Ethylene Production by Axenic Fruiting Cultures of Agaricus bisporus

Axenic cultures of Agaricus bisporus were used to show that the rise in ethylene production during fruiting derives from its own metabolism.     

Production and detection of muramidase and acetylglucosaminidase from Agaricus bisporus

The production and regulation of extracellular bacteriolytic enzymes of Agaricus bisporus are being studied to understand better the nutrition of this fungus and to identify factors that regulate the selectivity of mushroom compost as a growth medium. Both muramidase (EC.3.2.1.17) and N -acetyl-β- D -glucosaminidase (β-GlcNAcase, EC.3.2.1.30) have been detected in liquid cultures of A. bisporus , and in cultures fruiting in sterile and non-sterile compost. A turbidometric assay, based on the decrease in optical density of suspended Bacillus subtilis bacterial cell walls, was used to measure muramidase production by A. bisporus . A colorimetric assay was used to measure β-GlcNAcase. Both bacteriolytic enzyme activities were produced on a range of sole carbon sources, including killed freeze-dried B. subtilis cells. Muramidase activity was highest in axenic compost cultures. Bacteriolytic enzyme activity peaked as the first group of fruit bodies was harvested in both sterile and non-sterile compost.     

Fruiting of Agaricus bisporus

Several enzymes were assayed in extracts from mycelium-colonised compost during growth and fruiting of Agaricus bisporus (Lange) Imbach. Comparison of changes of enzyme levels in axenic and nonaxenic cultures and in cultures of non-fruiting strains indicated that they were associated directly with the fungal mycelium. Large changes were found in the amounts of laccase and cellulase which were correlated with fruit body development. Laccase concentration increased during mycelial growth and then declined rapidly at the start of fruiting. Cellulase activity could be detected throughout growth but increased at fruiting. No such changes were observed in xylanase, alkaline protease, laminarinase and acid and alkaline phosphatases. Activities of laccase and cellulase were measured in axenic cultures arrested at various stages of fruiting development. Such cultures showed that the changes in concentration of laccase and cellulase were associated with the enlargement of fruit bodies.     

Carboxylierungsreaktionen in Agaricus bisporus

Zusammenfassung In Gegenwart von ATP, Mn^2ü (oder Mg^2ü), CoA und Glutathion fixieren zellfreie Extrakte aus Sporophoren von Agaricus bisporus selbst in Abwesenheit exogener CO₂-Acceptoren in erheblichem Maße Kohlendioxyd. Entsprechend der Hemmbarkeit dieser CO₂-Fixierungsreaktionen durch Avidin und p-Chloromercuribenzoat handelt es sich bei diesen zum mindesten teilweise um durch Biotin-Enzyme katalysierte Vorgänge. Einer der endogenen CO₂-Acceptoren ist sehr washrscheinlich mit aus Isovaleriansäure gebildetem -Methylcrotonyl-CoA identisch.Die experimentellen Ergebnisse werden im Zusammenhang mit den bekannten physiologischen Effekten von CO₂ auf Fruktifikation und Morphogenese des Kulturchampignons diskutiert. Es scheint, daß Biotin-abhängige Carboxylierungsreaktionen eine der biochemischen Grundlagen für den Kontrolleinfluß von Kohlendioxyd im Lebenscyclus von A. bisporus darstellen.

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Carboxylation reactions in Agaricus bisporus I. The endogenous CO₂-acceptor

Summary Cell-free extracts of sporophores of Agaricus bisporus incorporate considerable amounts of carbon dioxide into organic acids even without added substrate, provided that ATP, Mn^2ü or Mg^2ü, Coenzyme A and glutathione are present. These CO₂-fixation reactions are inhibited by avidin and p-chloromercuribenzoate. It is therefore concluded that at least part of these reactions are katalyzed by biotin enzymes. One of the endogenous CO₂-acceptors is probably identical with -methylerotonyl-CoA, formed from isovaleric acid.The experimental results are diseussed in relation to the well-known physiological effects of carbon dioxide on fructification and morphogenesis in the cultivated mushroom. It is suggested that biotin-dependent carboxylations might represent the biochemical basis for the centrolling function of CO₂ in the life cycle of A. bisporus.

