Physiological and morphological changes in autolyzingAspergillus nidulans cultures

Physiological and morphological changes in carbon-limited autolyzing cultures of Aspergillus nidulans were described. The carbon starvation arrested conidiation while the formation of filamentous and "yeast-like" hyphal fragments with profoundly altered metabolism enabled the fungus to survive the nutritional stress. The morphological and physiological stress responses, which maintained the cellular integrity of surviving hyphal fragments at the expense of autolyzing cells, were highly concerted and regulated. Moreover, sublethal concentrations of the protein synthesis inhibitor cycloheximide or the mitochondrial uncoupler 2,4-dinitrophenol completely blocked the autolysis. In accordance with the propositions of the free-radical theory of ageing reactive oxygen species accumulated in the surviving fragments with a concomitant increase in the specific superoxide dismutase activity and a continuous decrease in cell viability. Glutathione was degraded extensively in carbon-starving cells due to the action of gamma-glutamyltranspeptidase, which resulted in a glutathione-glutathione disulfide redox imbalance during autolysis.     

Hyphal vocuolation and fragmentation inpenicillium chrysogenum

A link between vacuolation and fragmentation of Penicillium chrysogenum mycelia in stirred tank submerged fermentations is reported. Quantitative information on vocuolation and morphology was obtained by image analysis. In fed-batch fermentations the coincidence of the events of rapid vacuolation and the fall of the mean total and main hyphal lengths suggests that hyphal fragmentation is not necessarily due to "shear" alone. The physiological state of the hyphae, characterized by the proportions of vaccuoles, was found to have a significant influence on the breakage of mycelial hyphae, It was found that the fragmentation was greater when the hyphae became heavily vacuolated following nutrient limitation in the culture, i.e., during the switch from the rapid growth to the production phase. (c) 1994 John Wiley & Sons, Inc.     

Cytological analysis of mycelial incompatibility in Rosellinia necatrix

When the mycelia of Rosellinia necatrix encounter mycelia with a different genetic background, distinct barrage lines form. In this study, we observed hyphal interactions between compatible and incompatible R. necatrix pairs by means of light and electron microscopy. Although we observed perfect hyphal anastomosis in compatible pairs of isolates, the hyphae never anastomosed in incompatible pairs (i.e., the hyphae remained parallel or crossed over without merging). These behaviours appeared to result from the detection of or failure to detect one or more diffusible factors. The attraction to other hyphae in pairs of incompatible isolates was increased by supplementation of the growing medium with activated charcoal, although no anastomosis was observed and ultrastructural observation confirmed a lack of hyphal anastomosis. Programmed cell death (PCD) started with one of the two approaching hyphae. Heterochromatin condensation and genomic DNA fragmentation were not observed. Moreover, cell damage began with the tonoplast and continued with the plasma and nuclear membranes, suggesting that the PCD observed in heterogenic incompatibility of R. necatrix was a vacuole-mediated process.     

Viability, strength, and fragmentation of Saccharopolyspora erythraea in submerged fermentation.

Two fermentations of the commercially important erythromycin-producing filamentous bacterium Saccharopolyspora erythraea were conducted in defined media. One was glucose-limited and the other nitrate-limited. The viability of the hyphae was determined using the fluorescent stain BacLight (Molecular Probes, Eugene, OR). Also, the force required to strain hyphae to breakage was determined using micromanipulation and a sensitive force transducer. In both fermentations, fragmentation coincided with the appearance of regions in the mycelia with permeabilised membranes (considered nonviable). Under glucose-limitation, hyphal breaking force rose to 1,050 +/- 130 nN at the end of the growth phase and fell to an undetectable value as a result of glucose exhaustion. Under nitrate-limitation, hyphal breaking force fell from 900 +/- 160 nN during the growth phase to 550 +/- 40 nN in the stationary phase. In both cases image analysis showed that the dimensions of mycelia were of the same order, suggesting that the major factor influencing fragmentation was the appearance of nonviable regions (assumed to be weak). The location in which nonviable regions first appear within hyphae could not be determined because of their appearance coinciding with fragmentation.     

Quantification of autolysis in Penicillium chrysogenum by semiautomated image analysis

An image analysis method is described for the characterization of empty (autolyzed and inactive) regions within the mycelia of filamentous fungi. It extends a previous method that characterized only regions filled with cytoplasm or vacuoles (i.e., the active biomass). The method is semiautomatic, requiring some manual editing before automated measurements. When the method was used for samples from a batch fermentation of an industrial strain of Penicillium chrysogenum, the empty regions were observed to constitute up to 15% (by projected area) of the biomass during the growth phase. After nutrient exhaustion, however, the proportion of empty regions rose rapidly, eventually representing more than 50% of the biomass by the end of fermentation. The increase in the percentage of empty regions coincided with a decrease in biomass (as measured by dry cell weight) and a fall in penicillin titre. Further morphological analysis revealed that fragmentation of mycelia, particularly clumps, coincided with increases in the levels of empty regions. This new image analysis method gave additional information on hyphal differentiation and a measure of autolysis. It was also a useful indicator of the processes leading to autolysis.     

