Induction Of Sporulation Research Articles

A revised method for the observation of conidiogenous structures in fungi

Preparation of fungal material for microscopical examination of their conidiogenous structures is described below. A modification to the existing methods used coupled with a sporulation medium has proved to be simple to use and successful for the induction of sporulation and subsequent examination of conidiogenous structures in a range of xylariaceous species and in endophytic fungi.

GABA induces terminal differentiation ofDictyosteliumthrough a GABAB receptor

When prespore cells approach the top of the stalk in a Dictyostelium fruiting body, they rapidly encapsulate in response to the signalling peptide SDF-2. Glutamate decarboxylase, the product of the gadA gene, generates GABA from glutamate. gadA is expressed exclusively in prespore cells late in development. We have found that GABA induces the release of the precursor of SDF-2, AcbA, from prespore cells. GABA also induces exposure of the protease domain of TagC on the surface of prestalk cells where it can convert AcbA to SDF-2. The receptor for GABA in Dictyostelium, GrlE, is a seven-transmembrane G-protein-coupled receptor that is most similar to GABA(B) receptors. The signal transduction pathway from GABA/GrlE appears to be mediated by PI3 kinase and the PKB-related protein kinase PkbR1. Glutamate acts as a competitive inhibitor of GABA functions in Dictyostelium and is also able to inhibit induction of sporulation by SDF-2. The signal transduction pathway from SDF-2 is independent of the GABA/glutamate signal transduction pathway, but the two appear to converge to control release of AcbA and exposure of TagC protease. These results indicate that GABA is not only a neurotransmitter but also an ancient intercellular signal.

Bioassay method for testing Fusarium wilt disease tolerance in transgenic banana

Fusarium wilt caused by the fungus, Fusarium oxysporum f.sp. cubense is a major factor limiting commercial banana production in Malaysia. Field evaluation of banana plants for Fusarium wilt disease has not been effective as the infection period is slow and other variables such as distribution of inoculum concentration is difficult to control. Therefore, a study was carried out to develop a semi-qualitative bioassay for early and rapid susceptibility tests against Fusarium wilt disease in banana plantlets. The induction of sporulation and germination of Foc race 1 (VCG 01217) were firstly determined. Then, the effect of inoculum density of fungal spores on the magnitude of infection in Pisang Rastali (AAB) plantlets was carried out. The concentration of hydrogen peroxide (H 2 O 2 ) and other enzyme activities such as phenylalanine ammonia lyase (PAL), chitinase, glucanase, peroxidase (PER) and polyphenol oxidase (PPO) in infected roots of banana plants were determined to relate to the levels of tolerance or susceptibility to Fusarium wilt disease. Using similar conditions, transgenic Pisang Rastali (AAB) was tested to investigate the effectiveness of this bioassay. Hydrogen peroxide and phenylalanine ammonia lyase is the most sensitive compound and enzyme detectable after inoculated with Fusarium spores. The chitinase, β-1,3-glucanases, peroxidase and polyphenol oxidase enzymes activity of transgenic plants were higher than untransformed plantlets. The results obtained indicate that the use of these biochemical parameters has the potential to predict the levels of tolerance or susceptibility to Fusarium wilt disease in other banana cultivars.

Phosphorylation and functional analysis of the sporulation initiation factor Spo0A from Clostridium botulinum

The initiation of sporulation in aerobic Bacillus species is regulated by the phosphorelay consisting of several sensor histidine kinases, the Spo0F response regulator, the Spo0B phosphotransferase and the Spo0A transcription factor that upon phosphorylation represses genes for growth and activates the developmental process. Clostridium species lack both Spo0F and Spo0B and the identities of the sensor histidine kinases are unknown. The amino acid sequence of Spo0A is highly conserved in Clostridium botulinum relative to Bacillus subtilis but the cloned C. botulinum Spo0A was unable to complement a spo0A mutant of B. subtilis for sporulation. However, it was able to repress the abrB gene of B. subtilis. Active site mutations in Spo0A still repressed, indicating this activity was independent of phosphorylation. An orphan sensor histidine kinase of C. botulinum appeared to normally phosphorylate C. botulinum Spo0A and expression of this kinase in combination with C. botulinum Spo0A in B. subtilis was lethal, suggesting phosphorylation of C. botulinum Spo0A repressed essential growth genes as a prerequisite to sporulation but could not compensate for this effect by inducing sporulation. A chimera Spo0A consisting of a B. subtilis Spo0A response regulator domain fused to a C. botulinum DNA-binding domain was capable of restoring sporulation to a spo0A mutant of B. subtilis albeit at less than wild-type levels. The data suggest that induction of sporulation requires interactions of both domains of Spo0A with other conserved proteins and despite the high conservation of the amino acid sequence of C. botulinum Spo0A, some of these interactions have been lost.

