Effect of vitamin E on autolysis and sporulation of Aspergillus nidulans

The morphologic and physiologic effects of vitamin E, a powerful antioxidant, on the autolysis and sporulation of Aspergillus nidulans FGSC26 were studied. In carbon-depleted submerged cultures, reactive oxygen species (ROS) accumulated in the cells and, concomitantly, progressing autolysis was observed, which was characterized by decreasing dry cell masses and pellet diameters as well as by increasing extracellular chitinase activities. Vitamin E supplemented at a concentration of 1 g/L hindered effectively the intracellular accumulation of ROS, the autolytic loss of biomass, the disintegration of pellets, and the release of chitinase activities. In surface cultures, vitamin E inhibited autolysis of both A. nidulans FGSC26 and a loss-of-function FlbA autolytic phenotype mutant. In addition, supplementation of the culture medium with this antioxidant also had a negative effect on the sporulation of strain FGSC26 and the FadA ^G203R hypersporulating phenotype mutant. These results suggest that accumulation of ROS was involved in the initiation of both sporulation and autolysis in this filamentous fungus, but that FadA/FlbA signaling was not involved in this vitamin E-dependent regulation. Vitamin E can be recommended as a supplement in fermentations in which the disintegration of pellets and gross autolysis should be avoided.     

Size-Dependent Impact of Magnetic Nanoparticles on Growth and Sporulation of Aspergillus niger

Magnetic nanoparticles (MNPs) are becoming important DNA nanocarriers for genetic engineering of industrial fungi. However, the biological effect of MNPs on industrial fungi remains unknown. In this study, we prepared three kinds of magnetic nanoparticles with different sizes (i.e., 10 nm, 20 nm, and 200 nm) to investigate their impact on the growth and sporulation of the important industrial fungus Aspergillus niger. Transmission electron microscopy, X-ray diffraction analysis and Zeta potential analysis revealed that the three kinds of MNPs, including MNP10, MNP20 and MNP200, had uniform size distribution, regular Fe₃O₄ X-ray diffraction (XRD) patterns and similar Zeta potentials. Interestingly, although the three kinds of MNPs did not obviously inhibit growth of the fungus, the MNP20 at 500 mg/L strongly attenuated sporulation, leading to a remarkable decrease in spore numbers on culturing plates. Further investigation showed that MNP20 at the high concentration led to drastic chitin accumulation in the cell wall, indicating cell wall disruption of the MNP20-treated fungal cells. Moreover, the MNPs did not cause unusual iron dissolution and reactive oxygen species (ROS) accumulation, and the addition of ferrous ion, ferric ion or the reactive oxygen species scavenger N-acetyl-L-cysteine (NAC) had no impact on the sporulation of the fungus, suggesting that both iron dissolution and ROS accumulation did not contribute to attenuated sporulation by MNP20. This study revealed the size-dependent effect of MNPs on fungal sporulation, which was associated with MNP-induced cell wall disruption.     

Influence of fadA G203R and ΔflbA mutations on morphology and physiology of submerged Aspergillus nidulans cultures

Morphologic and physiologic changes taking place in carbon-limited submerged cultures of Aspergillus nidulans ΔflbA and fadA ^G203R strains were studied. Loss-of-function mutation of the flbA gene resulted in an altered germination with unusually thick germination tubes, “fluffy” pellet morphology, as well as a reduced fragmentation rate of hyphae during autolysis. In the fadA ^G203R mutant strain, conidiophores formed in the stationary phase of growth, and the size of pellets shrank considerably. There were no significant differences in the generation of reactive oxygen species (ROS) and in the specific catalase and superoxide dismutase activities by the tested mutants and the appropriate parental strains. Therefore, the participation of ROS or antioxidative enzymes in FadA/FlbA signaling pathways seems to be unlikely in submerged cultures. On the other hand, earlier increases in the extracellular protease and ammonia production were recorded with the ΔflbA strain, whereas the protease and ammonia production of the fadA ^G203R mutant lagged behind those of the wild-type strains. Similar changes in the time courses of the induction of γ-glutamyltranspeptidase and the degradation of glutathione were observed. These results suggest that FadA/FlbA signaling may be involved in the mobilization of protein and peptide reserves as energy sources during carbon starvation.     

