Agrobacterium-mediated transformation of the ectomycorrhizal basidiomycete Tricholoma matsutake that produces commercially valuable fruit bodies, matsutake

Using agroinfection with a T-DNA vector carrying a hygromycin resistance marker, the recombinants were generated for the first time from the ectomycorrhizal basidiomycete Tricholoma matsutake, which produces commercially valuable fruit bodies, matsutake, during association with Pinus sp. plants. The transformation system may be useful in the genetic analysis of T. matsutake.     

Polysaccharides from the fruit bodies of the basidiomycete Laetiporus sulphureus (Bull.: Fr.) Murr

The two main polysaccharides from the basidiomycetous fungus Laetiporus sulphureus were isolated, purified and characterized. The structural assignments were carried out using (13)C, (1)H, and (1)H,(13) HSQC nuclear magnetic resonance spectroscopy, methylation analysis, and Smith degradation. One was a linear beta-glucan having a (1-->3)-linked main chain, namely laminaran. The other was a fucomannogalactan, which consisted of a main chain of (1-->6)-linked alpha-D-galactopyranosyl residues, a part of them being substituted at O-2 by 3-O-D-mannopyranosyl-L-fucopyranosyl, alpha-D-mannopyranosyl and in a minor proportion, alpha-L-fucopyranosyl groups. This heteropolysaccharide is related to those of other Basidiomycetes heterogalactans, although it differs distinctly in its side-chain structures. Whereas part of the single-unit L-fucopyranosyl and/or 3-O-alpha-mannopyranosyl-L-fucopyranosyl residues are present as side chains of the other heterogalactans, additional alpha-D-mannopyranosyl units are present in our fucomannogalactan of L. sulphureus.     

Antibiotic inhibitors of the bacterial ribosome

[This corrects the article on p. 493 in vol. 32.].     

Radionuclide accumulation in fruit bodies of macromycetes

Coefficients of 137Cs accumulation and 90Sr were determined in macromycetes of different trophic groups (137Cs in 43 species and 90Sr in 19 species) in the conditions of droughty year (1992). Their variability in forest formations was determined in the period from 1992 to 1998. In the year with increased atmospheric humidity (1998), two-fold rise of 137Cs accumulation in fruit bodies was registered on average. The pollution of Boletus edulis correlates with photosynthetically active part of Betula pendula and Pinus silvestris closer than with soil pollution. This shows the possibility to indicate the pollution of short-living fruit bodies of fungi by the pollution of plants-symbiotrophs.     

Antibiotic production by bacterial biocontrol agents

Interest in biological control of plant pathogens has been stimulated in recent years by trends in agriculture towards greater sustainability and public concern about the use of hazardous pesticides. There is now unequivocal evidence that antibiotics play a key role in the suppression of various soilborne plant pathogens by antagonistic microorganisms. The significance of antibiotics in biocontrol, and more generally in microbial interactions, often has been questioned because of the indirect nature of the supporting evidence and the perceived constraints to antibiotic production in rhizosphere environments. Reporter gene systems and bio-analytical techniques have clearly demonstrated that antibiotics are produced in the spermosphere and rhizosphere of a variety of host plants. Several abiotic factors such as oxygen, temperature, specific carbon and nitrogen sources, and microelements have been identified to influence antibiotic production by bacteria biocontrol agents. Among the biotic factors that may play a determinative role in antibiotic production are the plant host, the pathogen, the indigenous microflora, and the cell density of the producing strain. This review presents recent advances in our understanding of antibiotic production by bacterial biocontrol agents and their role in microbial interactions. This revised version was published online in August 2006 with corrections to the Cover Date.     

Antibiotic activity of pteridophytes

The occurrence of antibiotic activity in the extracts of 114 species of pteridophytes (27 families, 61 genera) has been surveyed. The plants were extracted in water, methanol, 70% ethanol, acetone and ether and assayed against 3 gram-positive, I acid-fast and 5 gram-negative bacteria and 3 fungal plant pathogens. Sixty-seven ferns and 6 fern allies, representing 64% of the samples examined, were antibiotic ally active. The active substances were in most cases antibacterial and only 3 possessed antifungal activity. Thirty-three species were inhibitory to gram-positive bacteria alone, 9 exclusively against gram-negative bacteria and 15 to both. Twenty species were inhibitory to penicillin-resistant Staphylococcus aureus, 16 against Mycobacterium phlei, 24 against Salmonella typhi, 16 against Vibrio cholerae and 3 against Pseudomonas aeruginosa. Only Dryopteris coch-leata was active against both bacteria and fungi. Five other species of Dryopteris showed remarkable antibacterial activity. The ferns of “Adiantum group” were found to be particularly active against gram-positive bacteria. The polypodiaceous ferns constitute a rich group of which Microsorium alternifolium, Leptochillus decurrens, Polypodium irioides, Pyrrosia mannii and Phymatodes ebenipes deserve special mention. Several thelypteroid, davalloid and athyroid ferns were also fairly active.     