Differential Requirement for Phospholipase D/Spo14 and Its Novel Interactor Sma1 for Regulation of Exocytotic Vesicle Fusion in Yeast Meiosis

During sporulation and meiosis of budding yeast a developmental program determines the formation of the new plasma membranes of the spores. This process of prospore membrane (PSM) formation leads to the formation of meiotic daughter cells, the spores, within the lumen of the mother cell. It is initiated at the spindle pole bodies during meiosis II. Spore formation, but not meiotic cell cycle progression, requires the function of phospholipase D (PLD/Spo14). Here we show that PLD/Spo14 forms a complex with Sma1, a meiotically expressed protein essential for spore formation. Detailed analysis revealed that both proteins are required for early steps of prospore membrane assembly but with distinct defects in the respective mutants. In the Deltaspo14 mutant the initiation of PSM formation is blocked and aggregated vesicles of homogenous size are detected at the spindle pole bodies. In contrast, initiation of PSM formation does occur in the Deltasma1 mutant, but the enlargement of the membrane is impaired. During PSM growth both Spo14 and Sma1 localize to the membrane, and localization of Spo14 is independent of Sma1. Biochemical analysis revealed that Sma1 is not necessary for PLD activity per se and that PLD present in a complex with Sma1 is highly active. Together, our results suggest that yeast PLD is involved in two distinct but essential steps during the regulated vesicle fusion necessary for the assembly of the membranous encapsulations of the spores.

Fermentation of cellulose to acetic acid by Clostridium lentocellum SG6: induction of sporulation and effect of buffering agent on acetic acid production

To determine the growth, correlation between sporulation and acetic acid production and effect of buffering agent at high substrate cellulose concentrations of the strain Clostiridium lentocellum SG6. The strain SG6 was grown in cellulose mineral salt medium containing cellulose (Whatman No. 1 filter paper, Whatmore International Ltd., Maidstone, UK) or cellobiose. The strain fermented cellulose even after several transfers on cellobiose medium. The formation of endospores on third day onwards indicated the lowering of pH in the medium because of the formation of acetic acid. Maintaining the pH 7.2 at higher substrate concentrations resulted in increase of biomass, cellulose fermentation, acetic acid production, etc. The strain SG6, with its high fermentation yields and sporulating character can become a potential strain for acetic acid production and also as a probiotic strain in animal nutrition. The direct conversion of cellulosic biomass to acetic acid can eliminate expensive three-step saccharification, fermentation processes. The strain SG6 can ferment cellulose at high substrate concentrations.

Cultivation of Palmaria palmata (Palmariales, Rhodophyta) from isolated spores in semi-controlled conditions

Pilot scale studies were carried out to develop a methodology for the cultivation of fresh young fronds of Palmaria palmata to supply abalone hatcheries. This is the first report of cultivation of P. palmata in land-based tanks using isolated spores as seedstocks. Spores were released from field-collected tetrasporphytic fronds. A phenological study determined that tetrasporophytic fronds were fertile during the winter along the coast of northern France. This paper describes techniques for the induction of sporulation, for spore settlement and for incubation of germlings in 1.5-m-diameter tanks in semi-controlled conditions. The yield of released spores was in the range of 5000 to 25,000 g −1 of fertile sections. Mean spore density on substrate placed in hatching tanks was 500 spores cm −2. Spore germination occurred 3 days after inoculation and at this stage survival rate was high (80%). A few days after spore inoculation, both male and female gametophytes had germinated and a male to female ratio of approximately 1:1 was observed. Female gametophytic discs remained microscopic whereas males developed gametophytic fronds. Growth of plantlets was well correlated with the concentration of medium supplements. After 3 months of cultivation in tanks, survival rate was 35% and some plantlets measured more than 2 cm. At this stage, substrates covered with soft young fronds were transferred to an abalone hatchery to feed abalone juveniles.

