Beta-glucanases in the yeast Cryptococcus albidus var. aerius. Production and separation of beta-glucanases in asynchronous cultures.

beta-Glucanases were detected in cell-free extracts of the yeast Cryptococcus albidus var. aerius when grown on glucose as the sole carbon source. The production of beta-glucanases was followed in log-phase cells and stationary-phase cells; the maximal production of beta-(1 leads to 3) and beta-(1 leads to 6) glucanases takes place respectively in log-phase and stationary-phase cells. The results show that there are marked differences in the elution profiles on Sephadex G-50 of fractions containing beta-glucanase from cells grown for 12, 24, 48, 72, and 96 h. The possibility either of replacement changes in fractions containing beta-glucanase activity or of a different synthesis of each beta-glucanase during the growth of the yeast is discussed. The results suggest that all fractions containing beta-glucanases hydrolyze both beta-(1 leads to 3) and beta-(1 leads to 6) linkages. Evidence in support of the conclusion that a low molecular form of beta-glucanase has a molecular weight of 2100 +/- 100 is also shown.     

Cell wall-associated 1,4-beta-D-xylanase in Cryptococcus albidus var. aerius: in situ characterization of the activity.

1,4-beta-D-Xylanase (1,4-beta-D-xylan xylanohydrolase; EC 3.2.1.8) has been detected in both cell-free extracts and culture fluids of the yeast Cryptococcus albidus var. aerius grown on glucose as the only carbon source. Mild acid treatment of whole cells proved that the enzyme was extracellularly located. The activity remained almost completely linked to the wall after cell breakage, only being liberated in the presence of salt at high concentration. After release, the enzyme became very unstable and so has been characterized in situ in 'permeabilized' cells. The maximum production took place at the beginning of the exponential growth phase. The optimum pH and temperature for activity were 5.0 and 40 degrees C, respectively. The enzyme degraded xylan and xylo-oligosides by an endo-splitting mechanism giving xylobiose, xylotriose and xylose as the main end-products. Activation energy and kinetic constants for xylan degradation were determined. Several metal ions such as Ag+ and Hg2+ inhibited the enzyme. The possible function of this endo-xylanase in Cr. albidus var. aerius is discussed.     

Comparison of serological and chemical characteristics of capsular polysaccharides of Cryptococcus neoformans var. neoformans serotype A and Cryptococcus albidus var. albidus

The antigenic formula and chemical structure of capsular polysaccharide (CPS) of Cryptococcus albidus var. albidus (C. albidus) were studied in relation to those of C. neoformans var. neoformans serotype A (C. neoformans A). The results of slide agglutination tests with factor sera and reciprocal adsorption experiments showed that antigenic formula of C. albidus was the same as that of C. neoformans A. The soluble CPSs from the two species were obtained from culture supernatants by precipitation with ethanol followed by purification by chromatography on DEAE-cellulose column. The structural analyses of such CPSs from the two species showed that the antigenic CPS fractions consisted of a backbone of alpha(1-3)-linked D-mannopyranosyl residues with a single branch of beta(1-2)-xylose or glucuronic acid, and mostly with O-acetyl groups, in which side chains and O-acetyl groups were responsible for antigenic specificity. It was found that there was a minor difference between the CPS of C. neoformans A and that of C. albidus; in the former, unsubstituted mannose residues existed in a low frequency, but in the latter none. Moreover, the 1H-nuclear magnetic resonance spectra of partially hydrolyzed acidic fragments of the two CPSs indicated that two xylose side chains were present between glucuronic acid side chains. Taken together, it was suggested that these two species of C. neoformans A and C. albidus are closely related to each other in their CPSs.     

Xylan-degrading enzymes of the yeast Cryptococcus albidus. Identification and cellular localization

