Optospectroscopic detection of primary reactions associated with the graviperception of Phycomyces. Effects of micro- and hypergravity

The graviperception of sporangiophores of the fungus Phycomyces blakesleeanus involves gravity-induced absorbance changes (GIACs) that represent primary responses of gravitropism (Schmidt and Galland, 2000). GIACs (DeltaA(460-665)) of sporangiophores were measured in vivo with a micro-dual wavelength spectrometer at 460 and 665 nm. Sporangiophores that were placed horizontally displayed an instant increase of the GIACs while the return to the vertical position elicited an instant decrease. The GIACs are specific for graviperception, because they were absent in a gravitropism mutant with a defective madJ gene. During parabola flights hypergravity (1.8 g) elicited a decrease of the GIACs, while microgravity (0 +/- 3 x 10 (-2) g) elicited an instant increase. Hypergravity that was generated in a centrifuge (1.5-6.5 g) elicited also a decrease of the GIACs that saturated at about 5 g. The GIACs have a latency of about 20 ms or shorter and are thus the fastest graviresponses ever measured for fungi, protists, and plants. The threshold for eliciting the GIACs is near 3 x 10 (-2) g, which coincides numerically with the threshold for gravitropic bending. In contrast to gravitropic bending, which requires long-term stimulation, GIACs can be elicited by stimuli as short as 20 to 100 ms, leading to an extremely low threshold dose (acceleration x time) of about 3 x 10 (-3) g s, a value, which is four orders of magnitude below the ones described for other organisms and which makes the GIACs of Phycomyces blakesleeanus the most sensitive gravi-response in literature.     

Absence of Significant Light-Induced Changes in cAMP Levels in Sporangiophores of Phycomyces blakesleeanus

We were unable to find transient changes in the amount of cAMP or cGMP that had been proposed to mediate the light-growth response in sporangiophores of Phycomyces blakesleeanus.     

Induction of intracellular and extracellular beta-galactosidase activity in Phycomyces blakesleeanus

The inductive effect of different sugars on beta-galactosidase synthesis in Phycomyces blakesleeanus has been studied. The enzyme was inducible by galactose and fructose. When grown on these sugars the enzyme level was 10-20 times greater than when grown on glucose. We have detected both intra- and extracellular beta-galactosidase activity when Phycomyces blakesleeanus was grown on galactose, but only extracellular beta-galactosidase activity when grown on fructose plus lactose.     

Liposome-protoplast fusion in Phycomyces blakesleeanus

Abstract We describe here fusion between phospholipid vesicles (liposomes) and protoplasts to the fungus Phycomyces blakesleeanus . Both 6-carboxyfluorescein and the kanamycin resistance harboured by the plasmid have been transferred from liposomes to protoplasts of Phycomyces by the fusion technique.     

Characterisation and biosynthesis of D-erythroascorbic acid in Phycomyces blakesleeanus

D-Erythroascorbate and D-erythroascorbate glucoside have been identified in the Zygomycete fungus Phycomyces blakesleeanus. Ascomycete and Basidiomycete fungi also synthesise D-erythroascorbate instead of l-ascorbate, suggesting that D-erythroascorbate synthesis evolved in the common ancestor of the fungi. Both compounds accumulate in P. blakesleeanus at higher levels than observed in other fungal species. D-Erythroascorbate glucoside reduced dichlorophenolindophenol as effectively as L-ascorbate, but was more stable to autoxidation. D-Erythroascorbate glucoside predominated in spores and stationary phase mycelium. Free D-erythroascorbate accumulated during the exponential phase of mycelial growth and decreased to very low levels in the stationary phase. This suggests an association between growth and free D-erythroascorbate. P. blakesleeanus converted exogenous D-arabinose to D-erythroascorbate and its glucoside. A monomeric NAD-dependent D-arabinose dehydrogenase of 41 kDa was purified to near homogeneity. The enzyme oxidised D-arabinose, L-galactose, and L-fucose. Correspondingly, mycelium converted exogenous L-galactose and L-fucose to L-ascorbate and 6-deoxyascorbate, respectively. The antioxidant role of D-erythroascorbate and its glucoside is discussed.     

