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.

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.

A paracrystalline inclusion in Neurospora crassa. Induction by ethidium and acridine, isolation, and characterization.

A paracrystal indistinguishable from the one which occurs in the mitochondrial mutant abnormal-1 can be induced in wild-type Neurospora crassa after growth in either ethidium or euflavine. This paracrystal has been isolated and partially characterized. It appears to be composed of a single polypeptide (mol wt 68,000) which can be reversibly crystallized and dissociated by changes in the pH and ionic strength. When aggregated, the polypeptide forms oligomers which are arranged end-to-end into fibers. During the characterization of the polypeptide, it was found that the polypeptide's electrophoretic and immunological properties could be used as assays. Using these methods it was found that the polypeptide normally accumulates in a soluble form in the cytoplasm of wild-type Neurospora at the end of the log-phase of growth.

Mediated mutagenesis of dimethylnitrosamine in Neurospora crassa by various metabolic activation systems.

Four metabolic activation systems (growth mediated mycelium extract mediated, host mediated, and organ homogenate mediated) were used to study the mutagenic activity of dimethylnitrosamine (DMN) in both forward and reverse mutation systems in the ad-3 (adenine-3) region of Neurospora crassa. DMN was not mutagenic in Neurospora if conidia alone were treated. It was highly mutagenic, however, if conidia were treated with this compound under any of the four activation systems. Quantitative differences in DMN-induced mutation frequencies were observed between in vivo (growth and host mediated) and in vitro (mycelium extract and organ homogenate mediated) activations. The efficiency of the conversion of DMN to a mutagenic metabolite by the organs of rats and mice appeared to be in a reversed order between the host-mediated (liver greater than kidney greater than lung) and the in vitro organ homogenate-mediated (lung greater than kidney greater than liver) assays. Inductions of reverse mutations in strain N23 indicated that DMN induces base-pair substitution in N. crassa.

Reduction of ribosome activity and synthesis of stable RNA in Neurospora crassa.

The addition of cycloheximide (0.02 micrograms/ml) to exponentially growing cultures of Neurospora crassa causes a reduction in growth rate and a decrease in the rate of protein accumulation, due to a partial inhibition of protein synthesis, while RNA accumulation is unaffected for about 1 h. Thus, an increased RNA:protein ratio is established in the presence of the inhibitor. RNA that accumulates during treatment with cycloheximide has the same characteristics as that of the control cultures and this, together with the enhancement of the relative rate of synthesis of ribosomal proteins induced by cycloheximide, seems to indicate that more mature ribosomes are present in cycloheximide-treated cultures. The endocellular level of several amino acids begins to increase significantly only 60 min after cycloheximide addition. A possible explanation of the stimulation of ribosome production induced by cycloheximide is given in terms of the existence of a feed-back mechanism controlling ribosome synthesis.

Potassium transport in Neurospora. Evidence for a multisite carrier at high pH.

At low extracellular pH (4-6), net uptake of potassium by Neurospora is a simple exponential process which obeys Michaelis kinetics as a function of [K](o). At high pH, however, potassium uptake becomes considerably more complex, and can be resolved into two distinct exponential components. The fast component (time constant = 1.2 min) is matched quantitatively by a rapid loss of sodium; it is attributed to ion exchange within the cell wall, since it is comparatively insensitive to low temperature and metabolic inhibitors. By contrast, the slower component (time constant = 10.9 min) is inhibited markedly at 0 degrees C and by CN and deoxycorticosterone, and is thought to represent carrier-mediated transport of potassium across the cell membrane. This transport process exhibits sigmoid kinetics as a function of [K](o); the data can be fitted satisfactorily by two different two-site models (one involving a carrier site and a modifier site, the other an allosteric model). Either of these models could also accommodate the simple Michaelis kinetics at low pH.

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.

Isolation and properties of selenomethionine-resistant mutants of Neurospora crassa.

