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PubMed1994

Solubilization of lignin by the ruminal anaerobic fungus Neocallimastix patriciarum.

The ability of the ruminal anaerobic phycomycete Neocallimastix patriciarum to digest model lignin compounds and lignified structures in plant material was studied in batch culture. The fungus did not degrade or transform model lignin compounds that were representative of the predominant intermonomer linkages in lignin, nor did it solubilize acid detergent lignin that had been isolated from spear grass. In a stem fraction of sorghum, 33.6% of lignin was apparently solubilized by the fungus. Solubilization of ester- and either-linked phenolics accounted for 9.2% of the lignin released. The amounts of free phenolic acids detected in culture fluid were equivalent to the apparent loss of ester-linked phenolics from the sorghum substrate. However, the fungus was unable to cleave the ether bond in hydroxycinnamic acid bridges that cross-link lignin and polysaccharide. It is suggested that the majority of the solubilized lignin fraction was a lignin carbohydrate complex containing ether-linked hydroxycinnamic acids. The lignin carbohydrate complex was probably solubilized through dissolution of xylan in the lignin-xylan matrix rather than by lignin depolymerization.

Anaerobiosis↗

PubMed1963

Transformation of steroids by spores of microorganisms. I. Hydroxylation of progesterone by conidia of Aspergillus ochraceus.

Conidia of Aspergillus ochraceus convert progesterone into 11alpha-hydroxyprogesterone and 6beta, 11alpha-dihydroxyprogesterone. The conversion ability does not depend on the sporulation medium. Transformation depends on the strain and on the conidia concentration. Adaptation has never been observed. Age and storage of conidia, pH, aeration-agitation, nitrogen source, metal ions, chelating agents, and metabolic activators showed no great influence within wide limits. Mercuric chloride, p-chloromercuribenzoate, NaN(3), and KCN inhibit conversion. Glucose is necessary, but can be replaced completely by d(+)-xylose, and partially by some other carbon sources. The ratio mono-/di-hydroxyprogesterone is influenced by progesterone concentration and period of incubation; also, a mutant that accumulates only monohydroxyprogesterone has been produced. Conidia of A. ochraceus also hydroxylate a variety of steroids. Spores of certain streptomycetes, phycomycetes (mucors), ascomycetes, and deuteromycetes are active. Most reactions already observed with vegetative cells have been repeated with spores. In general, spores of a particular organism effect fewer reactions than its mycelium, and fewer products accumulate.

Aspergillus↗

PubMed1971

Fatty acid and sterol composition of Mucor genevensis in relation to dimorphism and anaerobic growth.

Fatty acid and sterol content and composition were determined for the dimorphic mold, Mucor genevensis, grown under a variety of experimental conditions. Fatty acids account for 6 to 9% of the dry weight of aerobically grown mycelium, and 70 to 80% of these are unsaturated. The organism contains gamma-linolenic acid which is characteristic for Phycomycetes, and in sporangiospores this compound represents 40% of the total fatty acids. Of the sterols found in mycelium, 80% is ergosterol, and stigmasterol was positively identified as one of the minor components. In anaerobically grown yeastlike cells, sterol content is less than 10% of the level found in aerobically grown cells, and fatty acids amount to less than 2% of the dry weight. These fatty acids are predominantly short chain and less than 10% are unsaturated. Yeastlike cells obtained under aerobic conditions by growth in the presence of phenethyl alcohol have fatty acid and sterol compositions characteristic of aerobically grown mycelium. It is concluded that the dimorphology of the organism is not directly related to lipid composition.

Aerobiosis↗

PubMed1975

Kynureninase-Type enzymes of Penicillum roqueforti, Aspergillus niger, Rhizopus stolonifer, and Pseudomonas fluorescens: further evidence for distinct kynureninase and hydroxykynureninase activities.