Photomorphogenesis in<Emphasis Type="Italic"> Phycomyces</Emphasis>: differential display of gene expression by PCR with arbitrary primers

The zygomycete fungus Phycomyces blakesleeanus develops two types of fruiting bodies of very different size, macrophores and microphores. Blue light stimulates macrophorogenesis and inhibits microphorogenesis. I have adapted a method based on the polymerase chain reaction with arbitrary primers to investigate the role of differential gene expression during photophorogenesis in Phycomyces. Several cDNAs for genes induced in vegetative mycelium have been observed, but only one gene induced by blue light has been detected. I have demonstrated the feasibility of this approach by the isolation of a cDNA segment for the heat-shock protein HSP100 that is induced by blue light at the onset of sporangiophore development. The heat-shock protein HSP100 is an ATP-binding protein that has the ability to disassemble protein complexes. In plants, the gene for HSP100 is induced by light. The cDNA segment for HSP100 obtained from Phycomyces is 686 bp long and the predicted amino acid sequence contains one of the ATP-binding sites.     

Indole-3-ethanol oxidase in Phycomyces blakesleeanus. Is indole-3-ethanol a “storage pool” for IAA?

Indole-3-ethanol (IEt) was extracted from Phycomyces blakesleeanus Bgff. and purified by TLC and HPLC. Identification was performed by mass spectrum. The HPLC-purified compound showed an UV-spectrum typical for indoles, with absorption maxima at 220 and 281 nm. The IEt content varied between 1.5 nmol (g fresh weight)⁻¹ and 5.6 nmol (g fresh weight)⁻¹. The observed variations were strongly correlated with certain developmental stages of the fungus. Furthermore, the decrease of IEt between 60 and 84 h of fungal development coincides with a high IEt oxidase activity. The product of the enzyme reaction was indole-3-acetaldehyde, which was identified by co-chromatography with an authentic standard in several TLC and HPLC systems and by chemical conversion to indole-3-acetaldoxime.     

Partial characterization of intracellular protease activity which participates in the inactivation of carbamyl phosphate synthase in Phycomyces blakesleeanus

We have partially characterized an intracellular fraction from Phycomyces blakesleeanus which shows proteolytic activity. The apparent thermal inactivation constant (Kd) was 0.12 min-1 at 50 degrees C. This proteolytic fraction was split into two active fractions by ultrafiltration using a membrane with an exclusion size of 30,000. Both fractions were inhibited by phenyl methyl sulphonyl fluoride. The Ki value for the fraction with molecular weight greater than 30,000 was 0.075 mM. The fraction with molecular weight less than 30,000 inactivated the Phycomyces CPS.     

Specific tropism caused by ultraviolet C radiation in Phycomyces

The giant sporangiophores of Phycomyces blakesleeanus turn towards blue and away from ultraviolet C sources (wavelength under 310 nm). We have isolated fifteen mutants with normal blue tropism but defective ultraviolet tropism. Wild-type sporangiophores described a double turn when exposed successively to blue and ultraviolet beams coming from the same side; under certain conditions, the mutants turned only to the blue. The new uvi mutations modified the behaviour in heterokaryosis and were lethal in homokaryosis, i.e., they affected essential cellular components. The responses of the wild type and one of the mutants were registered and evaluated with a computer-aided device. The mutant behaved normally under blue light, but took longer than the wild type to turn away from the ultraviolet source. With very weak ultraviolet stimuli (10(-8) and l0(-9) W m-2), the wild type turned towards the source, but the mutant did not respond. Calculations of absorbed-energy distributions in the sporangiophore showed that Phycomyces responds differently to similar spatial distributions of blue and ultraviolet radiations. Wild-type and mutant sporangiophores had the same high ultraviolet absorption due to gallic acid. We conclude that ultraviolet tropism is not just a modification of blue phototropism due to the high ultraviolet absorption of the sporangiophores. Phycomyces has a separate sensory system responsive to ultraviolet radiation, but not to blue light.     

