Trifluoromethanesulfonic acid-based proteomic analysis of cell wall and secreted proteins of the ascomycetous fungi Neurospora crassa and Candida albicans

Cell wall proteins from purified Candida albicans and Neurospora crassa cell walls were released using trifluoromethanesulfonic acid (TFMS) which cleaves the cell wall glucan/chitin matrix and deglycosylates the proteins. The cell wall proteins were then characterized by SDS–PAGE and identified by proteomic analysis. The analyses for C. albicans identified 15 cell wall proteins and six secreted proteins. For N. crassa, the analyses identified 26 cell wall proteins and nine secreted proteins. Most of the C. albicans cell wall proteins are found in the cell walls of both yeast and hyphae cells, but some cell type-specific cell wall proteins were observed. The analyses showed that the pattern of cell wall proteins present in N. crassa vegetative hyphae and conidia (asexual spores) are quite different. Almost all of the cell wall proteins identified in N. crassa have close homologs in the sequenced fungal genomes, suggesting that these proteins have important conserved functions within the cell wall.     

A RELATIONSHIP BETWEEN CELL WALL STRUCTURE AND COLONIAL GROWTH IN NEUROSPORA CRASSA

de Terra, Noël, and E. L. Tatum. (Rockefeller Inst., New York, N. Y.) A relationship between cell wall structure and colonial growth in Neurospora crassa . Amer.Jour. Bot. 50(7): 669–677. Illus. 1963.—Cell walls were isolated from morphological mutants of Neurospora crassa and from their corresponding wild-type strains. Acid hydrolysates prepared from these cell walls were then studied, qualitatively and quantitatively, with respect to their reducing sugar content. Paper chromatography revealed the presence of glucose and glucosamine in the cell walls of all strains studied. Quantitative analysis has shown that a group of 4 colonial mutants which strongly resemble each other in morphology all have significantly less glucose and more glucosamine per unit weight of cell wall than do their corresponding wild-type strains. These data strongly suggest that a particular type of morphological aberration in Neurospora is associated with similar changes in cell wall composition.     

Studies on the fungitoxicity of captan

The cytological changes in Neurospora crassa conidia following treatment with captan at the ED ₅₀ fungicidal dose have been examined by electron microscopy. With dormant conidia the only observable effect of captan was to produce a characteristic convoluted form of the nuclear membrane. It is suggested that this effect may be due to the reaction of captan with the sulphydryl groups of the nuclear proteins leading to an inhibition of cell division. The cytoplasmic membrane was unaffected by captan, confirming that this fungicide does not destroy cell permeability barriers. After incubation in Fries medium, captan-treated spores showed an almost complete loss of internal fine structure. Similar results were obtained with ‘protoplasts’ of N. crassa which showed disorganization of mitochondrial structure after captan treatment.     

Formation and regeneration of protoplasts of wild-typeNeurospora crassa

Trichoderma reesei was grown using purified cell walls ofNeurospora crassa as a primary source of carbon. The resulting culture medium contained an undefined mixture ofN. crassa cell-wall digesting enzymes. Protoplasts (cell lacking wall) were formed when youngN. crassa hyphae were treated withTrichoderma mixture. The vast majority of protoplasts resynthesized cell-wall material when washed free of cell-wall digesting enzyme; of these, about 40% regenerated a mycelium.     

Genetic variation in the orientation of nuclear spindles during the development of asci in Neurospora

The effect of a single mutant gene, peak-2, in Neurospora crassa is to alter the ascus from one in which the eight spores are linearly arranged to one in which the eight spores are arranged non-linearly. The same gene transferred to N. tetrasperma determines an ascus similar to that of the mutant N. crassa but in striking contrast to the normal ascus for N. tetrasperma, which includes four heterokaryotic spores, linearly arranged. A comparative cytological study has been made on asci from both species, including material from zygotes that are homozygous normal, heterozygous for peak-2, and homozygous for peak-2. The observed morphogenetic differences are closely related to differences in nuclear spindle orientation, which in turn are correlated with the geometry of the cell. The observations are shown to bear on the basis of pseudohomothallism in N. tetrasperma. Speculations on the importance of the cell wall in relation to spindle orientation are presented. Differences of expression of peak-2 in the different species are discussed.     

