Molecular architecture of fungal cell walls. An approach by use of fluorescent markers

Fluorescent probes have been applied to analyze the molecular architecture of fungal cell surfaces. Binding patterns of aniline blue and FITC-labeled wheat germ agglutinin (FITC-WGA) elucidated class-specific properties. Aniline-blue-induced fluorescence was distributed over the entire cell walls from Ascomycetes, but was confined to sporangial walls of Zygomocetes, hyphal tips and a few other sites in Basidiomycetes, while no fluorescence was found with sporangia and rhizoids of Chytridiales. FITC-WGA in Zygomycetes and in Ascomycetes was restricted to few sites (e.g. apex of hyphae), in Basidiomycetes and Chytridiales label was evenly associated with the entire surface of hyphal walls, or sporangia and rhizoids. For Oomycetes, Zygomycetes, Ascomycetes, and Basidiomycetes differences in the molecular architecture between apex and hyphal side walls were discerned, although the chemical nature of these differences is distinct for each class. Species specific differences, due to differences in binding patterns of several lectins are not apparent at fungal cell surfaces. The degree of intraspecies variation was found to be larger than interspecies diversification, suggesting changeableness of the molecular architecture of fungal cell walls. This is in contrast to assertions which we made by working on algae. There species-specific lectin binding patterns have been described.     

Localization of beta-(1,3)-glucanase in the mycelium of Sclerotium rolfsii.

The role of the lytic enzyme beta-(1,3)-glucanase in cell wall synthesis and its distribution in the mycelium of the fungus Sclerotium rolfsii were studied. Enzyme activity was determined after enzyme extraction with Triton X-100 from a cell wall preparation. Specific zones of immunofluorescence appeared in the hyphal tips, clamp connections, new septa, and lateral branching when a specific antiserum was used with the indirect method of the fluorescent antibody staining. Enzymatic activity in the cell wall preparation was inactivated by diethylpyrocarbonate. However, 69% of the total enzymatic activity was present in a latent form which was not affected by the ester. This result suggests that most of the beta-(1,3)-glucanase was present along the hyphal cell walls in a "masked" form. An active enzyme appeared only in those regions which showed immunofluorescence. The activity of glucan synthetase, an enzyme essential for wall formation, was higher in the branching funus grown on L-threonine-supplemented synthetic medium than in the synthetic medium-grown fungus.     

Lysis of cell walls ofMucor ramannianus möller by aStreptomyces sp.

A study has been made of some chemical and ultrastructural changes that occur in the hyphal, arthrospore and sporangiospore walls ofMucor ramannianus during lysis by a soil streptomycete.Arthrospore and hyphal walls, which were shown to contain chitin, chitosan, other polysaccharides and phosphate (principally as polyphosphate), were lysed by culture fluid of the streptomycete after this organism had been grown on the same material. Alcohol-insoluble material found in the supernatants of the incubation mixtures gave on hydrolysis glucosamine, galactose, mannose and fucose. No laminarinase activity was detected in these culture fluids. Culture fluids of the streptomycete after growth on chitin and chitosan were also found to lyse the walls of arthrospores and hyphae.Despite the chemical similarities the walls were very different in thin section.A major component in the sporangiospore walls was glucan and an active laminarinase was shown to be present in the culture fluids of the streptomycete after growth on them. Further, ultrathin sections showed that an inner fibrillar layer of the sporangiospore wall was lysed leaving an outer electron-dense layer.     