菌丝形态分化与头孢菌素C合成的关系

利用显微图像分析法对顶头孢霉菌的菌丝形态进行了定量研究,并统计分析了头孢菌素C发酵过程中的菌丝形态的变化规律,具体对菌丝长度、菌丝宽度和菌丝生长单位进行了定量分析,分析了菌丝形态分化与头孢菌素C合成的关系。研究表明头孢菌素C的合成是在细长菌丝分化成膨大菌丝片段后才启动的,头孢菌素C可能主要是在膨大菌丝分化成节孢子的过程中被合成。     

Purification and Characteristics of an Autolytic Chitinase of Piromyces communis OTS1 from Culture Medium

An autolysis chitinase was purified from the cultural medium of the anaerobic fungus Piromyces communis OTS1 by ammonium sulfate precipitation, affinity chromatography with regenerated chitin, chromato-focusing, gel filtration, and chromato-focusing again. The optimal pH and temperature were 6.0 and 50°C, respectively, for a 20-min assay. The chitinase was stable from pH 6.0 to 8.0, but was unstable at 70°C for 20 min. The molecular mass of chitinase was estimated by SDS-PAGE to be 44.9 kDa, and its pI was 4.4. The enzyme activity, which was of the ‘endo’ type, was inhibited by Hg²⁺ and allosamidin. The chitinase hydrolyzes chitin powder and fungal cell walls at a higher rate than an artificial chitin substrate. It can be concluded that extracellular chitinase is similar to cytosolic chitinase, but they are not the same protein. Received: 3 December 1996 / Accepted: 28 January 1997     

The Paxillin-like protein AgPxl1 is required for apical branching and maximal hyphal growth in A.gossypii

The development from young, slowly growing hyphae to fast growing hyphae in filamentous fungi is referred to as hyphal maturation. We have identified the Paxillin-like protein AgPxl1 in Ashbyagossypii as a developmental protein that is specifically required for hyphal maturation. The early development of A.gossypii strains lacking AgPxl1 is indistinguishable from wild-type. However, at later developmental stages the maximal hyphal extension rate is less than half compared to wild-type and apical branching is affected. Apical branching is characterised as the symmetric division of fast growing hyphal tips resulting in two sister hyphae. In Agpxl1Delta strains two thirds of the apical branching events lead to asymmetric sister hyphae where growth of one branch is either completely aborted or slowed down while extension of the other branch is not affected. This suggests that AgPxl1 plays a role in the organisation of growth and efficient division of growth upon apical branching in mature mycelia. The conserved C-terminal LIM domains are necessary for AgPxl1 function and also contribute to tip localisation. AgCLA4, a PAK-like kinase, is epistatic to AgPXL1 and robust localisation of AgPxl1 depends on AgCla4. This suggests that AgCla4 acts upstream of AgPxl1.     

Genetic diversity of isolates of Glomus mosseae from different geographic areas detected by vegetative compatibility testing and biochemical and molecular analysis

We detected, for the first time, the occurrence of vegetative incompatibility between different isolates of the arbuscular mycorrhizal fungal species Glomus mosseae. Vegetative compatibility tests performed on germlings belonging to the same isolate showed that six geographically different isolates were capable of self-anastomosing, and that the percentage of hyphal contacts leading to fusions ranged from 60 to 85%. Successful anastomoses were characterized by complete fusion of hyphal walls, protoplasm continuity and occurrence of nuclei in the middle of hyphal bridges. No anastomoses could be detected between hyphae belonging to different isolates, which intersected without any reaction in 49 to 68% of contacts. Microscopic examinations detected hyphal incompatibility responses in diverse pairings, consisting of protoplasm retraction from the tips and septum formation in the approaching hyphae, even before physical contact with neighboring hyphae. Interestingly, many hyphal tips showed precontact tropism, suggesting that specific recognition signals may be involved during this stage. The intraspecific genetic diversity of G. mosseae revealed by vegetative compatibility tests was confirmed by total protein profiles and internal transcribed spacer-restriction fragment length polymorphism profiles, which evidenced a higher level of molecular diversity between the two European isolates IMA1 and BEG25 than between IMA1 and the two American isolates. Since arbuscular mycorrhizal fungi lack a tractable genetic system, vegetative compatibility tests may represent an easy assay for the detection of genetically different mycelia and an additional powerful tool for investigating the population structure and genetics of these obligate symbionts.     