Mutants resistant to selenomethionine were isolated, and their properties studied. Mapping studies indicate that the mutation sites are located near the eth-1(r) locus in linkage group I, about ten map units away from the mating type locus. The sites of new mutation are either allelic to or very close to eth-1(r). They are resistant not only to selenomethionine but also to ethionine, while the ethionine-resistant mutant, eth-1(r), is sensitive to selenomethionine. The selenomethionine-resistant mutants are also temperature-sensitive mutants. However, they can grow at higher temperatures in medium containing 1 M glycerol.-It is very unlikely that the resistance is due to a change in the permeability of the membrane. Aryl sulfatase of se-met(r) mutants is not repressed by a high concentration of methionine (5 mM), although inorganic sulfate (2 mM) still can cause total repression. The gamma-cystathionase levels of the mutants are normal, but the S-adenosylmethionine synthetase levels are only one-tenth of that observed in the wild-type strain. The heat-stability of this enzyme in the mutant is also different from that of the wild-type enzyme suggesting that the mutation might affect the structural gene of S-adenosylmethionine synthetase.

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.

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.

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.

Stimulation by mammalian insulin of glycogen metabolism in a wall-less strain of Neurospora crassa.

Addition of bovine insulin to cells of the wall-less variant FGSC4761 of Neurospora crassa ("slime") produced several significant effects on glycogen metabolism. 1) Intracellular levels of the glycogen precursor UDP-glucose decreased 17-18% (P less than 0.01) within 30 min of insulin addition. 2) Cells grown with insulin possessed 40% more glycogen than did control cells. 3) The incorporation of 14C-labeled glucose into glycogen increased 41% after 30-min treatment with 100 nM bovine insulin (P less than 0.01). 4) Insulin treatment of the cells caused activation of the enzyme glycogen synthase from a glucose-6-phosphate-dependent form to an independent form. Half-maximum activation occurred with 2 nM insulin. These are similar to insulin-induced effects in some mammalian cells. In contrast, no insulin-induced effect on glucose transport could be demonstrated in these cells.

Effects of mammalian insulin on metabolism, growth, and morphology of a wall-less strain of Neurospora crassa.

Addition of mammalian insulin to a nutritionally rich, chemically defined culture medium affects Neurospora crassa "slime" (wall-less) cells, as indicated by enhancement of growth, extension of viability at the stationary phase of growth, alteration of morphology, and stimulation of glucose oxidation. Bovine, porcine, and recombinant human insulin had similar effects on growth and morphology, while proinsulin, reduced insulin, and several other proteins were inactive. Insulin added in the presence of excess antiinsulin antibody was without activity. Intact cells possessed high affinity insulin-binding sites, represented by a curvilinear Scatchard plot, suggesting that effects are mediated through insulin receptors on the cell surface. These findings establish a role for insulin or insulin-like molecules in regulating growth and metabolism in this fungal cell and demonstrate a close similarity to insulin effects on certain mammalian 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.

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.

Ageing of Neurospora crassa. VIII. Lethality and mutagenicity of ferrous ions, ascorbic acid, and malondialdehyde.

Ferrous ions were highly lethal and mutagenic to germinated conidia of Neurospora crassa. At comparable survival, treatment with 0.2 mM ferrous ions was 14- and 50-fold more mutagenic than ultra-violet irradiation or X-rays, respectively, in the reversion of an inositol auxotroph. Ascorbic acid alone (2 mM) was not reproducibly lethal and inhibited both the lethality and mutagenicity of ferrous ions. Bovine superoxide dismutase (SOD) completely inhibited the residual lethality of ferrous ascorbate. Protection by ascorbic acid and SOD indicates that superoxide radicals, generated by oxidation of Fe(II), are directly or indirectly mutagenic and lethal. Malondialdehyde (MDA) was lethal and appeared to be mutagenic; however, its action is probably different from that of superoxide. Therefore, superoxide-mediated production of endogenous MDA by way of peroxidation of polyunsaturated fatty acids is probably not an alternate mutagenic pathway, at least in the reversion of the allele of the inl locus examined. These results and the demonstration of superoxide-mediated decrease in the synthetic fidelity of DNA polymerase in vitro (Rana and Munkres, in preparation) warrant additional exploration of the hypothesis that endogenous cellular free radicals, generated by pre- and post-senescent metabolism, may enter into lethal and mutagenic reactions.

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.

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).