation of chromosome condensation in Physarum [II]. We asked whether such modifications, probably being devices for the modulation of DNA-histone interactions, also occurred in yeast and whether appearance of modified histone proteins varied during the vegetative cell cycle or during the sporulation process. We found incorporation of radioactive phosphate into yeast histone H2a and to a much smaller amount into H2b, but not into H3 and H4. The degree of incorporation did not change substantially during the vegetative growth cycle but increased considerably in cells preparing for sporulation. Radioactive sodium acetate was found to be incorporated into all core histone-bands. Analysis of unlabeled histones in acidurea gels, in which the acetylated variants of histones H4 and H3 can be separated, showed that these 2 histones in yeast exist as several subspecies acetylated to different degrees. Variations in the relative amounts of these subspecies were found during the mitotic cell cycle and less pronounced during the process of sporulation induction.

Endotrophic sporulation by the yeast Nadsonia fulvescens.

Studies of carbon requirements for sporulation by Nadsonia fulvescens showed an unexpected appearance of spores in the absence of an exogenous carbon source, when the cells were grown on ethanol prior to induction of sporulation, Dextrose-grown cells taken from either the logarithmic or stationary phase required a carbon source (dextrose, ethanol, or glycerol) for sporulation. An inhibitory effect by acetate upon sporulation was demonstrated, and it appears to be due to a repression mechanism.

Changes in the adenylate energy charge and the induction of sporulation in Saccharomyces diastaticus

The induction of sporulation in yeast is generally accompanied by a sharp increase in energy metabolism which is evidenced by a rise of the adenylate energy charge by that time. The energy charge can be held at a low level by limitation of the phosphate supply in the growth medium. Ascus formation remains unaffected by this treatment. This suggests that the rise in ATP production normally encountered during early sporulation is not essential for the initiation of sporulation.

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.

Complementation of Sporulation Mutations in Fused Protoplasts of Bacillus subtilis

Asporogenous and oligosporogenous mutants of Bacillus subtilis blocked at stages III, IV and V and carrying mutations at 18 different genetic loci have been tested for complementation. This was done by preparing protoplasts 3 h after induction of sporulation (t 3) and fusing them with wild-type protoplasts prepared in parallel. After incubation to t 10 the suspension was assayed for heat-resistant (80 °C, 40 min) colony-forming units. Mutations in 9 of the 18 loci were complemented, i.e. the spore count was increased > 1000-fold. All of the complemented mutants, except for one stage V mutant, were blocked at stage IV. Spo− mutants that were complementable by wild-type were also complementable by other Spo− mutants provided that they did not carry the same spo mutation. Complementation analysis was applied to three alleles in the spoIVC locus. The complementation pattern suggests that there are at least two cistrons in the locus and this agrees with the mapping data.

Mode of conjugation in homothallic cells of Schizosaccharomyces pombe.

When homothallic cells of Schizosaccharomyces pombe were plated on agar sporulation medium, a few residual divisions were usually observed before induction of conjugation and sporulation. Four cells resulting from the last two residual divisions were usually lined; the formed. Each four-lined-cell contained only one zygote by sistercell-or non-sistercell-copulation, and never two zygotes by two sistercell-copulations. Some of the remaining non-zygotic sistercells divided once again and resulted in the new formation of the four-lined-cell which contained only one zygote. The remaining non-zygotic cell of the four-lined-cell could conjugate with that of another four-lined-cell. These results suggest that the four-lined-cell is to be regarded as one unit with respect to zygote formation.

Germination properties as marker events characterizing later stages of Bacillus subtilis spore formation.

