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Spatial patterns in the fruiting bodies of the cellular slime mold Polysphondylium pallidum.

During morphogenesis in the slime mold Polysphondylium pallidum cell masses are periodically pinched off from the base of the developing sorogen. These masses round up and differentiate into secondary sorogens, which become radially ordered arrays of secondary fruiting bodies called whorls. Here we describe the morphogenesis of P. pallidum and characterize the spacing of whorls along the central stalk of the fruiting body and the spacing of sorocarps within whorls. We find both are highly regular. We propose that the linear spacing of whorls can be accounted for satisfactorily by a model that views the periodic release of cell masses from the base of the developing sorogen as the consequence of an imbalance between forces that orient amoebae toward the tip of the culminating sorogen, and cohesive forces between randomly moving cells in the basal region of the sorogen, which act as a retarding force. The orderly arrangement of fruiting bodies within whorls can be explained most easily by models that employ short-range activation and lateral inhibition.

Cell Differentiation↗

Lentinula edodes tlg1 encodes a thaumatin-like protein that is involved in lentinan degradation and fruiting body senescence.

Lentinan is an antitumor product that is purified from fresh Lentinula edodes fruiting bodies. It is a cell wall component, comprising beta-1,3-glucan with beta-1,6-linked branches, which becomes degraded during postharvest preservation as a result of increased glucanase activity. In this study, we used N-terminal amino acid sequence to isolate tlg1, a gene encoding a thaumatin-like (TL) protein in L. edodes. The cDNA clone was approximately 1.0 kb whereas the genomic sequence was 2.1 kb, and comparison of the two indicated that tlg1 contains 12 introns. The tlg1 gene product (TLG1) was predicted to comprise 240 amino acids, with a molecular mass of 25 kD and isoelectric point value of 3.5. The putative amino acid sequence exhibits approximately 40% identity with plant TL proteins, and a fungal genome database search revealed that these TL proteins are conserved in many fungi including the basidiomycota and ascomycota. Transcription of tlg1 was not detected in vegetative mycelium or young and fresh mushrooms. However, transcription increased following harvest. Western-blot analysis demonstrated a rise in TLG1 levels following harvest and spore diffusion. TLG1 expressed in Escherichia coli and Aspergillus oryzae exhibited beta-1,3-glucanase activity and, when purified from the L. edodes fruiting body, demonstrated lentinan degrading activity. Thus, we suggest that TLG1 is involved in lentinan and cell wall degradation during senescence following harvest and spore diffusion.

Amino Acid Sequence↗

Isolation and analysis of genes specifically expressed during fruiting body development in the basidiomycete Flammulina velutipes by fluorescence differential display.

Using fluorescence differential display, cDNAs specifically expressed at the primordial stage of fruiting body development were isolated from the basidiomycete, Flammulina velutipes. Seventy-five cDNAs were sequenced and compared with the amino-acid sequences of proteins in the database by BLASTX search. Significant similarity was found for 29 cDNAs coding for proteins with known function, GTP-binding protein, growth factor, ubiquitin-proteasome, cytochrome P450 and hydrophobin, all of which would be associated with fruiting body development. Seventeen cDNAs were not similar to proteins in the database and may represent unique genes that play specific roles in the process of fruiting in F. velutipes.

Agaricales↗

Isolation, crystallization, and characterization of a 16.5-kDa protein from fruit bodies of a lectin-deficient strain of Pleurotus cornucopiae.

Among mushrooms of Pleurotus cornucopiae two kinds of fruit bodies, lectin (PCL)-containing and -deficient, were found. The PCL-deficient fruit body was found to contain a 16.5-kDa protein, which was purified to homogeneity and crystallized. Properties of the protein were investigated in comparison with PCL. The protein consisted of four identical subunits each having a molecular mass of 16.5 kDa, which was close to that of PCL. Partial amino acid sequences of the two proteins were analyzed and some sequence homology was found, although the protein did not cross-react with anti-PCL serum and it was devoid of binding activity for mucin, an inhibitor of PCL. The 16.5-kDa protein was not found in vegetatively growing mycelia, indicating that the synthesis of the protein was developmentally regulated as was PCL. The results suggest that the 16.5-kDa protein was related to PCL.

Amino Acid Sequence↗

Cell motility is required for the transmission of C-factor, an intercellular signal that coordinates fruiting body morphogenesis of Myxococcus xanthus.