Observations on the effect of salinity and photon fluence rate on the induction of sporulation and rhizoid formation in the green alga Enteromorpha prolifera (Muller) J. Agardh (Chlorophyta, Ulvales)

Although most members of the genus Enteromorpha are important edible green algae, some species are also potentially economically valuable crops. Samples of E. prolifera were obtained from the Yoshino River estuary, Tokushima, Japan and cultured in laboratory conditions at 10°C, a salinity of 20 psu, under white light with 12 h light: 12 h dark cycle and at a photon fluence rate (PFR) of 40 μmol/m2 per s for 30 days. In the present study, the effect of salinity and PFR on the induction of reproductive cells and rhizoid formation were investigated. Synchronous formation of swarmers by thalli was induced in excised disks of 1.2 mm diameter after 2-5 days incubation. The optimum salinity for maturation of reproductive cells was between 5.0 and 52.0 psu, and between 13.2 and 45.3 psu for swarmer release, although the lower limit for swarmer release was 5.0 psu. Maturation of reproductive cells and swarmer release required a PFR higher than 16 μmol/m2 per s. The minimum PFR for swarmer release was 8 μmol/m2 per s. Many rhizoids were formed between 1.6 and 52.0 psu and photon fluence rates between 8 and 320 μmol/m2 per s. Rhizoids were formed in a polarized manner.

Induction of sporulation in Saccharomyces cerevisiae leads to the formation of N6-methyladenosine in mRNA: a potential mechanism for the activity of the IME4 gene.

N6-methyladenosine (m6A) is present at internal sites in mRNA isolated from all higher eukaryotes, but has not previously been detected in the mRNA of the yeast Saccharomyces cerevisiae. This nucleoside modification occurs only in a sequence- specific context that appears to be conserved across diverse species. The function of this modification is not fully established, but there is some indirect evidence that m6A may play a role in the efficiency of mRNA splicing, transport or translation. The S.cerevisiae gene IME4, which is important for induction of sporulation, is very similar to the human gene MT-A70, which has been shown to be a critical subunit of the human mRNA [N6-adenosine]-methyltransferase. This observation led to the hypothesis that yeast sporulation may be dependent upon methylation of yeast mRNA, mediated by Ime4p. In this study we show that induction of sporulation leads to the appearance of low levels of m6A in yeast mRNA and that this modification requires IME4. Moreover, single amino acid substitutions in the putative catalytic residues of Ime4p lead to severe sporulation defects in a strain whose sporulation ability is completely dependent on this protein. Collectively, these data suggest very strongly that the activation of sporulation by Ime4p is the result of its proposed methyltransferase activity and provide the most direct evidence to date of a physiologic role of m6A in a gene regulatory pathway.

Induction of sporulation and antibacterial activity in the aerial mycelium negative mutants of Streptomyces nasri

Induction of sporulation and antibacterial activity in 24 spontaneous (SP), ultraviolet (UV), nitrosoguanidine (NG) and ethidium bromide (ETH) aerial mycelium negative mutants (Bld + ) of Streptomyces nasri (Hashem & Diab) was investigated. Calcium and phosphate ions could overcome the problem of the development of the aerial mycelium negative mutants and restored sporulation and antibacterial activity. The positive role of calcium was further confirmed with the use of the chelating agent Ethylene glycol bis (β-aminoethyl ether)-N,N,N’,N’-tetraacetic acid (EGTA). Mutants demonstrated variable restoration of sporulation and antibacterial activity responses toward different concentrations of calcium and phosphate ions. Sporulation induction showed a parallelism to the antibacterial activity exerted by the mutants. On the other hand, some amino acids were found to have a positive effect on sporulation and antibacterial activity. Histidine was the best stimulant for both sporulation and antibacterial activity for SP, and NTG mutants and had the lowest effect on UV and no effect at all on ETH-mutants. Glycine induced sporulation for the UV and ETH-mutants, and phenylalanine and proline in the case of ETH-mutants. Cluster analysis showed a high degree of relatedness between NTG and ETH-mutants in their response to calcium addition. UV and ETHmutants were highly related when phosphate feeding is the criterion. Internal relatedness among each group also emerged when amino acid addition was evaluated.