The kynureninase-type enzymes of three fungi and one bacterium were isolated and examined kinetically for their ability to catalyze the hydrolysis of L-kynurenine and L-3-hydroxykynurenine. The phycomycete Rhizopus stolonifer was found to contain a single, constitutive enzyme with Km for L-3-hydroxykynurenine and L-kynurenine of 6.67 times 10-minus 6 and 2.5 times 10-minus 4 M, respectively. The ascomycetes Aspergillus niger and Penicillium roqueforti each contain an enzyme, induced by L-tryptophan, with similar Km for L-3-hydroxykynurenine and L-kynurenine ranging from 5.9 times 10-minus 5 to 14.3 times 10-minus 5 M, as well as a constitutive enzyme with Km for the two substrates of similar to 4 times 10-minus 6 M and 10-minus 4 M. The bacterium Pseudomonas fluorescens has a single, inducible enzyme with Km for L-3-hydroxykynurenine and L-kynurenine of 5 times 10-minus 4 and 7 times 10-minus 5 M. In addition, significant differences in maximal velocities (Vmax) were observed in two cases. The Vmax of the inducible activity from P. fluorescens was 4.5 times greater for L-kynurenine than L-3-hydroxykynurenine, whereas the Vmax of the constitutive activity from R. stolonifer was 2.5 times greater for L-3-hydroxykynurenine. It is concluded (i) that the constitutive activities are hydroxykynureninases involved in the biosynthesis of nicotinamide adenine dinucleotide from L-tryptophan, (ii) that the inducible activities are kynureninases involved in the catabolism of L-tryptophan to anthranilate, and (iii) that R. stolonifer and P. fluorescens, respectively, carry the most specific examples of each type of enzyme.

Ammonium Sulfate↗

PubMed1978

Regulation of macromolecular synthesis during hyphal germ tube emergence from Mucor racemosus sporangiospores.

Protein and RNA syntheses were examined during hyphal germ tube emergence from sporangiospores of a dimorphic phycomycete, Mucor racemosus. Both classes of macromolecules were synthesized immediately upon introduction of the dormant sporangiospores into nutrient medium. The specific rates of synthesis of both protein and RNA accelerated during initial germ tube emergence and reached a maximum when the emergence of new germ tubes ended. The specific rates of synthesis later decreased during further hyphal elongation. The distribution of ribosomes between active polysomes and monosomes and inactive subunits was determined by sucrose density gradient centrifugation, and the rate of amino acid addition to nascent polypeptide chains was calculated throughout the developmental sequence. The results showed that both the percentage of ribosomes active in protein synthesis and the velocity of ribosome movement along the mRNA were continuously adjusted throughout hyphal germ tube development. The free intracellular amino acid pools were measured throughout development. Alanine, glutamate, and aspartate were present at very high concentrations in the dormant spores but were rapidly depleted during hyphal germ tube emergence. The results of these studies are discussed in relation to hyphal germ tube development from yeast cells of Mucor and dormant spores of other fungal species.

Amino Acids↗

PubMed1980

Regulation of ornithine decarboxylase during morphogenesis of Mucor racemosus.

During the yeast-to-hyphae transition of the dimorphic phycomycete Mucor racemosus, there was a 30- to 50-fold increase in the activity of ornithine decarboxylase. Increased enzyme activity preceded the emergence of germ tubes and reached a maximum before conversion was completed. Subsequently, enzyme levels rapidly declined, despite the continuation of mycelial growth. Both putrescine and spermidine blocked the enzyme activity response. Protein synthesis was required for the increase in enzyme activity during morphogenesis. A combination of actinomycin D and netropsin inhibited ribonucleic acid synthesis but failed to inhibit the increase in ornithine decarboxylase activity. There was a twofold increase in the enzyme half-life during morphogenesis with either trichodermin or verrucarin to inhibit protein synthesis.

Carboxy-Lyases↗

PubMed1980

Glyoxylate cycle in Mucor racemosus.

The dimorphic phycomycete Mucor racemosus was grown in media containing acetate, glutamate, and peptone as carbon sources. The component enzymes of the glyoxylate bypass, isocitrate lyase and malate synthase, were present under these conditions throughout the growth cycles. Highest specific activities for each enzyme were found in media with acetate as the carbon source. In an enriched peptone medium containing glucose, neither activity was detected until glucose was exhausted from the medium. Treatment of acetate-grown cells with glucose resulted in a rapid decline in the specific activities of both enzymes. The importance of this cycle in acetate-grown cells was indicated by the ability of itaconic acid (100 mM) to inhibit the growth of M. racemosus in acetate but not glutamate media. Itaconate was also shown to be a potent inhibitor of isocitrate lyase activity in vitro.