Measurement of autolysis in submerged batch cultures of Penicillium chrysogenum

The process of cellular autolysis was studied in an industrial strain of Penicillium chrysogenum by a range of methods, including assessment of biomass decline, NH+4 release, changes in culture apparent viscosity, and by means of a quantitative assessment of changes in micromorphology using a computerized image analysis system. The pattern of total intracellular proteolytic and beta-1, 3-glucanolytic activity in the culture was also examined. The overall aim was to identify a suitable method, or methods, for examining the extent of autolysis in fungal cultures. Autolysis was studied in submerged batch processes, where DOT was maintained above 40% saturation (non-O2-limited), and, under O2-limited conditions. Both N and O2 limitation promoted extensive culture autolysis. Image analysis techniques were perhaps the most sensitive method of assessing the progress of autolysis in the culture. Autolytic regions within some hyphae were apparent even during growth phase, but became much more widespread as the process proceeded. The early stages of autolysis involved continued energy source consumption, increased carbon dioxide evolution rate, degradation of penicillin, and decreased broth filterability. Later stages involved widespread mycelial fragmentation, with some regrowth (cryptic growth) occurring in non-O2-limited cultures. Intracellular proteolytic activity showed two peaks, one during the growth phase, and the other during autolysis. Autolysis was also associated with a distinct peak in beta-1,3-glucanolytic activity, indicating that degradation of cell wall matrix polymers may be occurring during autolysis in this strain of P. chrysogenum.     

Effect of phenylacetic acid feeding on the process of cellular autolysis in submerged batch cultures of Penicillium chrysogenum.

The effects of feeding the 'toxic' penicillin precursor, phenylacetic acid (PAA) at varying rates, upon the process of cellular autolysis, was assessed in batch bioreactor cultures of an industrial strain of Penicillium chrysogenum. Five processes were fed at rates which resulted in extracellular concentrations of PAA ranging from zero (the control) to approximately ten times levels said to be optimal for penicillin biosynthesis. The culture response was assessed chemically and morphologically, using computerised image analysis. High concentrations of PAA reduced biomass and penicillin production, and were associated with increased cellular autolysis. However, the values of classical morphological indices (branch length, main hyphal length and hyphal growth unit) varied little in cultures which showed extensive autolysis and biomass loss. Lower precursor concentrations (0.01 to 1.0 g l-1) had little effect on biomass, penicillin, or upon the levels of autolysis compared with the control process. Therefore, precursor concentration controlled within the optimal range for penicillin production, has little impact upon differentiation or degradation within an industrial culture of P. chrysogenum. By contrast, exploitation of the toxicity of PAA is proposed as a means to bring forward or enhance autolysis, providing a reliable method of 'induction' with which to study the phenomenon in P. chrysogenum.     

Hyphal in vitro growth of the arbuscular mycorrhizal fungus Glomus mosseae is affected by chitinase but not by β-1,3-glucanase

Purified basic chitinase or #-1,3-glucanase or a combination of the two enzymes were applied to hyphae of the arbuscular mycorrhizal fungus Glomus mosseae grown in vitro. Chitinase applied to the hyphal tip produced an inhibition of hyphal extension, lysis of the apex and alterations of the growth pattern of the fungus. No effect was observed, however, when chitinase was applied to subapical parts of the hyphae or when glucanase was applied to any part of the hyphae. Application of a combination of the two enzymes to the hyphal tip produced an effect similar to that of chitinase alone.     

Genetic Diversity of Isolates of Glomus mosseae from Different Geographic Areas Detected by Vegetative Compatibility Testing and Biochemical and Molecular Analysis

We detected, for the first time, the occurrence of vegetative incompatibility between different isolates of the arbuscular mycorrhizal fungal species Glomus mosseae. Vegetative compatibility tests performed on germlings belonging to the same isolate showed that six geographically different isolates were capable of self-anastomosing, and that the percentage of hyphal contacts leading to fusions ranged from 60 to 85%. Successful anastomoses were characterized by complete fusion of hyphal walls, protoplasm continuity and occurrence of nuclei in the middle of hyphal bridges. No anastomoses could be detected between hyphae belonging to different isolates, which intersected without any reaction in 49 to 68% of contacts. Microscopic examinations detected hyphal incompatibility responses in diverse pairings, consisting of protoplasm retraction from the tips and septum formation in the approaching hyphae, even before physical contact with neighboring hyphae. Interestingly, many hyphal tips showed precontact tropism, suggesting that specific recognition signals may be involved during this stage. The intraspecific genetic diversity of G. mosseae revealed by vegetative compatibility tests was confirmed by total protein profiles and internal transcribed spacer-restriction fragment length polymorphism profiles, which evidenced a higher level of molecular diversity between the two European isolates IMA1 and BEG25 than between IMA1 and the two American isolates. Since arbuscular mycorrhizal fungi lack a tractable genetic system, vegetative compatibility tests may represent an easy assay for the detection of genetically different mycelia and an additional powerful tool for investigating the population structure and genetics of these obligate symbionts.     