Effects of media composition on submerged culture spores of the entomopathogenic fungus,Metarhizium anisopliae var. acridum Part 2: Effects of media osmolality on cell wall characteristics,carbohydrate concentrations,drying stability,and pathogenicity

This study evaluates osmolality of a submerged conidia-producing medium in relation to the following spore characteristics: yield, morphology (dimensions and cell wall structure), chemical properties of cell wall surfaces (charge, hydrophobicity, and lectin binding), cytoplasmic polyols and trehalose, and performance (drying stability and pathogenicity). Spore production was increased by the addition of up to 150 g l^?1 polyethylene glycol 200 (PEG). Spores from high osmolality medium (HOM spores) containing 100 g l^?1 PEG had thin cell walls and dimensions more similar to blastospores than submerged conidia or aerial conidia. However, a faint electron-dense layer separating primary and secondary HOM spores’ cell walls was discernable by transmission electron microscopy as found in aerial and submerged conidia but not found in blastospores. HOM spores also appeared to have an outer rodlet layer, unlike blastospores, although it was thinner than those observed in submerged conidia. HOM spores’ surfaces possessed hydrophobic microsites, which was further evidence of the presence of a rodlet layer. In addition, HOM spores had concentrations of exposed N-acetyl-β-d-glucosaminyl residues intermediate between blastospores and submerged conidia potentially indicating a masking of underlying cell wall by a rodlet layer. All spore types had exposed α-d-mannosyl and/or α-d-glucosyl residues, but lacked oligosaccharides. Similar to blastospores, HOM spores were less anionic than submerged conida. Although HOM spores had thin cell walls, they were more stable to drying than blastospores and submerged conidia. Relative drying stability did not appear to be the result of differences in polyol or trehalose concentrations, since trehalose concentrations were lower in HOM spores than submerged conidia and polyol concentrations were similar between the two spore types. HOM spores had faster germination rates than submerged conidia, similar to blastospores, and they were more pathogenic to Schistocerca americana than submerged conidia and aerial conidia.     

Iron limitation and its effect on membrane proteins and siderophore transport inNeurospora crassa

Summary Cells of the fungusNeurospora crassa were grown under iron-deficient and iron-sufficient conditions and their plasma membrane proteins were compared. Three strains were studied:N. crassa 74A (wild type), a siderophore-free mutantN. crassa (arg-5 ota aga) as well as a slime variant ofN. crassa which lacks a cell wall. Plasma membranes were purified, solubilized and analyzed by one-dimensional SDS/polyacrylamide gel electrophoresis yielding approximately 50 distinct protein bands with molecular masses in the range 14–160 kDa. Iron-sufficient and iron-deficient growth resulted in nearly identical plasma membrane protein profiles in all strains. Although minor alterations in the proportion of certain proteins could be detected, significant overproduction of certain membrane proteins during iron limitation could not be observed. Transport of⁵⁵ Fe-labeled siderophores seems to be correlated to the degree of iron limitation. For example, transport rates were enhanced five-fold after 16 h of growth in iron-deficient medium compared to growth in iron-sufficient medium. Extraction and HPLC measurement of siderophores from conidiospores yielded approximately 10^–15 mol/spore, indicating that germination tubes and young cells used for transport measurements are not iron-deficient. It is suggested that the putative transport systems for siderophores in fungal plasma membranes are constitutively expressed and enhanced uptake of siderophores during iron limitation is rather the result of cellular transport regulation mechanisms.     

