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Metabolism of adenosine 3',5'-cyclic monophosphate and induction of fruiting bodies in Coprinus macrorhizus.

The adenyl cyclase and phosphodiesterase metabolizing adenosine 3',5'-cyclic monophosphate (cyclic AMP) were detected in mycelia of strains of Coprinus macrorhizus which form fruiting bodies, but not in those of strains which do not form fruiting bodies. The adenyl cyclase synthesized cyclic AMP from adenosine triphosphate. The phosphodiesterase degr[UNK]ded cyclic AMP to adenosine-5'-monophosphate and was inhibited by adenosine-3'-monophosphate, theophylline, and caffeine. The strains which form fruiting bodies incorporated and metabolized cyclic AMP, but strains which do not form fruiting bodies did not. The possible participation of cyclic AMP in the induction of fruiting bodies is discussed.

Adenosine Monophosphate↗

Beta-D-Allose inhibits fruiting body formation and sporulation in Myxococcus xanthus.

Myxococcus xanthus, a gram-negative soil bacterium, responds to amino acid starvation by entering a process of multicellular development which culminates in the assembly of spore-filled fruiting bodies. Previous studies utilizing developmental inhibitors (such as methionine, lysine, or threonine) have revealed important clues about the mechanisms involved in fruiting body formation. We used Biolog phenotype microarrays to screen 384 chemicals for complete inhibition of fruiting body development in M. xanthus. Here, we report the identification of a novel inhibitor of fruiting body formation and sporulation, beta-d-allose. beta-d-Allose, a rare sugar, is a member of the aldohexose family and a C3 epimer of glucose. Our studies show that beta-d-allose does not affect cell growth, viability, agglutination, or motility. However, beta-galactosidase reporters demonstrate that genes activated between 4 and 14 h of development show significantly lower expression levels in the presence of beta-d-allose. Furthermore, inhibition of fruiting body formation occurs only when beta-d-allose is added to submerged cultures before 12 h of development. In competition studies, high concentrations of galactose and xylose antagonize the nonfruiting response to beta-d-allose, while glucose is capable of partial antagonism. Finally, a magellan-4 transposon mutagenesis screen identified glcK, a putative glucokinase gene, required for beta-d-allose-mediated inhibition of fruiting body formation. Subsequent glucokinase activity assays of the glcK mutant further supported the role of this protein in glucose phosphorylation.

Culture Media↗

The fruiting body and its caterpillar host of Cordyceps sinensis show close resemblance in main constituents and anti-oxidation activity.

Cordyceps (summer-grass, winter-worm), one of the most valued traditional Chinese medicines, is used commonly for the replenishment of body health. It consists of the dried fungus Cordyceps sinensis growing on caterpillar larvae. For medication, the fruiting body (fungus) and the worm (caterpillar) are used together. However, the pharmacological efficiency and the main constituents of the individual parts have not been determined. In the present study the water extracts from the fruiting body and worm of natural Cordyceps were analyzed for their content of nucleosides and polysaccharides; the results showed that the worm had chemical composition similar to the fruiting body. In addition, both the fruiting body and worm of Cordyceps showed similar potency in their anti-oxidation activities in the xanthine oxidase assay, the induction of hemolysis assay and the lipid-peroxidation assay. These results suggest that the function of the worm in Cordyceps is to provide a growth medium for the fruiting body, and that eventually, the worm is totally invaded by C. sinensis mycelia.

Animals↗

Elongation growth and gravitropic curvature in the Flammulina velutipes (Agaricales) fruiting body.

Differential elongation of stipe hyphae drives the gravitropic reorientation of Flammulina velutipes (Agaricales) fruiting bodies. The gravitropic curvature is strictly dependent on the presence of the transition zone between pileus and stipe. Elongation growth, providing the driving force for curvature, is also promoted by the pileus. Gravitropic curvature is successfully suppressed by clinostatic rotation, but the elongation rate is not affected. Explantation of fruiting body stipes lowers curvature and elongation rates corresponding to explant size reduction. In Flammulina, 25 mm length of transition zone explants is an efficient size for reproducible curvature and elongation during 48- to 72-h curvature tests. Submersion of specimens in aqueous medium causes cessation of the gravitropic curvature, but does not affect elongation. Thus the involvement of a diffusible factor in transmission of the curvature signal is probable. Splitting the fruiting body stipe in segments of 1/8 diameter does not suppress the gravitropic response, and the segments are individually reoriented to the vertical. It is concluded that the graviresponse of the Flammulina fruiting body is based on cellular perception of the gravistimulus and that a differential growth signal is transmitted in the stipe by a soluble factor that regulates hyphal elongation.

