Biocontrol Activity of Bacillus subtilis Isolated from Agaricus bisporus Mushroom Compost Against Pathogenic Fungi.

Bacillus subtilis strain B154, isolated from Agaricus bisporus mushroom compost infected by red bread mold, exhibited antagonistic activities against Neurospora sitophila. Antifungal activity against phytopathogenic fungi was also observed. The maximum antifungal activity was reached during the stationary phase. This antifungal activity was stable over a wide pH and temperature range and was not affected by proteases. Assay of antifungal activity in vitro indicated that a purified antifungal substance could strongly inhibit mycelia growth and spore germination of N. sitophila. In addition, treatment with strain B154 in A. bisporus mushroom compost infected with N. sitophila significantly increased the yield of bisporus mushrooms. Ultraviolet scan spectroscopy, tricine sodium dodecyl sulfate-polyacrylamide gel electrophoresis, matrix-associated laser desorption ionization time-of-flight mass spectrometry, and electrospray ionization tandem mass spectrometry analyses revealed a molecular weight consistent with 1498.7633 Da. The antifungal compound might belong to a new type of lipopeptide fengycin.

Fungal biodegradation of dibutyl phthalate and toxicity of its breakdown products on the basis of fungal and bacterial growth.

Phthalates are esters of phthalic acid that give flexibility to polyvinyl chloride. Diverse studies have reported that these compounds might be carcinogenic, mutagenic and/or teratogenic. Radial growth rate, biomass, hyphal thickness of Neurospora sitophyla, Trichoderma harzianum and Aspergillus niger, grown in two different concentrations of dibutyl phthalate (DBP) (500 and 1,000 mg/l) in agar and in submerged fermentation were studied. The inhibitory concentration (IC50) and the constant of biodegradation of dibutyl phthalate in Escherichia coli cultures were used to evaluate toxicity. The radial growth rate and thickness of the hypha were positively correlated with the concentration of phthalate. The pH of the cultures decreased as the fermentation proceeded. It is shown that these fungi are able to degrade DBP to non-toxic compounds and that these can be used as sole carbon and energy sources by this bacterium. It is demonstrated that the biodegradation of the DBP is directly correlated with the IC50. This is the first study that reports a method to determine the biodegradation of DBP on the basis of the IC50 and fungal growth, and the effect of this phthalate on the growth and thickness of hyphae of filamentous fungi in agar and in submerged fermentation.

Effect of ethanol on growth of Chrysonilia sitophila ('the red bread mould') and Hyphopichia burtonii ('the chalky mould') in sliced bread.

UNLABELLED: Contamination of food industrial environments and recontamination of finished products by Chrysonilia sitophila and Hyphopichia burtonii have long represented serious problems for the bakery industries. As one of the most common ways to slow down or avoid fungal spoilage on bakery products is the use of ethanol, in the present work the effect of this substance has been assessed on growth of two of the most frequently occurring associated moulds, C. sitophila and H. burtonii, by means of tests on both synthetic media and sliced bread. Test on synthetic media: H. burtonii was less markedly affected in lag-phase duration and radial growth rates by the addition of ethanol to DG18 and the reduction in incubation temperature than C. sitophila that failed to grow at the highest concentrations of ethanol tested (2·0 and 4·0% at 15°C; 4·0% at 25°C). Test on sliced bread: ethanol proved to be effective to prevent spoilage by C. sitophila even at the lowest concentration tested (0·8%, w/w), while higher concentrations (2·0%, w/w) were needed to prevent spoilage by H. burtonii. SIGNIFICANCE AND IMPACT OF THE STUDY: This study shows that ethanol could represent an effective barrier to prevent spoilage of bakery products by associated moulds such as Chrysonilia sitophila and Hyphopichia burtonii, whose growth on packed and sliced bread was inhibited at very low (0·8%) or medium (2·0%) ethanol concentrations, respectively. The results obtained represent a fundamental point of reference for the bakery industries, as they can apply them in the productive practice to avoid spoilage by C. sitophila and H. burtonii on their products.

Osmotin purified from the latex of Calotropis procera: biochemical characterization, biological activity and role in plant defense.