Functional stratification of the Spitzenkörper of Neurospora crassa

GS‐1 (ncu04189) is a protein required for the synthesis of β‐1,3‐glucan in Neurospora crassa. As chitin, β‐1,3‐glucan is a morphogenetically relevant component of the fungal cell wall. Previously, we showed that chitin synthases are delivered to the growing hyphal tip of N. crassa by secretory microvesicles that follow an unconventional route and accumulate in the core of the Spitzenkörper (Spk). Tagged with the green fluorescent protein (GFP), GS‐1 accumulated in the hyphal apex forming a dynamic and pleomorphic ring‐like structure (‘Spitzenring’) that corresponded to the Spk outer macrovesicular stratum and surrounded the inner core of chitin synthase‐containing microvesicles. TIRF microscopy revealed that GS‐1‐GFP reached the hyphal apex as a population of heterogeneous‐size particles that moved along defined paths. On sucrose density gradients, GS‐1‐associated particles mainly sedimented in a high density range 1.1272–1.2124 g ml^?1. Clearly, GS‐1 and chitin synthases of N. crassa are contained in two different types of secretory vesicles that accumulate in different strata of the Spk, a differentiation presumably related to the spatial control of cell‐wall synthesis.     

Reorganization of plasma membrane lipid domains during conidial germination

Neurospora crassa, a filamentous fungus, in the unicellular conidial stage has ideal features to study sphingolipid (SL)-enriched domains, which are implicated in fundamental cellular processes ranging from antifungal resistance to apoptosis. Several changes in lipid metabolism and in the membrane composition of N. crassa occur during spore germination. However, the biophysical impact of those changes is unknown. Thus, a biophysical study of N. crassa plasma membrane, particularly SL-enriched domains, and their dynamics along conidial germination is prompted.Two N. crassa strains, wild-type (WT) and slime, which is devoid of cell wall, were studied. Conidial growth of N. crassa WT from a dormancy state to an exponential phase was accompanied by membrane reorganization, namely an increase of membrane fluidity, occurring faster in a supplemented medium than in Vogel's minimal medium. Gel-like domains, likely enriched in SLs, were found in both N. crassa strains, but were particularly compact, rigid and abundant in the case of slime cells, even more than in budding yeast Saccharomyces cerevisiae. In N. crassa, our results suggest that the melting of SL-enriched domains occurs near growth temperature (30 °C) for WT, but at higher temperatures for slime. Regarding biophysical properties strongly affected by ergosterol, the plasma membrane of slime conidia lays in between those of N. crassa WT and S. cerevisiae cells. The differences in biophysical properties found in this work, and the relationships established between membrane lipid composition and dynamics, give new insights about the plasma membrane organization and structure of N. crassa strains during conidial growth.     

Characterization of the cell wall of the unicellular cyanobacterium Synechocystis PCC 6714

Cell walls free of cytoplasmic- and thylakoid membranes were isolated from Synechocystis PCC 6714 by sucrose density gradient centrifugation and extraction with Triton X-100. The Triton-insoluble cell wall fraction retained the multilayered fine structure. Peptidoglycan, proteins, polysaccharides, lipopolysaccharides, lipids and carotenoids were found as constituents of the cell wall. Polypeptide and lipid patterns of cell walls were completely different from that of the cytoplasmic/thylakoid membrane fraction. The purified cell walls contained about twelve outer membrane proteins. The two major polypeptides (M_r 67,000 and 61,000) were found to be associated with the peptidoglycan by ionic interactions.Myxoxanthophyll (major carotenoid), related carotenoid-glycosides and zeaxanthin were the predominating carotenoids of the cell wall of Synechocystis PCC 6714 over echinenone and -carotene. A polar unknown carotenoid was observed, the absorption spectrum of which resembled that of myxoxanthophyll. It was exclusively found in cell walls, but not in the cytoplasmic/thylakoid membrane fraction.Abbreviations Hep heptose - DGDG digalactosyldiglyceride - MGDG monogalactosyldiglyceride - SL sulfolipid - PC phosphatidylcholin - PG phosphatidylglyceride Dedicated to Prof. Dr. G. Drews on the occasion of his 60th birthday     

Small stable RNA of Neurospora crassa.

