Short-term dynamics of culturable bacteria in a soil amended with biotransformed dry olive residue.

Dry olive residue (DOR) transformation by wood decomposing basidiomycetes (e.g. Coriolopsis floccosa) is a possible strategy for eliminating the liabilities related to the use of olive oil industry waste as an organic soil amendment. The effects of organic fertilization with DOR on the culturable soil microbiota are largely unknown. Therefore, the objectives of this study were to measure the short-term effects of DOR and C. floccosa-transformed DOR on the culturable bacterial soil community, while at the same time documenting the bacterial diversity of an agronomic soil in the southeastern Iberian Peninsula. The control soil was compared with the same soil treated with DOR and with C. floccosa-transformed DOR for 0, 30 and 60 days. Impact was measured from total viable cells and CFU counts, as well as the isolation and characterization of 900 strains by fatty acid methyl ester profiles and 16S rRNA partial sequencing. The bacterial diversity was distributed between Actinobacteria, Alphaproteobacteria, Gammaproteobacteria, Betaproteobacteria, Bacilli, Sphingobacteria and Cytophagia. Analysis of the treatments and controls demonstrated that soil amendment with untransformed DOR produced important changes in bacterial density and diversity. However, when C. floccosa-transformed DOR was applied, bacterial proliferation was observed but bacterial diversity was less affected, and the distribution of microorganisms was more similar to the unamended soil.

Degradation of biogenic amines by vineyard ecosystem fungi. Potential use in winemaking.

AIMS: To evaluate the ability of grapevine ecosystem fungi to degrade histamine, tyramine and putrescine in synthetic medium and in wines. METHODS AND RESULTS: Grapevine and vineyard soil fungi were isolated from four locations of Spain and were subsequently identified by PCR. A total of 44 fungi were evaluated for in vitro amine degradation in a microfermentation system. Amine degradation by fungi was assayed by reversed-phase (RP)-HPLC. All fungi were able to degrade at least two different primary amines. Species of Pencillium citrinum, Alternaria sp., Phoma sp., Ulocladium chartarum and Epicoccum nigrum were found to exhibit the highest capacity for amine degradation. In a second experiment, cell-free supernatants of P. citrinum CIAL-274,760 (CECT 20782) grown in yeast carbon base with histamine, tyramine or putrescine, were tested for their ability to degrade amines in three different wines (red, white and synthetic). The highest levels of biogenic amine degradation were obtained with histamine-induced enzymatic extract. CONCLUSION: The study highlighted the ability of grapevine ecosystem fungi to degrade biogenic amines and their potential application for biogenic amines removal in wine. SIGNIFICANCE AND IMPACT OF STUDY: The fungi extracts described in this study may be useful in winemaking to reduce the biogenic amines content of wines, thereby preventing the possible adverse effects on health in sensitive individuals and the trade and export of wine.

Enhancement of antibiotic and secondary metabolite detection from filamentous fungi by growth on nutritional arrays.

AIMS: We asked to what extent does the application of the OSMAC (one strain, many compounds) approach lead to enhanced detection of antibiotics and secondary metabolites in fungi? Protocols for bacterial microfermentations were adapted to grow fungi in nutritional arrays. METHODS AND RESULTS: Protocols for microfermentations of non-sporulating fungi were validated using known antifungal-producing fungi. Detection of antifungal activity was often medium dependent. The effects of medium arrays and numbers of strains on detection of antifungal signals were modelled by interpolation of rarefaction curves derived from matrices of positive and negative extracts. Increasing the number of fermentation media for any given strain increased the probability of detection of growth inhibition of Candida albicans. Increasing biodiversity increased detection of antifungal phenotypes, however, nutritional arrays could partly compensate for lost antibiotic phenotypes when biodiversity was limiting. CONCLUSIONS: Growth and extraction in microtiter plates can enable a discovery strategy emphasizing low-cost medium arrays that can better exploit the metabolic potential of strains. SIGNIFICANCE AND IMPACT OF THE STUDY: Increasing fermentation parameters raise the probability of detecting bioactive metabolites from strains. The protocols can be used to pre-select strains and their growth conditions for scale up that will most likely yield antibiotics and secondary metabolites.