Characterization of blue-light and developmental regulation of the photolyase gene phr1 in Trichoderma harzianum.

Blue light and development regulate the expression of the phr1 gene of the filamentous fungus Trichoderma harzianum. The predicted product of phr1, the DNA repair enzyme photolyase, is likely to help protect Trichoderma, which grows in the soil as a mycoparasite or saprophyte, from damage upon emergence and exposure to ultraviolet-c. phr1 is transiently expressed in mycelium and conidiophores after illumination. phr1 mRNA also accumulates in conidiophores during development and spore maturation. As no other genes displaying rapid, direct light regulation have been described previously in this organism, we have characterized the fluence and time dependence of phr1 induction, and its relation to sporulation and photoreactivation. Induction is transient following a pulse, and, with slower decay, in continuous light. This implies that the photoreceptor, transducers or response are capable of adaptation. About two-fold more light is required to induce phr1 than conidiation, but this difference is modest, so both responses could use the same or similar chromophore. Adenosine 3':5'-cyclic monophosphate bypasses the requirement for light for sporulation, while atropine prevents sporulation even after photoinduction. Light regulation of phr1, however, is indifferent to both these effectors. Induction of photolyase expression behaves as a direct, rapid response to light, independent of the induction of sporulation. Indeed, illumination of mature spores increases their capacity for photoreactivation. Blue light seems to warn the organism against the harmful effects of short wave-lengths, inducing phr1 expression and sporulation by pathways that are, at least in part, distinct.

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.

Penicillin-binding protein sensitive to cephalexin in sporulation of Bacillus cereus

Cephalexin, cefaclor, cefadroxil, and cefotaxime strongly inhibited sporulation of Bacillus cereus ts-4 at 1 μg/ml. Cephalexin was most inhibitory on the sporulation of B. cereus when the antibiotic was added at 3 h after induction of sporulation by nutrient downshift technique. Examination of 4′,6-diamidino-2-phenylindole-stained cells by fluorescence-phase contrast microscopy showed that cephalexin inhibited the formation of asymmetric septum. By using [ 3H]penicillin, eight penicillin-binding proteins (PBPs) were detected from the cells of B. cereus ts-4. Among them, four PBPs were also detected in sporulating cells. Affinity of cephalexin to PBPs were measured indirectly by competition for subsequent binding of radioactive penicillin G. Cephalexin strongly bound to PBP 4 with molecular weight of 72,000 in sporulating cells.

Involvement of IMP dehydrogenase activity in induction of sporulation of Bacillus cereus

IMP dehydrogenase activity of B. cereus increased parallel to cell growth in YE-EMM, where B. cereus did not sporulate. When B. cereus was cultured in a modified G medium, a sporulation medium, the activity reached the highest level at 6 hr and decreased thereafter. After induction of sporulation by nutritional shift down in 1/100 G medium, the enzyme activity decreased to about 5% compared with exponentially growing cells at 1 hr of resuspension. The sporulation rate of B. cereus was over 90% in the modified G medium and 1/100 G medium. Sporulation was strongly inhibited by mycophenolic acid at 1 mM, when the drug was added at 0 and 1 hr of resuspension in 1/100 G medium. Intracellular GTP concentration of B. cereus decreased to the lowest level about 1 hr of resuspension. Although GTP increased to about 50% of the exponentially growing cells at 2 hr of resuspension in control cells, the concentration did not increase in the presence of 1 mM mycophenolic acid.

Starvation-independent sporulation in Myxococcus xanthus involves the pathway for beta-lactamase induction and provides a mechanism for competitive cell survival.