A protein, similar to osmotin- and thaumatin-like proteins, was purified from Calotropis procera (Ait.) R.Br latex. The isolation procedure required two cation exchange chromatography steps on 50mM Na-acetate buffer (pH 5.0) CM-Sepharose Fast Flow and 25 mM Na-phosphate buffer (pH 6.0) Resource-S, respectively. The protein purity was confirmed by an unique N-terminal sequence [ATFTIRNNCPYTIWAAAVPGGGRRLNSGGTWTINVAPGTA]. The osmotin (CpOsm) appeared as a single band (20,100 Da) in sodium dodecyl sulfate-polyacrylamide gel electrophoresis and as two spots in two-dimensional electrophoresis (pI 8.9 and 9.1). Both polypeptides were further identified by mass spectrometry as two osmotin isoforms with molecular masses of 22,340 and 22,536 Da. The CpOsm exerted antifungal activity against Fusarium solani (IC50=67.0 µg mL⁻¹), Neurospora sp. (IC50=57.5 µg mL⁻¹) and Colletotrichum gloeosporioides (IC50=32.1 µg mL⁻¹). However, this activity was lost when the protein was previously treated with a reducing agent (DTT, Dithiothreitol) suggesting the presence of disulfide bounds stabilizing the protein. The occurrence of osmotin in latex substantiates the defensive role of these fluids.

Setting the pace of the Neurospora circadian clock by multiple independent FRQ phosphorylation events.

Protein phosphorylation plays essential roles in eukaryotic circadian clocks. Like PERIOD in animals, the Neurospora core circadian protein FRQ is progressively phosphorylated and becomes extensively phosphorylated before its degradation. In this study, by using purified FRQ protein from Neurospora, we identified 43 in vivo FRQ phosphorylation sites by mass spectrometry analysis. In addition, we show that CK-1a and CKII are responsible for most FRQ phosphorylation events and identify an additional 33 phosphorylation sites by in vitro kinase assays. Whole-cell metabolic isotope labeling and quantitative MS analyses suggest that circadian oscillation of the FRQ phosphorylation profile is primarily due to progressive phosphorylation at the majority of these newly discovered phosphorylation sites. Furthermore, systematic mutations of the identified FRQ phosphorylation sites led to either long or short period phenotypes. These changes in circadian period are attributed to increases or decreases in FRQ stability, respectively. Together, this comprehensive study of FRQ phosphorylation reveals that regulation of FRQ stability by multiple independent phosphorylation events is a major factor that determines the period length of the clock. A model is proposed to explain how FRQ stability is regulated by multiple phosphorylation events.

A developmental cycle masks output from the circadian oscillator under conditions of choline deficiency in Neurospora.

In Neurospora, metabolic oscillators coexist with the circadian transcriptional/translational feedback loop governed by the FRQ (Frequency) and WC (White Collar) proteins. One of these, a choline deficiency oscillator (CDO) observed in chol-1 mutants grown under choline starvation, drives an uncompensated long-period developmental cycle ( approximately 60-120 h). To assess possible contributions of this metabolic oscillator to the circadian system, molecular and physiological rhythms were followed in liquid culture under choline starvation, but these only confirmed that an oscillator with a normal circadian period length can run under choline starvation. This finding suggested that long-period developmental cycles elicited by nutritional stress could be masking output from the circadian system, although a caveat was that the CDO sometimes requires several days to become consolidated. To circumvent this and observe both oscillators simultaneously, we used an assay using a codon-optimized luciferase to follow the circadian oscillator. Under conditions where the long-period, uncompensated, CDO-driven developmental rhythm was expressed for weeks in growth tubes, the luciferase rhythm in the same cultures continued in a typical compensated manner with a circadian period length dependent on the allelic state of frq. Periodograms revealed no influence of the CDO on the circadian oscillator. Instead, the CDO appears as a cryptic metabolic oscillator that can, under appropriate conditions, assume control of growth and development, thereby masking output from the circadian system. frq-driven luciferase as a reporter of the circadian oscillator may in this way provide a means for assessing prospective role(s) of metabolic and/or ancillary oscillators within cellular circadian systems.

Evaluation of phenylaminopyrimidines as antifungal protein kinase inhibitors.