During growth on wood beta-1,4-xylans the yeast Cryptococcus albidus produced at least two enzymes which convert the polysaccharide to xylose catabolized by the cells. The enzyme almost completely secreted into culture fluid was identified as an endo-1,4-beta-xylanase. The function of the extracellular beta-xylanase is to hydrolyze xylan to oligosaccharides, mainly to xylobiose and xylotriose, which enter the cell where they are split by the second identified enzyme, a cell-bound beta-xylosidae (xylobiase). Aryl beta-xylosidase activity detected in the culture fluid was snown to be due to low affinity of beta-xylanase for p-nitrophenyl beta-D-xylopyranoside. This property of beta-xylanase was preserved after purification of the enzyme by chromatography on DEAE-cellulose, CM-Sephadex and Biogel A 1.5 m or Biogel P 100. Purified beta-xylanase exhibited certain microheterogeneity after polyacrylamide gel electrophoresis. Both extracellular beta-xylanase and intracellular beta-xylosidase were produced in much lower amounts by the cells grown on glucose than by the cells grown on xylan. This suggested that they are not produced constitutively. The investigated strain was not able to grow on cellulose and the crude and purified beta-xylanase were unable to hydrolyze cellulose or its soluble derivatives.     

Effect of tunicamycin on xylanase secretion in the yeast Cryptococcus albidus

The yeast Cryptococcus albidus secretes a glycosylated xylanase (48 kDa) in the culture medium in response to beta-methylxyloside as inducer. Addition of tunicamycin to the medium results in the formation of a modified xylanase (40 kDa) which is depleted in carbohydrate content and whose enzymatic activity is 2.5 times less than that of the glycosylated xylanase. The secretion of xylanase was followed under both conditions by pulse-chase experiments. The half-time of secretion of the glycosylated and nonglycosylated forms was 5 and 2 h, respectively. Cell-associated xylanase activity was not detected when the cells were treated with the antibiotic. The absence of cell wall-associated xylanase, after tunicamycin treatment, was confirmed by immunolocalization with anti-xylanase antibodies at the electron microscopic level. The results suggest that the interactions of carbohydrate moiety within the cell wall retarded the secretion of the enzyme to the medium.     

Beta-Xylosidases and a nonspecific wall-bound beta-glucosidase of the yeast Cryptococcus albidus

Cryptococcus albidus grown on wood xylans possesses a soluble intracellular beta-xylosidase (EC 3.2.1.37) as an additional constituent of the xylan-degrading enzyme system of this yeast. The enzyme attacks linear 1,4-beta-xylooligosaccharides in an exo-fashion, liberating xylose from the non-reducing ends. The activity of the enzyme increases in the cells during growth on xylan and incubation with xylobiose or methyl beta-D-xylopyranoside which are the best inducers of extracellular beta-xylanase (EC 3.2.1.8). Various alkyl-,alkyl-1-thio- and aryl beta-D-xylopyranosides were excellent inducers of a different beta-xylosidase of Cryptococcus albidus. This enzyme is localized outside the plasma membrane and is principally associated with cell walls. Unlike the soluble intracellular beta-xylosidase, the wall-bound enzyme does not hydrolyze xylooligosaccharides. Evidence has been obtained that beta-xylosidase activity in the cell walls is not due to the presence of a specific aryl beta-xylosidase, but is exhibited by a nonspecific beta-glucosidase (EC 3.2.1.21) inducible by beta-D-xylopyranosides. The ratio of beta-glucosidase and beta-xylosidase activity in the cells and isolated cell walls from yeast induced by various beta-xylopyranosides and beta-glucopyranosides was very similar. Both wall-bound activities were inhibited in a similar pattern by inhibitors of beta-glucosidases, 1,5-gluconolactone and nojirimycin. This bifunctional enzyme does not bear any relationship to the utilization of xylans in Cryptococcus albidus.     

Ability of the antagonistic yeast Cryptococcus albidus to control Botrytis cinerea in strawberry

The yeast Cryptococcus albidus, originally isolated from mature strawberry fruits, was tested for antagonistic activity against Botrytis cinerea, the causal agent of grey mould in strawberries. Conidial germination and germ tube growth of conidia of B. cinerea were inhibited by a cell suspension of the antagonist in aqueous strawberry fruit pulp suspension (1%) after 6 and 24 hours of incubation. Application of a cell suspension (1 × 10⁶ cells/ml) on detached strawberry leaf disks incubated at 10°C reduced incidence and conidiophore density of B. cinerea by 86 and 99%, respectively, but effectiveness was reduced at higher temperatures. Treatments with C. albidus during bloom of strawberries reduced incidence of grey mould on ripe strawberry fruits after harvest by 33, 28 and 21% in three years of field trials. The effectiveness of the yeast was increased when formulation substances (alginate, xanthan and cellulose) were added to the cell suspension.     