Adaptative and developmental responses to stress in Aspergillus nidulan

Development in the ascomycete A. nidulans is principally determined by environmental signals. Adaptability to oxidative stimuli can derive in changes of growth patterns and /or the activation of sexual or asexual reproductive cycles but this model fungus might also respond to high osmotic or salt concentrations, the redox state, the availability and quantity of carbon or nitrogen sources and the degree or quality of illumination. Since each cell within the colony follows a single morphogenetic program at a time, all these environmental cues might be sensed and integrated into a limited number of intracellular signals which, finally, would activate the required morphogenetic program and repress the others. This signaling mainly occurs through stress response pathways. The present review aims to summarize the available knowledge on how these pathways transduce environmental stimuli to mediate morphological changes in Aspergillus nidulans.     

Cytochemical labeling for fungal and host components in plant tissues inoculated with fungal wilt pathogens.

Antibodies to detect pectin in present investigations attached to distinct fibrils in vessel lumina. In carnation infected with an isolate of Fusarium oxysporum f.sp., labeling of pathogen cells also occurred; in a resistant cultivar (cv.), it was coincident with proximate pectin fibrils and linked to altered fungal walls, which was the opposite in the susceptible cv., indicating that hindrance of pathogen ability to degrade pectin may be related to resistance. Labeling of the fungus in culture was nil, except in media containing pectin, showing that pectin is not native to the pathogen. Labeling of fungal walls for cellulose in elm (inoculated with Ophiostoma novo-ulmi) and carnation also occurred, linked to adsorbed host wall components. The chitin probe often attached to dispersed matter, in vessel lumina, traceable to irregularly labeled fungal cells and host wall degradation products. With an anti-horseradish peroxidase probe, host and fungal walls were equally labeled, and with a glucosidase, differences of labeling between these walls were observed, depending on pH of the test solution. Fungal extracellular matter and filamentous structures, present in fungal walls, predominantly in another elm isolate (Phaeotheca dimorphospora), did not label with any of the probes used. However, in cultures of this fungus, extracellular material labeled, even at a distance from the colony margin, with an anti-fimbriae probe.     

Enzyme function is regulated by its localization

To better understand how enzyme localization affects enzyme activity we studied the cellular localization of the glycosyltransferase MurG, an enzyme necessary for cell wall synthesis at the spore during sporulation in the bacterium Bacillus subtilis. During sporulation MurG was gradually enriched to the membrane at the forespore and point mutations in a MurG helical domain disrupting its localization to the membrane caused severe sporulation defects, but did not affect localization nor caused detectable defects during exponential growth. We found that this localization is dependent on the phospholipid cardiolipin, as in strains where the cardiolipin-synthesizing genes were deleted, MurG levels were diminished at the forespore. Furthermore, in this cardiolipin-less strain, MurG localization during sporulation was rescued by external addition of purified cardiolipin. These results support localization as a critical factor in the regulation of proper enzyme function and catalysis.     

Effect of Carbon and Nitrogen on the Cannibalistic Behavior of Bacillus subtilis

Bacillus subtilis is known to exhibit cannibalism under nutrient limitation to delay sporulation. Cells of B. subtilis in phosphate buffer solution (PBS) demonstrate an oscillatory behavior in cell number due to cannibalism. Since PBS did not contain any nutrients, the effect of carbon and nitrogen sources on the cannibalistic behavior is unclear. In this study, the effect of external carbon and nitrogen on the cannibalistic behavior of B. subtilis is presented. The studies demonstrated that when glucose as a carbon source was introduced into PBS in the absence of any other nutrients, the cannibalistic tendency was delayed. This delay increased with the increase in the amount of glucose present in the PBS. Thus, the cannibalism was observed to be very sensitive to the amount of carbon present in the medium. However, when the medium contained only ammonium sulfate as a nitrogen source and was devoid of any carbon, the effect on cannibalism was minimal. The study, therefore, demonstrated that cannibalism was more sensitive to carbon than nitrogen indicating that the phenomenon of cannibalism may be more dependent on the status of energy in the medium than on nitrogen assimilation.     

