Characteristics and N-terminal amino acid sequence of manganese peroxidase from solid substrate cultures of Agaricus bisporus.

Extracellular manganese peroxidase (MnP) was purified from the compost extract of Agaricus bisporus using anion exchange chromatography, gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Two forms (MnP1 and MnP2) were separated by isoelectric focusing and their isoelectric points were determined to be 3.25 (MnP1) and 3.3 (MnP2). Both forms had a molecular mass of 40 kDa. The first 25 amino acids of the N-terminal end of MnP1 sequence was found to share 68% identity with a Pleurotus ostreatus and a P. eryngii MnP. Lignin peroxidase was not detected during any of the steps in the purification process. In liquid cultures with both soluble and insoluble carbon sources in defined medium (D-glucose, glycerol, Whatman CC-41 microcrystalline cellulose or Solka-floc cellulose) MnP protein was detected in culture fluid by Western blot, but no MnP activity could be detected. A. bisporus appears to be in the group of ligninolytic fungi which do not produce lignin peroxidase.

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.

The molecular genetics of cultivated mushrooms.

The types, economic significance and methods of production of the principal cultivated mushrooms are described in outline. These organisms are all less than ideal for conventional genetic analysis and breeding, so molecular methods afford a particular opportunity to advance our understanding of their biology and potentially give the prospect of improvement by gene manipulation. The sequences described are limited to those found in GenBank by August 1999. The gene sequences isolated from the white button mushroom Agaricus bisporus, the shiitake Lentinula edodes, the oyster mushrooms Pleurotus spp., the paddy straw mushroom Volvariella volvacea and the enotake Flammulina velutipes are described. The largest group are genes from A. bisporus, which includes 29 for intracellular proteins and 12 for secreted proteins. In comparison, only a total of 26 sequences can be reported for the other cultivated species. A. bisporus is also the only cultivated species for which molecular karyotyping is already supported by reliable markers for all 13 of its chromosomes.

Tandem organization and highly disparate expression of the two laccase genes lcc1 and lcc2 in the cultivated mushroom Agaricus bisporus.

Two non-allelic laccase genes (lcc1 and lcc2) in Agaricus bisporus have been mapped to the same cosmid clone and are close together, in tandem. The intergenic region consists of 1562 bp between the stop codon of lcc1 and the start codon of lcc2. Differences between the 5' non-coding regions of the two genes suggest the potential for their differential regulation. By employing competitive RT-PCR and specific primer pairs that discriminate between lcc1 and lcc2, it has been shown that the level of lcc2 mRNA is approximately 300 times higher than that of lcc1 mRNA in malt extract liquid cultures; in compost cultures lcc2 mRNA is almost 7000 times more abundant than lcc1 mRNA.

Blue and yellow laccases of ligninolytic fungi.

Extracellular laccases from submerged cultures of Coriolus versicolor BKM F-116, Panus tigrinus 8/18, Phlebia radiata 79 (ATCC 64658), Phlebia tremellosa 77-51 and from cultures of Pa. tigrinus 8/18, Ph. radiata 79 and Agaricus bisporus D-649 grown on wheat straw (solid-state fermentation) were purified. All enzymes from submerged cultures had a blue colour and characteristic absorption and EPR spectra. Laccases from the solid-state cultures were yellow-brown and had no typical blue oxidase spectra and also showed atypical EPR spectra. Comparison of N-terminal amino acid sequences of purified laccases showed high homology between blue and yellow-brown laccase forms. Formation of yellow laccases as a result of binding of lignin-derived molecules by enzyme protein is proposed.

Correlation of exons with functional domains and folding regions in a cellulase from Agaricus bisporus.

The cellulase gene cel3 has been isolated from Agaricus bisporus and sequenced. The 5'-end of the cel3 transcript was determined by primer extension and S1 nuclease protection. Putative regulatory elements have been identified in the cel3 promoter and 3'-untranslated regions. The cel3 coding region is interrupted by six short introns, two of which separate the coding regions for the three modules in the CEL3 protein: cellulose-binding domain, linker region, and catalytic domain. Three of the remaining four introns are positioned in regions coding for loops between structural moieties. Intron positions are conserved between cel3 and other related cellulases.

The cel3 gene of Agaricus bisporus codes for a modular cellulase and is transcriptionally regulated by the carbon source.

