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PubMed1998

Aryl thioglycoside-based affinity purification of exo-acting cellulases.

The influence of ligand-coupling chemistry and mobile-phase composition on the interaction of exo-acting cellulases with an immobilized complementary ligand was investigated. p-Aminophenyl 1-thio-beta-D-cellobioside (APTC) was used as a representative affinity ligand to which exo-acting cellulases (cellobiohydrolases, CBHs) preferentially bind. A "crude" cellulase preparation from the fungus Trichoderma reesei served as an enzyme source. The adsorption properties of the two principal exo-acting CBHs in this preparation, CBH I and CBH II, are shown to be distinctly different under several scenarios. Their relative affinities, based on column elution behavior and partition equilibrium experiments, are shown to be highly dependent on the functional groups employed for ligand coupling, the extent of functional group hydrolysis, the composition of the mobile phase, and the inherent nature of the enzymes. The dependency on the chemistry of the supporting matrix was illustrated using agarose supports containing cyanate ester, N-hydroxy-succinimide, and epoxy functional groups. When compared under apparent optimal conditions, the affinity of CBH II for immobilized APTC was approximately 10-fold that of CBH I. However, selective adsorption of CBH I or CBH II can be achieved by adjusting experimental parameters.

Basidiomycota↗

PubMed1996

Sensitivity of fungi to nikkomycin Z.

Nikkomycin Z inhibited colony radial extension (Kr) and hyphal density in Geotrichum candidum, Trichoderma koningii, Mucor plumbeus, and Fusarium oxysporum, although the level of inhibition varied with the fungal species and was influenced by the concentration of nutrients in the culture medium. While G. candidum and M. plumbeus were sensitive to the inhibitory effects of nikkomycin Z, T. koningii and F. oxysporum were relatively tolerant of concentrations of the antibiotic up to 100 mumol. Nikkomycin Z also caused significant decreases in rates of hyphal extension (Emax) and swelling and bursting of hyphal apices in G. candidum and M. plumbeus. The sensitivity of G. candidum and M. plumbeus to the effects of the antibiotic was due in part to their ability to remove nikkomycin from a liquid culture medium; the nikkomycin-tolerant species T. koningii and F. oxysporum were unable to transport this nucleoside dipeptide antibiotic. Although both G. candidum and F. oxysporum appeared capable of removing the di- and tripeptides, dialanine and trialanine, from a liquid medium, rate of dipeptide uptake was greater in F. oxysporum, while rate of tripeptide uptake was greater in G. candidum. However, overall rate of uptake of peptides was greater in G. candidum than in F. oxysporum.

Amino Acid Sequence↗

PubMed1998

Effect of Soil Texture and Soil Sterilization on Susceptibility of Ovipositing Grasshoppers to Beauveria bassiana

The effect of conidial concentration, soil texture, and soil sterilization on the efficacy of Beauveria bassiana against ovipositing grasshoppers (Melanoplus sanguinipes) was investigated in a controlled environment. In the first experiment, mortality of female grasshoppers ovipositing into a sterile loamy-sand soil containing conidia of B. bassiana was measured. The prevalence of mortality increased as the concentration of conidia in soil increased, and a median lethal concentration of 10(4) colony-forming units (CFU) per gram of soil (dry weight) was observed. Conidia (10(2.9) to 10(3) CFU per abdomen) were recovered from the abdomens of grasshoppers ovipositing into sand containing 10(5.5) and 10(6) conidia per gram. Similar numbers of eggs were laid among treatments during the first oviposition period (1 to 7 days), but an effect of conidial concentration on eggs laid was observed during the second oviposition period (8 to 14 days). This was attributed to reduction in female numbers and not to reduction in fecundity independent of mortality. In a second experiment, grasshoppers oviposited into soils of three different textures (loamy-sand, sandy-loam, or clay-loam) that were amended with 10(5) B. bassiana conidia per gram and possessed either a viable or heat-killed microflora. There was no effect of soil texture on mortality of ovipositing grasshoppers, on the number of eggs laid, on positioning of egg pods in the soil profile, or on numbers of B. bassiana CFU recovered from female abdomens. However, a higher prevalence of mortality was observed for females ovipositing into the sterilized than nonsterilized sandy-loam and clay-loam soils. Substantial populations of fungi and bacteria were recovered from nonsterilized soils. The predominant fungi isolated from these soils were members of the genera Chrysosporium, Fusarium, Gliocladium, Penicillium, Rhizopus, and Trichoderma, whereas Bacillus, Paenibacillus, and Pseudomonas species were the most commonly isolated bacteria. This study demonstrates that ovipositing grasshoppers are susceptible to relatively low densities of conidia in soils of varying textures, but the soil microflora may have an adverse affect on the efficacy of B. bassiana in field soils. Copyright 1998 Academic Press. Copyright 1998 Academic Press

Journal Article↗

PubMed1996

Three-dimensional structure of Endo-1,4-beta-xylanase I from Aspergillus niger: molecular basis for its low pH optimum.