There are striking similarities between the developmental phenotypes of two different mutant classes of Myxococcus xanthus. The first class, mglA mutants, are nonmotile under all conditions tested. The second class, csgA mutants, are motile but belong to a class of signal-defective developmental mutants that cannot develop alone but will develop when mixed with intact wild-type cells. Nevertheless, both csgA and mglA mutants fail to aggregate properly or to sporulate when induced to form fruiting bodies. An mglA mutation and a csgA mutation affect expression of a panel of lacZ fusions to developmental genes in the same way, indicating that nonmotile cells and csgA cells arrest development at a similar stage. One explanation for the similarity of developmental phenotypes between these mutants is that motility is required for the csgA-mediated cell interaction. In support of this hypothesis, we report that C-factor, a protein purified from nascent wild-type fruiting bodies based on its ability to rescue csgA mutant fruiting body development, also rescues sporulation and expression of beta-galactosidase from developmentally controlled lacZ fusions in mglA strains, apparently without restoring their motility. Wild-type levels of active C-factor can be purified from mglA cells, yet intact mglA cells do not rescue csgA cells upon cell-cell mixing. Intact wild-type cells are unable to restore the sporulation and beta-galactosidase expression of mglA mutants. These results support the hypothesis that donor and responder cell motility is required for C-factor transmission between cells during development.(ABSTRACT TRUNCATED AT 250 WORDS)

Bacterial Proteins↗

Fruiting-body formation and myxospore differentiation and germination in Mxyococcus xanthus viewed by scanning electron microscopy.

Streaming cells, fruiting bodies, and single cells undergoing myxospore differentiation and germination were examined in the FB strain of Myxococcus xanthus by scanning electron microscopy. Myxospores differentiated in fruiting bodies differed in size, in kinetics of germination, in the fate of the myxospore capsule, and in the external structure of the walls of newly emerged cells when compared with myxospores differentiated in liquid medium after glycerol induction. Vegetative cells outgrowing from glycerol-induced myxospores were regularly pleomorphic, a condition that persisted through the first cell division.

Cell Aggregation↗

Lipids in fruiting bodies of the basidiomycete Oudemansiella mucida.

Dried fruiting bodies of the basidiomycete Oudemansiella mucida contain 29.1% total lipids. Their qualitative analysis revealed the presence of mono-, di-, triglycerides, sterols, free fatty acids and sterolesters. Quantitatively most significant were triglycerides (37.9%) and free fatty acids (29.7%). The phospholipid fraction contained phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine and phosphatidic acid. Gas chromatography showed the presence of a broad spectrum of fatty acids. The ratio between the neutral and polar fractions was 6 : 1, both having linoleic acid as the main component.

Anti-Bacterial Agents↗

Genetically related protein variants specifically associated with fruiting body maturation in Neurospora.

Electrophoretic analysis of fruiting body extracts from Neurospora reveals a characteristic protein, apparently absent in vegetative structures and ascospores, and which increases markedly in relative concentration after fertilization. Different wild-type species and strains studied have electrophoretic variants of this protein, two of which are shown to be controlled by members of an allelie pair.

Electrophoresis, Polyacrylamide Gel↗

Dictyostelium discoideum fruiting bodies observed by scanning electron microscopy.

The fruiting bodies of Dictyostelium discoideum, fixed in a vapor of 25% glutaraldehyde, were observed by scanning electron microscopy. The surface of the basal disk had rootlike ripples, sometimes with concentric rings of riblike striations at its periphery. The surface of the stalk had pleated and twisted ridges along its entire length, but no lateral projections. The spores were randomly orientated and had a crinkled surface. The surface of the lower part of the papilla had flattened ridges running horizontally. A slime coat remained in the region of transition between the stalk and sporangium, but not on the spores.

Dictyostelium↗

Improved methods of isolation and purification of myxobacteria and development of fruiting body formation of two strains.

By using baiting techniques and different purification methods, a high number of myxobacterial strains have been isolated as pure cultures from soil of different regions of China. Because myxobacterial cells do not disperse easily in liquid media, a medium containing an enzymatic hydrolysate of casein (CEH) medium have been used for purification and purity tests combined in a single step. The key method, in which isolates are reintroduced to sterile rabbit dung to induce fruiting bodies formation, facilitates purification of myxobacteria. Sterile rabbit dung pellets are used to mimic the natural growth substance of these organisms which has the advantage that characteristic fruiting bodies emerge, which is a key characteristics in the taxonomy of myxobacteria. In this study, the optimum program of isolation and purification of some myxobacteria strains has been established which will facilitate screening programs. Moreover, the development of fruiting body formation of strain BD20 (Chondromyces) and strain BD54 (Cystobacter) have been recorded in this study.