A 52-kDa protein, CEL3, has been separated from the culture filtrate of Agaricus bisporus during growth on cellulose. A PCR-derived probe was made, with a degenerate oligodeoxynucleotide derived from the amino acid sequence of a CEL3 CNBr cleavage product and was used to select cel3 cDNA clones from an A. bisporus cDNA library. Two allelic cDNAs were isolated. They showed 98.8% identity of their nucleotide sequences. The deduced amino acid sequence and domain architecture of CEL3 showed a high degree of similarity to those of cellobiohydrolase II of Trichoderma reesei. Functional expression of cel3 cDNA in Saccharomyces cerevisiae was achieved by placing it under the control of a constitutive promoter and fusing it to the yeast invertase signal sequence. Recombinant CEL3 secreted by yeast showed enzymatic activity towards crystalline cellulose. At long reaction times, CEL3 was also able to degrade carboxymethyl cellulose. Northern (RNA) analysis showed that cel3 gene expression was induced by cellulose and repressed by glucose, fructose, 2-deoxyglucose, and lactose. Glycerol, mannitol, sorbitol, and maltose were neutral carbon sources. Nuclear run-on analysis showed that the rate of synthesis of cel3 mRNA in cellulose-grown cultures was 13 times higher than that in glucose-grown cultures. A low basal rate of cel3 mRNA synthesis was observed in the nuclei isolated from glucose-grown mycelia.

Regulation of transcription of the cel1 gene in Agaricus bisporus.

Northern analysis showed that accumulation of Agaricus bisporus cel1 mRNA was regulated by two independent mechanisms: (i) induction by cellulose; and (ii) repression by glucose and other sugars. Isolated A. bisporus nuclei were transcriptionally active. Nuclei isolated from cellulose-grown mycelium synthesized six times more cel1 mRNA than nuclei from glucose-grown mycelium. The start point of transcription (tsp) was identified by primer extension and S1 nuclease analysis. Putative glucose-, and cAMP-responsive elements as well as regions with homology to promoter regions of other fungal cellulase genes were detected both upstream and downstream from the tsp of the cel1 gene.

CEL1: a novel cellulose binding protein secreted by Agaricus bisporus during growth on crystalline cellulose.

The cel1 gene of Agaricus bisporus encodes a protein (CEL1) that has an architecture resembling the multi-domain fungal cellulases, although the sequence of its putative catalytic core is not matched by any other in the protein and nucleic acid data bases. The N-terminal half of the putative catalytic domain of CEL1 was expressed in Escherichia coli as a fusion protein with glutathione-S-transferase. The fusion protein was used to raise a CEL1-specific antibody. CEL1 was detected as an extracellular 49.8 kDa protein in A. bisporus cellulose-grown cultures, where it bound strongly to cellulose. CEL1 was neither an endoglucanase, a cellobiohydrolase able to hydrolyze fluorogenic cellobiosides, a beta-glucosidase, a xylanase, nor a cellobiose: quinone oxidoreductase. CEL1 was present in some fractions of culture fluid separated by electrophoresis which released soluble sugars from crystalline cellulose.

Identification of two laccase genes in the cultivated mushroom Agaricus bisporus.

A cDNA library was constructed in lambda gt11 using mRNA from 11-d-old mycelium of Agaricus bisporus. Three clones containing laccase sequence were identified using an affinity-purified anti-laccase antibody. From one of these clones, a 333 bp sequence was used to identify further cDNA clones (including one which is close to full length) and a genomic clone. The coding sequences found were of two similar but not identical versions with differences at 36 out of 520 residues of deduced amino acid sequence. The laccase genes each encode a sequence expressed as a 2.3 kb mRNA, specifying a 520 residue polypeptide including a 19 amino acid residue signal peptide that is absent from the N terminus of the mature (extracellular) protein. The coding sequence of lcc1 is interrupted by 14 short introns. The lcc1 and lcc2 genes are not allelic as they do not segregate in uninucleate spores derived from a four-spored basidium. Comparison of the deduced amino acid sequences with that of the other fungal laccases that have been cloned, and with the very similar ascorbate oxidases from higher plants shows that whilst some sequence is absolutely conserved at and around the amino acid residues involved in copper binding, the overall sequence similarities are low.

Purification and characterization of a serine proteinase from senescent sporophores of the commercial mushroom Agaricus bisporus.

A proteinase has been purified from the stipes of senescent sporophores of the mushroom Agaricus bisporus. The proteinase was inhibited by PMSF. It has a broad pH optimum, 6.5-11.5, and a narrow substrate specificity, requiring both a hydrophobic amino acid in the P1 position and a minimum peptide chain length. The apparent molecular mass of the proteinase was 27 kDa when determined by SDS-PAGE and 14.1 kDa when measured by gel filtration. The isoelectric point of the proteinase was 9.0. Polyclonal antibodies have been raised to the proteinase. The proteinase from A. bisporus has similar properties to, and 60% N-terminal sequence identity with, proteinase K from the fungus Tritirachium album.