Selective delignification of wood by white-rot fungi

The white-rot fungi,Cerrena unicolor, Ganoderma applanatum, G. tsugae,Ischnoderma resinosum, andPerenniporia medullapanis, caused two distinct types of decay. Large areas of decayed wood were selectively delignified and a typical white-rot causing a simultaneous removal of all cell wall components was present. Preferential lignin degradation was intermittently dispersed throughout the decayed wood. Scanning and transmission electron microscopy were used to identify the micromorphological and ultrastructural changes that occurred in the cells during degradation. In delignified areas the compound middle lamella was extensively degraded without substantial alteration of the secondary wall. The S₂ layer of the secondary wall was least affected. The loss of middle lamellae resulted in extensive defibration of the cells. Sulfuric acid lignin determinations indicated that 95–98% of the lignin was removed. Wood sugar analyses using high pressure liquid chromatography demonstrated that hemicelluloses were removed in preference to cellulose when lignin was degraded. The results suggest that a highly diffusible ligninolytic system was responsible for the selective degradation of the wood. In simultaneously white-rotted wood, all cell wall layers were progressively removed from the cell lumen toward the middle lamella, causing erosion troughs or holes to form. Large voids filled with fungal mycelia resulted from a coalition of degraded areas. Chemical analyses of white-rotted wood indicated lignin, cellulose, and hemicellulose were removed in approximately the same amounts. Degradation was confined to areas around fungal hyphae.     

新型杀菌剂氟吗啉对黄瓜疫霉病菌细胞壁主要组分合成及分布的影响

应用同位素示踪和组织特异性荧光染色等生物化学方法研究了新型杀菌剂氟吗啉对黄瓜疫霉病菌细胞壁主要组分合成及分布的影响. 结果表明, 氟吗啉对细胞壁的主要组成物质纤维素和非纤维素葡聚糖的合成没有抑制作用, 但破坏细胞壁物质在菌丝正确位置的形成和分布, 扰乱菌丝的极性生长.     

Microbial degradation of polymer solids. I. Cellophane

Specimens of regenerated cellulose films (cellophane) have been inoculated with cellulolytic fungi and the resulting degradation monitored through change in physical properties. Investigation of structure–property relationship in cellophane have revealed that surface microfibrils are highly oriented parallel to the manufacturing machine direction, while those in the film interior exist in a somewhat disordered array. Cellulase enzymes have been observed to diffuse appreciable distances from their point of application and to attack disordered regions more readily than the crystallites. Damage to surface microfibrils appears to involve their being cleaved into short segments. The fungus studied in this work forms a network of hyphae that covers much of the specimen surface soon after growth starts; hyphae penetrate into the film and proceed to remove its core more rapidly than surface layers. Tensile strength declines progressively as this degradation occurs, but vapor barrier properties remain essentially unchanged over the period of these studies. The suggests that intrusion of hyphae, in combination with enzymatic digestion of cellulose, rapidly lowers load-bearing characteristics, but moisture permeation rates stay nearly constant owing to the presence of fungal material at sites where cellulose has been removed.     