The crystal structure of endo-1,4-beta-xylanase I from Aspergillus niger has been solved by molecular replacement and was refined to 2.4 A resolution. The final R-factor for all data from 6 to 2.4 A is 17.9%. The A. niger xylanase has a characteristic fold which is unique for family G xylanases (root-mean-square deviation = 1.1 A to Trichoderma reesei xylanase I, which has 53% sequence identity). It consists of a single domain composed predominantly of beta-strands. Two beta-sheets are twisted around a deep, long cleft, which is lined with many aromatic amino acid residues and is large enough to accommodate at least four xylose residues. The two conserved glutamate residues, Glu79 and Glu170, which are likely to be involved in catalysis, reach into this cleft from opposite sides. A niger xylanase I is of particular commercial interest because of its low pH optimum. A model is proposed which explains this low pH optimum compared to other members of xylanase family G.

Amino Acid Sequence↗

PubMed1999

Purification of active cysteine proteases by affinity chromatography with attached E-64 inhibitor.

Cysteine proteases are implicated in many regulatory and degradative processes in animal and plant cells. Many of the proteases are strongly inhibited by an irreversible inhibitor, trans-(epoxysuccinyl)-l-leucylamino-4-guanidinobutane (E-64) from Aspergillus japonicus. Here we report a method for purification of cysteine proteases by affinity chromatography on E-64. Attachment of the inhibitor to thiopropyl Sepharose through its epoxy group resulted in the loss of its irreversible activity but did not affect the specificity of interaction or its capability to bind cysteine proteases. Papain that served as a model cysteine protease was fully active after elution. We also provide evidence for purification of active proteases from a mixture of extracellular fluid of Botrytis cinerea- and Trichoderma harzianum-inoculated bean plants. Since the proteases are eluted with urea after the column is washed with 1 M NaCl, this procedure may provide highly efficient purification.

Aspergillus↗

PubMed1993

A fungal phylogeny based upon orotidine 5'-monophosphate decarboxylase.

Orotidine 5'-monophosphate decarboxylase protein sequences from 14 fungi, 1 slime mold, 2 mammals, and 3 bacteria are compared and aligned and shown to be homologous. Based on the optimal alignment of the fungal sequences, a phylogenetic tree is derived. Within the fungi, the fission yeast Schizosaccharomyces pombe shows a closer relationship to both basidiomycetes and phycomycetes than it does to orthodox ascomycetes (plectomycetes, pyrenomycetes, and budding yeasts). Intron conservation shows a close relationship between phycomycetes and basidiomycetes. The imperfect fungi Trichoderma and Cephalosporium are shown to be closely related to Neurospora. The predicted origin of the group of budding yeasts is dependent on the analytical method used.

Amino Acid Sequence↗

PubMed1992

Transformation of N epsilon-CBZ-L-lysine to CBZ-L-oxylysine using L-amino acid oxidase from Providencia alcalifaciens and L-2-hydroxy-isocaproate dehydrogenase from Lactobacillus confusus.