The Effects of Calcium and the Ionophore A23I87 on Modulation, Nitrogen Fixation and Growth of Soybeans

Tryptophan synthase in Phycomyces blakesleeanus. Part II: Activity of tryptophan synthase in Phycomyces blakesleeanus depending on the light and the content of zinc ions in the culture medium Five-day-old cultures of Phycomyces blakesleeanus show notice-able differences in the phenotype, depending on the culture conditions (permanent light, permanent dark, zinc deficiency, zinc sufficiency) and related to the distribution of tryptophan synthase activity between mycelium and sporangiophores. Permanent light and the presence of zinc ions in the medium during culturing have an antagonistic influence on the tryptophan synthase. The activity of the enzyme is being reduced in the sporangiophores and increased in the mycelium by the influence of light, while zinc ions in the culture medium increase the activity in the sporangiophores at simultaneous reduction in the mycelium. The importance of tryptophan synthase and tryptophan for the development of the fungus in relation to the metabolism of indole acetic acid is discussed.     

Isolation and characterization of Phycomyces blakesleeanus ferritin.

Ferritin was isolated from the fungus Phycomyces blakesleeanus and compared biochemically and immunologically with horse spleen ferritin. Phycomyces and horse spleen ferritins were shown to exhibit similar electrophoretic patterns on polyacrylamide gels. Both preparations yielded an identical single band on sodium dodecyl sulfate-containing polyacrylamide gels. Tryptic digests of Phycomyces ferritin yielded 17 ninhydrin-positive spots as compared to 26 for horse spleen ferritin tryptic digests. Phycomyces ferritin was immunologically unrelated to horse spleen ferritin.     

Modification of sexual development and carotene production by acetate and other small carboxylic acids in Blakeslea trispora and Phycomyces blakesleeanus

In Phycomyces blakesleeanus and Blakeslea trispora (order Mucorales, class Zygomycetes), sexual interaction on solid substrates leads to zygospore development and to increased carotene production (sexual carotenogenesis). Addition of small quantities of acetate, propionate, lactate, or leucine to mated cultures on minimal medium stimulated zygospore production and inhibited sexual carotenogenesis in both Phycomyces and Blakeslea. In Blakeslea, the threshold acetate concentration was <1 mmol/liter for both effects, and the concentrations that had one-half of the maximal effect were <2 mmol/liter for carotenogenesis and >7 mmol/liter for zygosporogenesis. The effects on Phycomyces were similar, but the concentrations of acetate had to be multiplied by ca. 3 to obtain the same results. Inhibition of sexual carotenogenesis by acetate occurred normally in Phycomyces mutants that cannot use acetate as a carbon source and in mutants whose dormant spores cannot be activated by acetate. Small carboxylic acids may be signals that, independent of their ability to trigger spore germination in Phycomyces, modify metabolism and development during the sexual cycle of Phycomyces and Blakeslea, uncoupling two processes that were thought to be linked and mediated by a common mechanism.     

Influence of pH on the allosteric properties of lactate dehydrogenase activity of Phycomyces blakesleeanus.

1. Lactate dehydrogenase from mycelium of Phycomyces blakesleeanus showed positive homotropic interactions with NADH at all pH values studied (pH 5.0-7.7). The calculated values for the first and last intrinsic association constants remained unaltered with pH, in contrast with the Hill coefficient value, which varied significantly, reaching its maximum values at pH 6.0 and 7.7. This suggests the hypothesis that pH regulates these homotropic effects by changes in the value of the intermediate intrinsic association constants. 2. From pH 7.2 to 7.7 lactate dehydrogenase exhibited, likewise, positive homotropic interactions with pyruvate. There were practically no changes in the first and last intrinsic association constants and in Hill coefficient values with pH. At pH values below 7.2 (pH 5.0-6.8) the enzyme showed high substrate inhibition, which was highly dependent on pH, NADH concentration and temperature. By way of substrate inhibition pH regulates, primarily, lactate dehydrogenase activity towards pyruvate, since the homotropic effects appear not to be dependent on pH. 3. Fructose 1,6-bisphosphate is a true allosteric effector of lactate dehydrogenase of Phycomyces blakesleeanus. it decreases positive co-operativity with NADH, and on the other hand pyruvate co-operativity turns into mixed co-operativity. In addition, the effector decreases the inhibitory effect caused by pyruvate.     