Ploidy differences in Cryptococcus albidus

Presumed haploid and diploid cultures ofCryptococcus albidus were analysed for their DNA content per cell. A ratio of approximately 1:2 was obtained by relating the DNA content per cell of the two phases to ploidy. As the diplophase formed neither longitudinally septated cells nor ballistoconidia, the earlier suggestions thatCryptococcus is closely related toTremella seems less likely. On the assumption that the metabasidia ofCryptococcus are gastromycetoid, a closer relationship between this genus and the tulasnelloid fungi appears more probable.Microbiology Research Group, South African Council for Scientific and Industrial Research, Pretoria, South Africa.     

Cloning and expression in Escherichia coli of a xylanase-encoding gene from the yeast Cryptococcus albidus.

In the yeast, Cryptococcus albidus, a comparison between the sequence of the xylanase (XLN)-encoding chromosomal gene (XLN) and the cDNA sequence reveals the presence of seven introns, ranging in length from 51 to 69 bp. One of their 5' splice site sequences is similar to the consensus sequence for yeast, while the other six resemble the consensus sequence for higher eukaryotes. Their 3' end splice site sequences are representative of the conserved sequence found in eukaryotes. Their putative branching point sequences are different from the well-known conserved sequence, 5'-TACTAAC, observed in yeast, but again resemble the mammalian one. The cDNA encoding XLN is expressed by Escherichia coli, under the control of the lacZ promoter. The gene product remains inside the cell and has a molecular size of 40 kDa, which matches the size of the nonglycosylated protein. When compared to the glycosylated enzyme, the nonglycosylated XLN from E. coli shows twofold less affinity for substrate and its Vmax is 100-fold lower. Moreover, the nonglycosylated XLN only acts on large xylan polymers and very slightly on xylohexaose.     

Cryptococcus antarcticus var. circumpolaris var. nov., a basidiomycetous yeast from Antarctica

Cryptococcus antarcticus Vishniac & Kurtzman var. circumpolaris Vishniac and Onofri var. nov. (Filobasidiales, Tremellomycetidae, Hymenomycetes), an anamorphic yeast with ca. 73% nDNA relatedness to Cryptococcus antarcticus var.antarcticus, differs in failure to assimilate raffinose, a lower maximum temperature for growth, fatty acid profile, and in a single nucleotide change in the D2 region of LSU rDNA. This revised version was published online in June 2006 with corrections to the Cover Date.     

Recognition of cytoplasmic yeast antigens of Cryptococcus neoformans var. neoformans and Cryptococcus neoformans var. gattii by immune human sera

The humoral immune response of patients infected with Cryptococcus neoformans var. neoformans and C. neoformans var. gattii to cytoplasmic (non-capsular) antigens from the two varieties of Cryptococcus has been investigated. Cytoplasmic antigens from C. neoformans (one clinical isolate and one acapsular mutant of var. neoformans and two clinical isolates from var. gattii) were subject to isoelectric focusing, SDS-PAGE and Western blotting; patients sera was then used in the immunoenzyme development of the Western blots. The humoral response from the 20 patients (all HIV+) infected with var. neoformans against the var. neoformans antigens was predominantly IgG based, with a large number of bands recognised; the most commonly recognised bands were at 26, 52, 74, 100, 115 and 144 kDa. The IgM response was less pronounced and the IgA response was practically non-existent. The humoral response of the sera from the 15 patients (all but one HIV-) infected with var. gattii against var. gattii antigens was also predominantly IgG based with bands at 37, 55, 65, 74, 94 and 115 kDa being most commonly recognised. Periodate treatment of cytoplasmic antigens reduced the intensity of antigen recognition, though it did not absolutely destroy reactivity to any individual antigen. Comparison of immunodevelopment of cytoplasmic antigens from both varieties grown at 25°C and 37°C revealed that culture temperature made no differences in the number of bands recognised although there were differences in the intensity of recognition. This is the first report on the pattern of serological recognition of the non-capsular antigens from the two varieties of Cryptococcus and it identifies a number of major antigenic components.     

Positional isomers of thioxylobiose, their synthesis and inducing ability for D-xylan-degrading enzymes in the yeast Cryptococcus albidus.