“Osmiophilic particles”—a fungal agent with characteristic distribution in white-and brown-rotted wood

When pine wood decayed by white- and brown-rot fungi was observed in TEM after fixation and staining with glutaraldehyde/osmium tetroxide/ uranylacetate and embedding in Spurr’s ultralow viscosity resin electron dense particles, called “osmiophilic particles,” a typical distribution for the two decay types could be observed: in white-rotted wood the particles could be found in and around the hyphae and on the lumen surface of the wood cell wall, mostly aggregated to thick clusters. During the whole course of decay the wood cell walls were free of the particles, but they were present on the corroded surfaces. In brown-rotted wood the “osmiophilic particles” also could be found in and around the hyphae, but in contrast the particles were distributed over all the wood cell wall layers from the early to late stages of decay. The distribution of the “osmiophilic particles” coincides with the place where the major cell wall degradation takes place: in white-rot the cell walls are degraded from the lumen to the middle lamella; in brown-rot a depolymerization and degradation of the carbohydrates takes place all over the wood cell wall. Since the “osmiophilic particles” can be found where the degradation takes place, it can be concluded that they are causally connected with wood decay. The fact that they also were found in, and some of them also around, hyphae grown on malt-agar or Sabouraud-dextrose-agar proves that they are produced by the fungi and cannot be degradation products. The possibility that they could be preparative artifacts can be excluded because uncolonized wood was free of “osmiophilic particles.” Since the “osmiophilic particles” are produced by the fungi and can be found in places where wood is decaying, it can be further concluded that they are a fungal agent that is involved in wood degradation, probably fungal enzymes. The observation that the large “osmiophilic particles,” which may have a size of up to 20 nm, are composed of globular subunits of a diameter of 2—3 nm also speaks for their enzymatic nature. To find out which type of enzyme they might be, the white-rot fungusTrametes hirsuta was grown on wood pulp with 7% lignin, on delignified wood pulp containing cellulose and hemicellulose, and on filterpaper (pure cellulose). The hyphae on wood pulp containing 7% lignin were surrounded by thick sheaths of “osmiophilic particles,” whereas with the hyphae grown on delignified wood pulp and pure cellulose only a few particles could be found. This makes it clear that the production of the “osmiophilic particles” is induced by lignin.     

Maniplating the phenolic acid content and digestibility of italian ryegrass (Lolium multiflorum)b y vacuolar-targeted epession of a fungal ferulic acid esterase

In grass cell walls, ferulic acid esters linked to arabinosyl residues in arabinoxylans play a key role in crosslinking hemicellulose. Although such crosslinks have a number of important roles in the cell wall, they also hinder the rate and extent of cell wall degradation by ruminant microbes and by fungal glycohydrolyase enzymes. Ferulic acid esterase (FAE) can release both monomeric and dimeric ferulic acids from arabinoxylans making the cell wall more susceptible to further enzymatic attack. Transgenic plants of Lolium multiflorum expressing a ferulic acid esterase gene from Aspergillus niger, targeted to the vacuole under a constitutive rice actin promoter, have been produced following microprojectile bombardment of embryogenic cell cultures. The level of FAE activity was found to vary with leaf age and was highest in young leaves. FAE expression resulted in the release of monomeric and dimeric ferulic acids from cell walls on cell death and this was enhanced severalfold by the addition of exogenous β-1,4-endoxylanase. We also show that a number of plants expressing FAE had reduced levels of cell wall esterified monomeric and dimeric ferulates and increased in vitro dry-matter digestibility compared with nontransformed plants.     

Prediction of the decay resistance of heat treated wood on the basis of its elemental composition

The effect of heat treatment temperature on the elemental composition of Scots pine sapwood (Pinus sylvestris) has been investigated in the range of temperatures between 220 and 250 °C. The results revealed an important increase of carbon content, while oxygen content significantly decreases. Independently of the heat treatment temperature, elemental composition is strongly correlated with the mass losses due to thermal degradations. Carbon content as well as O/C ratio seem to represent valuable markers to estimate wood degradation after heat treatment. Heat treated specimens were exposed to fungal decay using the brown rot fungus Poria placenta and the weight losses due to fungal degradation were determined. Correlations between weight losses recorded after fungal exposure and elemental composition indicated that carbon content or O/C ratio can be used to predict wood durability conferred by heat treatment allowing us to envisage the development of a proper method to evaluate the quality of heat treated wood and predict its durability. These results also confirm that chemical modifications of wood cell wall polymers are the main factors responsible for wood durability improvement against fungal decay after heat treatment.     

