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We investigated the ability of Trametes versicolor and Pycnoporous cinnabarinus to metabolize triclosan. T. versicolor produced three metabolites, 2-O-(2,4,4'-trichlorodiphenyl ether)-beta-D-xylopyranoside, 2-O-(2,4,4'-trichlorodiphenyl ether)-beta-D-glucopyranoside, and 2,4-dichlorophenol. P. cinnabarinus converted triclosan to 2,4, 4'-trichloro-2'-methoxydiphenyl ether and the glucoside conjugate known from T. versicolor. The conjugates showed a distinctly lower cytotoxic and microbicidal activity than triclosan did.

The yeast Trichosporon montevideense CBS 6721 expressed large amounts of cytochrome P450 after cultivation in a glucose-peptone medium. The P450, which could be detected in the cytosolic fraction after cell breakage and ultracentrifugation, was purified to electrophoretic homogeneity and migrated in SDS-PAGE with a M(r) of 43,000. As indicated by IEF, the preparation consisted of two different P450 isoforms with pI-values of 5.9 and 6.2, which were named P450MS1 and P450MS2 respectively. Both isoforms had a characteristic maximum at 446 nm in the reduced carbon monoxide difference spectra. Partial N-terminal sequencing of P450MS1 and P450MS2 demonstrated a high degree of sequence homology between the soluble P450 enzymes of T. montevideense CBS 6721 and their close relationship to the soluble P450 forms of Trichosporon spec. SBUG 752, T. cutaneum ATCC 58094 and to the P450s of the CYP55 family of Fusarium oxysporum and Cylindrocarpon tonkinense.

Penicillium canescens SBUG-M 1139 was shown to be able to grow using phenoxybutyric acid as the sole carbon source. The rapid conversion of the phenoxyalkanoic acid resulted in the formation of phenol, which was metabolized completely. These reactions were accompanied by an accumulation of the methyl ketone phenoxypropan-2-one. Furthermore, during the metabolism of phenoxybutyric acid, 4-phenoxy-2,3-dehydrobutyric acid, 4-phenoxy-3-hydroxybutyric acid, phenoxyacetic acid, and phenoxypropan-2-ol accumulated in minor amounts. Clearly, fungi can metabolize phenoxyalkanoic acids to produce methyl ketones in a manner analogous to that used for the conversion of short- or medium-chain fatty acids by fungi.

Candida is a heterogeneous genus which contains about a quarter of all yeast species. It includes not only species of uncertain affiliation but also unrelated strains whose phylogenetic relationships have not been resolved. A great variety of CoQ types are present in the genus, the mol % G + C ranges from 30-63%, and species that were found to sporulate have teleomorphic counterparts in 11 different genera. Candida species are mainly associated with plants, rotting vegetation, with insects which feed on plants or with food. In line with this, 71% of Candida species utilize xylose (wood degradation), 57% of species use cellobiose (cellulose degradation), 29% oxidize aliphatic hydrocarbons (components of plant cuticula), 27% of species degrade starch as a plant storage material, and 7% utilize methanol as a possible metabolite from pectin catabolism. 85% of species require individual vitamins produced mainly in plant materials. 65% of Candida species are not able to grow at temperatures of 37 degrees C. In comparison only relatively few species occur normally in humans and other warm blooded animals. About 16% of type strains and selected strains for comparative purposes (CBS) were isolated from human specimens. Perhaps up to 10% of Candida species may be of medical importance, though this has so far only been clearly demonstrated for less than 5% of currently known species.

Purified recombinant cytochrome P450 52A3 and the corresponding NADPH-cytochrome P450 reductase from the alkane-assimilating yeast Candida maltosa were reconstituted into an active alkane monooxygenase system. Besides the primary product, 1-hexadecanol, the conversion of hexadecane yielded up to five additional metabolites, which were identified by gas chromatography-electron impact mass spectrometry as hexadecanal, hexadecanoic acid, 1, 16-hexadecanediol, 16-hydroxyhexadecanoic acid, and 1, 16-hexadecanedioic acid. As shown by substrate binding studies, the final product 1,16-hexadecanedioic acid acts as a competitive inhibitor of n-alkane binding and may be important for the metabolic regulation of the P450 activity. Kinetic studies of the individual sequential reactions revealed high Vmax values for the conversion of hexadecane, 1-hexadecanol, and hexadecanal (27, 23, and 69 min-1, respectively), whereas the oxidation of hexadecanoic acid, 1, 16-hexadecanediol, and 16-hydroxyhexadecanoic acid occurred at significantly lower rates (9, 9, and 5 min-1, respectively). 1-Hexadecanol was found to be the main branch point between mono- and diterminal oxidation. Taken together with data on the incorporation of 18O2-derived oxygen into the hexadecane oxidation products, the present study demonstrates that a single P450 form is able to efficiently catalyze a cascade of sequential mono- and diterminal monooxygenation reactions from n-alkanes to alpha, omega-dioic acids with high regioselectivity.