Acetates↗

PubMed1987

Purification and properties of two isozymes of pyruvate kinase from Mucor racemosus.

The dimorphic phycomycete Mucor racemosus was found to contain up to five electrophoretic forms of pyruvate kinase (ATP: pyruvate 2-O-phosphotransferase, EC 2.7.1.40) depending on growth conditions. M. racemosus hyphal cells grown on glutamic acid as the carbon source contained only the fastest electrophoretic form, designated PK1, while yeast cells grown on glucose contained only the slowest electrophoretic form, PK5. Intermediate electrophoretic forms PK2, PK3, and PK4 as well as PK1 and PK5 were found in hyphal cells grown on media containing fructose or cellibiose. All five electrophoretic forms had molecular weights of ca. 230,000 as determined from plots of log Rm versus acrylamide gel concentration. Both PK1 and PK5 were purified to homogeneity and determined to be homotetramers, with subunit molecular weights of 54,000 and 58,100, respectively. The amino acid content of PK1 and PK5 was determined and found to be similar but not identical. Analysis of limited tryptic digests and cyanogen bromide cleavage fragments of PK1 and PK5 indicate that the subunits of the two isozymes are significantly different.

Amino Acids↗

PubMed1988

Novel Ca2+-activated neutral protease from an aquatic fungus, Allomyces arbuscula.

A Ca2+-activated neutral protease was purified to homogeneity from an aquatic Phycomycete fungus, Allomyces arbuscula. It requires millimolar concentrations of Ca2+ for activation (1.8 to 2 mM for 50% activation). Sr2+ can replace Ca2+ but at higher concentrations (4 mM for 50% activation). The enzyme is a dimer of 40-kilodalton subunits and contains six cysteine residues, three of which are revealed only after the addition of micromolar concentrations of Ca2+; the other three are free. Enzyme activity is strongly inhibited by SH-group inhibitors and some trypsin inhibitors (leupeptin and alpha-N-tosyl-L-lysine chloromethyl ketone). The enzyme lacks general trypsinlike specificity, since substrates containing tryptic cleavage sites are not cleaved nor is enzyme activity inhibited by other trypsin inhibitors. The enzyme has many functional similarities to the extensively characterized mammalian and avian Ca2+-activated neutral proteases but differs in its substrate specificity, inhibition by alpha-N-tosyl-L-phenylalanine chloromethyl ketone, and subunit structure. It is, nevertheless, presumed that this enzyme has a similar high order of specificity and is involved in the regulation of a specific growth function.

Calcium↗

PubMed1967

Organization of the tryptophan pathway: a phylogenetic study of the fungi.

The enzymes involved in tryptophan biosynthesis have been analyzed in a variety of fungal strains and a few other microorganisms. The same five biosynthetic reactions occur in all organisms tested, but differences have been found in the stability requirements for the enzymes, in their differential precipitation with ammonium sulfate, and in their sedimentation pattern after zone centrifugation. Based on the sedimentation behavior of anthranilate synthetase, phosphoribosyl-transferase, N-(5'-phosphoribosyl)-anthranilate isomerase, and indole-3-glycerophosphate synthetase, five different patterns of enzyme association could be recognized. The distribution of these patterns was used to evaluate several specific features of proposed phylogenetic relationships in the fungi. A closer relationship between Chytridiales and Aspergillales is postulated, eliminating the Zygomycetes and the Endomycetales as probable intermediates; the latter groups are considered to be sidelines. The data support the idea of a polyphyletic origin of the phycomycetes and suggest that anascosporogenous yeasts tested are related to the heterobasidiomycetes rather than to the Endomycetales. A possible sequence of changes leading to the various patterns of organization of the tryptophan pathway during the course of evolution is also proposed.

Aspergillus↗

PubMed1969

Organization of enzymes in the common aromatic synthetic pathway: evidence for aggregation in fungi.