The effects of diverse phenylaminopyrimidines (PAP), namely PAP-pyridines (type A), PAP-pyrazoles (type B) and PAP-thiazoles (type C), on Neurospora crassa Shear & Dodge has been investigated. The results revealed that type A strongly inhibit the in vitro growth of N crassa, whereas types B and C are much less active. A significant correlation was observed between the Neurospora growth inhibition and the intrinsic activity of type A compounds on the cyclin-dependent protein kinase p34(CDC2) of starfish, suggesting that the target of phenylaminopyrimidines in fungi is a cyclin-dependent protein kinase (CDK). The phenylaminopyrimidine-binding CDKs Phoss (major band) and CDC2 (minor band) involved in phosphorus uptake, glycogen synthesis and the cell cycle were identified from N crassa by affinity chromatography on phenylaminopyrimidine-sepharose. Comparative experiments with different protein kinases revealed the importance of the side chain of phenylaminopyrimidines for their target selectivity. A type B compound was found to selectively inhibit the MAP-kinase OS-2 involved in the osmoregulatory pathway of Neurospora.

FWD1-mediated degradation of FREQUENCY in Neurospora establishes a conserved mechanism for circadian clock regulation.

Phosphorylation of the Neurospora circadian clock protein FREQUENCY (FRQ) regulates its degradation and the proper function of the clock. The mechanism by which FRQ undergoes degradation has not been established. Here we show that FRQ is likely ubiquitylated in vivo, and its proper degradation requires FWD1, an F-box/WD-40 repeat-containing protein. In the fwd1 disruption strains, FRQ degradation is severely impaired, resulting in the accumulation of hyperphosphorylated FRQ. Furthermore, the circadian rhythms of gene expression and the circadian conidiation rhythms are abolished in these fwd1 mutants. Finally, FRQ and FWD1 interact physically in vivo, suggesting that FWD1 is the substrate-recruiting subunit of an SCF-type ubiquitin ligase responsible for FRQ ubiquitylation and degradation. Together with the recent finding that Slimb (the Drosophila homolog of FWD1) is involved in the degradation of the Period protein in flies, our results indicate that FWD1 regulates the degradation of FRQ in Neurospora and is an evolutionarily conserved component of the eukaryotic circadian clock.

Pochonins A-F, new antiviral and antiparasitic resorcylic acid lactones from Pochonia chlamydosporia var. catenulata.

Monorden (1) and the novel resorcylic acid lactones pochonins A (2), B (4), C (6), D (7), and E (8) as well as tetrahydromonorden (5) and pseurotin A (22) were isolated from cultures of the clavicipitaceous hyphomycete Pochonia chlamydosporia var. catenulata strain P 0297. Fermentation of P 0297 in bromide-containing culture media led to a shift in secondary metabolite production and yielded monocillins III (3) and II (9) as major metabolites besides monorden (1) as well as the novel compounds pochonin F (10) and a monocillin II glycoside (11) as minor metabolites. Most of these compounds showed moderate activities in a cellular replication assay against Herpes Simplex Virus 1 (HSV1) and against the parasitic protozoan Eimeria tenella. In contrast to the structurally related zearalenone derivatives none of the metabolites of strain P 0297 were found to be active in a fluorescence polarization assay for determination of modulatory activities on the human estrogenic receptor ERbeta. Beta-zearalenol (17), but not zearalenone (15) and alpha-zearalenol (16), showed antiherpetic effects. We report the production, isolation, and structure elucidation of compounds 1-11 and their biological characterization.

Ee-CBP, a hevein-type antimicrobial peptide from bark of the spindle tree (Euonymus europaeus L.).

Ee-CBP, a hevein-type antimicrobial peptide was isolated from the bark of the spindle tree (Euonymus europaeus L.). This 4992.5 Da protein exhibited a very strong antifungal activity against five different fytopathogenic fungi that were tested. Concentrations required to inhibit the growth of Botrytis cinerea in agar diffusion assays and microtiterplate assays were 5 micrograms/ml and 1 microgram/ml, respectively. Comparative tests further indicated that Ee-CBP is a more potent antifungal protein than Ac-AMP2, an antimicrobial peptide from seeds of Amaranthus caudatus L. when tested with the same fungus.