Cells of the yeast strain Debaryomyces vanrijiae SBUG 770, grown with glucose, converted biphenyl to 4-hydroxybiphenyl as the major metabolite. In addition, 2-hydroxybiphenyl was formed in minor amounts. No further degradation of these substances was detected. However, these monohydroxylated derivatives were oxidised by alkane-grown cells in the presence of the co-metabolic substrate, tetradecane. Under these conditions 2-hydroxybiphenyl was oxidised to 2,5-dihydroxybiphenyl, and 4-hydroxybiphenyl was rapidly metabolised by formation of two major metabolites. One was identified as 3,4-dihydroxybiphenyl. Characterisation of the second product as 4-phenylmuconolactone points to a further metabolism of the initially formed dihydroxylated biphenyl via ortho-ring fission.

The yeast Trichosporon spec. SBUG 752 isolated from soil produced cytochrome P450 during the stationary phase of growth on glucose. After cell disruption and ultracentrifugation, large amounts of P450 (250 pmol/mg protein) were found in the cytosolic fraction. In contrast, no P450 was detectable in the microsomes. Similar results were also obtained from some other yeast species of the genus Trichosporon. After purification to electrophoretic homogeneity, the P450 from Trichosporon spec. SBUG 752 migrated in SDS-PAGE with an apparent Mr of 43,000. Final purification by isoelectric focusing yielded two different isoenzymes in their spectrally active state-P450TS1 and P450TS2-having pI values of 5.9 and 6.2, respectively. Partial N-terminal amino acid sequencing revealed a high degree of sequence homology between P450TS1 and P450TS2 and their close relationship to the soluble P450 forms of the CYP55 family which are known to act as nitric oxide reductases in some filamentous fungi. The P450TS1 from Trichosporon spec. SBUG 752 catalyzed nitric oxide reduction under anaerobic conditions in an NADH- and NADPH-dependent manner-an activity not yet described for yeasts. These results demonstrate the existence of soluble P450 forms in yeasts exhibiting functional and structural characteristics similar to those of the P450 forms of the CYP55 family., (Copyright 1998 Academic Press.)

We characterized the ability of a yeast to cleave the aromatic structure of the dioxin-like compound dibenzofuran. The yeast strain was isolated from a dioxin-contaminated soil sample and identified as Trichosporon mucoides. During incubation of glucose-pregrown cells with dibenzofuran, six major metabolites were detected by high-performance liquid chromatography. The formation of four different monohydroxylated dibenzofurans was proven by comparison of analytical data (gas chromatography-mass spectrometry) with that for authentic standards. Further oxidation produced 2, 3-dihydroxydibenzofuran and its ring cleavage product 2-(1-carboxy methylidene)-2,3-dihydrobenzo[b]furanylidene glycolic acid, which were characterized by mass spectrometry and 1H nuclear magnetic resonance spectroscopy. These two metabolites are derived from 2-hydroxydibenzofuran and 3-hydroxydibenzofuran, as shown by incubation experiments using these monohydroxylated dibenzofurans as substrates.

By varying selected environmental factors, the degradation and mineralization of biaryl compounds by the bacterium Ralstonia pickettii in soil and compost were investigated. An optimized soil moisture and enhanced bioavailability by using the nonionic surfactant Tween 80 were of great importance for the degradation rates of biaryl compounds like biphenyl and 4-chlorobiphenyl by cells of Ralstonia picketti SBUG 290 inoculated into soil. Additionally, degradation of these compounds by the investigated strain in soil was strongly dependent upon the medium of precultivation. Also the influence of temperature and soil pH-value was tested. In contrast to the used soil, the autochthonous flora of the compost seemed to have a higher physiological activity. All investigated compounds (biphenyl, 4-chlorobiphenyl and dibenzofuran) were degraded quickly in compost. Inoculation with the investigated bacterium did not enhance the degradation rates significantly.