Centrifugation in sucrose density gradients of partially purified extracts from six species of fungi, i.e., Rhizopus stolonifer, Phycomyces nitens, Absidia glauca (Phycomycetes), Aspergillus nidulans (Ascomycetes), Coprinus lagopus, and Ustilago maydis (Basidiomycetes), indicate that the five enzymes catalyzing steps two to six in the prechorismic acid part of the polyaromatic synthetic pathway sediment together. The sedimentation coefficients for these enzymes are very similar in the six species and are comparable to those previously observed for the multienzyme complexes (arom aggregates) of Neurospora crassa and Saccharomyces cerevisiae. These results are interpreted as indicating the presence in each of these fungi of arom aggregates, presumably encoded by arom gene clusters similar to those in N. crassa and S. cerevisiae. Evidence has also been obtained for the presence in two species (A. nidulans and U. maydis) and the absence in the other four species of a second dehydroquinase isozyme which is distinguishable from the synthetic activity on the basis of both thermostability tests and S values. This second dehydroquinase, which is apparently involved in the catabolism of quinic acid via a pathway similar to that in N. crassa, is inducible in A. nidulans (as it is in N. crassa), but constitutive in U. maydis. These comparative findings are discussed in relation to the organization, evolution, and possible functional relationships of synthetic and catabolic aromatic pathways in fungi.

Alcohol Oxidoreductases↗

PubMed1989

Transformation in fungi.

Transformation with exogenous deoxyribonucleic acid (DNA) now appears to be possible with all fungal species, or at least all that can be grown in culture. This field of research is at present dominated by Saccharomyces cerevisiae and two filamentous members of the class Ascomycetes, Aspergillus nidulans and Neurospora crassa, with substantial contributions also from fission yeast (Schizosaccharomyces pombe) and another filamentous member of the class Ascomycetes, Podospora anserina. However, transformation has been demonstrated, and will no doubt be extensively used, in representatives of most of the main fungal classes, including Phycomycetes, Basidiomycetes (the order Agaricales and Ustilago species), and a number of the Fungi Imperfecti. The list includes a number of plant pathogens, and transformation is likely to become important in the analysis of the molecular basis of pathogenicity. Transformation may be maintained either by using an autonomously replicating plasmid as a vehicle for the transforming DNA or through integration of the DNA into the chromosomes. In S. cerevisiae and other yeasts, a variety of autonomously replicating plasmids have been used successfully, some of them designed for use as shuttle vectors for Escherichia coli as well as for yeast transformation. Suitable plasmids are not yet available for use in filamentous fungi, in which stable transformation is dependent on chromosomal integration. In Saccharomyces cerevisiae, integration of transforming DNA is virtually always by homology; in filamentous fungi, in contrast, it occurs just as frequently at nonhomologous (ectopic) chromosomal sites. The main importance of transformation in fungi at present is in connection with gene cloning and the analysis of gene function. The most advanced work is being done with S. cerevisiae, in which the virtual restriction of stable DNA integration to homologous chromosome loci enables gene disruption and gene replacement to be carried out with greater precision and efficiency than is possible in other species that show a high proportion of DNA integration events at nonhomologous (ectopic) sites. With a little more trouble, however, the methodology pioneered for S. cerevisiae can be applied to other fungi too. Transformation of fungi with DNA constructs designed for high gene expression and efficient secretion of gene products appears to have great commercial potential.

DNA, Fungal↗

PubMed1976

Somatic nuclear division in the sporidia of Ustilago violacea. III. Ultrastructural observations.

The paper provides detailed ultrastructural observations on nuclear division in the smut fungus Ustilago violacea and is based on previous light-microscopic work outlining the division in living and stained cells. The division as in many other Basidiomycetes is not intranuclear, but occurs within a partially disrupted membrane. The division takes place after migration of most of the nucleus into the bud cell, after limited breakdown of the nuclear membrane, and after the formation of a spindle between two spindle-pole bodies (SPB). The remaining part of the nucleus containing the nucleolus is left behind in the parent cell and degenerates there. The SPB, as in other Basidiomycetes, is a dome-shaped relatively structureless body, quite distinct from the flat plaques of many Ascomycetes and the elaborat centrioles of Phycomycetes. The SPB divides shortly before migration into the daughter cell and invariably is located at the apex of the migrating nucleus. Nuclear division is completed when the two masses of chromatin clustered about each of the SPB's are separated as the spindle elongates. One daughter neculeus reforms in the bud and the other is reformed in the mother cell. Cells fixed and stained by conventional light-microscopic methods were examined in the light of the electron-microscopic observations to determine whether these procedures induce artefacts. Aceto-orcein and Giemsa when used cold were found to produce relatively artefact-free preparations. However, previous results in which the cells were warmed gently in these stains are now seen to contain artefacts in the form of contracted chromatinic granules often arranged in chains. These artefacts may provide useful information but clearly they must be interpreted cautiously until the nature of the changes induced by heating are known.