Status of nuclear division in arbuscular mycorrhizal fungi during in vitro development

Summary The number of nuclei in spores and along hyphae of an arbuscular mycorrhizal fungiGigaspora margarita was measured in digital images of fluorescence arising from mithramycin stained cultures. Typical dormant spores (250 m diameter) contained 2000 nuclei. Eight hundred nuclei were mobilized during the first 3 days of germination. The number of nuclei in the spores nearly returned to the initial number after 22 days of hyphal growth. The average relative DNA content in the nuclei of dormant spores and in the nuclei of spores incubated for 22 days was comparable, as judged from fluorescence intensity. Hyphal elongation occurred with 460 nuclei per cm under a special set of in vitro conditions that promote extensive hyphal growth of arbuscular mycorrhizal fungi. We found an average total of 26000 hyphal nuclei per germinating spore after 22 days. The specific DNA polymerase inhibitor aphidicolin did not inhibit spore germination but it rapidly reduced the rate of hyphal growth and arrested growth after 4 days. No nuclei were produced de novo during this time. These results demonstrate thatG. margarita replicates nuclear DNA and undergoes nuclear division when grown in vitro even in the absence of a plant host.     

Analysis of wall glucans from yeast, hyphal and germ-tube forming cells of Candida albicans

Acid-soluble and alkali-insoluble glucan fractions were prepared from yeast, hyphal and germ-tube forming cells of Candida albicans. Alkali-insoluble glucan was also extracted from purified yeast cell walls. Paper chromatography of partial acid hydrolysates confirmed that the glucan preparations contained beta(1----3)- and beta(1----6)-chains but no mixed intra-chain beta(1----3)/(1----6) linkages. Methylation and 13C-NMR analyses showed that the acid-soluble glucan consisted of a highly branched polymer composed mainly (67.0% to 76.6%) of beta(1----6)-linked glucose residues. The alkali-insoluble glucan from yeast and hyphal cells contained from 29.6% to 38.9% beta(1----3) and 43.3% to 53.2% beta(1----6) linkages. Alkali-insoluble glucan from germ-tube forming cells consisted of 67.0% beta(1----3) and 14% beta(1----6) linkages. Branch points accounted for 6.7%, 12.3% and 17.4% of the residues in the alkali-insoluble glucan of yeast, germ-tube forming and hyphal cells, respectively.     

甲霜灵对大豆疫霉野生菌株及突变菌株生长发育影响的细胞学研究

本文利用电子显微镜技术研究了内吸性杀菌剂甲霜灵(Metalaxyl)对大豆疫霉Phytophthoras ojae野生菌株和突变菌株的形态学及超微结构的影响。结果表明:不同浓度甲霜灵处理后可导致野生菌株和突变菌株发生一系列不同的变化。低浓度(1μg/mL)处理后,野生菌株在培养基上的生长即可受到抑制,菌丝呈现不规则的肿胀、过度分枝;菌丝细胞壁不规则加厚,菌丝细胞内液泡增加,脂肪粒累积,细胞器排列紊乱,原生质最终坏死。随浓度的升高,野生菌株立即停止生长,菌丝干瘪坏死。而突变菌株只在高浓度(10μg/mL)甲霜灵处理后顶端菌丝出现少量较小的分枝,菌丝细胞壁无增厚现象,但细胞内脂肪粒大量积累,明显高于敏感性菌株;突变菌株在高浓度甲霜灵压力下仍继续生长。     

Effect of papulacandin B on the cell wall and growth of Geotrichum lactis

Addition of the antifungal antibiotic papulacandin B to an exponential culture of Geotrichum lactis inhibited incorporation of glucose into the alkali-insoluble and alkali-soluble glucan fractions of the hyphal wall, although the rate of growth was practically unaltered. Synthesis of other cell wall components (i.e. galactomannan and chitin) was not affected. Papulacandin B also induced the proliferation of branches along the hyphae which continued to branch dichotomously resulting in a 'colonial' pattern of growth. Aculeacin A, another antifungal antibiotic that inhibited beta-glucan synthesis also caused morphological alterations similar to those described for papulacandin B. Inhibition of beta-glucan synthesis and the altered growth pattern persisted for several hours after removal of the antibiotic. Recovery of beta-glucan synthesis and restoration of the normal pattern of growth occurred simultaneously. Growth of G. lactis in L-sorbose medium also led to inhibition of beta-glucan synthesis and dichotomous branching.     

