Determination of the effect of polyamines on an oil-degrading strain of Yarrowia lipolytica using an odc minus mutant.

Yarrowia lipolytica is an ascomycetous dimorphic yeast with immense potential for industrial applications, including bioremediation of crude oil-contaminated environments. It has been shown that a dimorphic marine isolate of Y. lipolytica (var. indica) has significant capacity to degrade fatty acids and alkanes, when in its yeast morphology. It has also been demonstrated that polyamines play an important role in the yeast-to-mycelium transition of different strains of Y. lipolytica that are unable to utilize those carbon sources. To determine the role of polyamines on their capacity to utilize oils and hydrocarbons, on the dimorphic transition, and also on other characteristics of the var. indica strain of Y. lipolytica, we proceeded to obtain ornithine decarboxylase minus (odc-) mutants. These mutants behaved as yeasts independently of the concentrations of putrescine added. Further, they conserved the oil-degrading capacity of the parent strain. The odc- mutant can thus be used in fatty acid degradation, and oil spill remediation with distinct advantages.

Transcriptomic analysis of basidiocarp development in Ustilago maydis (DC) Cda.

Previously, we demonstrated that when Ustilago maydis (DC) Cda., a phytopathogenic basidiomycete and the causal agent of corn smut, is grown in the vicinity of maize embryogenic calli in a medium supplemented with the herbicide Dicamba, it developed gastroid-like basidiocarps. To elucidate the molecular mechanisms involved in the basidiocarp development by the fungus, we proceeded to analyze the transcriptome of the process, identifying a total of 2002 and 1064 differentially expressed genes at two developmental stages, young and mature basidiocarps, respectively. Function of these genes was analyzed with the use of different databases. MIPS analysis revealed that in the stage of young basidiocarp, among the ca. two thousand differentially expressed genes, there were some previously described for basidiocarp development in other fungal species. Additional elements that operated at this stage included, among others, genes encoding the transcription factors FOXO3, MIG3, PRO1, TEC1, copper and MFS transporters, and cytochromes P450. During mature basidiocarp development, important up-regulated genes included those encoding hydrophobins, laccases, and ferric reductase (FRE/NOX). The demonstration that a mapkk mutant was unable to form basidiocarps, indicated the importance of the MAPK signaling pathway in this developmental process.

Dimorphism and hydrocarbon metabolism in Yarrowia lipolytica var. indica.

Yarrowia lipolytica is able to metabolize high Mr hydrophobic natural compounds such as fatty acids and hydrocarbons. Characteristically, strains of Y. lipolytica can grow as populations with variable proportions of yeast and filamentous forms. In the present study, we describe the dimorphic characteristics of a variant designated as Y. lipolytica var. indica isolated from petroleum contaminated sea water and the effect of cell morphology on hydrocarbon metabolism. The variant behaved as a yeast monomorphic strain, under conditions at which terrestrial Y. lipolytica strain W29 and its derived strains, grow as almost uniform populations of mycelial cells. Using organic nitrogen sources and N-acetylglucosamine as carbon source, var. indica was able to form mycelial cells, the proportion of which increased when incubated under semi-anaerobic conditions. The cell surface characteristics of var. indica and W29 were found to be different with respect to contact angle and percent hydrophobicity. For instance, percent hydrophobicity of var. indica was 89.93 ± 1.95 while that of W29 was 70.78 ± 1.1. Furthermore, while all tested strains metabolize hydrocarbons, only var. indica was able to use it as a carbon source. Yeast cells of var. indica metabolized hexadecane with higher efficiency than the mycelial form, whereas the mycelial form of the terrestrial strain metabolized the hydrocarbon more efficiently, as occurred with the mycelial monomorphic mutant AC11, compared to the yeast monomorphic mutant AC1.

Synthesis of cell wall microfibrils in vitro by a "soluble" chitin synthetase from Mucor rouxii.

A "soluble" form of chitin synthetase was separated from a membrane-rich fraction by exposure to the enzyme substrate (uridine diphosphate N-acetyl-D-glucosamine) and activator (N-acetyl- D-glucosamine). The solubilized enzyme catalyzed the synthesis of chitin microfibrils similar, if not identical, to those formed in vivo by the fungus. Cell wall microfibrils were thus abundantly formed in the absence of a living cell or its membranes.