     

Germination of Basidiospores of Agaricus bisporus

The type of dormancy and conditions necessary for germination of Agaricus bisporus basidiospores were studied. Basidiospores failed to germinate on starvation agar and required the presence of carbon and nitrogen sources (asparagine and/or glucose) in the medium. Upon 3-week storage, basidiospores germinated after 4–5 days. Heat shock (20 min at 45°C) and decreased temperature facilitated activation of germination. Heterocyclic compounds stimulating germination of endogenously dormant spores, such as furfural, failed to activate germination. The data obtained suggested an endogenous dormancy of A. bisporus basidiospores differing from zygospores of Mucorales. Basidiospores contained 17–19% lipids with a composition of fatty acids differing from those of the pileus and stipe of the fruiting body. The soluble carbohydrates of the cytosol amounted to 12% dry spore weight and consisted of mannitol (74%) and trehalose (26%). Unlike basidiospores stored at 2°C, basidiospores stored for 5 months at 20°C lost their ability to germinate, which correlated with a decrease in the content of trehalose.     

Germination of basidiospores of Agaricus bisporus

The type of dormancy and conditions necessary for germination of Agaricus bisporus basidiospores (BS) were studied. BS failed to germinate on starvation agar and required the presence of carbon and nitrogen (asparagine and/or glucose) sources in the medium. Upon 3-week storage, BS germinated after 4-5 days. Heat shock (20 min at 45 degrees C) and the decreased temperature facilitated activation of germination. Heterocyclic compounds stimulating germination of endogenously dormant spores, such as furfural, failed to activate germination. The data obtained suggested an endogenous dormancy of A. bisporus BS differing from zygospores of Mucorales. BS contained 17-19% lipids with a composition of fatty acids differing from those of pileus and stipe of the fruiting body. The soluble carbohydrates of the cytosol amounted to 12% dry spore weight and consisted of mannitol (74%) and trehalose (26%). Unlike BS stored at 2 degrees C, the BS stored for 5 months at 20 degrees C lost their ability to germinate, which correlated with a decrease in the content of trehalose.     

The lipids of Agaricus bisporus.

A comparison of the lipid composition of the vegetative and reproductive stages of Agaricus bisporus revealed no major qualitative differences, although quantitative divergence exist. The glycolipids consisted of acylglucoses, acylmannitol, acyltrehalose and a glucosyloxyfatty acid. Two of the acylglucoses corresponded to a tetra-acylglucose and to either a di- or a triacylglucose. The phospholipids were distinctive in that phosphatidylcholine could not be detected. Phosphatidylethanolamine and phosphatidylserine were the major phosphoglycerides. Examination of the neutral lipids revealed the expected array of acylglycerols, free and esterified sterols, and free fatty acids. A substantial amount (26 to 33%) of the fatty acids of the neutral lipids from both sporophore and mycelium were apparently of chain length greater than C18. Linoleic acid was a minor component of the total neutral-lipid fatty acids but comprised about one-half of the total free fatty acids.     

NMR lipid profile of Agaricus bisporus

Lipids extracted from freeze dried and powdered cultivated Agaricus bisporus by the Bligh and Dwyer method, were subjected to 1D-proton and 2D-COSY NMR analysis. The diacylglycerophospholipids, mono-, di- and tri-glycerides, ether lipids, sphingolipids and steroidal lipids were studied qualitatively and quantitatively. Our findings suggested that (a) ethanolamines and cholines were the predominant diacylphospholipids, (b) sterols, mainly ergosterol, were present in relatively large quantities and (c) the phospholipid fatty acid composition consisted almost exclusively of linoleic acid. This type of detailed data on lipid composition was accurately and rapidly obtained in one step, without chemical modification of the sample. Additional information on four classes of lipid, including their fatty acid composition was obtained after separating the total lipid extract by NH₂-aminopropyl Certify II Bond Elut solid phase chromatography and analysing the NMR spectra of each class of lipids. The results demonstrated the potential of the method for the study of plant metabolism, development and taxonomy.     