Sequential phsophorylation of histone subfractions in the Chinese hamster cell cycle.

Ion exchange chromatography and preparative electrophoresis were used to examine the phosphorylation of histone f1 and f3 subfractions in synchronized Chinese hamster cells (line CHO). Three discrete f1 phosphorylation events were demonstrated to occur in sequence during the cell cycle. The first event (f1G1) commenced in G1 2 hours prior to entry of cells into S phase; the second event (f1s) commenced simultaneously with initiation of DNA synthesis; and the third event (f1M) commenced when cells entered mitosis. F1M phosphorylation occurred simultaneously with the phosphorylation of histone f3 (which is not phosphorylated during G1, S, or G2). Fractionation of f1 and f3 revealed no differences in these sequential phosphorylation patterns among the various f1 and f3 subfractions, indicating that these phosphorylations are general biochemical events of the cell cycle. Phosphorylated (f1G1) was found to accumulate in cells as they traversed THEIR CELL CYCLE. F1s was phosphorylated to twice the extent of f1G1, but f1s did not accumulate in the cells as they passed through interphase. F1M was phosphorylated to about 4 times the extent of the first phosphorylated form (f1G1). A model of the relationship of histone phosphorylation to the cell cycle is presented which suggests that (a) f1G1 phosphorylation is involved with chromatin structural changes necessary for cell proliferation; (b) f1s phosphorylation is involved with DNA replication; (c) F1M and f3 phosphorylations are involved in chromosome condensation.     

beta-1,3-Glucan in Developing Cotton Fibers: Structure, Localization, and Relationship of Synthesis to That of Secondary Wall Cellulose

Evidence is presented for the existence of a noncellulosic β-1,3-glucan in cotton fibers. The glucan can be isolated as distinct fractions of varying solubility. When fibers are homogenized rigorously in aqueous buffer, part of the total β-1,3-glucan is found as a soluble polymer in homogenates freed of cell walls. The proportion of total β-1,3-glucan which is found as the soluble polymer varies somewhat as a function of fiber age. The insoluble fraction of the β-1,3-glucan remains associated with the cell wall fraction. Of this cell wall β-1,3-glucan, a variable portion can be solubilized by treatment of walls with hot water, a further portion can be solubilized by alkaline extraction of the walls, and 17 to 29% of the glucan remains associated with cellulose even after alkaline extraction. A portion of this glucan can also be removed from the cell walls of intact cotton fibers by digestion with an endo-β-1,3-glucanase. The glucan fraction which can be isolated as a soluble polymer in homogenates freed of cell walls is not associated with membranous material, and we propose that it represents glucan which is also extracellular but not tightly associated with the cell wall. Enzyme digestion studies indicate that all of the cotton fiber glucan is β-linked, and methylation analyses and enzyme studies both show that the predominant linkage in the glucan is 1 → 3. The possibility of some minor branching at C-6 can also be deduced from the methylation analyses. The timing of deposition of the β-1,3-glucan during fiber development coincides closely with the onset of secondary wall cellulose synthesis. Kinetic studies performed with ovules and fibers cultured in vitro show that incorporation of radioactivity from [¹⁴C]glucose into β-1,3-glucan is linear with respect to time almost from the start of the labeling period; however, a lag is observed before incorporation into cellulose becomes linear with time, suggesting that these two different glucans are not polymerized directly from the same substrate pool. Pulse-chase experiments indicate that neither the β-1,3-glucan nor cellulose exhibits significant turnover after synthesis.     