Biotransformations were developed to oxidize N epsilon-carbobenzoxy(CBZ)-L-lysine and to reduce the product keto acid to L-CBZ-oxylysine. Lysyl oxidase (L-lysine: O2 oxidoreductase, EC 1.4.3.14) from Trichoderma viride was relatively specific for L-lysine and had very low activity with N epsilon-substituted derivatives. L-Amino acid oxidase (L-amino acid: O2 oxidoreductase [deaminating], EC 1.4.3.2) from Crotalus adamanteus venom had low activity with L-lysine but high activity with N epsilon-formyl-, t-butyoxycarbonyl(BOC)-, acetyl-, trifluoroacetyl-, or CBZ-L-lysine. L-2-Hydroxyisocaproate dehydrogenase (EC 1.1.1.-) from Lactobacillus confusus catalyzed the reduction by NADH of the keto acids from N epsilon-acetyl-, trifluoroacetyl-, formyl- and CBZ-L-lysine but was inactive with the products from oxidation of L-lysine, L-lysine methyl ester, L-lysine ethyl ester or N epsilon-t-BOC-L-lysine. Providencia alcalifaciens (SC9036, ATCC 13159) was a good microbial substitute for the snake venom oxidase and also provided catalase (H2O2:H2O2 oxidoreductase EC 1.11.1.6). N epsilon-CBZ-L-Lysine was converted to CBZ-L-oxylysine in 95% yield with 98.5% optical purity by oxidation using P. alcalifaciens cells followed by reduction of the keto acid using L-2-hydroxyisocaproate dehydrogenase. NADH was regenerated using formate dehydrogenase (formate: NAD oxidoreductase, EC 1.2.1.2) from Candida boidinii. The Providencia oxidase was localized in the particulate fraction and catalase activity was predominantly in the soluble fraction of sonicated cells. The pH optima and kinetic constants were determined for the reactions.

Alcohol Oxidoreductases↗

PubMed1992

The PYR1 gene of the plant pathogenic fungus Colletotrichum graminicola: selection by intraspecific complementation and sequence analysis.

A spontaneous uridine-requiring auxotroph of Colletotrichum graminicola was recovered by selection for resistance to 5-fluoro-orotic acid. The auxotroph lacked orotate phosphoribosyl transferase (OPRTase) and was complemented with a clone from a cosmid library of C. graminicola DNA. A 3.1 kb HindIII-SalI fragment was subcloned from the cosmid and it could efficiently transform the auxotrophic strain to uridine prototrophy and integrate by site-specific recombination. This DNA fragment contains an open reading frame that is similar to OPRTase genes of the fungi Sordaria macrospora, Trichoderma reesei, Podospora anserina, and Saccharomyces cerevisiae. Based on the sequence similarities and the ability to restore uridine prototrophy, we conclude that the fragment contains the C. graminicola gene for OPRTase, which we have named PYR1. Our results demonstrate that cloning by complementation is feasible in C. graminicola, that the gene for OPRTase from C. graminicola can be useful as a selectable marker in transformation of the fungus, and that the OPRTase gene product is similar to OPRTase from other fungi.

Amino Acid Sequence↗

PubMed1991

Electrophoretic karyotype of cellulolytic Penicillium janthinellum strains.

The genome of the cellulolytic fungus Penicillium janthinellum BIOURGE was resolved completely by rotating field electrophoresis. The gel pattern revealed 8-10 different chromosomes. On the basis of data for yeast chromosome size standards from Schizosaccharomyces pombe and Saccharomyces cerevisiae, chromosome sizes in the range from 2.0 to 8 mb were estimated. By Southern hybridization with heterologous probes, the chromosomal locations of rDNA, the elongation factor EF1, actin and ubiquitin have all been mapped. In addition, first results with heterologous and homologous probes for cellulolytic genes from Trichoderma reesei and P. janthinellum are presented. In contrast to the other probes, signals obtained with cellulase genes are clearly distributed over several chromosomes.

Blotting, Southern↗

PubMed1991

High efficiency transformation of Tolypocladium geodes conidiospores to phleomycin resistance.

A convenient and efficient transformation system has been developed for the filamentous fungus Tolypocladium geodes. In contrast to most of the commonly described techniques requiring prior preparation of protoplasts or spheroplasts, this method leads to high efficiency transformation of T. geodes conidiospores following moderate lytic enzyme treatment. Competent cells so obtained are still resistant to osmotic pressure and can be stored frozen without loss of viability. The highest transformation frequency (3-5 x 10(3) transformants per microgram of DNA) was obtained with plasmid pUT737 containing the Sh ble gene conferring phleomycin resistance under the control of a strong promoter isolated from Trichoderma reesei. Southern hybridization revealed multiple integration sites of plasmid DNA into the T. geodes nuclear DNA despite the absence of homology between the transforming DNA and the recipient genome. Instability could not be detected for the phleomycin phenotype during more than five generations of mitotic growth under non-selective conditions.

Blotting, Southern↗

PubMed1993

Cloning and sequencing of the 3-phosphoglycerate kinase (PGK) gene from Penicillium citrinum and its application to heterologous gene expression.