Bacteriological Techniques↗

Pleurotus fruiting bodies contain the inhibitor of 3-hydroxy-3-methylglutaryl-coenzyme A reductase-lovastatin.

In the fruiting bodies of the fungus Pleurotus ostreatus, also called the oyster mushroom, we found a competitive inhibitor of 3-hydroxy-3-methylglutaryl-coenzyme A reductase-lovastatin. The appearance of the inhibitor during the development of fruiting bodies was followed and lovastatin determined in the vegetative mycelium, in the primordia, as well as in different parts of sporocarps of different sizes. Less lovastatin was found in stripes as compared to pili or in mature stages in the lamellae and basidiospores.

Cell Extracts↗

Concerted regulation of all hyphal tips generates fungal fruit body structures: experiments with computer visualizations produced by a new mathematical model of hyphal growth.

Filamentous hyphal growth is inherently suited to kinetic analysis, and in many respects the fungal mycelium can be viewed as a very mechanical biological system, which lends itself to mathematical modelling. The mathematics of hyphal tip extension growth are well-established. However, even though a hyphal growth equation can be written with confidence, and we have a good understanding of the effects of tropisms on growth, it is not easy to form a mental picture of the behaviour of large populations of hyphal tips. What is required, and what we believe we have produced, is a mathematical model that is sufficiently sophisticated to produce a realistic visualization of fungal hyphal growth. This provides us with a cyberfungus that can be used for experimentation on the theoretical rules that might govern hyphal patterning, hyphal interactions, and tissue formation and organ development by actually visualizing the virtual hyphal growth patterns that result from different regulatory scenarios. From a series of model experiments the most significant observation is that complex fungal fruit body shapes can be simulated by applying the same regulatory functions to all of the growth points active in a structure at any specific time. No global control of fruit body geometry is necessary. No localized regulation is necessary. The shape of the fruit body emerges from the concerted response of the entire population of hyphal tips, in the same way, to the same signals.

Computer Graphics↗

Transport and localization of protein S, a spore coat protein, during fruiting body formation by Myxococcus xanthus.

Protein S, the most abundant soluble protein synthesized by Myxococcus xanthus FB during early fruiting body formation, accumulates in the soluble fraction of developing cells, reaching a peak at about 24 h; at late stages of fruiting body formation, protein S is found on the surface of spores (M. Inouye et al. Proc. Natl. Acad. Sci. U.S.A. 76:209-213, 1979). In this study, the transport and localization of protein S were investigated. Cells were fractionated to give osmotic shock, membrane, cytoplasmic, and spore fractions. The various fractions were then analyzed for protein S. Protein S was first detected in the cytoplasmic fraction at about 3 to 6 h of development. However, transport of protein S through the cytoplasmic membrane was not observed until 15 to 18 h of development. Thus, protein S is unusual among translocated proteins in that it accumulates as a soluble cytoplasmic protein before translocation. Biosynthesis of protein S ceased after 48 h; by 72 h, protein S was only found on the surface of spores. Pulse-chase experiments were performed to determine the transport kinetics of protein S. The results showed that in 24-h developing cells, the transport of protein S across the cytoplasmic membrane was rapid, occurring in less than 2 min. However, transport across the outer membrane was slow, requiring 10 to 15 min. Pulses of 15 s with [35S]methionine failed to reveal any short-lived precursor form in immunoprecipitated material separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Isoelectric focusing also failed to detect any precursor form of protein S. Thus, protein S appears to be translocated in the absence of a cleaved signal peptide.

Bacterial Proteins↗

FrzCD, a methyl-accepting taxis protein from Myxococcus xanthus, shows modulated methylation during fruiting body formation.

The frizzy (frz) genes of Myxococcus xanthus are required to control directed motility during vegetative growth and fruiting body formation. FrzCD, a protein homologous to the methyl-accepting chemotaxis proteins from enteric bacteria, is modified by methylation in response to environmental conditions. Transfer of cells from rich medium to fruiting medium initially caused rapid demethylation of FrzCD. Subsequently, the amount of FrzCD increased, but most remained unmethylated. At about the time of mound formation (9 h), most of the FrzCD was converted to methylated forms. Dispersal of developing cells (10 h) in buffer led to the demethylation of FrzCD, whereas concentration of these cells caused methylation of FrzCD. Some mutants which were unable to form fruiting bodies still modified their FrzCD during incubation under conditions of starvation on a surface.

