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.

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.

Disruption of gene YlODC reveals absolute requirement of polyamines for mycelial development in Yarrowia lipolytica.

Polyamines are required for cellular growth and differentiation. In mammals and fungi they are synthesized via a pathway involving ornithine decarboxylase (ODC), which transforms ornithine into putrescine. We have cloned and disrupted the gene coding for ODC in Yarrowia lipolytica to analyze the role of polyamines in dimorphism of this fungus. Substrate- and cofactor-binding motifs, as well as two putative PEST boxes were identified in the amino acid sequence. A single transcript 1.7 kb in size was identified by Northern hybridization, and confirmed by rapid amplification of cDNA ends (RACE). Null mutants lacked ODC activity and behaved as polyamine auxotrophs. When low levels of polyamines were supplied to the null mutant, only yeast-like, but not mycelial growth was sustained. This phenomenon was confirmed by introduction of the YlODC gene under the control of an inducible promoter into the null mutant.

Cycloheximide-induced inhibition of chitin synthesis, and decay of apical vesicles and chitosomes in Phycomyces blakesleeanus.

Addition of cycloheximide rapidly inhibited protein synthesis in Phycomyces blakesleeanus. In contrast, chitin biosynthesis decreased with biphasic kinetics displaying a slow and a rapid decay phases. Electron microscopic studies revealed a decrease in the number of apical vesicles and chitosomes after cycloheximide addition; and no change in wall thickness. It is proposed that the slow phase of decay in chitin biosynthesis represents the exhaustion of the pool of chitosomes which transport the chitin synthase necessary to maintain apical wall growth; whereas the second one corresponds to inactivation of the enzyme, which is short lived in vivo. Data also rule out a change in the polarization of wall synthesis induced by cycloheximide, as suggested in other systems.

Completion of the sexual cycle and demonstration of genetic recombination in Ustilago maydis in vitro.

The heterobasidiomycetes responsible for plant smuts obligatorily require their hosts for the completion of the sexual cycle. Accordingly, the sexual cycle of these fungi could so far be studied only by infecting host plants. We have now induced Ustilago maydis, the causative agent of corn smut, to traverse the whole life cycle by growing mixtures of mating-compatible strains of the fungus on a porous membrane placed on top of embryogenic cell cultures of its host Zea mays. Under these conditions, mating, karyogamy and meiosis take place, and the fungus induces differentiation of the plant cells. These results suggest that embryogenic maize cells produce diffusible compounds needed for completion of the sexual cycle of U. maydis, as the plant does for the pathogen during infection.

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

Control of filament formation in Candida albicans by polyamine levels.

Candida albicans, the most common fungal pathogen, regulates its cellular morphology in response to environmental conditions. The ODC gene, which encodes ornithine decarboxylase, a key enzyme in polyamine biosynthesis, was isolated and disrupted. Homozygous null Candida mutants behaved as polyamine auxotrophs and grew exclusively in the yeast form at low polyamine levels (0.01 mM putrescine) under all conditions tested. An increase in the polyamine concentration (10 mM putrescine) restored the capacity to switch from the yeast to the filamentous form. The strain with a deletion mutation also showed increased sensitivity to salts and calcofluor white. This Candida odc/odc mutant was virulent in a mouse model. The results suggest a model in which polyamine levels exert a pleiotrophic effect on transcriptional activity.

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.

Increased activity of wall hydrolytic enzymes may explain the phenotype of Saccharomyces cerevisiae fragile mutants.

Fragile mutants of Saccharomyces cerevisiae require osmotic stabilizers and lyse in hypotonic solutions. A single recessive mutation, srb1, is responsible for their phenotype, but the cause of cell lysis remains uncertain. We have analyzed three possible mechanisms for this behavior: comparative amounts of wall per cell; their chitin content; and the relative activity of wall hydrolytic enzymes activated by osmotic shock. We found normal amounts of wall and higher amounts of chitin in the fragile mutants. Determination of lytic enzymes by radiolabel of the reducing ends of wall polysaccharides gave results suggesting that fragile mutants produce increased amounts of stretch-activated wall hydrolytic enzymes, which may be responsible for their lysis in hypotonic media. These enzymes normally may play a role in cell wall growth and shaping.

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.

Correlation between the regulation of sterigmatocystin biosynthesis and asexual and sexual sporulation in Emericella nidulans.

We analyzed the regulation of sterigmatocystin biosynthesis in wild type and mutant strains of Emericella nidulans (= Aspergillus nidulans). A positive correlation between both asexual and sexual sporulation and synthesis of the mycotoxin was observed. Those conditions which favored sporulation stimulated sterigmatocystin formation, and vice versa. Both processes were stimulated by light in a veA+ genetic background. In contrast, they were inhibited by diaminobutanone, an inhibitor of ornithine decarboxylase. The effect of this inhibitor was partially reverted by putrescine addition. Partial supplementation of specific requirements to auxotrophic mutants allowed normal vegetative growth, but interfered with asexual sporulation and sterigmatocystin biosynthesis. Synthesis of the mycotoxin was neither affected in a brlA mutant or in developmental mutants blocked at later steps in sporulation. As in wild type strain, diaminobutanone inhibited sterigmatocystin biosynthesis and cleisthotecia formation in the brlA mutant, and its effect was reverted by addition of putrescine. The inhibitor also affected the transcription of brlA. Our results indicate that sporulation and the synthesis of sterigmatocystin are co-regulated at a step previous to the brlA execution point.

The fungus Ustilago maydis, from the aztec cuisine to the research laboratory.