The structure of laccase protein and its synthesis by the commercial mushroom Agaricus bisporus.

Agaricus bisporus secretes abundant laccase activity into the medium during mycelial growth. SDS-PAGE analysis of extracellular laccase protein, purified from compost extract, showed a predominant band of 65 kDa molecular mass, together with lesser amounts of smaller polypeptides. The main polypeptide was purified electrophoretically. Amino acid sequence analysis of the N-terminal region of the main polypeptide was used to specify the sequence of a 15-residue chemically synthesized peptide (N-terminal peptide). Rabbit antibodies were raised against pure laccase, electrophoretically purified main polypeptide and the synthetic N-terminal peptide. Electrophoretically purified main polypeptide antibody was further purified by affinity chromatography on laccase-CNBr-Sepharose. Western blot analysis showed that the antigenic behaviour of laccase in compost extract, culture filtrate from malt-extract culture, and the purified enzyme from both sources, differed. The patterns of bands revealed are most simply explained by generation of (proteolytically) partially cleaved enzyme molecules in the culture medium, possibly combined with differences in extent of glycosylation. [35S]Methionine incorporation and immunoprecipitation were used to follow laccase synthesis in cultures grown on malt extract. After short-term labelling, a single polypeptide of 68 kDa apparent molecular mass was immunoprecipitated from both mycelial extracts and the culture medium. When poly(A)-containing RNA from malt-extract-grown mycelium was translated in vitro in rabbit reticulocyte lysate, a single polypeptide of about 57 kDa molecular mass was immunoprecipitated, consistent with the previously measured carbohydrate content of 15% for the pure enzyme. After treatment with N-glycanase, the polypeptide showed an increase in mobility during SDS-PAGE consistent with a reduction in molecular mass of about 5 kDa, indicating about equal amounts of N- and O-linked carbohydrate. C-terminal labelling of pure laccase was attempted by transpeptidation with carboxypeptidase Y. Although some minor bands were labelled, the main polypeptide was not, indicating that the C-terminus of the enzyme may be blocked.

Isolation and characterization of a cellulose-growth-specific gene from Agaricus bisporus.

The edible basidiomycete, Agaricus bisporus, produces extracellular endoglucanase. Endoglucanase production is induced by cellulose and repressed by fructose in A. bisporus grown on minimal medium, and is regulated in activity during fruiting body development. An anti-endoglucanase antibody was used to isolate cellulase-related genes. Three main polypeptides of 38, 58, and 60 kDa were immunoprecipitated by the antibody from products of in vitro cell-free translation of mRNAs isolated from cellulose-grown mycelium. No cross-reaction was detected with the translated products from fructose-grown mycelium. This antibody was used to immunoscreen a lambda ZAPII-cDNA expression library made from mRNA isolated from cellulose-grown mycelium. Two cDNA cross-reacting clones, pSRc110 and pSRc200, were isolated. Clones pSRc110 and pSRc200 cross-hybridized and had the same restriction map. Clone pSRc200 hybrid selected an mRNA that on cell-free translation produced a 38-kDa polypeptide. The cDNA fragment from pSRc200 hybridized to a 1.3-kb mRNA from cellulose-grown mycelium. No hybridization was observed when using fructose-grown mycelium mRNA. Thus, the gene (cel1) expressing the 1.3-kb mRNA, is differentially regulated by the carbon source of the culture medium. The cell gene was isolated in a 8.9-kb EcoRI genomic fragment after hybridization to pSRc200. Sequences similar to those in the egl1 and cbh2 genes from Trichoderma reesi were found upstream from the ATG start codon in cel1. Nine short intervening sequences disrupt the cel1 coding sequence, and a strong bias against codons ending with G and A was observed.(ABSTRACT TRUNCATED AT 250 WORDS)

An investigation of dye reduction by food-borne bacteria.

The rates of reduction of seven redox dyes by 13 bacterial strains were measured and found to vary greatly between different bacterium/dye combinations. Phenazine ethosulphate and toluidine blue were the most rapidly reduced dyes by the majority of bacteria and resorufin and 2-hydroxy-1,4-naphthoquinone were reduced slowly, if at all. There was also considerable variation in the rates of reduction with any single dye/organism combination. Glucose stimulated the rates of endogenous dye reduction in about half of the organisms. For Bacillus cereus, Pseudomonas fluorescens and Escherichia coli, dye reduction was stimulated by a range of exogenous substrates but lactose, the primary available carbon and energy source in milk, had little effect. In Lactococcus lactis, dye reduction was stimulated by sugars but not by organic acids. Oxygen successfully competed with dye reduction in organisms containing respiratory chains, but with membrane fractions, dye reduction was more rapid than oxygen consumption. All the organisms showed little cytosolic dye reduction, except L. lactis which showed substantial rates of reduction of some dyes by this fraction. With the membrane fraction of E. coli and Ps. fluorescens, cyanide inhibited NADH and succinate-dependent dye reduction, Antimycin A inhibited lactate and succinate and rotenone had no significant effect, but inhibition was not always observed with membrane from both organisms.