The gene coding for 3-phosphoglycerate kinase (PGK) in ML-236B (compactin)-producing Penicillium citrinum was isolated from the recombinant phage lambda library using the corresponding Aspergillus nidulans pgk gene as a probe. The P. citrinum pgk gene has an open reading frame of 1,254 bp, encoding a protein of 417 amino acids with a predicted molecular weight of 44,079 daltons. The position of the two introns, 59 and 60 bp respectively, was deduced from an homology comparison with the sequence of the A. nidulans pgk gene. The PGK protein of P. citrinum shows extensive high homology to the PGKs of four other fungi: P. chrysogenum (93%), A. nidulans (84%), Trichoderma reesei (78%) and Saccharomyces cerevisiae (68%). Almost total conservation is found in P. citrinum of residues thought to be important for the structure and function of the yeast enzyme. The strong codon preference found has greater similarity to that in other filamentous fungi than in yeast. A DNA fragment encompassing the pgk gene was shown to hybridize a 1.35-kb poly(A)+RNA, sufficient to encode the PGK polypeptide. A fused gene, pgk-hpt, containing the putative pgk promoter and the open reading frame of the Escherichia coli hygromycin B phosphotransferase (hpt) gene was constructed, and was successfully used to transform P. citrinum to a hygromycin B (HmB)-resistant phenotype.

Amino Acid Sequence↗

PubMed1992

Genomic organization of a cellulase gene family in Phanerochaete chrysosporium.

Southern blot and nucleotide sequence analysis of Phanerochaete chrysosporium BKM-F-1767 genomic clones indicate that this wood-degrading fungus contains at least six genes with significant homology to the Trichoderma reesei cellobiohydrolase I gene (cbh1). Using pulsed-field gel electrophoresis to separate P. chrysosporium chromosomes, the six cellulase genes were found to hybridize to at least three different chromosomes, one of which is dimorphic. The organization of these genes was similar in another P. chrysosporium strain, ME 446. It is clear that, unlike T. reesei, the most well-studied cellulolytic fungus, P. chrysosporium contains a complex, cbh1-like gene family.

Amino Acid Sequence↗

PubMed1978

Studies on growth inhibition by lectins of Penicillia and Aspergilli.

It has previously been shown in our laboratory that wheat germ agglutinin (WGA) binds to Trichoderma viride and inhibits growth of this fungus. Here we report on the effect of WGA, soybean agglutinin (SBA) and peanut agglutinin (PNA) on Penicillia and Aspergilli. Binding of the lectins to the fungi was examined with the aid of their fluorescein isothiocyanate (FITC) conjugated derivatives. FITC-WGA bound to young hyphal walls of all species, in particular to the hyphal tips and septa, in agreement with the chitinous composition of the cell walls of the two genera. Hyphae of all species examined were labelled, though in different patterns, by FITC-SBA and FITC-PNA, suggesting the presence of galactose residues on their surfaces. Young conidiophores, metulae (of the Penicillia), vesicles (of the Aspergilli), sterigmata and young spores, were also labelled. The three lectins inhibited incorporation of [3H]acetate, N-acetyl-D-[3h]glucosamine and D-[14C]galactose into young hyphae of Aspergillus ochraceus, indicating interference with fungal growth. Inhibition of spore germination by the three lectins was also observed. Preincubation of the lectins with their specific saccharide inhibitors prevented binding and the inhibitory effects. We conclude that lectins are useful tools for the study of fungal cell surfaces, and may also serve as an important aid in fungal classification. The present findings also support the suggestion that one role of lectins in plants is protection against fungal pathogens.

Aspergillus↗

PubMed1984

Distribution of moulds on stored grains in households in an area affected by endemic nephropathy in Yugoslavia.

The distribution of moulds on stored maize, wheat and beans in families affected and unaffected by nephropathy in the endemic area (Endemic nephrophaty - Balkan kidney disease) of middle Posavinain Yugoslavia was studied. Among 3 700 samples of stored grains, no significant difference was noted in the contamination by moulds of samples taken from affected and unaffected families. The predominant moulds on all the samples were 'storage moulds' from the genera Penicillium and Aspergillus, 'field moulds' among which Alternaria and Fusarium were encountered most frequently, and the 'spoilage moulds' Absidia, Mucor, Rhizopus, Trichoderma and Trichothecium.

Balkan Nephropathy↗

PubMed1996

High incidence of Aspergillus flavus and aflatoxins in stored groundnut in Ghana and the use of a microbial assay to assess the inhibitory effects of plant extracts on aflatoxin synthesis.