Summary Small stable RNAs in wild-type Neurospora crassa were investigated by analyzing the cell contents of long term ³²P_i labeled cultures in thin slab polyacrylamide gels. Because of the rigid fungal cell wall and the potency of nucleases the degradation of RNA in opening the cells was rather extensive. Some of these degradation problems were circumvented by using a slime strain of N. crassa which lacks a rigid cell wall. Our findings show that N. crassa. like many other eukaryotes, contains a number of small stable RNA molecules. We also found that the ribosomal RNA, the so called 5.8S, migrates slower on polyacrylamide gels than the 6S RNA of E. coli, which contains 184 nucleotides. The relative migration of the molecules was not changed when the samples were denatured prior to electrophoresis. The mobility of the Neurospora rRNA molecule suggested a chain length of 220 nucleotides. Fingerprinting of a T₁ ribonuclease digest indicated a chain length of 212 nucleotides. Because of the unusually large size of the so-called 5.8S rRNA we found it more appropriate to refer to this molecule as a 7S rRNA. It seems that the N. crassa 7S rRNA is the largest low molecular weight ribosomal RNA studied thus far.     

Studies on the fungitoxicity of captan

The cellular distribution of ³⁵S after incubating labelled captan with Neurospora crassa conidia has been determined. Nearly all the ³⁵S in the spores is bound to the water-soluble and protein fractions. Thin-layer chromatography of the hot-water extract of spores has shown that ³⁵S occurs largely in oxidized glutathione (GSSG) and in a product tentatively identified as a thiazolidine derivative of glutathione. It is suggested that this derivative, which only forms above pH 6·5, is produced by reaction between glutathione (GSH) and the thiocarbonyl chloride liberated on the decomposition of captan. Captan toxicity could not be completely reversed by pretreatment with thiols and disulphides capable of penetrating the cell membrane, confirming the previous hypothesis that fungitoxicity is due to irreversible changes following the oxidation of the protein thiols to disulphides.     

THE FINE STRUCTURE OF STREPTOMYCES VIOLACEORUBER (S. COELICOLOR) : III. The Walls of the Mycelium and Spores

A study of thin sections of hyphae of Streptomyces violaceoruber in the electron microscope showed that the structure of the walls and the mode of formation of cross-walls are similar to those of Gram-positive bacteria. A beaded structure was seen in some regions of the wall, and the significance of this observation is discussed in relation to previous studies of the fine structure of bacterial cell walls. Elements of the intracytoplasmic membrane system appear to be involved in the process of cross-wall formation. The walls of the hyphae of the aerial mycelium divide into two layers before the spores are formed, and only the inner component of the wall grows inwards to form the cross-walls and so delimit the spores. The outer component remains intact for a time and acts as a sheath around the developing spores. Finally the sheath breaks and the spores are liberated. This process is contrasted with the formation of endospores in eubacteria. When the spores germinate, the walls of the germ tubes are continuous with those of the spores.     

Ion-exchange properties of the cell wall of reindeer lichen <Emphasis Type="Italic">Cladonia rangiferina</Emphasis>

Ion-exchange properties of cell walls were investigated in reindeer lichen Cladonia rangiferina (L.) F. H. Wigg. In order to isolate cell walls, we used living parts of podetia as well as young parts (four upper internodes of podetia) and old parts (from the 4th to the 8th internode). We studied functional dependences of cell wall ion-exchange capacity on pH in the range from 2 to 12 and constant ionic strength of solution equal to 10 mM. It was found that three-dimensional structure of C. rangiferina cell walls comprised three types of ionogenic groups, which determine ion-exchange properties of the cell walls. They are amino groups with pK_a of about 3, carboxyl groups with pK_a of about 7, and phenolic OH-groups with pK_a of about 10. The content of groups of each type and their ionization constants were determined, and it was shown that, in the cell walls of young parts, the content of amino groups and carboxylic groups was greater than in old parts of podetia (by 1.5 and 2.0 times, respectively). It was found that with age the content of nitrogen and the proportion of deacetylated amino groups in the cell walls changed from 34% (young parts of podetia) to 40% (old parts of podetia). It was shown that in C. rangiferina N-acetyl glucosamine and glucosamine are not the main monomers of cell wall polymers because both in thalli and in the cell walls isolated therefrom the content of total nitrogen was less than 1%.     