Antibodies as probes for the study of location and metabolism of (1→3), (1→4)-β-D-glucans

Polyclonal antibodies, raised against ((1→3), (1→4)-β-D-glucans from oat ( Avena sativa L.) caryopsis, were used to investigate the location and the metabolism of mixed-linked β-D-glucans. The binding of these antibodies to the cell walls of oat coleoptiles was shown by an indirect fluorescence method. Distinct fluorescent regions were observed along the inner layers of the walls of each cell. The preimmune serum or antibodies pretreated with oat caryopsis β-D-glucans did not react with the cell walls. Glucan antibodies were bound to the walls of other Poaceae coleoptiles as well as to those from oat mesocotyls and roots, whereas they were not bound to the walls of some dicotyledons tested. The relative glucan content of the cell walls of oat coleoptiles as determined by β-D-glucanase (EC 3.2.1.73) treatment was maximum between day 3 and 4 after soaking, but it declined during further elongation. A rapid decrease in glucan content was observed in excised coleoptiles when auxin or β-D-glucanase was present. There was a clear correlation between the glucan content expressed on a basis of cell wall polysaccharides and the amount of the antibodies bound to the cell walls. These results indicate that the antibodies are useful probes to detect and determine (1→3), (1→4)-β-D-glucans of cell walls.     

Phytochrome-dependent modulation and re-induction of growth of the first rhizoid in Dryopteris paleacea Sw

Phytochrome-dependent growth in Dryopteris paleacea Sw. was investigated in young, developing gametophytes with respect to formation and differentiation of rhizoids. Under continuous red light (Rc), the first rhizoids grew synchronously by tip elongation at a constant rate of 240 μm · d⁻¹ until formation and outgrowth of the second rhizoid. Cessation of growth of the first rhizoids and outgrowth of the second rhizoids showed a correlation in time assumed to be mediated by intercellular signaling. The first rhizoids showed two modes of response to actinic irradiations: (i) modulation of rhizoid growth, and (ii) re-induction of growth in non-growing rhizoids. In the former, the promotory effect of actinic irradiations on rhizoids pre-cultured under Rc determined both the time for which rhizoids continued to grow after transfer into darkness and the final rhizoid length. In the latter, re-induced growth was studied using non-growing rhizoids which were obtained after irradiation with a far-red light (FR) pulse at the end of the pre-culture in Rc and transfer into darkness for 3 d to stop growth. Re-induction of growth occurred with a lag phase of 36 to 48 h after formation of the FR-absorbing form of phytochrome (Pfr) by a red light (R) pulse. From the incomplete R/FR reversibility it is evident that, here, coupling of Pfr to signal transduction is possible within minutes. Re-induction of growth possesses the advantage that the effect of actinic irradiations can be studied as an all-or-none response at the level of single gametophytes in future experiments. The present results clearly indicate that the developmental stage of the whole gametophyte, i.e. temporal and spatial patterns undergone during development, affects the regulation of rhizoid growth by the external factor light. Received: 8 June 1998 / Accepted: 22 December 1998     

DNA synthesis following gross alterations of the nucleocytoplasmic ratio in the ciliate Stentor coeruleus

Incorporation of externally supplied and injected ³H-thymidine into DNA was measured autoradiographically. Starved stentors synthesized no DNA, in contrast to well-fed animals, but replication commenced in some cases if they were fed. Grafting starved and well-fed stentors together rapidly induced DNA synthesis in the starved partner. Suppression of synthesis in the well-fed macronucleus was not observed. Well-fed cytoplasm alone induced DNA synthesis in starved stentors, and starved cytoplasm grafted to starved animals also induced synthesis after a lag. Starved animals with the beaded macronucleus reduced to 2 nodes commenced DNA replication after 6 hr; however, initiation was prevented if the normal nuclear complement was restored before the fourth hour.The macronucleus was required to render starved cytoplasm capable of supporting DNA synthesis, but once potentiated the cytoplasm alone could initiate replication in a starved nucleus. Initiation required RNA synthesis, shown by actinomycin sensitivity.This nucleic acid analysis suggests that decreasing the nucleocytoplasmic ratio elicits RNA synthesis in the remaining macronucleus. The RNA codes for proteins involved in DNA synthesis which are synthesized in the cytoplasm and enter the nucleus to initiate DNA replication.     