Basidiomycota↗

Stigmatella aurantiaca fruiting body formation is dependent on the fbfA gene encoding a polypeptide homologous to chitin synthases.

Stigmatella aurantiaca is a prokaryotic organism that undergoes a multicellular cycle of development resulting in the formation of a fruiting body. For analyzing this process, mutants defective in fruiting body formation have been induced by transposon mutagenesis using a Tn5-derived transposon. About 800 bp upstream of the transposon insertion of mutant AP182 which inactivates a gene (fbfB) involved in fruiting, a further gene (fbfA) needed for fruiting body formation was detected. Inactivation of fbfA leads to mutants which form only non-structured clumps instead of the wild-type fruiting body. The mutant phenotype of fbfA mutants can be partially suppressed by mixing the mutant cells with cells of some independent mutants defective in fruiting body formation. The fbfA gene is transcribed after 8 h of development as determined by measuring the induction of beta-galactosidase activity of a fbfA-delta(trp)-lacZ fusion gene and by Northern (RNA) analysis using an insertion encoding a stable mRNA. The predicted polypeptide FbfA shows a homology of about 30% to NodC of rhizobia, an N-acetylglucosamine-transferase which is involved in the synthesis of the sugar backbone of lipo-oligosaccharides. These induce the formation of the root nodules in the Papilionaceae. Besides the predicted molecular mass of 45.5 kDa, the hydropathy profile reveals a structural relationship to the NodC polypeptide.

Amino Acid Sequence↗

Effect of glucose on the fruiting body formation and adenosine 3',5'-cyclic monophosphate levels in Coprinus macrorhizus.

The formation of fruiting bodies in the monokaryotic fis(c) strain and a dikaryon of Coprinus macrorhizus was inhibited by growth in high-glucose media. In high-glucose media the characteristic burst of adenosine 3',5'-cyclic monophosphate (cAMP) accumulation during fruiting-body formation was absent. Enzymatic activity assays revealed that mycelia grown in high-glucose media contained relatively lower amounts of adenylate cyclase and cAMP-phosphodiesterase than mycelia grown in low-glucose media. The synthesis of inducible d-serine deaminase and tryptophanase was repressed in high-glucose media. A mutant (gluR) in which the glucose repression of fruiting-body formation is affected was isolated by selection in high-glucose media. The mutation caused the cAMP levels to be no longer affected by glucose and affected ability to synthesize the inducible d-serine deaminase and tryptophanase. The gluR mutant was partially dominant in dikaryons. It is suggested that cAMP may play important roles in inducing fruiting bodies and in controlling inducible enzyme synthesis in C. macrorhizus.

Adenylyl Cyclases↗

Morphogenesis of Stigmatella aurantiaca fruiting bodies.

Scanning electron micrographs of intermediate stages of fruiting body formation in the myxobacterium Stigmatella aurantiaca suggest that fruiting body formation can be divided into several stages distinguishable on the basis of the motile behavior of the cells. Aggregates formed at sites where cells glide as groups in circles or spirals. Thus, each aggregate was surrounded by a wide band of cells. Several streams of cells were pointed toward and connected to the wide band of cells at the base of the aggregate, suggesting directed cell movement toward the aggregate. The pattern of cells at the base of taller, more mature aggregates suggested that groups of cells enter the aggregate from the surrounding band of cells by changing the pitch of their movement, thus creating an ascending spiral. Stalk formation was characterized by a distinctly different pattern, which suggested that single cells emerge from the band of cells and move toward the aggregate, under it, and then vertically to create the stalk. At this stage, the aggregate appeared to be torn from the substrate as it was lifted off the surface. The cells in the completed stalks were well separated, and most had their long axes pointed in a vertical direction. A great deal of the stalk material appeared to be slime in which the cells were embedded and through which they were presumably moving in the live material. Some suggestions regarding factors that may direct the observed morphogenetic movements are discussed.