Biosynthesis of beta-glucans by cell-free extracts from Saccharomyces cerevisiae.

Cell-free extracts from Saccharomyces cerevisiae catalyzed the incorporation of glucosyl residues from UDP-[U-14C]glucose into beta-1,3-glucans which contained a significant proportion of beta-1,6-glycosidic linkages. When GDP-[U-14C]glucose was used as substrate only trace amounts of glucose were incorporated. Activity of beta-glucan synthetase was distributed among membrane and cell wall fractions, specific activity being higher in this latter. Beta-glucan synthesized by membrane and cell wall fractions contained 0.6% and 2.5% of beta-1,6-glycosidic linkages respectively. A marked decrease in the activity of beta-glucan synthetase occurred as the cells aged. Significant activity of glycogen synthetase was detected only in cells which had reached the stationary phase of growth.

Regulation of nitrate reductase at the transcriptional and translational levels in Escherichia coli.

Nitrate reductase from Escherichia coli is induced by nitrate and derepressed by oxygen removal after a lag phase. Elimination of inducer, shift to aerobic conditions and addition of actinomycin D causes the decline in the rate of its synthesis, which eventually may stop. Kinetic analysis of the sensitivity of the biosynthetic process to oxygen, chloramphenicol, actinomycin D and rifampicin gave results which we interprete as evidence that oxygen (and possibly nitrate) affect simultaneously both the transcriptional and translational processes.

Dissociation of chitosomes by digitonin into 16 S subunits with chitin synthetase activity.

Digitonin exerts profound effects on chitosomes (microvesicular structures with chitin synthetase activity isolated from the fungus Mucor rouxii). At low concentrations, it stimulates chitin synthetase (UDP-2-acetamido-2-deoxy-D-glucose: chitin 4-beta acetamidodeoxy-D-glucosyltransferase, EC 2.4.1.16) activity; at higher concentrations, it inhibits it. Digitonin also causes disintegration of the chitosome and the release of a homogeneous population of chitosome subunits with chitin synthetase activity. These chitosome subunits have a sedimentation coefficient of 16 S, compared to 105 S for whole chitosomes, as determined by centrifugation in sucrose density gradients, and measure 7--12 nm in diameter. After dissociation, chitin synthetase remains in a zymogenic state, and requires treatment with a protease for activation. No change in sedimentation coefficient of chitosome subunits was observed after proteolytic activation. The product synthesized by the chitosome subunits was characterized by X-ray diffractometry ad alpha-chitin and was by the criterion indistinfuishable from chitin made by preparations of undissociated chitosomes. However, in the electron microscope, the chitin microfibrils made from chitosome subunits were, in general, much shorter than those produced by undissociated chitosomes and often exhibited a needle-like appearance.

The inhibitory protein of chitin synthetase from Mucor rouxii is a chitinase.

The 'inhibitor' of chitin synthetase previously isolated from the cytosol of Mucor rouxii was found to be a chitinase. Paper chromatographic analysis of reaction products showed that the overall rate of chitin synthesis was unaffected by the 'inhibitor'. The observed reduction in chitin synthesis was due to depolymerization of chitin, mostly dimers (diacetyl-N,N'chitobiose). The chitinase was much more effective against nascent chitin, i.e., chitin being made in a chitin synthetase incubation mixture, than against preformed chitin.

Purification and properties of an inhibitory protein of chitin synthetase from Mucor rouxii.

A soluble protein inhibitor of chitin synthetase (UDP-2-acetamindo-2-deoxy-D-glucose:chitin 4-beta-acetamidodeoxyglucosyltransferase, EC 2.4.1.16) was isolated from the cytoplasm of Mucor rouxii. By gel filtration, the molecular weight of the inhibitor was estimated to be 17 500. The inhibitor was effective against crude or purified (chitosome) preparations of chitin synthetase. Unlike the chitin synthesis inhibitor from Saccharomyces spp., the inhibitor from M. rouxii does not operate by blocking the proteolytic activation of chitin synthetase zymogen but by inhibiting the operation of activated enzyme. Presumably, the inhibitor forms part of the regulatory mechanism of chitin synthesis in the cell.

Selective inhibition of cytosine-DNA methylases by polyamines.

