Kanji Takeo

Inhibitory effect of essential oils on apical growth of Aspergillus fumigatus by vapour contact

The inhibitory effect of seven essential oils on the apical growth of hyphae of Aspergillus fumigatus was studied using a bio cell tracer by vapour contact in a sealed vessel. Based on the inhibitory pattern, these essential oils were classified into three groups. The first group, composed of citron, lavender and tea tree oils, stopped the apical growth in a loading dose of 63 μg ml−1 air, but allowed the regrowth of the hyphae after removal of the vapour, indicating fungistatic action. The second group, consisting of perilla and lemongrass oils, stopped the apical growth in a loading dose of 6.3 μg ml−1 air, and did not allow the regrowth after gaseous contact at 63 μg ml−1 air, indicative of fungicidal action. The third group, consisting of cinnamon bark and thyme oils, retarded the growth in a dose of 6.3 μg ml−1 air, stopped it in a dose of 63 μg ml−1 air, and incompletely suppressed regrowth of the hyphae. Gas chromatographic analysis revealed that vapours of essential oils were absorbed on fungal mycelia and agar medium most abundantly by the first group, followed by the second and third groups, reflecting the volatility of the respective groups. Suppression of the apical growth by vapour contact was ascribed to the direct deposition of essential oils on fungal mycelia, together with an indirect effect via the agar medium absorbed.

Fungal Ammonia Fermentation, a Novel Metabolic Mechanism That Couples the Dissimilatory and Assimilatory Pathways of Both Nitrate and Ethanol ROLE OF ACETYL CoA SYNTHETASE IN ANAEROBIC ATP SYNTHESIS

Fungal ammonia fermentation is a novel dissimilatory metabolic mechanism that supplies energy under anoxic conditions. The fungus Fusarium oxysporum reduces nitrate to ammonium and simultaneously oxidizes ethanol to acetate to generate ATP (Zhou, Z., Takaya, N., Nakamura, A., Yamaguchi, M., Takeo, K., and Shoun, H. (2002) J. Biol. Chem. 277, 1892–1896). We identified the Aspergillus nidulans genes involved in ammonia fermentation by analyzing fungal mutants. The results showed that assimilatory nitrate and nitrite reductases (the gene products of niaD and niiA) were essential for reducing nitrate and for anaerobic cell growth during ammonia fermentation. We also found that ethanol oxidation is coupled with nitrate reduction and catalyzed by alcohol dehydrogenase, coenzyme A (CoA)-acylating aldehyde dehydrogenase, and acetyl-CoA synthetase (Acs). This is similar to the mechanism suggested in F. oxysporum except A. nidulans uses Acs to produce ATP instead of the ADP-dependent acetate kinase of F. oxysporum. The production of Acs requires a functional facA gene that encodes Acs and that is involved in ethanol assimilation and other metabolic processes. We purified the gene product of facA (FacA) from the fungus to show that the fungus acetylates FacA on its lysine residue(s) specifically under conditions of ammonia fermentation to regulate its substrate affinity. Acetylated FacA had higher affinity for acetyl-CoA than for acetate, whereas non-acetylated FacA had more affinity for acetate. Thus, the acetylated variant of the FacA protein is responsible for ATP synthesis during fungal ammonia fermentation. These results showed that the fungus ferments ammonium via coupled dissimilatory and assimilatory mechanisms.

Simple detection method for distinguishing dead and living yeast colonies

A rapid and simple assay was developed for detection of yeast colonies containing dying or dead cells. Methylene blue, phloxin B, rose bengal and trypan blue at concentrations of 5-10 micromol l(-1) were shown to stain non-viable cells in colonies of Saccharomyces cerevisiae, Schizosaccharomyces pombe, Candida albicans and Filobasidium capsuligenum without staining or affecting the viability of living cells of the colonies.

Pleoanamorphic life cycle of Exophiala (Wangiella) dermatitidis

The anamorph life cycle of the black yeastExophiala (Wangiella) dermatitidis is described. The fungus is dimorphic, yeast cells being the prevalent form of propagation. The fungus is strongly hydrophilic, probably completing its anamorph life cycle in submersion. Adaptation to dry conditions is slow. Types of conidiogenesis comprise annellidic, phialidic and sympodial reproduction, in addition to isotropic development. Phialoconidia fail to germinate under the conditions tested, and thus may have a function other than dispersal. Sterile, multicellular bodies resembling aCapronia teleomorph are described.

Antisporulating and respiration‐inhibitory effects of essential oils on filamentous fungi

Summary. Sporulation of four species of filamentous fungi, namely Aspergillus fumigatus, Fusarium solani, Penicillium expansum and Rhizopus oryzae, was suppressed by gaseous contact with citron, lavender and thyme oils and, to a lesser extent, by that of perilla and tea tree oils. Lemongrass and cinnamon bark oils were scarcely active. The antisporulating effect of the essential oils was not observed when they were applied as a solution, indicating that their vapours were the active form. Moreover, exposure of fungal cultures to vapours of the active essential oils caused curling of the tips of aerial hyphae (R. cryzae) or incomplete development of conidiophores (A. fumigatus). These antisporulating effects of the vapourizing essential oils seemed to be correlated with their respirationinhibitory activity, rather than with their growthinhibitory activity.