At various stages during spore formation sporangia were shocked by cold treatment or with toluene, and the germination requirements of the prespores were examined. Up to 5 h after induction of sporulation (t5) germination was spontaneous; i.e., it occurred without any added germinants. After t5, during stages V and VI, the capacity for spontaneous germination diminished progressively, and the spores acquired a need for externally added germinants. At t6 this need was satisfied by either L-alanine or a mixture of KCl, glucose, and fructose. By t8, the latter response had disappeared. The spores germinated only with L-alanine, and the response was much slower. Experiments with chloramphenicol showed that the germination properties of the spores appearing between t6 and t8 were the expression of events in protein synthesis that had occurred before t5. Although the germination requirements developed at about the same time as heat resistance, they could be dissociated from heat resistance in wild-type and mutant cells. The germination properties of the developing spores are additional marker events characterizing the later stages of sporulation, as follows: (i) spontaneous germination (up to the end of stage IV); (ii) germination requirements that are satisfied by KCl-glucose-fructose or L-alanine (stage V); and (iii) slow germination response with L-alanine only (stage VI).

Temporal dissociation of late events in Bacillus subtilis sporulation from expression of genes that determine them.

During sporulation in replacement medium, resistance to toluene to heating at 65 degrees C, to lysozyme, and to heating at 80 degrees C appeared in sequence between 4 and 8 h after the induction of sporulation (i.e., between t4 and t8). The addition of sufficient chloramphenicol at t4.5 to prevent protein synthesis nevertheless allowed the emergence of all of these types of resistance except lysozyme resistance. The numbers of spores with these types of resistance (lysozyme resistance again excepted) increased about fourfold when phenylmethylsulfonyl fluoride (an inhibitor of serine protease activity) was also present. Thus, the observed increases in resistance in the 2 h after the addition of chloramphenicol resulted from the utilization of preformed protein elements. Dipicolinate did not seem to be a determining factor in the development of any of these forms of resistance. Electron micrographs showed that inhibition of protein synthesis did not prevent deposition of the outer layers of the spores. Lysozyme resistance developed differently; synthesis of the relevant proteins began later (t5), and continued synthesis was necessary up to t8. Some processing of proteins made earlier was a prerequisite for lysozyme resistance. Therefore, it appears that from the viewpoint of regulation, the expression of the genes and the production of the proteins for resistance to toluene, heating at 65 degrees C, and heating at 80 degrees C are all stage IV sporulation events, although the resistance properties themselves appear only during stages V and VI. Lysozyme resistance is the only real late event among those examined. The germination characteristics of the spores, which are also late events, are discussed in this context, as they too are dependent on proteins that are synthesized much earlier.

Conversion of Mitotic to Meiotic Cells in Saccharomyces cerevisiae. I. Efficient Induction of Meiosis

An efficient method has been developed for the synchronous and high frequent induction of meiosis in Saccharomyces cerevisiae. An exponential-phase cell population was treated with 0.1 M hydroxyurea (HU) for various periods, then it was transferred to a sporulation medium. The 5-h treatment with HU caused the accumulation of oversized pairs containing a parent cell and bud with a dividing nucleus. After transfer to the sporulation medium, all the buds developed into daughter cells in the first few hours, then both parent and daughter cells synchronously underwent meiotic division. In this synchronous system, meiosis occurred in more than 95 % of the cells, but the frequency of ascus formation and the spore number per ascus were not improved in comparison with the control cultures. Our results indicate that the ability of cells to undergo meiosis depends primarily on the size attained prior to sporulation induction.

The Physiology of Mutants Derepressed for Sporulation in Saccharomyces cerevisiae

Summary: The spd 1 mutants of Saccharomyces cerevisiae are affected in the initiation of sporulation since they sporulate under conditions in which the wild-type does not. They are unable to grow on a range of non-fermentable carbon substrates, but can grow on ethanol. On others, including acetate, glycerol, lactate and pyruvate, the mutants sporulate abundantly, even in rich media. The mutation does not affect the uptake of glycerol or the synthesis of the enzymes concerned with its entry into general metabolic pathways. It probably affects a central metabolic function concerned with the metabolism of some, but not all, non-fermentable carbon sources. The spd 1 mutation is also expressed in haploids. When haploid or diploid cells are transferred to media containing only non-fermentable carbon substrates they become arrested in the G1 phase of the cell cycle before, or at, the execution point for the cdc 28 mutation. In diploids homozygous for the spd 1 gene these conditions also lead to a lowering of the repression by the nitrogen source. The spd 1 mutants are therefore probably not affected directly in events unique to the initiation of sporulation, but in areas of metabolism closely connected with both the arrest of cells in the G1 phase of the cell cycle and the control of induction of sporulation.