Conditions for fruit body formation in the white rot basidiomycete Phanerochaete chrysosporium

In Phanerochaete chrysosporium fruit body formations is subject to strong catabolite repression by glucose in the presence of physiological levels of nitrogen. Walseth cellulose was found to be the best source of carbon for the induction of fruit body and consequent basidiospore synthesis. Ejected basidiospores collected from cultures grown under these conditions for two weeks are contaminated with neither conidia nor mycelial fragments and are therefore suitable for genetic analysis of recombination. Under conditions of nitrogen limitation, the glucose catabolite repression of fruit body synthesis was relieved. Exogenous adenosine 3,5-monophosphate but not other related nucleotides, also relieved glucose catabolite repression of fruit body formation.     

Amyloid-like properties of bacterial inclusion bodies

Bacterial inclusion bodies are major bottlenecks in protein production, narrowing the spectrum of relevant polypeptides obtained by recombinant DNA. While regarded as amorphous deposits formed by passive and rather unspecific precipitation of unfolded chains, we prove here that they are instead organized aggregates sharing important structural and biological features with amyloids. By using an Escherichia coli beta-galactosidase variant, we show that aggregation does not necessarily require unfolded polypeptide chains but rather depends on specific interactions between solvent-exposed hydrophobic stretches in partially structured species. In addition, purified inclusion bodies are efficient and highly selective nucleation seeds, promoting deposition of soluble homologous but not heterologous polypeptides in a dose-dependent manner. Finally, inclusion bodies bind amyloid-diagnostic dyes, which, jointly with Fourier transform infra red spectroscopy data, indicates a high level of organized intermolecular beta-sheet structure. The evidences of amyloid-like structure of bacterial inclusion bodies, irrespective of potential applications in bioprocess engineering, prompts the use of bacterial models to explore the molecular determinants of protein aggregation by means of simple biological systems.     

Protease inhibitors clitocypin and macrocypin are differentially expressed within basidiomycete fruiting bodies

Clitocypin and macrocypin are cysteine protease inhibitors of the mycocypin family which is unique to basidiomycetes. We have established that Clitocybe nebularis and Macrolepiota procera each contain genes for both macrocypin and clitocypin. Both are expressed in M. procera but only clitocypin in C. nebularis. Further analysis of mycocypin expression at the mRNA and protein levels in mature fruiting bodies of M. procera revealed that clitocypin is expressed evenly throughout the fruiting body, while the level of expression of macrocypins varies, and, at the protein level, is much higher in the veil fragments and the ring. The expression patterns of various mycocypins were determined in Coprinopsis cinerea, using promoters linked to a reporter gene. The expression profile of the clitocypin promoter was similar to that of the constitutive promoter gpdII from Agaricus bisporus, while that of the macrocypin 4 promoter was limited to the outer edges of the fruiting body throughout development. In addition, the activity of the macrocypin 3 promoter was different, indicating different regulation of expression for different macrocypin genes. The complex, tissue specific expression patterns for mycocypin genes suggest different biological roles for the products, either in regulation of endogenous proteases or in defense against pathogens or predators.     

Isolation of cell-free bacterial inclusion bodies

Background  

Bacterial inclusion bodies are submicron protein clusters usually found in recombinant bacteria that have been traditionally considered as undesirable products from protein production processes. However, being fully biocompatible, they have been recently characterized as nanoparticulate inert materials useful as scaffolds for tissue engineering, with potentially wider applicability in biomedicine and material sciences. Current protocols for inclusion body isolation from Escherichia coli usually offer between 95 to 99% of protein recovery, what in practical terms, might imply extensive bacterial cell contamination, not compatible with the use of inclusion bodies in biological interfaces.     

Antiviral activity of aqueous extracts and polysaccharide fractions from mycelium and fruit bodies of higher fungi

Sixty preparations of basidiomycetes (Ganoderma, Lentinus, Pleurotus, Laetiporus, Polyporus, Inonotus, Flammulina, Grifola, Trametes) were investigated with respect to their toxicity for Vero cells and antiviral activity. The antiviral activity was estimated with the use of the West Nile virus and type 2 Herpes simplex. It was shown that 11 preparations of Ganoderma, Lentinus and Pleurotus completely inhibited the infective activity in doses not lower than 1000 TCD50 (the West Nile virus) and 100 PPU (type 2 Herpes simplex). The antiviral activity of the preparations was likely due to the content of polysaccharides or their derivatives in the composition. It increased with increasing of the quantity of the total polysaccharide fraction or its concentration.     