We have advanced the hypothesis that polyamines affect DNA methylation and thus promote the expression of developmentally controlled genes. We demonstrate that the activity of cytosine-DNA methyltransferases HpaII, HhaI, HaeIII and SssI is inhibited by physiological concentrations of polyamines. On the other hand, activity of the adenine-DNA methyltransferase EcoRI, and restriction enzymes HpaII, HhaI, HaeIII and EcoRI, is insensitive to polyamine concentrations up to 40 mM. Our results indicate that the effect of polyamines on cytosine-DNA methyltransferases is rather selective and suggest a possible mode of action in vivo.

Chitin synthesis as target for antifungal drugs.

Human mycoses have become a threat to health world-wide. Unfortunately there are only a limited number of antimycotic drugs in use. Promising targets for drugs specific against fungi are those affecting chitin synthesis. Chitin is absent in vertebrates, and is essential for fungal wall integrity. A thorough knowledge of the mechanism of chitin synthesis is required to design specific inhibitors. We review here our current understanding of the process, and the most promising drugs that inhibit it. Chitin is made by chitin synthases requiring specific microvesicles, the chitosomes, for intracellular transport. Fungi contain several chitin synthases, some of which may be essential at a certain stage. This phenomenon is important to take into account for drug design. The most widely studied chitin synthase inhibitors are polyoxins and nikkomycins that probably bind to the catalytic site of chitin synthases. These are not equally susceptible to the drugs. In Saccharomyces cerevisiae the order of sensitivity is: Chs3p>Chs1p>Chs2p. Main problems for their succesful use in vivo are: low permeability, and different susceptibility of fungal species, and variable responses in animal models. Chemical modifications have been proposed to make more potent derivatives. Other synthetic or natural compounds are also promising as possible inhibitors, but their properties are less well known. Rational drug design has proceeded only on the basis of existing inhibitors, because the structure of the active site of chitin synthase is unknown. Undoubtedly, determination of this, and the biosynthetic mechanism will reveal unexpected drug targets in the future.

Chitin biosynthesis and structural organization in vivo.

Many organisms utilize chitin as a structural component of the protective cell walls or exoskeletons which surround them. These structures are light and resistant composites with specific structural and mechanical properties which allow them to fulfill their protective role. Chitin, in the form of microfibrils, is immersed in a matrix of proteins and other polysaccharides. Chitin microfibrils provide the high strength which allows them to resist tensions and modulus. The cementing compounds protect chitin from chemical attack; keep the microfibrils separate, preventing fracture; and provide support to tensions. The resulting structures adopt specific forms which are conserved during growth and are transmitted in a hereditary fashion. Synthesis of these complex structures involves the following steps: (i) synthesis of chitin either intracellularly or at the interphase with the extracellular medium; (ii) transport of the chitin molecules to the extracellular space; (iii) chemical modification of part of the noncrystallized chitin and association with other molecules; (iv) crystallization of the unmodified chitin which is covered by the rest of the components. The resulting supramolecular structure acquires viscoelastic mechanical properties; (v) maturation of the composite through formation of secondary covalent bonds among its components, and deposition of different substances.

Reaggregation and binding of cell wall proteins from Candida albicans to structural polysaccharides.

Urea or hot sodium dodecyl sulphate extracted a significant amount of the same proteins from the matrix of the cell wall of the yeast form and mycelial cells of Candida albicans. Gel filtration analysis of the urea-extracted proteins revealed that they occurred in the form of large complexes which were unaffected by up to 8 M urea. Among them, proteins en route to becoming covalently associated within the wall scaffold were identified by their reaction with specific antibodies. When urea was removed by dialysis, some of these proteins specifically reassociated into large aggregates which bound strongly with ConA, whereas others remained soluble in smaller associated products. The ability of some of these proteins to bind to the insoluble wall polysaccharides was also assessed. No self-assembling proteins were able to bind to glucans and/or chitin. Specificity of the binding to polysaccharides made of beta-bound glucosyl or N-acetylglucosaminyl residues was determined by the competitive effect of several disaccharides. Whereas laminaribiose and diacetylchitobiose were strong inhibitors of protein binding to both glucan and chitin, lactose, maltose and sucrose were ineffective.

The role of polyamines in fungal cell differentiation.

