Transformation of Curvularia lunata IM 2901 with pAN7-1 influences selected physiological properties of the fungus.

Genetic analysis of Curvularia lunata IM 2901 transformants, previously obtained by electroporation with plasmid pAN7-1, was carried out. Isolates displayed several differences in hygromycin B resistance and their physiology. It was shown that plasmid pAN7-1 was integrated in different copy numbers and at different positions in the genome of the strains studied. Both the wild type and pAN7-1 isolates, when growing in liquid media, produced an extracellular emulsifying agent. The transformants differed in their growth kinetics, intensity of surfactant production and in the efficiency of cortexolone 11beta-hydroxylation, in comparison with the wild type. The micro-organisms varied in susceptibility to the lytic enzyme complex (Novozyme 234), which indicated the presence of differences in their cell wall composition and/or in architecture caused by an integrated plasmid pAN7-1.

The effect of cell wall components on glycine-enhanced sterol side chain degradation to androstene derivatives by mycobacteria.

beta-Sitosterol side chain degradation by Mycobacterium sp. NRRL MB 3683 results in the formation of androstene derivatives and is increased in the presence of glycine. As the sterol transformation is carried out inside the cell, higher product accumulation could indicate faster diffusion of highly hydrophobic substrate through the cell wall permeability barrier. Cell wall preparations were obtained to analyse the effect of glycine on peptidoglycan components. Peptidoglycan is known to be the target for glycine action. In glycine-treated preparations, the molar ratio of diaminopimelic acid:muramic acid, the marker compounds of tetrapeptides and glycan strands respectively, was about 60% lower than in the control. This indicates a possible reduction in cross-linking between peptide units and the destruction of peptidoglycan. Unexpectedly, glycine also caused changes in the relative proportion of mycolic acids to other lipids occurring in the strain used for this study. The enhancement of beta-sitosterol side chain degradation is likely to result from disturbing the integrity of the cell wall components responsible for the permeability barrier in mycobacteria.

Removal of anthracene and phenanthrene by filamentous fungi capable of cortexolone 11-hydroxylation.

Nine fungal strains showing ability of cortexolone hydroxylation to epicortisol and/or cortisol were screened in this work for anthracene and phenanthrene elimination (250 mg/l). All of the strains (Cylindrocladium simplex IM 2358, C. simplex IM 2358/650, Monosporium olivaceum IM 484, Curvularia lunata IM 2901, C. lunata IM 2901/366, C. tuberculata IM 4417, Cunninghamella elegans IM 1785, C. elegans IM 1785/21Gp, C. elegans IM 1785/10Gi) significantly removed anthracene and phenanthrene. During incubation with anthracene formation of intermediate products was observed. The amount of the main intermediate product, identified as 9, 10-anthraquinone, was not greater than 22.2% of the anthracene introduced to the fungal cultures. C. elegans IM 1785/21Gp was the best degrader of both anthracene and phenanthrene, removing 81.6 and 99.4% of these compounds after 7 days, respectively. Phenanthrene removal by C. elegans IM 1785/21Gp was preceded by PAHs accumulation in mycelium and growth inhibition. Elimination of phenanthrene started after one day of incubation and was related to the fungus growth.

Deleterious effects of androstenedione on growth and cell morphology of Schizosaccharomyces pombe.

Androgens (androstenedione and testosterone) belong to the most important compounds in human steroidogenesis. The 17 beta-hydroxysteroid dehydrogenase responsible for interconversion of the oxygenic group on C-17 of androgens ring is involved in steroid hormone synthesis. The fission yeast Schizosaccharomyces pombe 972 h- was found to contain constitutive 17 beta-hydroxysteroid dehydrogenase that was able to reduce androstenedione to testosterone and oxidize testosterone to androstenedione. The reductive pathway was found to be predominant while the oxidative one was carried out with much lower activity. Exogenous androstenedione, contrary to testosterone, inhibited S. pombe growth and stimulated the formation of aberrant swollen cells with slighter cell wall sensitivity to the action of the lytic enzyme Novozym. It is postulated that the 17 beta-hydroxysteroid dehydrogenase prevents the deleterious effects of androstenedione on the morphology and growth of the yeast's cells by androstedione reduction to testosterone.

The use of cryopreserved apical protoplasts from Curvularia lunata for electrotransformation.

An electroporation method, utilizing cryopreserved protoplasts, has been developed for the steroid 11-hydroxylating fungus Curvularia lunata strain IM 2901. Protoplasts released from the apical parts of 24- and 48-h-old mycelia were suspended in cryopreservation buffer and stored at -75 degrees C for several weeks. The thawed and freshly prepared (control) protoplasts were electroporated with pAN 7-1 plasmid carrying the Escherichia coli hygromycin B resistance gene (hph) under the control of Aspergillus nidulans sequences. The electroporation efficiency of the control protoplasts with plasmid pAN 7-1 was 7.5 and 12.0 transformants per microgram DNA (protoplasts liberated from 24- and 48-h-old mycelia, respectively). Protoplasts released from the younger mycelium were more stable according to their reversion ability to mycelial form and transformation efficiency. After 16 weeks of cryopreservation the yield of electroporation was 61.3% of the control value. All isolated electrotransformants proved to be stable for a period of > 4 months even without selective pressure.

Isolation and characterization of regulatory mutants from Schizosaccharomyces pombe involved in thiamine-regulated gene expression.