Possible benefits of kalilo plasmids to their Neurospora hosts.

Neurospora mitochondrial plasmids are ubiquitous in natural populations, yet many of them are lethal to their host strains or seem to impose a molecular genetic load. Five pairs of strains of Neurospora tetrasperma and N. crassa with and without kalilo-like plasmids were tested under a variety of situations. The purpose was to find possible beneficial effects of plasmids that might offset their disadvantages. We found that, in all cases tested, plasmids conferred an advantage to growth at temperatures close to the top of the range for this fungus. Also, the plasmids improved fertility, as measured by perithecial production. Negative results were obtained for heavy metal resistance and ascospore germination. The results generate the hypothesis that plasmids may have adaptive significance to their hosts.

Enhancement of Neurospora VS ribozyme cleavage by tuberactinomycin antibiotics.

Several examples of inhibition of the function of a ribozyme or RNA-protein complex have shown that certain antibiotics can interact specifically with RNA. There are, however, few examples of antibiotics that have a positive, rather than a negative, effect on the function of an RNA. We have found that micromolar concentrations of viomycin, a basic, cyclic peptide antibiotic of the tuberactinomycin group, enhance the cleavage of a ribozyme derived from Neurospora VS RNA. Viomycin decreases by an order of magnitude the concentration of magnesium required for cleavage. It also stimulates an otherwise insignificant transcleavage reaction by enhancing interactions between RNA molecules. The ability of viomycin to enhance some RNA-mediated reactions but inhibit others, including translation and Group I intron splicing, demonstrates the potential for natural selection by small molecules during evolution in the 'RNA world' and may have broader implications with respect to ribozyme expression and activity in contemporary cells.

Selection and analysis of superoxide dismutase mutants of Neurospora.

A survey of 12 genetically distinct, heat-sensitive mutants of Neurospora revealed three (un-1, un-3, and un-17) that are specifically deficient in the superoxide dismutase (SOD) isozymes SOD-2 (mitochondrial), SOD-3 (mitochondrial), SOD-4 (exocellular), respectively. Genetic analysis of the three mutants indicates that the enzyme deficiencies are probably the cause of the heat-sensitive phenotype. The phenotypes of the mutants are (1) no growth at the normally optimal temperature 35 degrees C and comparatively inferior growth at 15-30 degrees C; (2) inferior resistance to the oxidants paraquat or oxygen; (3) female sterility; and (4) inferior conidial viability and longevity. Paraquat or O2 inhibition is alleviated respectively by desferrioxamine-Mn (a SOD mimic) and tocopherol. Diverse antioxidants, including tocopherol, are therapeutic for the heat-sensitive and female-sterile phenotypes, and for inferior growth of wild type at stressfully high temperatures. The results support previous theories that heat stress is a form of oxyradical/oxidant stress and that antioxidant enzymes such as SOD are essential for normal growth, development, and longevity. Since the three genes may encode the three enzymes and are not closely linked to either one another or the family of antioxidant-enzyme regulatory genes Age-1, the latter apparently trans-regulate their expression.

Respiration and gene expression in germinating ascospores of Neurospora tetrasperma.

Cellular proteins were not synthesized by germinating ascospores of Neurospora tetrasperma until 90 min after spore activation. Nevertheless, immediately after activation these ascospores developed a cyanide-sensitive respiration which increased throughout this 90-min period. At 90 min the respiratory rates accelerated rapidly, protein synthesis was initiated, and transcripts for a subunit of the mitochondrial ATPase, employed here as a representative mRNA, began to accumulate.

Spontaneous mutation at the mtr locus of Neurospora: the spectrum of mutant types.

We have isolated 135 strains of Neurospora which have mutations at the mtr locus resulting from independent spontaneous events. mtr is the structural gene for the neutral amino acid permease. The mutants have been characterized by their reversion behavior (both spontaneous and induced) and by hybridization studies of restriction digests of their DNA. About half of the mutants (54%) appear to result from single base-pair substitutions. Thirty-four percent have deletions, including some which extend into neighboring genes. Most of the remaining mutants have insertions. Several of the insertions are tandem duplications of 400-1000 bp and these mutants are unstable, reverting to mtr+ with a high frequency. When tandem-duplication mutants go through a cross, they are modified: the mutant progeny are fully stable. This modification is probably due to RIP (repeat-induced point mutation). This process has an important bearing on the comparison of germinal to somatic mutation.