Dual-pump coherent anti-Stokes Raman scattering (CARS) has been demonstrated for the simultaneous measurement of gas-phase temperature and concentrations of molecular nitrogen and oxygen. A polarization technique was used to vary the relative intensities of the two CARS signals and expand the dynamic range of the relative concentration measurements. Detailed temperature and oxygen mole fraction measurements were performed in the stabilization region of a hydrogen-nitrogen jet diffusion flame. These results indicate that there is a region below the nozzle exit where significant amounts of oxygen are found on the fuel side of the peak flame temperature profile.

Laccase, a ligninolytic enzyme, was secreted by each of the white rot fungi Trametes versicolor and Pycnoporus cinnabarinus during growth in a nitrogen-rich medium under agitated conditions. After addition of 2-hydroxydibenzofuran to cell-free supernatants of the cultures, yellow precipitates were formed. These precipitates were poorly soluble in water and therefore readily separated from the supernatant. The products formed were more hydrophobic than the substrate, as indicated by their longer retention times on a reverse phase high-performance liquid chromatography column. Mass spectrometric analysis of the purified products indicated the formation of oligomers. Analysis of the mixture of products by gas chromatography and mass spectrometry after derivatization with diazomethane suggested the formation of at least three dimeric and nine trimeric products. Carbon-carbon and carbon-oxygen bonds were identified in the dimers and trimers, respectively. The nuclear magnetic resonance spectrum of the main dimer suggested coupling of the two monomers at the carbon one position.

The processing of yeast cells for scanning electron microscopy by conventional sequential fixation with glutaraldehyde and osmium tetroxide and subsequent dehydration and critical point-drying caused pronounced deformation and visible shrinkage in all basidiomycetous and ascomycetous yeast strains studied. The mean cell diameter decreased to nearly 60 and 70%, respectively. After an additional sequential fixation with 1% tannic acid and 0.5% uranyl acetate the cell shrinkage was significantly reduced, but the most important result was a considerable reduction of wrinkling and deformation of the yeast cells.

Incubation of phenol-induced cells of the yeast Candida maltosa SBUG 700 with mono- and dichlorophenols resulted in the formation of metabolites of the substrates and of further metabolites not related to the degradation pathway of the substrates. These additional compounds, identified as 4-hydroxyphenylacetic acid (4-HPA), phenylacetic acid (PA), indolylacetic acid (IA) and indolylethanol (i.e.) by means of HPLC and GC/MS, were not excreted in incubation experiments with glucose. The excretion of these metabolites of aromatic amino acid metabolism is not caused by toxic effects of the phenol derivatives, but seems to be a result of carbon and nitrogen starvation of yeast cells.

Trichosporon beigelii SBUG 752 was able to transform diphenyl ether. By TLC, HPLC, GC, GC-MS, NMR- and UV-spectroscopy, several oxidation products were identified. The primary attack was initiated by a monooxygenation step, resulting in the formation of 4-hydroxydiphenyl ether, 2-hydroxydiphenyl ether and 3-hydroxydiphenyl ether (48:47:5). Further oxidation led to 3,4-dihydroxydiphenyl ether. As a characteristic product resulting from the cleavage of an aromatic ring, the lactone of 2-hydroxy-4-phenoxymuconic acid was identified. The possible mechanism of ring cleavage to yield this metabolite is discussed.

Using the right to die and the United States Supreme Court case of Cruzan v. Director, Missouri Department of Health as exemplars, this article explores the notion of third-order decisionmaking. If first order decisionmaking is about what should happen, and second-order decisionmaking is about who should decide what should happen, then third-order decisionmaking is about who should decide who decides. This turns out to be an apt characterization of constitutionalism, which is centrally concerned with the allocation of responsibility for making decisions about the allocation of responsibility. Deference to erroneous second-order decisions, as in the Cruzan case itself, may merely be an example of this central feature of constitutionalism.