Isomeric S-linked 2-thioxylobiose 10, 3-thioxylobiose 17, and 4-thioxylobiose 19 were conveniently prepared by SN2 displacement of suitable triflylglycoses with the sodium salt of 2,3,4-tri-O-acetyl-1-thio-beta-D-glucopyranose, either in N,N-dimethylformamide, or in oxolan in the presence of a sodium complexing agent. Allyl 3,5-O-isopropylidene-2-O-trifluoromethanesulfonyl-beta-D-lyxofu ranoside was a convenient electrophilic precursor for 10, which was smoothly obtained after a short sequence of deprotection involving conversion to the 1-propenyl glycoside. 1,2:5,6-Di-O-isopropylidene-3-O-trifluoromethylsulfonyl-alpha-D-++ +allofuranose and 1,2,3-tri-O-benzoyl-4-O-trifluoromethylsulfonyl-beta-L-arabinop yranose were the respective precursors for 17 and 19. 4-Thioxylobiose has a highly stimulatory effect on the synthesis of enzymes of the xylanolytic system in the yeast Cryptococcus albidus when applied to the cells in the presence of the natural disaccharide inducer (1----4)-beta-D-xylobiose.     

Diversity in the yeast Cryptococcus albidus and related species as revealed by ribosomal DNA sequence analysis

Evidence accumulated from studies based on physiological, biochemical, and molecular characteristics has pointed to the heterogeneity of the ubiquitous anamorphic basidiomycetous yeast species Cryptococcus albidus (Saito) Skinner, with its current varieties and synonyms. The taxonomic status of this species has not been reappraised because different studies, mostly involving limited numbers of strains, have not been integrated. To assess species diversity within the clade containing Cryptococcus albidus and other phylogenetically related Cryptococcus and Filobasidium species, we determined ribosomal DNA (rDNA) sequences of 69 strains from the 5' end of the 26S gene, D1/D2 region, and in some cases, the non-coding ITS2 region. Analysis of the sequence data together with available physiological, biochemical, and molecular characteristics, showed the segregation of C. albidus into at least 12 species, leading to the elevation of former varieties to the rank of species (C. aerius, C. diffluens), the reinstatement of synonyms (C. liquefaciens, C. terricola), and the proposal of new species (C. arrabidensis, C. chernovii, C. cylindricus, C. oeirensis, C. phenolicus, C. saitoi, C. uzbekistanensis, C. wieringae). The overall analyses of the results argue in favour of the use of rDNA sequence data to improve species delineation when integrated with other available physiological and molecular characteristics.     

Translation mobility of water isolated in the cells of lyophilized Cryptococcus albidus var. diffluens yeasts by the NMR impulse method

The method of NMR spin echo in a combination with the impulse gradient of the magnetic field was used to study the self-diffusion of water isolated in the cells of lyophylized Criptococcus. A relative fall of the amplitude of spin echo (factor R) in relation to the value of magnetic field impulse gradient (g), their duration (delta) and temporary distance between them (delta). The studies were carried out in the temperature range of 20 degrees divided by 80 degrees C. It has been shown that the fraction of isolated mobile water is located in intracellular permeable compartments with an average size of 0.6.10--4 cm at 20 degrees C. The coefficient of selfdiffusion of isolated water of Criptococcus (0.25.10--5 cm2S--1 at 20 degrees C) and the activation energy of selfdiffusion (4.4 ccal/mole at 20 degrees divided by 60 degrees C) were determined, permeability of walls of water-containing compartments taken into account.     

Simulated in situ competitive ability and survival of a representative soil yeast,Cryptococcus albidus

Microcosms containing an air-dried autoclaved loamy sand (Eufala A) with low salt and organic content were inoculated with a representative (obligately aerobic, encapsulated) soil yeast, Cryptococcus albidus var. albidus ^T ATCC 10666, singly (for growth rate and survival determinations) and together with the bacterial biota native to Eufala A. The yeast competed successfully with the more rapidly growing bacteria in the presence of added water from 1% (5.7% of field capacity) to 14% (80% of field capacity) but grew for shorter times than when grown alone; times correlated with the lag phase of the bacterial biota. When well-watered (10 and 14%) competition cultures were allowed to dry and used as inoculum for subcultures, the yeast made significant growth only at 1% added water but survived at the higher moisture concentrations. The competitive ability of Cr. albidus confirms the previously reported advantages of the cryptococcal capsule in hydration and desiccation and, together with lengthy survival, suggests that the importance of such yeasts in the biogeochemistry of arid soils has been seriously underestimated.     