Cell Division↗

Expression of the urease gene of Agaricus bisporus: a tool for studying fruit body formation and post-harvest development.

Fruit body initials of Agaricus bisporus contain high levels of urea, which decrease in the following developmental stages until stage 4 (harvest) when urea levels increase again. At storage, the high urea content may affect the quality of the mushroom, i.e. by the formation of ammonia from urea through the action of urease (EC 3.5.1.5). Despite the abundance of urea in the edible mushroom A. bisporus, little is known about its physiological role. The urease gene of A. bisporus and its promoter region were identified and cloned. The coding part of the genomic DNA was interrupted by nine introns as confirmed by cDNA analysis. The first full homobasidiomycete urease protein sequence obtained comprised 838 amino acids (molecular mass 90,694 Da, pI 5.8). An alignment with fungal, plant and bacterial ureases revealed a high conservation. The expression of the urease gene, measured by Northern analyses, was studied both during normal development of fruit bodies and during post-harvest senescence. Expression in normal development was significantly up-regulated in developmental stages 5 and 6. During post-harvest senescence, the expression of urease was mainly observed in the stipe tissue; expression decreased on the first day and remained at a basal level through the remaining sampling period.

Agaricus↗

Isolation of genes specifically expressed in the fruit body of the edible basidiomycete Lentinula edodes.

In order to isolate the genes expressed specifically and abundantly in the mature fruit body of Lentinula edodes, the cDNAs derived from the gill of the fruit body were compared with the cDNAs from the mycelia by differential screening. Consequently, six clones were identified as fruit-body-specific genes (fbg03, 08, 13, 14, 16, and 21). The deduced amino acid sequence of fbg14 (Le.cypfb) had significant homology with the cytochrome P450 protein. The transcriptional level of fbg16, which showed 29.9% identity with the riboflavin aldehyde-forming enzyme of Agaricus bisporus, was highest among all of the fbg clones. This result indicates that the promoter region of fbg16 may become a powerful candidate for the expression signal of the vector for the gene manipulation in the mature fruit body.

Amino Acid Sequence↗

Determination of specific volatile organic compounds synthesised during Tuber borchii fruit body development by solid-phase microextraction and gas chromatography/mass spectrometry.

Fruit body development is a particular phase of the Tuber life cycle, characterised by the aggregation of different types of hyphae, i.e., vegetative hyphal cells and highly specialised reproductive hyphae (asci). In order to identify the volatile organic compounds (VOCs) produced in different stages of the Tuber borchii ripening fruit body, solid-phase microextraction with gas chromatography and mass spectrometry was used. The volatile organic compounds were extracted using a DVB/CAR/PDMS 50/30 microm fiber placed for 10 min at room temperature in the truffle headspace. The results obtained reveal 49 compounds each of which was present only in a particular stage of maturation. 1-octen-3-ol, aromadendrene, alpha-farnesene and other terpenoid compounds were of particular interest, and their possible biological roles are discussed. The production of aromadendrene in the completely unripe fruit body suggests the existence of communication events in the early stage of ascomata formation between the fungus and the host plant. alpha-Farnesene could represent a chemotactic attractant to saprophytic organisms in order to disperse the fungal spores in the environment. The identification of the VOCs produced by truffles during their maturation could give information about the processes underlying this phase of Tuber life cycle.

Ascomycota↗

Radiocaesium activity concentrations in the fruit-bodies of macrofungi in Great Britain and an assessment of dietary intake habits.