The course of chloroplast DNA replication and its relationship to other reproductive processes in the chloroplast and nucleocytoplasmic compartment during the cell cycle of the algaScenedesmus quadricauda

Summary DNA containing structures (cellular, chloroplast and mitochondrial nuclei) were stained with the fluorochrome DAPI. Fluorescence intensity, as a measure of DNA content, was estimated during the mitotic cycle in synchronized populations of the chlorococcal alga,Scenedesmus quadricauda. In cells yielding eight daughter cells, three consecutive steps in chloroplast DNA increase occurred over one mitotic cycle. The first step was performed shortly after releasing the daughter cells, the second and third steps occurred consecutively during the first half of the mitotic cycle. Commitment to chloroplast DNA replication was chronologically separated from commitment to division of chloroplast nuclei, revealing that these two chloroplast reproductive steps were under different control mechanisms. The replication of chloroplast DNA occurred at a different time to that of cell-nuclear DNA. The coordination of chloroplast reproductive processes and those in the nucleocytoplasmic compartment were governed by the mutual trophic and metabolic dependency of these compartments rather than by any direct or feedback control controlled by either of them.Abbreviations DAPI 46-diamidino-2-phenylindole - ptDNA DNA in chloroplast nuclei - nucDNA DNA in cell nuclei     

Novel Structural Features in Candida albicans Hyphal Glucan Provide a Basis for Differential Innate Immune Recognition of Hyphae Versus Yeast

The innate immune system differentially recognizes Candida albicans yeast and hyphae. It is not clear how the innate immune system effectively discriminates between yeast and hyphal forms of C. albicans. Glucans are major components of the fungal cell wall and key fungal pathogen-associated molecular patterns. C. albicans yeast glucan has been characterized; however, little is known about glucan structure in C. albicans hyphae. Using an extraction procedure that minimizes degradation of the native structure, we extracted glucans from C. albicans hyphal cell walls. ¹H NMR data analysis revealed that, when compared with reference (1→3,1→6) β-linked glucans and C. albicans yeast glucan, hyphal glucan has a unique cyclical or “closed chain” structure that is not found in yeast glucan. GC/MS analyses showed a high abundance of 3- and 6-linked glucose units when compared with yeast β-glucan. In addition to the expected (1→3), (1→6), and 3,6 linkages, we also identified a 2,3 linkage that has not been reported previously in C. albicans. Hyphal glucan induced robust immune responses in human peripheral blood mononuclear cells and macrophages via a Dectin-1-dependent mechanism. In contrast, C. albicans yeast glucan was a much less potent stimulus. We also demonstrated the capacity of C. albicans hyphal glucan, but not yeast glucan, to induce IL-1β processing and secretion. This finding provides important evidence for understanding the immune discrimination between colonization and invasion at the mucosal level. When taken together, these data provide a structural basis for differential innate immune recognition of C. albicans yeast versus hyphae.     

Effect of restrictive conditions on the growth and morphology of a temperature-sensitive mannose-requiring mutant of Aspergillus nidulans

When incubated at 45 degrees C in the absence of added mannose, pregrown hyphae of a temperature-sensitive, mannose-relief mutant (mnrA455) of Aspergillus nidulans grew normally for a short time (4-5 h) before exhibiting an abnormal morphology consisting of the production by hyphae of discrete spherical swellings called balloons. These swellings could be up to 10 microns in diameter and were produced either at or behind the hyphal apex. Often only one swelling was produced in association with each hyphal tip, but in a significant minority of cases (approximately 19.6%) a second balloon was produced in close association with the first. Hyphal tip extension slowed before and during balloon formation, but growth at individual tips did not usually stop when a balloon began to be formed in the same hypha. All tip extension ceased after approximately 8 h in cultures maintained at 45 degrees C. However, normal growth resumed 45-60 min after transfer of such a culture to the permissive temperature of 37 degrees C even after 48 h at 45 degrees C. Electron microscopic examination indicated that balloons consistently had thicker walls than the surrounding hyphae but that no accumulation of cytoplasmic vesicles was apparent within them. This indicates that a modification of wall structure, probably including deposition of new wall material, was caused by a mannose deficiency, but that this altered wall synthesis and attendant hyphal swelling was not due to diversion of the normal vesicle-mediated tip-extension system to the side walls of hyphae.     