Ion exchange properties of plant root cell walls

Acid-base properties and the swelling capacity of wheat, lupin and pea root cell walls were investigated. Roots of seedlings and green plants of different age were analysed by the potentiometric method. The ion exchange capacity (S _i) and the swelling coefficient (K ^cw) of root cell walls were estimated at various pH values (from 2 to 12) and at different ionic strength (between 0.3 and 1000 mM). To analyse the polysigmoid titration curves pH_i = f (S _i), the Gregor's equation was employed. It was shown that the Gregor's model fits well the experimental data. The total number of the cation exchange (S _t ^cat) and the anion exchange (S _t ^an) groups were determined in the root cell walls. The number of the functional group of each type (S ^j) was estimated, and the corresponding values of pK _a ^j were calculated. It was shown that for all types of cation exchangeable groups arranged in the cell wall structure the acid properties are enhanced by the increasing concentration of electrolyte. For each ionogenic group the coefficients of Helfferich's equation [pK _a ^j = f (C ^K+)] were determined. It was found that the swelling of root cell walls changes with pH, C ^K+ and strongly depends on plant species. Within the experimental pH and C ^K+ range the swelling coefficient changes as follows: lupin > pea > wheat. The obtained results show that for the plant species under investigation the differences in the swelling coefficients originate from (a) the differences in the cross-linking degrees of polymeric chains arranged in the cell wall structure, (b) the differences in the number of carboxyl groups and (c) the differences in the total number of functional groups. Based on the estimated swelling coefficients in water it could be inferred that for wheat the cross-linking degree of the polymeric chains in the root cell walls is higher than those for lupin or pea. It has been emphasized that the calculated parameters (S ^j, pK _a ^j, K ^cw), the equation {pK _a ^j = f (C^K+)} and the dependencies {K ^cw = f (C^K+, pH)} allow to estimate quantitatively the changes in the ion exchange capacity of the root cell walls in response to the changes in an ionic composition of an outer solution. The results of these estimations allow to suggest that (a) the root apoplast is a compartment where the accumulation of cations takes place during the first stage of cation uptake from an outer medium, and (b) the accumulation degree is defined by pH and ionic composition of an outer solution. On the basis of the literature review and the results of the present experimental study it was proposed that the changes in the cell wall swelling in response to variances of environmental or experimental conditions could lead to a change of the water flow through a root apoplast. It has been supported that there is direct relationship between the swelling of root cell walls and the water flow within the plant root apoplast.     

Barrier function of the cell wall during uptake of nickel ions

Cell walls were isolated from roots of six plant species to study their ion-exchange capacity for nickel ions (S _Ni) at Ni²⁺ concentration of 10⁻³ M. The S _Ni values varied depending on the plant species from 50 to 150 μmol Ni²⁺ per gram dry wt; the sorption capacity increased in a row: Poaceae < Chenopodiaceae < Fabaceae. At pH 5 the sorption capacity of cell walls for nickel ions was determined by the presence of carboxyl groups of polygalacturonic acid in the polymeric cell-wall matrix. In all cases the ion-exchange capacity of cell walls was higher at pH 8 than at pH 5, indicating that Ni²⁺ binds also to a carboxyl group different from that of polygalacturonic acid. Irrespective of plant species, the presence of EDTA in the solution diminished drastically the absorption capacity of cell walls for Ni²⁺. It is concluded that the presence of 10⁻³ M EDTA weakens the defense properties of cell walls. The sequestration of Ni²⁺ in the cell wall can be considered as an effective means of plant cell defense against elevated concentrations of nickel ions in the external medium.     