Induction of sporulation in Bacillus brevis by peptide antibiotics.

THE regulatory mechanisms of the process of bacterial sporulation are unknown, although some authors have speculated that sporulation and the synthesis of peptide antibiotics are somehow connected1–4. Paulus and coworkers proposed that tyrocidine and gramicidin D, both synthesised by Bacillus brevis, may interact with the transcriptional process during sporogenesis5. They have isolated a mutant of B. brevis which is unable to synthesise gramicidin D and which produces defective spores. The defects could be cured by exogenous gramicidin D, suggesting that this peptide is involved in sporogenesis6. In this report we demonstrate that sporulation of early vegetative growing B. brevis cells can be induced by exogenous tyrothricin, a mixture of tyrocidine and gramicidin D, provided that the cells are exposed to a culture medium lacking the nitrogen source. The induction of sporulation occurs concomitantly with a significant increase in RNA synthesis. The results suggest that sporulation and the action of these two peptides are causally connected. Both peptides may play a positive regulatory role in cell differentiation.

Induction of sporulation in developmental mutants of Bacillus subtilis.

Sporulation of the standard strain of Bacillus subtilis can be induced by decoyinine (a specific inhibitor of guanosine monophosphate synthesis) in the presence of excess ammonium ions, rapidly utilizable carbohydrates and phosphate. Certain developmental mutants unable to sporulate in usual sporulation media can also be induced to sporulate under these conditions. Among the inducible strains are mutants blocked in the citrate cycle, in phosphoenolpyruvate carboxykinase or one of the glycerolphosphate dehydrogenases (NAD dependent or independent). A mutant lacking phosphoglycerate kinase activity and able to grow on glucose and L-malate but not on either carbon source alone, can also be induced to sporulate in a medium containing both glucose and malate. Among other sporulation mutants whose biochemical deficiency is not known, some are weakly inducible whereas many, blocked at sporulation stages O, II, III, or IV, are not inducible. The results show that the functions of the citrate cycle (used for ATP synthesis) and of gluconeogenesis (used for glycerol phosphate and D-glucosamine-6-phosphate synthesis), normally needed for sporulation under conditions of carbohydrate deprivation, are no longer required when sporulation is induced by decoyinine in the presence of carbohydrates. Mutants in which sporulation is highly inducible can be used as a sensitive tool for the detection of other inducing compounds because their spontaneous (uninduced) background of spores is very low.

Induction of sporulation by inhibitory purines and related compounds

Sporulation of Bacillus subtilis can be induced, in the presence of excess ammonia, glucose and phosphate, by many purine derivatives under conditions of partial growth inhibition. Some of the compounds are known inhibitors of purine nucleotide synthesis. For most compounds the effect is counteracted by adenine and guanine. Partial growth inhibition by amethopterin (methotrexate) causes sporulation in the absence of purines but not in their presence. Unable to induce sporulation at any concentration are inhibitors of DNA, RNA, and protein synthesis as well as base or amino acid analogs that are incorporated into these polymers.

The Relationship Between DNA Replication and the Induction of Sporulation in Bacillus subtilis

6-(p-Hydroxyphenylazo)uracil (HPUra), which is thought to be a specific inhibitor of DNA replication in many Gram-positive bacteria, has been used to investigate the relationship between DNA replication and the induction of sporulation in Bacillus subtilis. Gene frequency analysis of samples removed from sporulating cultures indicates that sporulating cells terminate their final round of DNA replication at the same time as they escape from the inhibitory effect of HPUra. If the rate of DNA replication during starvation is slowed down by the use of sub-inhibitory concentrations of HPUra, the onset of this escape is delayed. When cells containing only completed chromosomes are transferred from a rich growth medium to a poor sporulation medium they initiate new rounds of DNA replication in the sporulation medium and then subsequently sporulate. The results are consistent with the hypothesis of Mandelstam & Higgs (1974) that induction of sporulation occurs 15 to 20 min after the initiation of a round of DNA replication.