Glucose control of staphylococcal enterotoxin A synthesis and location is mediated by cyclic AMP.

The regulation of staphylococcal enterotoxin A (SEA) synthesis in a defined medium was studied using continuous culture techniques. SEA production was repressed by glucose and repression could be overcome by addition of exogenous cyclic AMP. As well as this classical catabolite repression control, addition of glucose to de-repressed steady-state cultures resulted in rapid disappearance of toxin from the medium (also mediated by loss of cyclic AMP). When the toxin dissappeared from the medium, it was taken up again by the bacteria without apparent modification.

Direct immunoprecipitation of protein.

Many biochemical experiments depend on the measurement of the amount of an enzyme (or other protein) independently of any enzymatic activity it may have. When working with cell-free extracts or other complex mixtures this is not simple to do, and most procedures that have general application involve the use of antibodies. Two distinct strategies have been used to good effect. The amount of antibody bound can be made to reflect amount of antigen, as in an ELISA assay (ref. 1 and Vol. 1 of this series) or in a "Western blot" (ref. 2 and Chapters 28 and 29 in this volume). Alternatively, the antibody can be used effectively to purify the antigen protein. The method described here is of the latter sort and has advantages of simplicity and rapidity both over ELISA and blotting techniques, on the one hand, and indirect precipitation techniques on the other.

Isoelectric focusing under denaturing conditions.

The introduction of methods for the electrophoretic analysis of proteins under denaturing conditions (1,2) is something of a landmark in the development of methodologies for the analysis of proteins. Not only was analysis giving good correlation to molecular weight possible with small samples of complex mixtures of proteins, but in addition, analysis of normally insoluble proteins became possible in a relatively simple manner. These methods were based on solubilizing protein in sodium dodecyl sulfate (SDS) such that the intrinsic charge differences between proteins were overwhelmed by the relatively strong charge of the dodecyl sulfate ion, which resulted in very good correlation (for most, but not all, proteins) between electrophoretic mobility and molecular weight (see Chapter 6 in Vol. 1 of this series).

Northern blotting.

Northern blotting is a way of detecting specific RNA sequences, homologous to a hybridization probe, within a complex RNA sample. In this procedure RNA is first separated by electrophoresis under denaturing conditions (see Chapter 1 ), followed by blotting onto a suitable filter. Specific sequences are then detected by hybridization. The RNA species are both immobilized on the filter and denatured so that when the filter is immersed in a solution containing a labeled nucleic acid probe, the probe binds to RNA of complementary base se?quence (hybridizes), specifically labeling the position on the filter that this RNA sequence has been blotted to from the gel. This defines the size of the RNA molecule complementary to the probe and can be used to compare relative amounts of the RNA species in different samples.

Electrophoresis of RNA denatured with glyoxal or formaldehyde.

The first successful method for electrophoretic analysis of the full size range of cellular RNA molecules was described by Loening (1), and its introduction allowed for major advances, most particularly in the molecular biology of eukaryotic organisms. The method had, nevertheless, two significant disadvantages in that the gels (composed of acrylamide at very low concentrations) were mechanically fragile, and the migration of RNA molecules did not necessarily reflect their size because RNA secondary structure was not disrupted.

The complexity of poly(A)+ RNA in the alga Chlorella fusca.

RNA excess hybridization with radioactively labelled complementary DNA (cDNA) was used to reveal the complexity of poly(A)+ RNA from Chlorella fusca var. vacuolata. RNA was tested from cells during photosynthetic exponential growth and during adaptation to heterotrophic growth in the dark on acetate. Both RNA populations were resolved into abundant, intermediate and rare sequence classes. Abundant sequences (200-400 copies per cell) constituted a significantly larger proportion of total poly(A)+ RNA in acetate-adapting cells than in exponentially growing autotrophic cells. Both types of RNA contained a rare sequence class of complexity consistent with a composition of about 20 000 different sequences. This indicated a substantially greater complexity of genes expressed in this alga and Euglena than in fungi and slime moulds. Heterologous hybridization, and hybridization with fractionated cDNA, showed that the majority of differences between RNA populations from exponentially growing and adapting cells were changes in relative abundance of groups of sequences, rather than presence of different sets of sequences in the two populations.