The genomic structure of an industrial yeast strain Trichosporon spec. LS3 was compared with Trichosporon adeninovorans type strain CBS 8244. The cot values, the portion of single copy sequences (CBS 8244: 10.9 x 10(9) D, LS3: 10.6 x 10(9) D) as well as of repetitive sequences (CBS 8244: 6.0 x 10(9) D, LS3: 5.5 x 10(9) D) per haploid genome and genome complexity of these strains (CBS 2844: 16.9 x 10(9) D, LS3: 16.1 x 10(9) D) have been analysed. Both strains show a high genome complexity. The mitochondrial DNA content was measured and compared. No plasmidal DNA was identified for both strains. A survey of the data of genomic DNA made it possible to postulate for each of both strains a haploid set of chromosomes. The intraspecific reassoziation values of the nuclear DNA from the strain LS3 and CBS 8244 are interpreted to confirm classification of these strains based on physiological and genetical properties into one species Trichosporon adeninovorans.

Oxygen is one of the most important environmental factors for microorganisms. Many metabolic reactions of aerobic or facultative anaerobic bacteria are influenced by varying oxygen concentrations. A lot of enzyme reactions in respiration processes, catabolism, anabolism and gene expression depend upon oxygen. Other enzymes such as nitrogenase or hydrogenases can be inhibited by increasing oxygen levels. Also complex metabolic processes including anaerobic respiration and fermentations are regulated by oxygen. Finally toxic oxygen derivatives have to be eliminated by living cells to overcome damage of cell constituents. In this way also bacteria which are included into the nitrogen cycle in the nature are influenced by oxygen. The different strategies of microorganisms to protect their nitrogenases for oxygen inactivation and the regulation of dissimilative nitrate reduction by oxygen are demonstrated in detail.

Two fungal strains of the genus Sporothrix, isolated from industrial waste water, were characterized with respect to some morphological and physiological properties. After comparison with other species of the genus Sporothrix, the fungal strains are described as Sporothrix nivea, n. sp. Among 28 carbon sources tested, such as sugars, polysaccharides, alcohols, organic acids, and hydrocarbons, 23 are utilized. The most remarkable property of Sporothrix nivea is the utilization of both n-alkanes and methanol.

77 Ascomycetous, basidiomycetous as well as imperfect yeast strains of 46 different species and 20 genera were tested for growth with the substrates n-octane, n-hexadecane, and phenol. Of 59 yeast strains with ascomycetous cell wall structure 33 grew on hydrocarbons and 32 on phenol. No yeast strain out of 26 which are unable to use n-alkanes as a source of carbon and energy grew on phenol. In comparison with the latter 32 out of 33 n-hexadecane assimilating yeasts were also capable of using phenol. All n-octane utilizing yeasts of this group also assimilate phenol as a carbon source for growth. The correlation of the hydrocarbon assimilation with the phenol assimilation seems to be not so strong in the basidiomycetous yeasts. 7 out of 18 strains from this group grew on n-hexadecane and 13 on phenol. Furthermore, it could be shown that the use of hydrocarbons and phenol (as well as methanol) is strongly correlated with the coenzyme Q structure of the respective yeast strain. The results are discussed with respect to the particular chemical properties of the substrates used and the fact that coenzyme Q structure is considered to be an important marker of evolutionary relationships among yeasts.

Dimorphic fungi can grow as mycelial phase and as yeast phase. The change of growth form is effected by an altered programme in gene expression, which is induced either in certain stages of ontogenesis or by environmental factors. Therefore it is necessary to distinguish ontogenetically conditioned (irreversible) and environment conditioned (reversible) dimorphism. The first is characteristic for dimorphic Ustomycetes, Basidiomycetes and related anamorphs as well as for Taphrinales; the second for the majority of dimorphic Ascomycetes, Endomycetes, and related anamorphs. The integration of dimorphic fungi in the systems of filamentous fungi and of yeasts--which originally have been elaborated rather independently--was difficult in many cases. In recent times, the study of certain ultrastructural and biochemical-physiological characters has much facilitated the taxonomic arrangement of dimorphic fungi and has allowed to demonstrate some phylogenetic relations among filamentous, dimorphic, and yeast fungi. The authors hold the concept that yeasts have evolved from filamentous resp. dimorphic fungi by neotenic simplification.