Radiocaesium activity concentrations in the fruit-bodies of some species of macrofungi are higher than in many other foodstuffs. The consumption of fruit-bodies contributes significantly to radiocaesium intake of humans in some countries. In the United Kingdom, the collection of wild fungi has generally been considered to be of minor importance and there are few data on consumption rates or radiocaesium activity concentrations in most edible species. Samples of commonly eaten species in Great Britain have been collected to assess radiocaesium contamination levels and geographical variation. Concurrently, surveys of consumption habits were conducted. A total of 425 samples representing 37 different species were collected. Significantly higher radiocaesium activity concentrations occurred in mycorrhizal compared to saprotrophic or parasitic species. The highest 137Cs activity concentration of 30.5 kBq kg-1 dry wt. was determined in a sample of Hydnum repandum collected in Wales. The transfer of radiocaesium from soil to fungal fruit-bodies was highly variable, ranging over three orders of magnitude within individual species. A number of approaches to quantifying radiocaesium transfer from soil to fungal fruit-bodies were used. Although these were in general agreement with previously measured values in other countries, all the approaches gave variable results. Over 200 people responded to the dietary habits questionnaire. The median intake rate was 0.75 kg year-1 (fresh wt.) and 60% of respondents consumed only one species (generally Agaricus campestris). However, intakes of up to 26 kg year-1 were recorded and a total of 82 species were consumed. The intake of 137Cs was determined by the amount of mycorrhizal fungi in the diet rather than the total intake of fungi. Assuming median recorded 137Cs activity concentrations in each fungal species, the estimated annual committed effective dose for over 95% of respondents was < 1 microSv. Hence, currently, the consumption of wild fungi in the UK would not be expected to significantly increase the dose above that attributable to the normal diet of most consumers. However, the results of this study demonstrate that, in the event of any future accidental release of radiocaesium, the potential ingestion dose received from the consumption of wild fungi would need to be considered.

Cesium Radioisotopes↗

Phenoloxidase activity and fruiting body formation Schizophyllum commune.

Studies of induced haploid fruiting in the Basidiomycete Schizophyllum commune suggested that polyphenoloxidase enzymes may participate in the formation of fruiting bodies. Reported inhibitors of phenoloxidases were found to affect both the activity of these enzymes and the fruiting response. Fertile mycelia were found to give a positive Bavendamm reaction in situ, and cell-free extracts from fertile mycelia exhibited polyphenoloxidase activity when studied spectrophotometrically. Sterile mycelia, on the other hand, were devoid of these activities. Various kinds of mutant homo- and dikaryotic mycelia were studied, and the results suggested a correlation between phenoloxidase activity and fruiting body formation in S. commune.

Aminobenzoates↗

fbfB, a gene encoding a putative galactose oxidase, is involved in Stigmatella aurantiaca fruiting body formation.

Stigmatella aurantiaca is a gram-negative bacterium which forms, under conditions of starvation in a multicellular process, characteristic three-dimensional structures: the fruiting bodies. For studying this complex process, mutants impaired in fruiting body formation have been induced by transposon insertion with a Tn5-derived transposon. The gene affected (fbfB) in one of the mutants (AP182) was studied further. Inactivation of fbfB results in mutants which form only clumps during starvation instead of wild-type fruiting bodies. This mutant phenotype can be partially rescued, if cells of mutants impaired in fbfB function are mixed with those of some independent mutants defective in fruiting before starvation. The fbfB gene is expressed about 14 h after induction of fruiting body formation as determined by measuring beta-galactosidase activity in a merodiploid strain harboring the wild-type gene and an fbfB-delta trp-lacZ fusion gene or by Northern (RNA) analysis with the Rhodobacter capsulatus pufBA fragment fused to fbfB as an indicator. The predicted polypeptide FbfB has a molecular mass of 57.8 kDa and shows a significant homology to the galactose oxidase (GaoA) of the fungus Dactylium dendroides. Galactose oxidase catalyzes the oxidation of galactose and primary alcohols to the corresponding aldehydes.

Amino Acid Sequence↗

Interactions of heterologous mycelia colonized in the substrate govern fruit body production in the cultivated homobasidiomycete Pholiota nameko.

The spawn of cultivated mushrooms are generally produced, propagated, and distributed to growers as a mycelial culture without genetic purification, in which phenotypic variants frequently occur. We investigated how heterologous mycelia present in a spawn influence fruit body production in the cultivated basidiomycete Pholiota nameko. The 'di-mon' dual cultivation of protoplast clones produced mosaic fruit bodies, which could result from the 'di-mon' mating. In the 'di-di' dual cultivation of heterologous strains with different fruiting times, authentic fruit bodies of each dikaryon and chimera showing a feature combining characteristics of the two dikaryons emerged simultaneously. Mycelia isolated from the chimera produced all three types of fruit bodies, indicating unlikeliness of the occurrence of anastomosis. These results suggest that mycelia colonized in the substrate interact with each other and coordinately promote fruit body production in P. nameko. This phenomenon masks a clonal variability that may be surfaced through multiplication and distribution of the spawn, occasionally bringing about abnormal fruiting.