Characterization and amino acid sequence of IRT-4, a novel TEM-type enzyme with a decreased susceptibility to β-lactamase inhibitors

Abstract A novel procedure was used to purify a cytosolic chitinase from Candida albicans to electrophoretic homogeneity. The results represent the first demonstration of the purification of a fungal intracellular chitinase using the criterion of a single band detected following silver-staining of a polyacrylamide gel run under denaturing conditions. Purified chitinase had pH and temperature optima of 5.0 and 50°C, respectively. Inhibition of enzyme activity by allosamidin was pH-dependent occuring maximally at pH 8.0. Phospholipids had similar marked and highly specific effects on the activities of both the purified soluble enzyme and a solubilized microsomal chitinase from C. albicans . Evidence is provided for the existence of a complex chitinolytic system in this organism.     

Tip growth of <Emphasis Type="Italic">Neurospora crassa</Emphasis> under glucose deprivation

Growth parameters of vegetative hyphae and isolated tip fragments of the mycelial fungus N. crassa were studied after complete substitution of an easily metabolized carbon source (glucose) for a non-metabolized one (sorbitol). The images of growing tips were recorded at 20–30-min intervals. Using original image processing software, geometrical parameters of the hyphal trees (length and number of branches, area of convex polygons circumscribed about the hyphal trees, etc.) were determined and growth characteristics, such as rate of tip elongation (V) and the ratio of the total hyphal length to the number of growing tips (termed “hyphal growth unit”, HGU), were calculated. It is shown that after 4–5-h growth in sorbitol-enriched media growth characteristics of intact hyphae did not differ significantly from the corresponding parameters of hyphae growing in glucose-enriched media. In isolated tip fragments (about 800-μ m long), the values of V were lower than those in intact hyphae but did not depend on the carbon source in the nutrient media. However, in such fragments growing in sorbitol-enriched media the number of branches decreased, while the HGU value and the number of large intracellular vacuoles increased. Staining of cells with a standard chitin probe, Calcofluor White (10 μg/ml), did not reveal any considerable differences in hyphal cell walls and septa in tip fragments grown in the presence of different carbon sources. Possible mechanisms of the dependence of the tip growth parameters on the glucose deficiency are discussed.     

Anastomosis formation and nuclear and protoplasmic exchange in arbuscular mycorrhizal fungi

We observed anastomosis between hyphae originating from the same spore and from different spores of the same isolate of the arbuscular mycorrhizal fungi Glomus mosseae, Glomus caledonium, and Glomus intraradices. The percentage of contacts leading to anastomosis ranged from 35 to 69% in hyphae from the same germling and from 34 to 90% in hyphae from different germlings. The number of anastomoses ranged from 0.6 to 1.3 per cm (length) of hyphae in mycelia originating from the same spore. No anastomoses were observed between hyphae from the same or different germlings of Gigaspora rosea and Scutellospora castanea; no interspecific or intergeneric hyphal fusions were observed. We monitored anastomosis formation with time-lapse and video-enhanced light microscopy. We observed complete fusion of hyphal walls and the migration of a mass of particles in both directions within the hyphal bridges. In hyphal bridges of G. caledonium, light-opaque particles moved at the speed of 1.8 +/- 0.06 microm/s. We observed nuclear migration between hyphae of the same germling and between hyphae belonging to different germlings of the same isolate of three Glomus species. Our work suggests that genetic exchange may occur through intermingling of nuclei during anastomosis formation and opens the way to studies of vegetative compatibility in natural populations of arbuscular mycorrhizal fungi.     

Chemical modification studies of the active centre of Candida albicans chitinase and its inhibition by allosamidin.

Allosamidin, a glycoside antibiotic, is shown to be a strong, competitive inhibitor of semi-purified chitinase from yeast cells of Candida albicans. The inhibitory potency of allosamidin was pH-dependent, with IC50 values of 280 nM at pH 5.0 and 21 nM at pH 7.5. At higher, micromolar, concentrations, allosamidin inactivated this chitinase in a time- and concentration-dependent manner. Kinetic studies of this inactivation provided evidence for the formation of a reversible complex between allosamidin and chitinase, characterized by Kinact = 5 microM, followed by irreversible modification of the enzyme with velocity constant k2 = 4.6 x 10(-3) s-1. Chemical modification studies with the use of group-specific reagents suggested the presence of Glu/Asp carboxyl group(s) at or near the active site, that were important for enzyme activity. The carboxyl-specific reagent, 1-ethyl-3(3-dimethylaminopropyl)-carbodiimide, inactivated the chitinase in a single step process, with apparent second-order rate constant of 0.014 M-1 s-1.