Groundnut samples from 21 selected markets in the 10 regions of Ghana yielded high levels of the aflatoxigenic fungus Aspergillus flavus on half-strength potato dextrose agar. The fungus was associated with 31.7 and 12.8%, respectively, of all damaged and undamaged kernels assayed. Only 0.24% of total kernels assayed yielded A. parasiticus. Other fungi detected from total kernels assayed were A. niger (34%), A. candidus (1.45%), A. tamarii (3.93%), A. ochraceous (5.26%), Fusarium spp. (1.7%) Penicillium spp. (5.19%), a Mucor sp. (2.3%), a Trichoderma sp. (0.2%), Rhizopus stolonifer (12%) and certain unidentifiable fungi (11.72%). Total aflatoxin levels ranging from 5.7 to 22, 168 ppb were identified with damaged kernel samples. The mycotoxin was not detected in 50% of undamaged kernel samples tested and very low levels mostly ranging from 0.1 to 12.2 ppb were associated with the undamaged samples that tested positive for aflatoxins. In a novel in vitro microbial assay to determine the effectiveness of certain plant extracts against aflatoxin synthesis, extracts from Xylopia aethiopica, Monodera myristica, Cinnamomum verum and Piper nigrum permitted fungal growth in 1.5% potato-dextrose broth while completely suppressing NOR formation. These extracts, however, could not suppress NOR formation in a yeast extract sucrose medium.

Aflatoxins↗

PubMed1983

Saprophytic fungi isolated from the hair of domestic and laboratory animals with suspected dermatophytosis.

Hair samples from domestic and laboratory animals with suspected dermatophytosis were examined for the presence of saprophytic fungi. A nutritionally poor base medium, developed by the author, was used in the isolation and identification of the saprophytes. Three hundred and ninety-four specimens were examined of which 246 were from dogs, 75 from cats, 30 from horses, 19 from cows, 12 from guinea pigs, 5 from rats, 2 from parakeets, 2 from chinchillas and one each from a goat, a mink and a lesser panda (Ailurus fulgens). Moulds classified in 32 genera were isolated. The commonest in order of frequency were members of the genera Penicillium, Cladosporium, Aspergillus, Mucor, Aureobasidium, Alternaria, Scopulariopsis, Trichoderma and Trichothecium. The yeasts that were isolated were not identified. Aureobasidium pullulans was isolated significantly more often (chi 2 test p less than 0.025) from the dog samples than those from cats, Cladosporium spp. in the samples from dogs than horses, Mucor spp. from the cow samples than horses, Penicillium spp. from the dog samples than those of cats or horses. Skin infections caused by any of the contaminants were not encountered.

Animals↗

PubMed1986

Mycoflora and mycotoxin-producing fungi of air-dust particles from Egypt.

Using the dilution-plate method, 27 genera and 64 species were collected from 20 air-dust samples on glucose - (24 genera and 57 species) and cellulose - (21 genera and 45 species) Czapek's agar at 28 degrees C. There are basic similarities between the mycoflora of air-dust on the two media and the most prevalent species were Aspergillus niger, A. flavus, A. ochraceus, A. terreus, A. versicolor, Penicillium chrysogenum, P. funiculosum, Alternaria alternata, Cladosporium herbarum, Fusarium oxysporum, Rhizopus stolonifer and Trichoderma viride. Chaetomium globosum, Stachybotrys chartarum, Humicola grisea and Arthrobotrys oligospora were common only on cellulose agar plates. Extracts of mycelium from 25 isolates were tested with brine schrimp (Artemia salina); of these 23 displayed varying degrees of toxicity. Thin layer chromatographic analysis of 12 isolates of Aspergillus flavus revealed that 4 strains were producing detectable aflatoxin. Zearalenone production was noted for 3 out of 5 strains of Fusarium oxysporum and 2 out of 5 strains of F. solani.

Aflatoxins↗

PubMed1988

Mycotoxin-producing potential of fungi isolated from red kidney beans.

The predominant fungi present in samples of reject and retail red kidney beans were Aspergillus glaucus, Penicillium spp. and Alternaria spp. Together with A. ochraceus, A. flavus, Fusarium spp., and Trichoderma, these isolates from the reject beans were screened for numerous mycotoxins by TLC. The most consistently produced mycotoxins were penicillic acid (from A. ochraceus and Penicillium spp.) and Alternaria toxins (tenuazonic acid and alternariol). A. glaucus strains were tested for cytotoxicity in three tissue culture cell lines with positive results.

Aspergillus↗