High diversity and morphological convergence among melanised fungi from rock formations in the Central Mountain System of Spain.

Melanised fungi were isolated from rock surfaces in the Central Mountain System of Spain. Two hundred sixty six isolates were recovered from four geologically and topographically distinct sites. Microsatellite-primed PCR techniques were used to group isolates into genotypes assumed to represent species. One hundred and sixty three genotypes were characterised from the four sites. Only five genotypes were common to two or more sites. Morphological and molecular data were used to characterise and identify representative strains, but morphology rarely provided a definitive identification due to the scarce differentiation of the fungal structures or the apparent novelty of the isolates. Vegetative states of fungi prevailed in culture and in many cases could not be reliably distinguished without sequence data. Morphological characters that were widespread among the isolates included scarce micronematous conidial states, endoconidia, mycelia with dark olive-green or black hyphae, and mycelia with torulose, isodiametric or moniliform hyphae whose cells develop one or more transverse and/or oblique septa. In many of the strains, mature hyphae disarticulated, suggesting asexual reproduction by a thallic micronematous conidiogenesis or by simple fragmentation. Sequencing of the internal transcribed spacers (ITS1, ITS2) and 5.8S rDNA gene were employed to investigate the phylogenetic affinities of the isolates. According to ITS sequence alignments, the majority of the isolates could be grouped among four main orders of Pezizomycotina: Pleosporales, Dothideales, Capnodiales, and Chaetothyriales. Ubiquitous known soil and epiphytic fungi species were generally absent from the rock surfaces. In part, the mycota of the rock surfaces shared similar elements with melanised fungi from plant surfaces and fungi described from rock formations in Europe and Antarctica. The possibility that some of the fungi were lichen mycobionts or lichen parasites could not be ruled out.

Candelalides A-C: novel diterpenoid pyrones from fermentations of Sesquicillium candelabrum as blockers of the voltage-gated potassium channel Kv1.3.

[figure: see text] Blockers of the voltage-gated potassium channel Kv1.3 are potential immunosuppressants. Candelalides A-C are three novel diterpenoid pyrones that block this channel. The structure, stereochemistry, and activity against Kv1.3 are described.

Arundifungin, a novel antifungal compound produced by fungi: biological activity and taxonomy of the producing organisms.

Echinocandins, the lipopeptide class of glucan synthase inhibitors, are an alternative to ergosterol-synthesis inhibitors to treat candidiasis and aspergillosis. Their oral absorption, however, is low and they can only be used parenterally. During a natural product screening program for novel types of glucan synthesis inhibitors with improved bioavailability, a fungal extract was found that inhibited the growth of both a wild-type Saccharomyces cerevisiae strain and the null mutant of the FKS1 gene (fks1::HIS). The mutant strain was more sensitive to growth inhibition, suggesting that the fungal extract could contain an inhibitor of glucan synthesis. A novel acidic steroid, named arundifungin, was purified from a fungal extract obtained from a liquid culture of Arthrinium arundinis collected in Costa Rica. Arundifungin caused the same pattern of hallmark morphological alterations in Aspergillus fumigatus hyphae as echinocandins, further supporting the idea that arundifungin belongs to a new class of glucan synthesis inhibitors. Moreover, its antifungal spectrum was comparable to those of echinocandins and papulacandins, preferentially inhibiting the growth of Candida and Aspergillus strains, with very poor activity against Cryptococcus. Arundifungin was also detected in nine other fungal isolates which were ecologically and taxonomically unrelated, as assessed by sequencing of the ITS1 region. Further, it was also found in two more Arthrinium spp from tropical and temperate regions, in five psychrotolerant conspecific isolates collected on Macquarie Island (South Pacific) and belonging to the Leotiales, and in two endophytes collected in central Spain (a sterile fungus belonging to the Leotiales and an undetermined coelomycete).

Coprophilin: an anticoccidial agent produced by a dung inhabiting fungus.