Biosynthese von Mannitol in Agaricus bisporus

Zusammenfassung Zellfreie Extrakte aus Fruchtkörpern von Agaricus bisporus katalysieren eine NADPH-abhängige Reduktion freier Fructose zu Mannitol. In vivo werden neben diesem Zucker auch andere Monosen in den Hexit eingebaut; die entsprechenden Inkorporationsraten sind jedoch gering (für Mannose 11%, Glucose 7% und Xylose 2%, bezogen auf diejenige von Fructose = 100%). Auch die Mannitolbildung aus Glucose erfolgt über Fructose als Zwischenprodukt, und ein alternativer Syntheseweg, Reduktion von Glucose zu Sorbitol und dessen Epimerisierung zu Mannitol beinhaltend, scheint nicht realisiert zu werden, obschon es gelang, Spuren von Sorbitol gaschromatographisch nachzuweisen. Im Kulturchampignon ist demnach freie Fructose als obligater Präkursor von Mannitol zu betrachten.Die experimentellen Resultate werden im Zusammenhang mit unseren gegenwärtigen Kenntnissen über den Kohlenhydratstoffwechsel von A. bisporus diskutiert.

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Biosynthesis of mannitol in Agaricus bisporus

Summary In cell-free extracts of fruiting bodies of A. bisporus mannitol is shown to be synthesized by a NADPH-dependent reduction of free fructose. In vivo other monoses are also incorporated into the mannitol skeleton, but to a much lesser extent. Formation of this hexitol from glucose proceeds through fructose as an intermediate, whereas mannitol synthesis by a pathway involving reduction of glucose to sorbitol and epimerization of the latter to the polyol in question does not seem to occur, although it was shown that sorbitol exists in the common mushroom. Therefore, fructose would appear to be the obligate precursor of mannitol in this fungus. The experimental results are integrated into the picture of our present knowledge of carbohydrate metabolism in A. bisporus.

     

RAPD discrimination of Agaricus bisporus mushroom cultivars

Cultivars of the white button mushroom Agaricus bisporus are difficult to differentiate, which has made strain protection problematic for this crop species. We have used RAPDs to discriminate between 26 strains of A. bisporus, 24 of which were commercial cultivars, and to characterise the genetic relatedness of these strains. Using 20 primers, 211 RAPD markers were identified and used in hierarchical cluster, patristic distance and parsimony analyses. All strains could be differentiated using the aggregated primer data. Although no one primer could differentiate all 26 strains, several individual primers yielded unique fingerprints for a variety of strains. The greatest differences (up to 28% variation) were observed in comparisons with or between two wild collections of A. bisporus. Quondam cultivars, commercial brown and off-white varieties proved more variable than the widely grown 'hybrid' types. Of the 15 hybrid varieties analysed, only one differed substantially (20% or more variable). The patristic and parsimony analyses both demonstrated the gross similarity of the hybrids, many of which appear to be essentially derived varieties from two original hybrid cultivars. RAPD analyses can assist mushroom strain identification and could play a role in the protection of novel cultivars.     

The cel4 gene of Agaricus bisporus encodes a beta-mannanase

Mannases have industrial uses in food and pulp industries, and their regulation may influence development of the mushrooms of commercially important basidiomycetes. We expressed an Agaricus bisporus cel4 cDNA, which encodes a mannanase, in Saccharomyces cerevisiae and Pichia pastoris. CEL4 had no detectable activity on cellulose or xylan. This gene is the first isolated from this economically important fungus to encode a mannanase. P. pastoris secreted about three times more CEL4 than S. cerevisiae. The removal of the cellulose-binding domain of CEL4 lowered the secreted specific activity by P. pastoris by approximately 97%. The genomic sequence of cel4 was isolated by screening a cosmid library of A. bisporus C54-carb8. The open reading frame was interrupted by 12 introns. The level of extracellular CEL4 increases dramatically at the postharvest stage in compost extracts of A. bisporus fruiting cultures. In laboratory liquid cultures of A. bisporus, the activity of CEL4 detected in the culture filtrate reached a maximum after 21 days. The levels of CEL4 broadly mirrored the levels of enzyme activity. In the Solka floc-bound mycelium, CEL4 protein showed a maximum after 2 to 3 weeks of culture and then declined. Changes in CEL4 activity during fruiting-body development suggest that hemicellulose utilization plays an important role in sporophore formation. The availability of the cloned gene will further studies of compost decomposition and the extracellular enzymes that fungi deploy in this process.     