Polyamines are low molecular weight polycations which are present in all organisms, both procaryotic and eucaryotic (1). The most widely distributed polyamines are putrescine, spermidine and spermine; nevertheless, a large number of fungal species are devoid of spermine. Polyamines are essential for growth, and mutants affected in their synthesis become auxotrophic. Regarding their physiological roles, it has been demonstrated that polyamine starvation leads to reduction in the synthesis of nucleic acids and proteins. It has been concluded that polyamines are essential for macromolecule synthesis, although their precise mode of action remains unknown (2) (Table 1). Because of their net charge, it has been suggested that polyamines bind to macromolecular anions such as nucleic acids and phospholipids, stabilizing their structure. Levels of polyamines, as well as those of the first enzyme in the biosynthetic route: ornithine decarboxylase (ODC), increase during the phases of active growth and differentiation in distinct eucaryotic systems. In fungi the role of polyamines in cell differentiation remains debatable since no clear cut correlation between their levels and development has been demonstrated. This lack of correlation may be due to the fact that most polyamines present in the cell are inside the vacuole or bound to all polyanions, only a small amount remaining free to fulfill other tasks associated with development (3).

Molecular and genetic control of chitin biosynthesis in fungi.

Chitin is the most important structural component of the cell walls of fungi. Its synthesis involves the transfer of N-acetylglucosaminyl residues from the universal donor UDPGlcNAC to the growing chain. This reaction is catalyzed by an ill-defined enzyme called chitin synthetase. By use of diverse techniques, including reverse genetics, it has been possible to isolate mutants affected in chitin biosynthesis in vitro and in vivo. These studies have permitted the identification of several genes that code for the catalytic components of the enzyme, and probably for ancillary reactions. In general, two types of genes have been identified in fungi. The so-called CHS genes, from which two families, and possibly three classes exist, code for chitin synthetases activated by proteases. All fungi thus far studied contain more than one CHS gene, which are normally dispensable. The second class are larger, essential genes coding for the catalytic polypeptide of chitin synthetases non-activated by proteolysis, and probably made of more than one polypeptide. These are labeled CSD2 or CAL1. It may be hoped that our knowledge of chitin synthetase will make them the most suitable targets for new strategies to control fungal infections.

Monomorphic Nonpathogenic Mutants of Ustilago maydis.

ABSTRACT We have developed conditions which promote the dimorphic transition of haploid cells of Ustilago maydis in vitro by controlling the pH of the media. At low pH (below 5.0) mycelial growth occurs, whereas at neutral pH yeastlike growth takes place. We screened for mutants unable to form mycelium at low pH and obtained 26 mutants. These mutants have been characterized by their cell and colony morphology in different media. Mutations in 18 strains were found to be recessive when these strains were crossed with the wild type. Other crosses indicated that they were affected in genes other than a and b. Crosses between mutants suggest that the mutations fall in at least two complementation groups. In addition, mutants were characterized by their pathogenicity to corn seedlings. Mutations which were recessive for pathogenicity were also recessive for morphogenesis in vitro.

Carboxin-resistant mutant of ustilago maydis is impaired in its pathogenicity for zea mays

We analyzed the pathogenicity of chitin synthetase (chs) disruptants of Ustilago maydis obtained with the carboxin-resistant or the hygromycin-resistant cassettes. We found that only chitin synthetase (chs) mutants obtained by gene disruption with the carboxin resistance cassette lost their virulence to maize (Zea mays) seedlings. Carboxin is a systemic fungicide that inhibits respiration by preventing the oxidation of succinate. We demonstrated that carboxin-resistant transformants were affected in the levels of succinate dehydrogenase and respiratory activities when compared with hygromycin-resistant disruptants. We propose that loss of virulence in the carboxin-resistant transformants is owing to loss of respiratory fitness, which probably represents an important component of virulence in this fungus.http://link. springer-ny.com/link/service/journals/00284/bibs/39n5p291.html

Identification of the structural component in the cyst wall of Entamoeba invadens.

Cyst walls of Entamoeba invadens were isolated and purified. Both whole cysts and purified walls appeared intensely fluorescent when stained with Calcofluor white M2R. Examination of positive replicas of purified cyst walls with the electron microscope revealed the presence of a microfibrillar structure. The main sugars detected in acid hydrolysates from the walls were hexosamines. X-ray diffraction analysis of purified cyst walls demonstrated that the crystalline polymer present was chitin.