Myxococcus xanthus is a Gram-negative, soil-dwelling bacterium with a complex life cycle which includes fruiting body formation and sporulation in response to starvation. This developmental process is slow, requiring a minimum of 24-48 h, and requires cells to be at high cell density on a solid surface. It is known that, in the absence of starvation, vegetatively growing cell suspensions can form 'glycerol spores' when exposed to high levels of glycerol, usually 0.5 M. The cells differentiate from rods to resistant spheres rapidly (2-4 h) and synchronously. We have found that the chromosomally encoded beta-lactamase of M. xanthus can be induced by numerous beta-lactam antibiotics as well as by non-specific inducers including glycine and many D-amino acids. In addition, D-cycloserine, phosphomycin, and hen egg-white lysozyme also induce beta-lactamase in this bacterium. Unexpectedly, agents which induce beta-lactamase can induce 'glycerol spores'; all of the agents tested which induce glycerol spores (glycerol, DMSO, ethylene glycol) also induce beta-lactamase. During the induction of sporulation, beta-lactamase activity increases, reaching a peak during the morphological transition from rod-shaped cells to spherical spores. These spores are viable and resistant to many treatments which disrupt vegetatively growing rods but are not as resistant as fruiting body spores. The concomitant induction of beta-lactamase and starvation-independent sporulation suggests that these processes share a common signal-transduction pathway. These results also suggest that starvation-independent sporulation may be an adaptation of cells in order to resist agents that damage peptidoglycan structure and therefore threaten cell survival.

Photoreactivation of UV‐lnactivated Spores of Trichoderma harzianum

Abstract— Photoreactivation in the filamentous soil fungus Trichoderma harzianum is of interest because its blue, UVA photoreceptors (cryptochromes) may share homology with DNA photolyases. Furthermore, this organism antagonizes, by mycoparasitism, a number of soil‐borne pathogens. Photoreactivation is thus important as one of the factors that may contribute to survival in the field. Exposure of asexually produced spores (conidia) to UVC inhibits germination. Nongerminating spores either do not swell or are inhibited later in germination, swelling but failing to put out a germ tube. Both types of inhibition can be reversed by photoreactivation with visible and UVA (320‐400 nm) light, restoring high germination percentages. Conidia of mutants lacking the normal greenish pigmentation are more sensitive to UVC (200‐280 nm) than wild‐type conidia but photoreactivation still occurs. The action spectrum for photoreactivation indicates that T. harzianum has a DNA photolyase with a pterin as second chromophore. The most effective wavelengths for photoreactivation correspond to valleys, rather than peaks, in the action spectrum for photoinduction of sporulation. Furthermore, mutants with defects in photoinduction of sporulation (dimY) are not defective in photoreactivation. Induction of sporulation and DNA photorepair, while sharing parts of the blue/UVA spectrum, are different, by spectroscopic, kinetic and genetic criteria.

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.

Mass cultivation of the agar-producing alga Gracilaria chilensis (Rhodophyta) from spores

Gracilaria chilensis has been extensively cultivated in Chile by means of vegetative propagation. This method consists of outplanting pieces of thalli, which are anchored by several methods to muddy or sandy substrata at densities of about 1 kg (wet weight) m −2. Here we describe a new technique for mass cultivation of Gracilaria on ropes inoculated with spores. The paper describes: (i) induction of sporulation and fixation on polypropylene ropes; (ii) evaluation of spore settlement on the ropes; (iii) incubation of the germlings in tanks with agricultural fertilizers and (iv) outplanting of the ropes with plantlets to a pilot stand of about 5000 m 2 in an estuary in Central Chile. The technique will allow the establishment of a commercial farm using the stock of spores contained in 30–40 kg cystocarpic Gracilaria chilensis ha −1, compared with the 10000 kg of seaweed necessary for planting 1 ha using the traditional methodology.

Phosphate-starvation-inducible proteins in Bacillus subtilis: a two-dimensional gel electrophoresis study

A two-dimensional (2-D) gel electrophoresis study of Bacillus subtilis strain 168 identified 20 proteins that are strongly induced in response to phosphate starvation. The induction of nine of these phosphate-starvation-induced (Psi) proteins was dependent on a functional PhoR protein. PhoR is the histidine sensor-kinase component of a phosphate-concentration-sensing two-component regulatory system which, together with its partner response regulator PhoP, controls the expression of genes in the Pho regulon. Genes encoding PhoR-dependent Psi proteins are therefore likely to be members of the Pho regulon. Spo0A approximately P, the response regulator of the signal transduction pathway required for the induction of sporulation, has previously been shown to negatively affect the induction of the Pho regulon by repressing the phoP-phoR operon. The induction pattern of some PhoR-dependent Psi proteins was altered in a spo0A mutant such that their synthesis continued for longer than was found with the wild-type. The most abundant Psi protein, Psi1-3, was characterized by N-terminal sequencing of internal peptide fragments and shown to have a high similarity to an Escherichia coli protein which is involved in phosphate uptake during phosphate starvation.