Basidiomycota↗

PubMed1981

Orbital phycomycosis.

3 cases with rhinoorbital phycomycosis and a 4th case, in which it was considered retrospectively, are presented in hematological patients. 2 of the 4 patients survived. In the first 3 cases, which appeared in a period of 2 months in the Hematology Unit, an epidemiological study was made, showing that the cause of the infections was contamination of the air-conditioning systems of this unit by numerous Phycomycetes and Mucor pusillus.

Adult↗

PubMed1977

Pulmonary phycomycetoma in a patient with diabetes mellitus.

Phycomycetes are ubiquitous saprophytic fungi sharing with other fungi a propensity for invasion and disease production in immunologically compromised hosts. Diabetes mellitus, in particular diabetic ketoacidosis, is a common clinical setting for phycomycosis. A pulmonary phycomycetoma was diagnosed in a diabetic patient from material obtained by bronchial brushing was treated successfully with a combination of surgery and amphotericin B.

Diabetes Complications↗

PubMed1980

Infection with Absidia corymbifera in bank voles (Clethrionomys glariolus).

Bank voles, Clethrionomys glariolus, kept under laboratory conditions were highly susceptible to both naturally-acquired and experimental infection with the phycomycete Absidia corymbifera. The infection was manifested as an acute lethal disease characterized by fungal invasion of the kidneys, central nervous system and, less frequently, other tissues. Natural infection was probably acquired via the respiratory route.

Acute Disease↗

PubMed1982

Cephalic phycomycosis: a report of eight cases.

Phycomycosis is the preferred terminology to define a fungal disease which may be devastating and fatal. It is caused by a nonseptate hyphae, class phycomycetes and genus (Rhizopus, Mucor, Absidia). Phycomycosis in man is usually associated with debilitating diseases such as: diabetes mellitus, leukemia and immunosuppressive conditions. The cephalic phycomycosis has two forms: 1. rhino-orbital cerebral which may be fatal, and 2. rhino-paranasal sinuses form which usually has a benign clinical course. From 1943 to 1967, only 45 cases of the cephalic form were described with a mortality rate of 50%. Since then several series have been added to the literature with improved survival, probably due to the addition of amphotericin B to the therapy. Even with modern therapy, the mortality rate is still about 30%. Modern technology C.T. scan is very helpful to establish orbital and intracranial extension. When intracranial involvement is present, the prognosis is dismal. Our series of 8 patients is reported.

Adult↗

PubMed1982

Entomophthorales infection of the maxillofacial region.

Phycomycosis infections caused by the fungi Rhizopus and Mucor are commonly termed mucormycosis. Mucormycosis infections exhibit a rapidly progressive course characterized by angioinvasion and necrosis. The rhinocerebral form has been described thoroughly in the literature. However, there exists another order of Phycomycetes, the Entomophthorales, which rarely cause clinical disease. Their infection is indolent and is associated with granulomatous inflammation and a protracted clinical course. Presented here is a rare case of suspected Canidiobolus coronato infection occurring in the maxillofacial region of a previously healthy male. The authors believe this to be the first reported case occurring in the United States. Because of the poorly documented natural history of this infection and the patient's relative intolerance to parenteral antifungal therapy, a multidisciplinary therapeutic approach was designed. It consisted of long-term, low-dosage amphotericin-B, hyperbaric oxygen and sequential surgical debridements. This unusual disease entity must now be considered in the differential diagnosis of granulomatous disorders of the head and neck.

Amphotericin B↗