Antibiotic properties of the strains of the basidiomycete Lentinus edodes (Berk.) sing]

Antibiotic properties of the extracts from the fermentation broth and mycelium of 15 strains of the edible and medicinal basidiomycete L. edodes were studied and it was shown that the extracts were active against grampositive and gramnegative bacteria, yeasts and mycelial fungi, including dermatophytes and phytopathogens. The strains differed by the set of the organisms susceptible to the action of the extracts. Strains of L. edodes combining marked antibiotic properties and high yields of water soluble polysaccharides were screened. The active compounds were detected by preparative TLC. Two of them were identified with UV- and mass spectrometry as lentinamycin B and erytadenine (lentinacin). Lentinamycin B was found to be the main component responsible for the antibiotic activity of the L. edodes strains.     

Protein quality in bacterial inclusion bodies

A common limitation of recombinant protein production in bacteria is the formation of insoluble protein aggregates known as inclusion bodies. The propensity of a given protein to aggregate is unpredictable, and the goal of a properly folded, soluble species has been pursued using four main approaches: modification of the protein sequence; increasing the availability of folding assistant proteins; increasing the performance of the translation machinery; and minimizing physicochemical conditions favoring conformational stress and aggregation. From a molecular point of view, inclusion bodies are considered to be formed by unspecific hydrophobic interactions between disorderly deposited polypeptides, and are observed as "molecular dust-balls" in productive cells. However, recent data suggest that these protein aggregates might be a reservoir of alternative conformational states, their formation being no less specific than the acquisition of the native-state structure.     

Construction and deconstruction of bacterial inclusion bodies

Bacterial inclusion bodies (IBs) are refractile aggregates of protease-resistant misfolded protein that often occur in recombinant bacteria upon gratuitous overexpression of cloned genes. In biotechnology, the formation of IBs represents a main obstacle for protein production since even favouring high protein yields, the in vitro recovery of functional protein from insoluble deposits depends on technically diverse and often complex re-folding procedures. On the other hand, IBs represent an exciting model to approach the in vivo analysis of protein folding and to explore aggregation dynamics. Recent findings on the molecular organisation of embodied polypeptides and on the kinetics of inclusion body formation have revealed an unexpected dynamism of these protein aggregates, from which polypeptides are steadily released in living cells to be further refolded or degraded. The close connection between in vivo protein folding, aggregation, solubilisation and proteolytic digestion offers an integrated view of the bacterial protein quality control system of which IBs might be an important component especially in recombinant bacteria.     

Antibiotic activity of the pyrenocines

Pyrenocine A, a phytotoxin produced by Pyrenochaeta terrestris (Hansen) Gorenz, Walker and Larson, possesses general antibiotic activity against plants, fungi, and bacteria. Effective doses for 50% inhibition (ED50s) are 4 micrograms/mL for onion seedling elongation; 14, 20, 20, and 25 micrograms/mL for the germination of asexual spores of Fusarium oxysporum f. sp. cepae, Fusarium solani f. sp. pisi, Mucor hiemalis, and Rhizopus stolonifer, respectively. Pyrenocine A also inhibits the linear mycelial growth of both P. terrestris and F. oxysporum with ED50s calculated as 77 and 54 micrograms/mL, respectively. Gram-positive bacteria are more susceptible to pyrenocine A than Gram-negative bacteria. ED50s are estimated as 30, 45, and 200 micrograms/mL for the inhibition of growth of Bacillus subtilis, Staphylococcus aureus, and Escherichia coli, respectively, with Pseudomonas aeruginosa resistant to those concentrations tested. Pyrenocine A acts primarily as a biostatic rather than a biocidal agent with all organisms tested showing some degree of recovery when released from pyrenocine A. Pyrenocines B and C show little antibiotic activity in the bioassays performed.     

Hymenial area of agaric fruit bodies consumed by Collembola

On the gill surfaces of agaric fruit bodies fed on by Collembola, the hymenium layer was consumed and many fecal pellets containing many basidiospores were observed. The hymenial area consumed by a collembolan (=Hypogatsrura denisana) varied from 1% to 92%, correlating with the density of collembolans on each fruit body among 11 agaric species. In Lactarius quietus, the hymenial area consumed by collembolans varied from 30% to 69% during the 5 days during which sampling took place. A weak correlation was found between the ratio of the hymenial area and the density of collembolans on the fruit bodies, and this fact suggested that other factors influenced the ratio of the hymenial area consumed by collembolans.