Molecular expression of xylanase gene in Cryptococcus albidus

In the yeast Cryptococcus albidus, the utilization of xylan as compared to xylose requires at least an inducible endoxylanase enzyme, secreted in the culture medium. The endoxylanase induction was monitored by immunoprecipitation of in vivo and in vitro synthesized products. The mature endoxylanase is a highly glycosylated enzyme with an apparent molecular weight of 48 000. Upon chemical deglycosylation with trifluoromethanesulfonic acid, the molecular weight was reduced to 40 000. Addition of tunicamycin to the culture medium resulted in the synthesis of a modified polypeptide having a molecular weight of 40 000. Poly(A)-containing RNA isolated from the yeast was translated in the rabbit reticulocyte protein-synthesizing system. The appearance of a translatable xylanase mRNA was observed in xylan-grown cells but not in xylose-grown cells. The polypeptide identified as xylanase had a molecular weight of 44 000. This suggests that the xylanase is synthesized as a precursor, containing a peptide signal sequence of 35 residues.     

Production,purification and partial characterization of an endopolygalacturonase fromCryptococcus albidus var.albidus

Cryptococcus albidus var.albidus produced an extracellular endo-polygalacturonase (poly (1,4--d-galacturonide) glycanohydrolase EC 3.2.1.15) when grown in a synthetic medium containing one of a variety of pectic substances or galacturonic acid. The highest level of enzyme activity (15.5 VU-ml^–1) was obtained after 72 h of growth on 1.0% low-methoxyl pectin. The enzyme, purified by gel filtration (Sephadex G-100) after repeated ammonium sulphate precipitation and dialysis, showed only one band by polyacrylamide gel electrophoresis and had the following properties: mol wt (MW_r) 41000 dal; isoelectric point (pl) = 8.10 ± 0.10; optimum temperature and pH for activity around 37°C and pH 3.75, respectively; pH stability in the pH range 4.0 to 8.0; complete heat inactivation after 10 min at 55°C; Km and Vmax values 5.7· 10^–1 mg·ml^–1 and 5.1 · 10^–1 mmoles·min^–1, respectively.     

Ethylene production from l-methionine by Cryptococcus albidus

Cryptococcus albidus IFO 0939 was selected from microorganisms producing ethylene from l-methionine in a culture medium. When methionine was excluded from the culture medium of C. albidus, there was little production of ethylene. Ethylene production in a methionine-containing culture medium occurred for a brief period at the end of the growth phase. 2-oxo-4-methylthiobutyric acid (KMBA), a deaminated product of methionine, accumulated in the culture filtrate. An ethylene-forming enzyme was partially purified from C. albidus by means of DEAE-Sepharose CL-6B ion exchange chromatography, and a cell-free ethylene-forming system was constructed. Using this system, the precursor of ethylene was found to be KMBA and essential factors were NAD(P)H, Fe³⁺, EDTA and oxygen.     

Monitoring growth and lipid production of new isolated oleaginous yeast Cryptococcus aerius UIMC65 on glucose and xylose cultures

Process monitoring is one of the most important factors affecting production efficiency at industrial scale bioprocesses. In the present work, Flow-cytometric analysis has been employed to monitor and determine neutral lipid cell droplets, granularity and size of the cells of the new oleaginous yeast, Cryptococcus aerius UIMC65. It has been shown that, differences of fluorescent intensity as well as side and forward scatter light properties have close correlations with the differences in lipid production by these yeast cells. The lipid content-related fluorescent intensity versus forward scatter parameter has been used to monitor and compare different subpopulations during growth phases on both glucose and xylose in batch cultures. Flow cytometric results have revealed that the observed differences in the proportion of each subpopulation were related to the specific growth phase and lipid content of the cells. The highest lipid content and lipid productivity were attained at 82.62%, 4.47 g/L (at 72 h) and 78.41%, 6.21 g/L (at 60 h) on glucose and xylose growth cultures, respectively. The highest biomass, lipid yield and biomass yield were found to be 7.92 g/L (on glucose culture, at 60 h), 20.92% (on glucose culture, at 48 h) and 50.71% (on glucose culture, at 24 h), respectively.