Mutations affecting the biosynthesis of S-adenosylmethionine cause reduction of DNA methylation in Neurospora crassa.

A temperature-sensitive methionine auxotroph of Neurospora crassa was found in a collection of conditional mutants and shown to be deficient in DNA methylation when grown under semipermissive conditions. The defective gene was identified as met-3, which encodes cystathionine-gamma-synthase. We explored the possibility that the methylation defect results from deficiency of S-adenosylmethionine (SAM), the presumptive methyl group donor. Methionine starvation of mutants from each of nine complementation groups in the methionine (met) pathway (met-1, met-2, met-3, met-5, met-6, met-8, met-9, met-10 and for) resulted in decreased DNA methylation while amino acid starvation, per se, did not. In most of the strains, including wild-type, intracellular SAM peaked during rapid growth (12-18 h after inoculation), whereas DNA methylation continued to increase. In met mutants starved for methionine, SAM levels were most reduced (3-11-fold) during rapid growth while the greatest reduction in DNA methylation levels occurred later. Addition of 3 mM methionine to cultures of met or cysteine-requiring (cys) mutants resulted in 5-28-fold increases in SAM, compared with wild-type, at a time when DNA methylation was reduced approximately 40%, suggesting that the decreased methylation during rapid growth in Neurospora is not due to limiting SAM. DNA methylation continued to increase in a cys-3 mutant that had stopped growing due to methionine starvation, suggesting that methylation is not obligatorily coupled to DNA replication in Neurospora.     

THE CHEMICAL COMPOSITION OF THE HYPHAL WALLS OF THE FUNGUS ALLOMYCES

Aronson , Jerome M., and Leonard Machlis . (U. California, Berkeley.) The chemical composition of the hyphal walls of the fungus Allomyees. Amer. Jour. Bot. 46(4): 292–300. Illus. 1959.—The hyphal walls of Allomyces macrogynus were isloated by both alkaline digestion methods and by sonic oscillation. Both types of preparations showed the walls to consist of chitin, glucan, and ash. In addition, the mechanically isolated walls contained a protein fraction, the properties and significance of which were not determined. Hemicellulose-type polysaccharides, pectic substances, ether soluble lipids, and constituents giving rise to 3–0-α-earboxyethyl hexosamine were not found to be present in the walls. The walls of plants grown for 60–70 hr. under the prescribed conditions contain approximately 60% chitin, 15% glucan, 10% ash, and 10% protein intimately associated with the walls. The percentage of wall material in a mycelium, as well as the percentage of chitin in the walls, increases with the chronological age of the mycelium. These percentages were not, however, affected by variations in the composition of the nutrient medium. The chitin in the walls could be hydrolyzed in the presence of chitinase; lysozyme, however, had no detectable effect on the walls.     

Die Zellwand der Pilze als Korsett

Form follows function: The fungal cell wall as a support structure Within the domain of Eukarya, the fungi form a seperate kingdom. The typical formation of branched mycelia from single hyphae is based on cell wall production at the growing hyphal tip. There, excretory vesicle fuse with the membrane releasing cell wall synthesis enzymes like chitin synthase forming the polymer of N‐acetyl glucosamin, the backbone of fungal cell walls. In addition, glucan synthases form the structural component β‐1.3‐glucan. Via β‐1,6‐glucan, cell wall proteins can be linked to the maturing cell wall, and α‐1,3‐glucan can form a matrix within the cell wall, but also a slimy matrix secreted into the medium. A layer of hydrophobins allows for growth into the air, but also facilitates formation of macroscopic structures like mushrooms.