Basidiomycota↗

Apoptotic effects of extract from Antrodia camphorata fruiting bodies in human hepatocellular carcinoma cell lines.

The fruiting body of Antrodia camphorata is well known in Taiwan as a traditional medicine for treating cancer and inflammation. The purpose of this study was to evaluate the apoptotic effects of ethylacetate extract from A. camphorata (EAC) fruiting bodies in two human liver cancer cell lines, Hep G2 and PLC/PRF/5. Treatment with EAC decreased the cell growth of Hep G2 and PLC/PRF/5 cells in a dose dependent manner. In Fas/APO-1 positive-Hep G2 cells, EAC increased the expression level of Fas/APO-1 and its two forms of ligands, membrane-bound Fas ligand (mFasL) and soluble Fas ligand (sFasL), in a p53-indenpendent manner. In addition, EAC also initiated mitochondrial apoptotic pathway through regulation of Bcl-2 family proteins expression, release of cytochrome c, and activation of caspase-9 both in Hep G2 and PLC/PRF/5 cells. Furthermore, EAC also inhibited the cell survival signaling by enhancing the amount of IkappaBalpha in cytoplasm and reducing the level and activity of NF-kappaB in the nucleus, and subsequently attenuated the expression of Bcl-X(L) in Hep G2 and PLC/PRF/5 cells. EAC therefore decreased the cell growth and induced apoptosis both in Hep G2 and PLC/PRF/5 cells.

Antineoplastic Agents, Phytogenic↗

The pro1(+) gene from Sordaria macrospora encodes a C6 zinc finger transcription factor required for fruiting body development.

During sexual morphogenesis, the filamentous ascomycete Sordaria macrospora differentiates into multicellular fruiting bodies called perithecia. Previously it has been shown that this developmental process is under polygenic control. To further understand the molecular mechanisms involved in fruiting body formation, we generated the protoperithecia forming mutant pro1, in which the normal development of protoperithecia into perithecia has been disrupted. We succeeded in isolating a cosmid clone from an indexed cosmid library, which was able to complement the pro1(-) mutation. Deletion analysis, followed by DNA sequencing, subsequently demonstrated that fertility was restored to the pro1 mutant by an open reading frame encoding a 689-amino-acid polypeptide, which we named PRO1. A region from this polypeptide shares significant homology with the DNA-binding domains found in fungal C6 zinc finger transcription factors, such as the GAL4 protein from yeast. However, other typical regions of C6 zinc finger proteins, such as dimerization elements, are absent in PRO1. The involvement of the pro1(+) gene in fruiting body development was further confirmed by trying to complement the mutant phenotype with in vitro mutagenized and truncated versions of the pro1 open reading frame. Southern hybridization experiments also indicated that pro1(+) homologues are present in other sexually propagating filamentous ascomycetes.

Acyl Carrier Protein↗

C-signal: a cell surface-associated morphogen that induces and co-ordinates multicellular fruiting body morphogenesis and sporulation in Myxococcus xanthus.

In Myxococcus xanthus, morphogenesis of multicellular fruiting bodies and sporulation are co-ordinated temporally and spatially. csgA mutants fail to synthesize the cell surface-associated C-signal and are unable to aggregate and sporulate. We report that csgA encodes two proteins, a 25 kDa species corresponding to full-length CsgA protein and a 17 kDa species similar in size to C-factor protein, which has been shown previously to have C-signal activity. By systematically varying the accumulation of the csgA proteins, we show that overproduction of the csgA proteins results in premature aggregation and sporulation, uncoupling of the two events and the formation of small fruiting bodies, whereas reduced synthesis of the csgA proteins causes delayed aggregation, reduced sporulation and the formation of large fruiting bodies. These results show that C-signal induces aggregation as well as sporulation, and that an ordered increase in the level of C-signalling during development is essential for the spatial co-ordination of these events. The results support a quantitative model, in which aggregation and sporulation are induced at distinct threshold levels of C-signalling. In this model, the two events are temporally co-ordinated by the regulated increase in C-signalling levels during development. The contact-dependent C-signal transmission mechanism allows the spatial co-ordination of aggregation and sporulation by coupling cell position and signalling levels.

Bacterial Proteins↗