Ustilago maydis is a plant pathogen fungus responsible for corn smut. It has a complex life cycle. In its saprophitic stage, it grows as haploid yeast cells, while in the invasive stage it grows as a mycelium formed by diploid cells. Thus, a correlation exists between genetic ploidy, pathogenicity and morphogenesis. Dimorphism can be modulated in vitro by changing environmental parameters such as pH. Studies with auxotrophic mutants have shown that polyamines play a central role in regulating dimorphism. Molecular biology approaches are being employed for the analysis of fundamental aspects of the biology of this fungus, such as mating type regulation, dimorphism or cell wall biogenesis.

The ornithine decarboxylase gene from Candida albicans. Sequence analysis and expression during dimorphism.

The gene (CaODC) coding for ornithine decarboxylase, a key enzyme in polyamine biosynthesis, was cloned from Candida albicans by PCR and characterized. The deduced protein contains 470 amino acids together with the substrate- and co-factor-binding sequences which define the ornithine decarboxylases of eukaryotic species. It shows a high homology with other ODCs, mainly those from Saccharomyces cerevisiae and Neurospora crassa. A putative PEST sequence, which correlates quite well with those described for other fungal ODCs, could be identified in the protein. The mRNA of the gene is 2.4 kb in size and by primer extension a long leader sequence was found at -558 bases upstream of the predicted start of translation. An identical single ODC gene was identified in three different C. albicans strains. During the dimorphic switch, a transient initial increase in ODC activity was observed. This increase was not accompanied by a rise in the levels of the transcript, suggesting that ODC activity levels may be regulated post-transcriptionally during differentiation.

Relationship between aflatoxin biosynthesis and sporulation in Aspergillus parasiticus.

Regulation of aflatoxin biosynthesis during differentiation of Aspergillus parasiticus was analyzed by using a drug that inhibits the development of the fungus and mutants affected in sporulation. Diaminobutanone, a competitive inhibitor of ornithine decarboxylase, repressed spore germination. If added after spore germination had occurred, it blocked sporulation completely and suppressed aflatoxin biosynthesis, but was only partially inhibitory of mycelial growth. Putrescine partially counteracted the inhibitory effect of the drug on both sporulation and aflatoxin biosynthesis. Analysis of mutants affected in sporulation confirmed the existence of a relationship between sporulation and aflatoxin formation. A nonsporulating mutant was unable to synthesize aflatoxins. In a sectorial mutant, the sporulating sector synthesized aflatoxins normally, whereas the asporogenous sector was unable to do so. It is suggested that regulation of aflatoxin biosynthesis is correlated with the sporulation process.

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.

Differences in DNA methylation patterns are detectable during the dimorphic transition of fungi by amplification of restriction polymorphisms.

A modification of the amplified fragment length polymorphism technique was developed for the determination of DNA methylation in dimorphic fungi representative of three of the major fungal taxa: Mucor rouxii, a zygomycete; Yarrowia lipolytica, an ascomycete; and Ustilago maydis, a basidiomycete. DNA obtained from the yeast or mycelial stages of the fungi was digested with a mixture of EcoRI, and one of the isoschizomers MspI and HpaII, whose ability to cleave at the sequence CpCpGpG is affected by the methylation state. The resulting fragments were ligated to primers and subjected to a double round of amplification by the polymerase chain reaction, radiolabeled in the second round, and separated by polyacrylamide gel electrophoresis. Comparison of patterns revealed differences indicative of fragments whose methylation state did or did not change during the dimorphic transition. These results indicate the usefulness of the method for the study of DNA methylation, demonstrate the universality of DNA methylation in fungi, and confirm that differential DNA methylation occurs during fungal morphogenesis.

Umchs5, a gene coding for a class IV chitin synthase in Ustilago maydis.

A fragment corresponding to a conserved region of a fifth gene coding for chitin synthase in the plant pathogenic fungus Ustilago maydis was amplified by means of the polymerase chain reaction (PCR). The amplified fragment was utilized as a probe for the identification of the whole gene in a genomic library of the fungus. The predicted gene product of Umchs5 has highest similarity with class IV chitin synthases encoded by the CHS3 genes from Saccharomyces cerevisiae and Candida albicans, chs-4 from Neurospora crassa, and chsE from Aspergillus nidulans. Umchs5 null mutants were constructed by substitution of most of the coding sequence with the hygromycin B resistance cassette. Mutants displayed significant reduction in growth rate, chitin content, and chitin synthase activity, specially in the mycelial form. Virulence to corn plantules was also reduced in the mutants. PCR was also used to obtain a fragment of a sixth chitin synthase, Umchs6. It is suggested that multigenic control of chitin synthesis in U. maydis operates as a protection mechanism for fungal viability in which the loss of one activity is partially compensated by the remaining enzymes.

Polyamines in growth and dimorphism of Paracoccidioides brasiliensis.

Putrescine and spermidine were the only polyamines found in Paracoccidioides brasiliensis, a dimorphic fungus pathogenic for humans. Free polyamines (putrescine > spermidine) increased during the first 24 h of yeast growth, with a second peak at 42 h, and also during the first 12 h of mycelium-to-yeast transition (spermidine > putrescine). Conjugated and bound polyamines were also quantified. 1, 4-Diamino-2-butanone decreased free putrescine and spermidine accumulation by inhibiting the activity of ornithine decarboxylase. The increase in free polyamines corresponds to bud emergence in yeast growth and to the mycelium-to-yeast transition of P. brasiliensis.