L.J.L.D. van Griensven

Population Dynamics of Scytalidium thermophilum in Mushroom Compost and Stimulatory Effects on Growth Rate and Yield of Agaricus bisporus

SUMMARY: Mycelial growth of Agaricus bisporus on sterilized compost is strongly stimulated by pre-incubating the compost with the thermophilic fungus Scytalidium thermophilum. This stimulatory effect is not species specific, for either A. bisporus or S. thermophilum. Normal mushroom compost is almost completely colonized with S. thermophilum. In experimental composts a positive relation was found between the logarithm of mushroom yield of A. bisporusand the density of S. thermophilum. S. thermophilum provides for compost selectivity: it protects against negative effects of compost bacteria on mycelial growth of A. bisporus. S. thermophilum is inactivated by the growth of A. bisporusmycelium.

Isolation of developmentally regulated genes from the edible mushroom Agaricus bisporus.

From a cDNA library, constructed from mushroom primordia, nine cDNAs were isolated which were either induced or specifically expressed during fruit body development and maturation of the basidiomycete Agaricus bisporus. These cDNAs varied in size from 372 to 1019 bp and hybridized to transcripts of 400-1600 nt. Four of the cDNAs were only expressed in the generative phase of the life cycle while the other five cDNAs were strongly induced but had low steady-state mRNA levels in vegetatively grown mycelium of the hybrid strain Horst U1. An apparent full-length cDNA could be identified by sequence analysis and specified a putative protein homologous to the delta-subunit of the mitochondrial ATP synthase complex of Saccharomyces cerevisiae and Neurospora crassa. For one of the partial cDNAs, significant homology was found with a family of cell division control proteins, while another partial cDNA appeared to encode a cytochrome P450. All cDNAs, except the presumed cytochrome-P450-specifying cDNA (cypA), hybridized with single copy genes scattered over the Agaricus genome. For the cypA gene, the presence of several additional copies was shown by heterologous hybridizations. Based on changes in expression levels of the fruit-body-induced genes during development coinciding with alterations in morphological appearance of mushrooms, four stages of development were distinguished during growth and maturation of A. bisporus fruit bodies.

Different temporal and spatial expression of two hydrophobin-encoding genes of the edible mushroom Agaricus bisporus

In a search for genes that are only expressed in fruit bodies of the basidiomycete Agaricus bisporus, two cDNAs, hypA and hypB that encode hydrophobins have been isolated previously. In this study, the structure of hypB is resolved and it is shown that the two genes are differentially expressed, indicating that the encoded hydrophobins serve different functions in A. bisporus mushrooms. hypB encodes a polypeptide (HYPB) of 119 aa that shows little sequence identity with HYPA apart from the characteristic arrangement of eight cysteines found exclusively in hydrophobins. The temporal and spatial expression of the two hydrophobin-encoding genes during fruit body development was compared using Northern analysis and in situ hybridization. Accumulation of hypA mRNA was found in tissue fractions consisting of undifferentiated white hyphae. In situ hybridization showed that the highest hypA mRNA levels are not found in the outermost cell layers of the pileipellis but in the cell layers adjacent to that. The highest level of expression of hypB occurs early in development when the primordium differentiates into densely packed, randomly oriented cap hyphae and loosely packed, vertically oriented stipe hyphae. In mature mushrooms, a strong accumulation of hypB transcripts was found only in the transitional zone between cap and stipe tissue, demonstrating that transcription regulation of hypB is clearly distinct from hypA.

Nitrogen assimilating enzymes in the white button mushroom Agaricus bisporus

Agaricus bisporus has the enzymic potential to assimilate ammonia by the activities of glutamine synthetase (EC 6.3.1.2), NADp-dependent glutamate dehydrogenase (EC 1.4.1.2) and NADP-dependent glutamate dehydrogenase (EC 1.4.1.4). It also contains glutamate synthase (EC 1.4.7.1) and a number of transaminating activities like glutamate-oxaloacetate transaminase (EC 2.6.1.1), glutamate-pyruvate transaminase (EC 2.6.1.2) and alanine-glyoxylate transaminase (EC 2.6.1.44). A. bisporus showed good growth in a defined buffered medium on glucose as a carbon source and a number of organic nitrogen compounds or ammonia as a nitrogen source. No growth was observed using nitrate as a nitrogen source. A. bisporus was not able to use organic nitrogen containing substances as a sole nitrogen and carbon source. Specific activities of the ammonia assimilating enzymes showed some variation when mycelia were cultivated on different nitrogen sources. Highest specific activities for glutamine synthetase, NAD-dependent glutamate dehydrogenase and NADP-dependent glutamate dehydrogenase were found when mycelia were grown on glutamate as a nitrogen source. Lowest values were found when the mycelia were grown on ammonia or glutamine. The specific activities of the ammonia assimilating enzymes showed no variation during maturation of the sporophores.

Nucleotide sequence and expression of the gene encoding NADP(+) dependent glutamate dehydrogenase (gdhA) from Agaricus bisporus

The gene encoding NADP+-dependent glutamate dehydrogenase (gdhA) was isolated from anAgaricus bisporus recombinant phageλ library. The deduced amino acid sequence would specify a 457-amino acid protein that is highly homologous in sequence to those derived from previously isolated and characterized genes coding for microbial NADP+-GDH. The open reading frame is interrupted by six introns. None of the introns is located at either one of the positions of the two introns conserved in the corresponding open reading frames of the ascomycete fungiAspergillus nidulans andNeurospora crassa. Northern analysis suggests that theA. bisporus gdhA gene is transcriptionally regulated and that, unlike the case in ascomycetes, transcription of this gene is repressed upon the addition of ammonium to the culture.