Characterization of the Neurospora crassa DHN melanin biosynthetic pathway in developing ascospores and peridium cells

Neurospora crassa contains all four enzymes for the synthesis of DHN (dihydroxynaphthalene), the substrate for melanin formation. We show that the DHN melanin pathway functions during N. crassa female development to generate melanized peridium and ascospore cell walls. N. crassa contains one polyketide synthase (PER-1), two polyketide hydrolases (PKH-1 and PKH-2), two THN (tetrahydroxynaphthalene) reductases (PKR-1 and PKR-2), and one scytalone dehydratase (SCY-1). We show that the PER-1, PKH-1, PKR-1 and SCY-1 are required for ascospoer melanization. We also identified the laccase that functions in the conversion of DHN into melanin via a free radical oxidative polymerization reaction, and have named the gene lacm-1 (laccase for melanin formation-1). In maturing perithecia, we show that LACM-1 is localized to the peridium cell wall space while the DHN pathway enzymes are localized to intracellular vesicles. We present a model for melanin formation in which melanin is formed within the cell wall space and the cell wall structure is similar to “reinforced concrete” with the cell wall glucan, chitin, and glycoproteins encased within the melanin polymer. This arrangement provides for a very strong and resilient cell wall and protects the glucan/chitin/glycoprotein matrix from digestion from enzymes and damage from free radicals.     

Ion-exchange properties of cell walls of red seaweed <Emphasis Type="Italic">Phyllophora crispa</Emphasis>

Research into ion-exchange properties of cell walls isolated from thallus of red seaweed Phyllophora crispa was carried out. Ion-exchange capacity and the swelling coefficient of the red alga cell walls were estimated at various pH values (from 2 to 12) and at constant ionic strength of a solution (10 mM). It was established that behavior of cell walls as ion-exchangers is caused by the presence in their matrix of two types of cation-exchange groups and amino groups. The amount of the functional group of each type was estimated, and the corresponding values of pK _a were calculated. It can be assumed that ionogenic groups with pK _a ∼5 are carboxyl groups of uronic acids, and ionogenic groups with pK _a ∼7.5 are carboxyl groups of the proteins. Intervals of pH in which cation-exchange groups are ionized and can take part in exchange reactions with cations in the environment are defined. It was found that protein was a major component of cell wall polymeric matrix because its content was 36%.     

Ion-exchange properties of the cell walls isolated from suspension-cultured plant cells

Ion-exchange capacity of the cell walls isolated from suspension-cultured Panax japonicus, Polyscias filicifolia and Dioscorea deltoidea cells was analyzed at pH 2.8–12 and constant ionic strength (100 mM). The cell walls of all cultures contain three types of ion-exchange groups: primary amino groups (pK _a < 3), carboxyl groups of polygalacturonic acid (pK _a 3.71), and carboxyl groups of hydroxycinnamic acids (pK _a 7.62). Amount of primary amino groups ranges from 500 (D. deltoidea) to 710 (P. japonicus) µmol/g cell wall dry weight, carboxyl groups with pK _a 3.71—from 570 (D. deltoidea) to 670 (P. filicifolia), carboxyl groups with pK _a 7.62—from 270 (P. filicifolia) to 370 (P. japonicus) µmol/g cell wall dry weight. The comparison of the data obtained by elemental and functional analyses demonstrated that the cell walls of all cultures are characterized by high content of pectins (~40% by weight) and structural proteins (~17–30% by weight), but do not contain phenolic OH–groups, which presumably signifies the absence of lignin in them.     

Role of cell wall bound calcium in Neurospora crassa

Cell wall bound calcium constitutes a significant fraction (25%) of total mycelia calcium in Neurospora crassa. Wall bound calcium increases as a function of growth and calcium concentration, while cell wall bound calcium decreases in Ca-free medium. Removal of wall bound calcium causes its rapid replacement from intracellular pool, inhibited by verapamil, nifedipine, concanamycin A, and wortmanin in a vacuolar mutant (Vma-5), but is unaffected by trifluoropyrazine, and calmidizoluim in a calcineurin mutant (Cnb-1) of N. crassa. Ca²⁺ removal from surface with EGTA resulted in leakage of periplasmic enzymes invertase and alkaline phosphatase. Scanning and transmission electron microscopy revealed gross abnormalities represented by giant vacuoles. Toxic metal ions bound to wall fraction by displacing calcium. Our data underline the physiological importance of wall bound calcium in N. crassa.