Mutants from Schizosaccharomyces pombe deficient in the regulation of thiamine-repressible acid phosphatase have been isolated. Mutants expressing derepressed levels of the enzyme in the presence and absence of thiamine map in three genes, tnr1, tnr2 and tnr3. mRNA levels of the pho4 gene (coding for thiamine repressible acid phosphatase) and another thiamine-regulatable gene, thi3 (coding for a thiamine biosynthetic enzyme and corresponding to nmt1) are constitutively synthesized in the mutants. The mutants also exhibit constitutive thiamine transport which is thiamine repressible in wild type. The tnr3 mutants reveal a 10-20-fold higher intracellular thiamine level than tnr1 and tnr2 mutants and wild type. Mutants expressing repressed levels of thiamine-repressible acid phosphatase map in gene thi1. No or little amounts of pho4- and nmt1-specific mRNA can be detected. These mutants are impaired in thiamine uptake and are thiamine auxotrophic due to the inability to synthesize the thiazole moiety of the thiamine molecule. All tested tnr and thi1 alleles are recessive, and thi1 mutations are epistatic over tnr mutations. We assume that the thi1 and tnr genes are involved in thiamine-mediated transcription control.

Thiamine in Schizosaccharomyces pombe: dephosphorylation, intracellular pool, biosynthesis and transport.

We have investigated the thiamine metabolism in Schizosaccharomyces pombe and shown that: (1) Thiamine-repressible acid phosphate, coded for by the gene pho4, dephosphorylates thiamine phosphates indicating that the enzyme acts as a thiamine phosphate phosphatase. (2) In vivo synthesized thiamine is present intracellularly mainly as thiamine diphosphate. Starving cells for glucose decreases the intracellular thiamine pool. (3) The genes thi2, thi3 and thi4 control thiamine biosynthesis and probably code for thiamine biosynthetic enzymes. Thi3, which is involved in the synthesis of the pyrimidine moiety of the thiamine molecule, is allelic to the thiamine repressible gene nmt1. (4) Thiamine uptake is a thiamine regulated process, probably occurs by active transport and is controlled by the gene ptr1.

Determination of cytochrome P-450 in Cunninghamella elegans intact protoplasts and cell-free preparations capable of steroid hydroxylation.

Cytochrome P-450 was shown to be involved in 11 alpha-, and 11 beta-hydroxylation of Substance S in intact C. elegans protoplasts. The steroid transformation was inhibited by carbon monoxide, the inhibitory effect being dependent on CO concentration. The function of cyt P-450 in intact protoplasts was confirmed by the estimation of strong absorption at 450 nm in the CO difference spectrum. The presence of antimycin A was necessary to prevent the reduction of the cytochrome oxidase and its interference with the cyt P-450 in the spectrophotometric analysis. The intracellular content of cyt P-450 could be increased from 5.25 pM/mg protein to 26.88 pM/mg protein when the steroid inducer was present in the medium at each stage of protoplast preparation and during cyt P-450 determination. The enriched microsomal fraction obtained from the crude extract of ruptured protoplasts contained the steroid 11 alpha-hydroxylase system of C. elegans. The activity of 11 beta-hydroxylase could not be detected under the conditions of the experiment. The localization of steroid hydroxylases of C. elegans in microsomes was confirmed by cyt P-450 detection in the 9600 x g supernatant. Membranous fractions (pellets 1100 x g and 9600 x g) of the concanavaline A stabilized protoplasts, carrying the marker plasma-membrane-bound and mitochondrial ATPases, did not show maximum absorption at 450 nm in the CO difference spectrum.

Protoplast release from fungi capable of steroid transformation.

Protoplasts were obtained from Hyphoderma roseum (Fries) and Cunninghamella elegans (Lendner), fungi capable of steroid 11-hydroxylation. The lytic enzyme preparation was derived from Trichoderma viride CBS 354-33. Homogeneous protoplast suspension, free of mycelial debris and cell wall fragments, transformed cortexolone and 6 alpha-fluorocortexolone-16,17-acetonide to the same products as the intact mycelium of the microorganisms. Liberation of protoplasts and their stabilization during steroid transformation was the most effective in 0.8 M MgSO4; still, this compound impaired steroid hydroxylation. Consequently, the concentration of the transformation product formed was nearly the same as in sucrose, mannitol, and sorbitol, compounds which caused no inhibition but which were less effective stabilizers.

Regulation of steroid 16alpha-hydroxylation in Streptomyces olivoviridis.

The steroid 16alpha-hydroxylase of Streptomyces olivoviridis was shown to be constitutive, but synthetized only in small amounts in the absence of cortisol. The induction of the enzyme with the steroidal substrate resulted in a distinct increase in the 16alpha-hydroxylation rate. This increase in activity, however, could be demonstrated only in later phases of culturing, after the growth of the mycelium was completed. The possible mechanisms controlling the activity of the enzyme during the growth phase of the culture are discussed.

Adenylate energy charge and the steroid 11 alfa-hydroxylase activity in Monosporium olivaceum during growth and starvation.

During growth of Monosporium olivaceum its energy charge, E.C., (i.e. the adenylates ratio ATP + 0.5 ADP/ATP + ADP + AMP) increased from an initial value of 0.59 up to 0.85 after 25 hr of growth and then decreased to 0.51. The increase of energy charge was followed by the decrease of the activity of the 11 alpha-hydroxylase of cortexolone. This occured very clearly in the starved mycelium. Highest hydroxylation activity was observed when the lowest E.C. level (0.39-0.33) was reached.

Changes in the cellular content of the pool constituents of Monosporium olivaceum -- a steroid hydroxylating mould.

Changes of the metabolic pool constitutents of Monosporium olivaceum -- a mould capable of steroid hydroxylation were examined. The experiments were carried during growth and starvation of the microorganism. The highest activity of the 11alpha-hydroxylase was observed in the mycelium which contained the lowest level of free amino acids, glucose, and mannitol. It is suggested that the inhibition of biosynthetic processes and the decrease of the respiration rate, the activity of the NAD(P)H regenerating systems maintained, provide the optimal physiological conditions for the activity of the steroid hydroxylases.