Coprophilin, a decalin pentanedienoic acid methyl ester, was isolated from an unidentified fungus by bioassay guided separation. It inhibited (MIC = 1.5 microM) the growth of Eimeria tenella in an in vitro assay. The isolation, structure elucidation, absolute stereochemistry and biology are described.

Oreganic acid, a potent inhibitor of ras farnesyl-protein transferase.

A sulfated tricarboxylic acid fungal metabolite is an inhibitor of human farnesyl-protein transferase (FPTase). The compound, designated as oreganic acid, has a molecular weight of 494, an empirical formula of C22H38O10S and inhibits FPTase with an IC50 value of 14 nM. Oreganic acid is a selective inhibitor of FPTase because it does not inhibit human geranylgeranyl-protein transferase type I (GGPTase-I). It is not a time-dependent inhibitor, reversibly inhibits FPTase, is competitive with respect to farnesyl diphosphate and non-competitive with respect to the Ras acceptor peptide. The structure of oreganic acid resembles that of farnesyl diphosphate and most likely inhibits FPTase by mimicking farnesyl diphosphate at the active site of the enzyme.

Discovery of an angiotensin II binding inhibitor from a Cytospora sp. using semi-automated screening procedures.

Cytosporin A, B and C, three antagonists of [125I]-angiotensin II binding to rat adrenal glands were discovered in fermentations of an endophytic Cytospora sp. during routine screening using semi-automated procedures. The most potent of these displayed an IC50 of 1.5-3 microM and was specific for angiotensin II AT2.

L-735,334, a novel sesquiterpenoid potassium channel-agonist from Trichoderma virens.

A novel oleic acid ester of the carotane sesquiterpene 14-hydroxy CAF-603 was isolated from Trichoderma virens grown in a solid brown rice-based medium, a solid millet-based medium, or a mannitol-based liquid medium. Its structure was determined on the basis of ms and nmr analysis. It retains distinct biological activity on the high conductance calcium-activated potassium channel, unlike its analogues 14-hydroxy CAF-603, CAF-603 3-oleate, or CAF-603 3-linoleate.

Barceloneic acid A, a new farnesyl-protein transferase inhibitor from a Phoma species.

Three new diphenyl ethers, barceloneic acids A, B, and barceloneic lactone [1, 2, and 3, respectively] were isolated from a fermentation extract of a fungus of the genus Phoma. The structures of compounds 1-3 were determined by a combination of spectroscopic and single-crystal X-ray diffraction methods. The effect of these compounds on the inhibition of farnesyl-protein transferase (FPTase) was evaluated and results are presented. Barceloneic acid A [1] is a novel and modest inhibitor of FPTase with an IC50 value of 40 microM.

The discovery of australifungin, a novel inhibitor of sphinganine N-acyltransferase from Sporormiella australis. Producing organism, fermentation, isolation, and biological activity.

Potent antifungal activity was detected in fermentation extracts of Sporormiella australis and two related components were isolated from solid fermentations using silica gel and high speed countercurrent chromatography. The most active antifungal component, australifungin, contained a unique combination of alpha-diketone and beta-ketoaldehyde functional groups. Australifungin exhibited broad spectrum antifungal activity against human pathogenic fungi with MICs against Candida spp., Cryptococcus neoformans, and Aspergillus spp. between 0.015 and 1.0 microgram/ml. Mode of action studies revealed that australifungin interfered with fungal lipid metabolism by specifically inhibiting sphingolipid synthesis at the step converting sphinganine to ceramide.

Discovery, biosynthesis, and mechanism of action of the zaragozic acids: potent inhibitors of squalene synthase.