Bacterial Proteins↗

Lead and cadmium in fruiting bodies of macrofungi in the vicinity of a lead smelter.

The concentrations of lead and cadmium in the fruiting bodies of mycorrhizal and edible macrofungi (Basidiomycetes, Agaricales) were measured in the vicinity of a lead smelter. Very high concentrations of both metals were found 1 km from the stack, and elevated concentrations were observed up to a distance of 4.5 km. The dependence of the concentrations on the distance from the stack were approximated by an exponential function, the extrapolation of which showed that only at a distance of 6 km did the concentrations approach those reported from an area in the Krusné Hory Mountains with a medium level of air pollution. The concentrations of lead were slightly higher in the caps than in the stems of the fungal fruiting bodies from the sites close to the smelters; at a distance of 1.7 km they were the same, and at greater distances the lead concentrations in the caps were slightly lower than those in the stems. The cap/stem concentration ratio for cadmium does not exhibit any change with distance. The different behaviour of the two metals can be explained in terms of the lower mobility of Pb2+ ions. Because of their lead and cadmium contents, edible mycorrhizal fungi picked within a distance of 0.6 km of the stack can be toxic to man if eaten often in quantities that are usual in Czechoslovakia.

Agaricales↗

Primary fluorescence (autofluorescence) of fruiting bodies of the wood-rotting fungus Fomes fomentarius.

Autofluorescence of fruiting bodies of the wood-rotting fungus Fomes fomentarius has been observed and is described among native macrofungi for the first time. The strongest yellow autofluorescence with blue excitation was displayed by pith sets, a weaker yellow, yellow-green to yellow-red fluorescence was due to generative thin-walled hyphae while the weakest yellow-reddish fluorescence was emitted by thick-walled skeletal hyphae (though their parts may emit a more intensive yellow fluorescence). This yellow, yellow-green to yellow-red autofluorescence was assessed to be more intensive than the emission described so far in bacteria and fungi (except for lysed hyphae of the fungus Trametes versicolor). With green excitation all F. fomentarius cells emitted strong red autofluorescence.

Fluorescence↗

Identification of an activator protein required for the induction of fruA, a gene essential for fruiting body development in Myxococcus xanthus.

Myxococcus xanthus exhibits social behavior and multicellular development. FruA is an essential transcription factor for fruiting body development in M. xanthus. In the present study, the upstream promoter region was found to be necessary for the induction of fruA expression during development. A cis-acting element required for the induction was identified and was located between nucleotides -154 and -107 with respect to the transcription initiation site. In addition, it was found that two binding sites exist within this element of the fruA promoter. By using DNA affinity column chromatography containing the cis-acting element, a fruA promoter-binding protein was purified. The purified protein was shown by N-terminal sequence analysis to be identical to MrpC, a protein identified previously by transposon insertion mutagenesis as an essential locus for fruiting body development [Sun, H. & Shi, W. (2001) J. Bacteriol. 183, 4786-4795]. Furthermore, fruA mRNA was not detectable in the mrpC::km strain, demonstrating that MrpC is essential for fruA expression. Moreover, mutational analysis of the binding sites for MrpC in the fruA promoter indicates that binding of MrpC activates transcription of fruA in vivo. This report provides evidence for a direct molecular interaction involved in temporally regulated gene expression in M. xanthus.

Amino Acid Sequence↗

Laetiporic acids, a family of non-carotenoid polyene pigments from fruit-bodies and liquid cultures of Laetiporus sulphureus (Polyporales, Fungi).

The non-isoprenoid polyene laetiporic acid A, recently described from fruit-bodies of the wood-rotting fungus Laetiporus sulphureus, was found to be the major orange pigment also in mycelium grown in liquid culture. Its formation was variable, ranging from 0.1 to 6.7 mg/g dry weight in three strains, all of which were identified as L. sulphureus by ITS rDNA sequence analysis. A second pigment, 2-dehydro-3-deoxylaetiporic acid A, is also described and fully characterized by NMR spectroscopy. Two further minor pigments, laetiporic acids B and C, were produced in liquid culture. These resemble laetiporic acid A but are enlarged by two and four carbon atoms, respectively, resulting in chromophores with 11 or 12 instead of 10 conjugated double bonds as described for laetiporic acid A. Since fruit-bodies of L. sulphureus are edible, laetiporic acids might hold potential as food colourants.

Molecular Structure↗