Intracellular infection of the cultivated mushroom Agaricus bisporus by the mycoparasite Verticillium fungicola var. fungicola

Infection of the cultivated mushroom Agaricus bisporus by the mycoparasite Verticillium fungicola var. fungicola was studied using light microscopy and TEM. Light microscopy indicated that V. fungicola hyphae grew on and in the hyphae of A. bisporus. Appressoria of Verticillium were also seen. TEM revealed the presence of V. fungicola hyphae inside the hyphae of A. bisporus.

Growth kinetics of Agaricus bisporus mycelium on solid substrate (mushroom compost)

Summary: Growth of Agaricus bisporus was studied on sterile compost and on sterile compost pre-grown with the thermophilic fungus Scytalidium thermophilum. Early growth was characterized by depressed, slowly growing hyphae. The mycelial radius extended exponentially in this stage. Mycelium on sterile compost switched to a fluffy growth type of radially orientated hyphae that extended at a linear rate, K r, of 5mm d-1. On compost pre-grown with S. thermophilum, early growth continued up to 30 mm, then extension became linear at K r = 7·2 mm d-1. If cultures were well ventilated, K r was higher. A new growth function was derived to combine both growth phases. The width of the peripheral growth zone, w, of A. bisporus mycelium was 3 mm. The exponential specific growth rate, μ, was calculated from K r/w = 2·4 d-1, which is much higher than a previous estimate of 0·19 d-1. In addition to growth-promoting effects, S. thermophilum had inhibitor, effects; the period of early growth was reduced if S. thermophilum was inactivated in pre-grown compost. Under commercial conditions, maximum growth rates are not exploited. The introduction of selected strains of thermophilic fungi in compost and the development of a new kind of spawn inoculum of A. bisporus might lead to improvements in commercial practice.

Purification and characterization of NADP-dependent glutamate dehydrogenase from the commercial mushroom Agaricus bisporus

The nicotinamide adenine dinucleotide phosphate (NADP)-dependent glutamate dehydrogenase (NADP-GDH) of Agaricus bisporus, a key enzyme in ammonia assimilation, was purified to apparent electrophoretic homogeneity with 27% recovery of the initial activity. The molecular weight of the native enzyme was 330 kDa. The enzyme is probably a hexamer, composed of identical subunits of 48 kDa. The isoelectric point of the enzyme was found at pH 4.8. The N-terminus appeared to be blocked. The enzyme was specific for NADP(H). The Km-values were 2.1, 3.2, 0.074, 27.0, and 0.117mM for ammonia, 2-oxoglutarate, NADPH, L-glutamate, and NADP respectively. The pH optima for the amination and deamination reactions were found to be 7.6 and 9.0, respectively. The temperature optimum was 33°C. The effect of several metabolites on the enzymes activity was tested. Pyruvate, oxaloacetate, ADP, and ATP showed some inhibitory effect. Divalent cations slightly stimulated the aminating reaction. Antibodies raised against the purified enzyme were able to precipitate NADP-GDH activity from a cell-free extract in an anticatalytic immunoprecipitation test. Analysis of a Western blot showed the antibodies to be specific for NADP-GDH.

Molecular characterization of the glnA gene encoding glutamine synthetase from the edible mushroom Agaricus bisporus.

Abstract The gene encoding glutamine synthetase (glnA) was isolated from an Agaricus bisporus H39 recombinant λ phage library. The deduced A. bisporus glutamine synthetase amino acid sequence contains 354 residues. The amino acid sequence is very similar to that derived from the gene coding for glutamine synthetase of the yeast Saccharomyces cerevisiae. The open reading frame is interrupted by four introns. Northern analysis revealed that transcription of the gene is repressed upon addition of ammonium to the culture but the repression was not as strong as that of the gene encoding NADP+-dependent glutamate dehydrogenase (gdhA). Enzyme activities are low in the presence of ammonium, glutamine and albumin and do not correlate with the mRNA levels revealed by Northern analysis. This suggests that glutamine synthetase expression in A. bisporus is also post-transcriptionally regulated by the nitrogen source.

15N-NMR study of ammonium assimilation in Agaricus bisporus

Ammonium assimilation was studied by feeding [15N]ammonium to actively growing mycelium of Agaricus bisporus. Products of ammonium assimilation were analysed using 15N-NMR. Participation of glutamine synthetase, glutamate synthase and NADP-dependent glutamate dehydrogenase was determined by inhibiting glutamine synthetase with phosphinothricin and glutamate synthase with azaserine. Our results clearly indicate that, under the conditions used, ammonium assimilation is mainly catalysed by the enzymes of the glutamine synthetase/glutamate synthase pathway. No indications were found for participation of NADP-dependent glutamate dehydrogenase. Furthermore, 15N-labelling shows that transamination of glutamate with pyruvate to yield alanine is a major route in nitrogen metabolism. Another major route is the formation of N-acetylglucosamine. Compared to the formation of N-acetylglucosamine there was only a limited formation of arginine.