The zaragozic acids (ZAs), a family of fungal metabolites containing a novel 4,6,7-trihydroxy-2,8-dioxobicyclo[3.2.1]octane-3,4,5-tricarboxylic acid core, were discovered independently by two separate groups screening natural product sources to discover inhibitors of squalene synthase. This family of compounds all contain the same core but differ in their 1-alkyl and their 6-acyl side chains. Production of the ZAs is distributed over an extensive taxonomic range of Ascomycotina or their anamorphic states. The zaragozic acids are very potent inhibitors of squalene synthase that inhibit cholesterol synthesis and lower plasma cholesterol levels in primates. They also inhibit fungal ergosterol synthesis and are potent fungicidal compounds. The biosynthesis of the zaragozic acids appears to proceed through alkyl citrate intermediates and new members of the family have been produced through directed biosynthesis. These potent natural product based inhibitors of squalene synthase have potential to be developed either as cholesterol lowering agents and/or as antifungal agents.

Chaetomella acutiseta produces chaetomellic acids A and B which are reversible inhibitors of farnesyl-protein transferase.

Chaetomellic acids A and B, isolated from Chaetomella acutiseta, are specific inhibitors of farnesyl-protein transferase that do not inhibit geranylgeranyl transferase type 1 or squalene synthase. Chaetomellic acids A and B are reversible inhibitors, resemble farnesyl diphosphate and probably inhibit FPTase by substituting for farnesyl diphosphate. Chaetomellic acid production appears to be widespread within the genus Chaetomella.

A novel inositol mono-phosphatase inhibitor from Memnoniella echinata. Producing organism, fermentation, isolation, physicochemical and in vitro biological properties.

A novel inositol mono-phosphatase inhibitor, L-671,776 (1), was discovered from a culture of the hyphomycete, Memnoniella echinata (ATCC 20928). 1 has a molecular weight of 388 and a molecular formula of C23H32O5. The mode of inhibition is non-competitive, with a Ki of 450 microM. It shows no inhibition of myo-inositol 1,4-bisphosphate 1-phosphatase or myo-inositol 1,4,5-triphosphate 5-phosphatase, although it weakly inhibits myo-inositol 1,4,5-triphosphate 3-kinase (IC50 = 3 mM). It elevates inositol monophosphates in rat parotid slices (EC50 approximately 3 mM), but abolishes agonist effects. It also produces short-lived contraction of guinea pig trachea at 300 microM.

L-696,474, a novel cytochalasin as an inhibitor of HIV-1 protease. I. The producing organism and its fermentation.

A novel cytochalasin, L-696,474, (18-dehydroxy cytochalasin H) that inhibits HIV-1 protease was discovered in fermentations of a bark-inhabiting Ascomycete, Hypoxylon fragiforme. The product was first identified from extracts of an agar medium. Fermentation studies on a number of media indicated that the product can be made on several solid and liquid media. Optimum production was obtained from growth in a complex medium composed of glycerol, glucose, citrate, Ardamine, soybean meal, tomato paste, and inorganic salts. Other Hypoxylon spp., related species of Xylariales, and other fungi known to produce cytochalasins, were also surveyed for their ability to make L-696,474. Only one other Hypoxylon fragiforme isolate was found to make this novel cytochalasin; none of the other cultures surveyed made L-696,474 or any other compounds which inhibit HIV-1 protease.

L-696,474, a novel cytochalasin as an inhibitor of HIV-1 protease. III. Biological activity.

L-696,474, an inhibitor of the HIV-1 protease, was discovered in extracts of the fungal culture Hypoxylon fragiforme (MF5511; ATCC 20995). L-696,474 is a novel cytochalasin with a molecular weight of 477 and an empirical formula of C30H39NO4. L-696,474 inhibited HIV-1 protease activity with an IC50 of 3 microM and the mode of inhibition was competitive with respect to substrate (apparent Ki = 1 microM). Furthermore, L-696,474 was not a slow-binding inhibitor. The inhibition due to L-696,474 was also independent of the HIV-1 protease concentration. L-696,474 was inactive against pepsin, another aspartyl protease; stromelysin, a zinc-metalloproteinase; papain, a cysteine-specific protease or human leucocyte elastase, a serine-specific protease. Two other novel cytochalasins (L-697,318 and L-696,475) isolated from the same culture were inactive against the HIV-1 protease. Commercially available cytochalasins B, C, D, E, F, H and J were inactive while cytochalasin A was as active as L-696,474 against the HIV-1 protease.