Environmentally controlled dimorphic cycle in a fission yeast

The fission yeast Schizosaccharomyces pombe shows bipolar growth and is a convenient model for studying cell polarity and polar growth. This paper shows that the related Schiz. japonicus var. japonicus can switch to unipolar growth and can exist in both yeast and mycelial phases. On solid media, the yeast phase is unstable and prone to switch to the mycelial form, which shows unipolar growth by tip elongation. The hyphae can colonize the body of the substrate (true mycelium) or just its surface (pseudo-mycelium). The yeast-to-mycelium transition and the growth of the mycelium are regulated by a nutritional gradient and are associated with extensive vacuolation. The mycelium can convert into arthroconidia or return to the yeast phase in response to environmental changes. These environmentally controlled morphological transitions make Schiz. japonicus var. japonicus an attractive model for the investigation of cell polarity and morphogenesis.

Determination of ploidy in Cryptococcus neoformans by flow cytometry

We determined the ploidy of Cryptococcus neoformans (28 strains) isolated from patients and nature. The cellular DNA content of these strains, which stained with propidium iodide in comparison to that of two authentic haploid strains, was determined by flow cytometry. All the strains exhibited diphasic histograms. In case of the authentic haploid strains, the first peak was centred around channel 9, and the second peak around channel 18. Most strains exhibited this type of histogram. Some strains exhibited another type of histogram: the first peak was centred around channel 18, and the second one around channel 35. In flow cytometry, the channel number is correlated with the intensity of fluorescence, namely, in proportion to the channel number the DNA content in the cells increases. The cellular DNA content of the second type of histograms showed twice that of the authentic haploid strains, and thus, five of 28 isolates were concluded to be diploid, and the others haploid.

Cell-surface hydrophobicity and lipolysis as essential factors in human tinea nigra

Summary. Hydrophobic adhesion of cells of the black yeast Hortaea werneckii (Horta) Nishimura & Miyaji, causative agent of human tinea nigra, was established by microbial adhesion to hexadecane (MATH) and adhesion to polystyrene, and compared with adhesion of other species of black yeasts. Additional ecophysiological tests were performed. Hortaea werneckii cells proved to have a high degree of hydrophobicity (98.5% MATH). The species is unable to degrade keratin, but shows significant lipolytic activity. It is concluded that H. werneckii is a commensal, that shows lipophilic adhesion to human skin and survives by the assimilation of excretion products.

Microtubules and actin cytoskeleton in Cryptococcus neoformans compared with ascomycetous budding and fission yeasts.

Actin cytoskeleton and microtubules were studied in a human fungal pathogen, the basidiomycetous yeast Cryptococcus neoformans (haploid phase of Filobasidiella neoformans), during its asexual reproduction by budding using fluorescence and electron microscopy. Staining with rhodamine-conjugated phalloidin revealed an F-actin cytoskeleton consisting of cortical patches, cables and cytokinetic ring. F-actin patches accumulated at the regions of cell wall growth, i. e. in sterigma, bud and septum. In mother cells evenly distributed F-actin patches were joined to F-actin cables, which were directed to the growing sterigma and bud. Some F-actin cables were associated with the cell nucleus. The F-actin cytokinetic ring was located in the bud neck, where the septum originated. Antitubulin TAT1 antibody revealed a microtubular cytoskeleton consisting of cytoplasmic and spindle microtubules. In interphase cells cytoplasmic microtubules pointed to the growing sterigma and bud. As the nucleus was translocated to the bud for mitosis, the cytoplasmic microtubules disassembled and were replaced by a short intranuclear spindle. Astral microtubules then emanated from the spindle poles. Elongation of the mitotic spindle from bud to mother cell preceded nuclear division, followed by cytokinesis (septum formation in the bud neck). Electron microscopy of ultrathin sections of chemically fixed and freeze-substituted cells revealed filamentous bundles directed to the cell cortex. The bundles corresponded in width to the actin microfilament cables. At the bud neck numerous ribosomes accumulated before septum synthesis. We conclude: (i) the topology of F-actin patches, cables and rings in C. neoformans resembles ascomycetous budding yeast Saccharomyces, while the arrangement of interphase and mitotic microtubules resembles ascomycetous fission yeast Schizosaccharomyces. The organization of the cytoskeleton of the mitotic nucleus, however, is characteristic of basidiomycetous yeasts. (ii) A specific feature of C. neoformans was the formation of a cylindrical sterigma, characterized by invasion of F-actin cables and microtubules, followed by accumulation of F-actin patches around its terminal region resulting in development of an isodiametrical bud.

Pathogenicity of strains of the black yeast Exophiala (Wangiella) dermatitidis: an evaluation based on polymerase chain reaction.

Summary. Strains of Exophiala dermatitidis, mainly originating from patients with systemic neurotropic mycosis in Asia and from the lungs of patients with cystic fibrosis (CF) in Europe, were analysed by ribotyping of the small subunit rDNA and by random amplification of polymorphic DNA (RAPD). A characteristic banding pattern for the species was found after restriction analysis of amplified fragments V9 and ITS4. The small subunit rDNA gene of five strains was about 1800 base pairs (bp) long, while in 16 strains its length was about 3000 bp Using RAPD, seven populations could be distinguished. European CF strains as well as Asian strains from systemic mycoses are mainly distributed over two populations, one of which contained both CF strains and a systemic strain. It is concluded that the two clinical pictures are caused by genetically similar strains. The differences in pathogenicity may be explained by immunological differences in the hosts or by differences in exposure to the fungal propagules.