Blue light photoreception in Neurospora circadian rhythm: evidence for involvement of the flavin triplet state.

The mechanism of the photoreceptor acting on the circadian conidiation rhythm of Neurospora crassa was studied, with the following results: (1) the efficiency of 8-haloflavins as sensitizers increased with their triplet yields. (2) Phase shifts were not abolished by removal of oxygen prior to illumination. (3) Oxygen inhibited phase shifts when introduced into the cultures after light treatment. It is proposed that the blue light photoreceptor for the circadian clock of Neurospora crassa acts (1) from its triplet state, but (2) not via singlet oxygen; (3) signal transduction involves (an) oxygen-sensitive intermediate(s).

Pharmacogenetics of cyclic guanylate, antioxidants, and antioxidant enzymes in Neurospora.

Previous studies indicate that antioxidant enzyme regulatory gene mutants (Age-) of Neurospora are defective in cellular longevity and development, numerous antioxienzymes, and cAMP, and that dietary cGMP confers normal longevity. Here it is shown that cGMP also phenotypically cures the developmental and enzymatic defects. At least 5 of the 12 known antioxidant enzyme deficiencies are normalized by cGMP.cGMP-mediated enzyme induction apparently requires gene transcription: actinomycin D is inhibitory. The mutants' inferior growth and development are stimulated by feeding cGMP, the cyclic nucleotide phosphodiesterase inhibitor theophylline, antioxidant enzymes, and antioxidants or by omission of both ferrous and cupric ions normally added to the culture medium. The analysis indicates that the inferior growth and development probably is a consequence of the formation of toxic hydroxyl radicals. The mutants are rather specific, conditional cGMP auxotrophs and respond to physiological concentrations of that nucleotide: cAMP tends to be ineffective. The observations indicate that: i) genetic regulation of antioxidant enzymes is a determinant of not only cellular longevity, but also normal growth and development; and ii) the enzymes do indeed provide defense against free radical/oxidant toxicity. The hypothesis that cGMP is a second messenger in regulation of the expression of antioxidant enzyme genes is consistent with experimental data, but remains to be verified.

Cycloheximide inhibits light-induced phase shifting of the circadian clock in Neurospora.

Cycloheximide inhibits light-induced phase shifting of the circadian clock and protein synthesis in Neurospora. Light resetting is not inhibited in mutants whose protein synthesis is resistant to cycloheximide. When light and cycloheximide are presented together at various circadian phases, the final phase shift is always determined by cycloheximide. This dual-treatment phase response curve approach may be useful for other studies using pharmacological treatments to analyze clock pathways. Taken together, the results suggest that synthesis of a protein (or proteins) is involved in the phototransduction pathway of the circadian clock in Neurospora.

Sensitivity to cyclosporin A is mediated by cyclophilin in Neurospora crassa and Saccharomyces cerevisiae.

Cyclosporin A, a cyclic fungal undecapeptide produced by Tolypocladium inflatum, is a potent immunosuppressive drug originally isolated as an antifungal antibiotic. Cyclosporin A (CsA) is widely used in humans to prevent rejection of transplanted organs such as kidney, heart, bone marrow and liver. The biochemical basis of CsA action is not known: its primary cellular target has been suggested to be calmodulin, the prolactin receptor or cyclophilin, a CsA-binding protein originally isolated from the cytosol of bovine thymocytes. Cyclophilin has been shown to be a highly conserved protein present in all eukaryotic cells tested and to be identical to peptidyl-prolyl cis-trans isomerase, a novel type of enzyme that accelerates the slow refolding phase of certain proteins in vitro. We demonstrate that in the lower eukaryotes N. crassa and S. cerevisiae, cyclo philin mediates the cytotoxic CsA effect. In CsA-resistant mutants of both organisms, the cyclophilin protein is either lost completely or, if present, has lost its ability to bind CsA.