Development of a regenerable cell culture system that senses and releases dead cells

We developed a rapidly regenerable cell culture system in which the cell culture substrate detects cell death and selectively releases the dead cells. This culture material was achieved by combining a detector that responds to the signal from the dead cells and an actuator to release the dead cells. Benzo-18-crown-6-acrylamide (BCAm) with a pendant crown ether receptor was used as the sensor to recognize cellular signals and N-isopropylacrylamide (NIPAM) was used as the actuator. This copolymer of NIPAM and BCAm can respond to potassium ions and change its nature from hydrophobic to hydrophilic at the culture temperature of 37 degrees C. Living cells concentrate potassium ion internally; when cells die, potassium ions are released. The polymer surface recognizes the potassium ions released from the dead cells, the NIPAM hydrates, and the dead cells are selectively detached. This in vitro culture system is a novel one in which artificial culture materials work cooperatively with cellular metabolism by responding to this signal from the cells, thereby realizing in vitro tissue regeneration partly mimicking the mechanisms of in vivo homeostasis.     

A structured model for vegetative growth and sporulation inBacillus thuringiensis

A mathematical model has been developed for the δ-endotoxin producingBacillus thuringiensis. The structure of the model involves the processes taking place during vegetative growth, those leading to the initiation of sporulation under conditions of carbon and/or nitrogen limitation, and the sporulation events. The key features in the model are the pools of compounds, such as PRPP, IMP, ADP/ATP, GDP/GTP, pyrimidine nucleotides, NAD/NADH2, amino acids, nucleic acids, cell wall, and vegetative and sporulation proteins. These, along with σ-factors that control the nature of RNA-polymerase during the different phases, effectively stimulate the vegetative growth and sporulation. The initiation of sporulation is controlled by the intracellular concentration of GTP. Results of simulation of vegetative growth, initiation of sporulation, spore protein formation, and production of δ-endotoxin under C- or N-limitation are presented.

     

Saccharomyces cerevisiae entrapped in chrysotile increases life-span for up to 3 years

Long-term viability of microorganisms has seldom been reported but is important from the technological and scientific point of view. In this work we show that Saccharomyces cerevisiae, a well known biocatalyst, remains viable and active in fermentation experiments for up to 3 years, in the absence of nutrients, when supported on chrysotile fibers. This long-term viability is ascribed to a latency state which the cells enter after about 4 months storage, induced by entrapment of the cells within the chrysotile fibers. Adhered chrysotile fibers do not penetrate the cell. TEM results show that the fibers are adhered only to the external cellular wall layer, and that no damage is caused to the cell wall structure. No fibers were ever found inside the cells. The entrapping fibers could be observed as a distinctive, well preserved silk nest in preparations in which the cells were not fixed chemically. No degradation of the chrysotile adhered fibers was observed. The entrapment is ascribed to the chrysotile flexibility and the size of the cells, which maximize adhesion by electrostatic and van der Waals interactions between the fibers and the cell surface polysaccharides.     

Characterization of blue-light and developmental regulation of the photolyase gene phr1 in Trichoderma harzianum