Homology models of four Agaricus bisporus tyrosinases

Partially purified tyrosinase from the white button mushroom Agaricus bisporus is available commercially and is a widely used experimental model for the study of tyrosinase. The structure of an H₂L₂ tetrameric form of the mushroom enzyme was recently determined by X-ray crystallography. In this structure the two H subunits originate from the PPO3 gene, and the two L subunits are formed by a protein of unknown function with a lectin-like fold. However, the X-ray structures and oligomeric states of the mushroom PPO1, PPO2, PPO4, and PPO5 gene products remain unknown. Commercial mushroom tyrosinase powder is a mixture containing several or all of these tyrosinases, so knowledge of their structures should provide insight regarding interpretation of experimental data generated using commercial preparations of the enzyme. The PPO3 structure (H-subunit) was used as a template to generate homology models for the structures of the other four tyrosinases, and the resulting structural models were evaluated. Due to the moderate to high percentage of sequence identity (∼37-76%) between PPO3 and the other four tyrosinases, the backbone conformations of the predicted structures are very similar to that of PPO3. The alpha carbons of the six copper-coordinating histidines in the active site are positioned properly in the predicted structures, but their side chains are not oriented optimally for copper binding in some cases. Thus, the models are likely to provide an accurate representation of the actual tertiary structures, but they may have limited use in studies involving docking of substrates or inhibitors in the active site. Comparison of the homology models to the structure of molluscan hemocyanin enabled a prediction of the orientation of the enzyme's C-terminal domain over the active site in the latent enzyme.     

双孢蘑菇遗传多样性分析

应用AFLP指纹技术对双孢蘑菇的20个野生菌株和5个栽培品种的遗传多样性进行了研究。AFLP指纹揭示出20个野生异核体菌株所固的基因型。5个商业品种表现出比较一致的AFLP指纹,但也显示出它们之间的一些差别。由单孢分离获得的同核体菌株携带着部分异核体菌株的AFLP指纹;由同一子实体分离得到的大部分单孢菌株是异核体菌株,它们具有与其亲本一致的AFLP指纹。UPGMA分析揭示出2个与地理分布（美洲、欧洲）和相对应的组。研究结果表明：（1）在野生菌株之间存在着明显的遗传差异;（2）大多数单孢分离的菌株具有与母本一致的遗传物质;（3）野生菌株间的遗传变异大于栽培品种间的变异;（4）在双孢蘑菇的遗传多样性分析中,AFLP技术是非常有效的工具。     

2-amino-2-deoxy-D-erythrose in Agaricus bisporus

2-Amino-2-deoxy-D-erythrose was isolated from the cell wall of the fruit body of Agaricus bisporus. The structure of the amino sugar was determined by mass spectrography and 1H-NMR spectrography of its acetylated derivative and by paper chromatographic comparisons with authentic 2-amino-2-deoxy-D-erythrose. This amino sugar is a component of the glycoprotein fraction from the cell wall. Its content in the glycoprotein increased markedly, especially during the ripening stage of the fruit body.     

Double-stranded ribonucleic acid in Agaricus bisporus.

Double-stranded ribonucleic acid present in virus-infected mushrooms of Agaricus bisporus has been resolved through polyacrylamide gel electrophoresis into six molecular-weight forms. Identification of these six double-stranded ribonucleic acids in mushrooms by this procedure has proven to be a useful and diagnostic method for viral infection in the cultivated mushroom.