Blue light and development regulate the expression of the phr1 gene of the filamentous fungus Trichoderma harzianum. The predicted product of phr1, the DNA repair enzyme photolyase, is likely to help protect Trichoderma, which grows in the soil as a mycoparasite or saprophyte, from damage upon emergence and exposure to ultraviolet-c. phr1 is transiently expressed in mycelium and conidiophores after illumination. phr1 mRNA also accumulates in conidiophores during development and spore maturation. As no other genes displaying rapid, direct light regulation have been described previously in this organism, we have characterized the fluence and time dependence of phr1 induction, and its relation to sporulation and photoreactivation. Induction is transient following a pulse, and, with slower decay, in continuous light. This implies that the photoreceptor, transducers or response are capable of adaptation. About two-fold more light is required to induce phr1 than conidiation, but this difference is modest, so both responses could use the same or similar chromophore. Adenosine 3':5'-cyclic monophosphate bypasses the requirement for light for sporulation, while atropine prevents sporulation even after photoinduction. Light regulation of phr1, however, is indifferent to both these effectors. Induction of photolyase expression behaves as a direct, rapid response to light, independent of the induction of sporulation. Indeed, illumination of mature spores increases their capacity for photoreactivation. Blue light seems to warn the organism against the harmful effects of short wave-lengths, inducing phr1 expression and sporulation by pathways that are, at least in part, distinct.     

Characteristics of black zones associated with delignified wood

Several white-rot fungi cause two micromorphologically distinct types of decay. White-rot fungi typically cause a simultaneous removal of all cell wall components in close proximity to fungal hyphae. This type of degradation results in erosion troughs and holes in the cell walls. In addition, a selective removal of lignin and hemicellulose can occur intermittently throughout the decayed wood. Selectively delignified wood can be characterized by the complete removal of middle lamellae, resulting in a defibration of cells and exposure of cellulosic macrofibrils within cell walls. A common observation of decay in the field is the association of dark zones with selectively delignified wood. Swollen hyphae containing pigmented materials were commonly associated with the dark zones in wood delignified byHeterobasidion annosum, Ganoderma applanatum, G. tsugae, Ischnoderma resinosum, Perenniporia medulla-panis, orDichomitus squalens. Dark zones examined using scanning electron microscopy in conjunction with energy dispersive X-ray analysis were found to contain high concentrations of manganese. Multielement analyses using inductively coupled plasma atomic emission spectrometry indicated approximately a 50-fold increase of Mn in black zones. Pigmented substances containing Mn were readily decolorized with acids. Since large concentrations of Mn were only found in selectively delignified wood, manganese may influence the selective degradation of lignin.     

Effect of Fusarium oxysporum f. sp. lycopersici on the degradation of humic acid associated with Cu, Pb, and Ni: an in vitro study

The intent of this work was to gain further insight on the fungus-assisted degradation/solubilization of humic acid and the related changes in metal-binding profiles. In the experimental design, Aldrich reagent humic acid (HA) or HA enriched with Cu, Pb, and Ni (HA(Me)) was added to Fusarium oxysporum f. sp. lycopersici cultures in vitro. The cultures were supplied by different carbon- and nitrogen-containing nutrients (glucose, Glc, or glutamate, Glu and ammonium, NH₄⁺, or nitrate, NO₃⁻, ions, respectively) in order to examine their possible effect on HA and HA(Me) decomposition. During the first 48 h of fungus growth, gradual acidification to pH 2 was observed in medium containing Glc + NH₄⁺, while for other cultures, alkalinization to pH 9 occurred and then, the above conditions were stable up to at least 200 h. Size exclusion chromatography (SEC) with UV/Vis detection showed progressive degradation and solubilization of both HA and HA(Me) with the increasing time of fungus growth. However, the molecular mass distributions of HA-related soluble species were different in the presence of metals (HA(Me)) as referred to HA and were also influenced by the composition of growth medium. The solubilization of Pb, Cu, and Ni and their association with HA molecular mass fractions were studied using inductively coupled plasma mass spectrometry (ICP-MS) detection. Under acidic conditions, relatively high concentrations of low-molecular-mass metallic species were found in culture supernatants, while in alkaline media, metal solubilization was generally poorer. In contrast to low pH culture, SEC-ICP-MS results obtained in alkaline supernatants indicated metal binding to degradation products of humic substances of MM > 5 kDa. In summary, the results of this study suggest that fungus-assisted degradation of HA and HA(Me) might be controlled using appropriate N- and C- sources required for fungus growth, which in turn would affect molecular mass distribution of soluble metallic species thus potentially influencing their actual bioaccessibility. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.