Gravitropism in Phycomyces: threshold determination on a clinostat centrifuge

The absolute sensitivity of sporangiophores of Phycomyces blakesleeanus to centrifugal acceleration was determined on a clinostat centrifuge. The centrifuge provides centrifugal accelerations ranging from 10(-4) to 6 x g. The rotor of the centrifuge, which accommodates 96 culture vials with single sporangiophores, is clinostatted, that is, turning "head over", at slow speed (1 rev min(-1)) while it is running. The negative gravitropism of sporangiophores is characterized by two components: a polar angle, which is measured in the plane of bending, and an aiming-error angle, which indicates the deviation of the plane of bending from the vector of the centrifugal acceleration. Dose-response curves were generated for both angles with centrifugations lasting 3, 5, and 8 h. The threshold for the polar angle depends on the presence of statoliths, so-called octahedral protein crystals in the vacuoles. The albino strain C171 carAcarR (with crystals) has a threshold near 10(-2) x g while the albino strain C2 carAgeo-3 (without crystals) has a threshold of about 2 x 10(-1) x g. The threshold for the aiming error angle is ill defined and is between 10(-2) and 10(-1) x g. The threshold for the polar angle of the wild type NRRL 1555 (with crystals) is near 8 x 10(-2) x g.     

Mutants of Phycomyces blakesleeanus Defective in Acetyl-CoA Synthetase

Nine mutants of the filamentous fungus Phycomyces blakesleeanus have been isolated on the basis of their resistance to fluoroacetate. None of the isolates uses acetate as the sole carbon source. Genetic complementation experiments revealed that all the mutants belong to the same complementation group. Biochemical analysis indicated that the acetate-induced acetyl-CoA synthetase activity is abolished in all nine mutants, thus suggesting that they are affected in the gene coding for acetyl-CoA synthetase (facA).     

Mossbauer effect studies in the fungus Phycomyces.

Mossbauer spectra of 57- Fe have been observed from different parts (mycelia, spores, sporangiophores) of the fungus Phycomyces blakesleeanus grown in an agar medium isotopically enriched with 57- Fe. The spectra indicate that the iron within Phycomyces exists primarily in two chemical states: one which is the same as that of the iron in the growth medium and the other in the form of ferritin, an iron-storage protein. The amount of iron in the former state is observed to decrease relative to the amount of iron in the latter state in going from mycelia to the sporangiophores to the sporangia themselves. Thus, the conversion of iron from the chemical state of the nutrient to ferritin has been monitored for different parts of the phycomyces. In addition, our spectra indicate that at low temperatures the iron atoms clustered within a ferritin molecule are antiferromagnetically coupled. The size of these clusters is inferred from their superparamagnetic behavior at low tempertures and comparison with horse ferritin indicates that the phycomyces ferritin iron clusters are smaller by a factor of two.     

Microbial transformation of steroids--II. Transformations of progesterone, testosterone and androstenedione by Phycomyces blakesleeanus

Phycomyces blakesleeanus transformed progesterone, testosterone and androstenedione into mixtures of products. Five monohydroxylated metabolites were obtained in reasonable yields from the progesterone transformation. Only 7 alpha- and 15 beta-hydroxyprogesterone have been reported previously from this organism. We find that it gives these two metabolites and also 6 beta-, 14 alpha- and 15 alpha-hydroxyprogesterone as major products. Five compounds were also purified from testosterone transformation mixtures. Two of these were monohydroxylated, two were ring A dehydrogenation products, and two were oxidised at C-17. The products were identified as 6 beta-hydroxytestosterone, 7 alpha-hydroxytestosterone, androsta-1,4-diene-3,17-dione (1-dehydroandrostenedione), 17 beta-hydroxyandrosta-1,4-diene-3-one (1-dehydrotestosterone) and androstenedione. All five metabolites were produced in reasonable yields, although hydroxylation was the minor transformation in this case. Only two significant products were formed from androstenedione. Both were reduced at C-17; one was also monohydroxylated. They were testosterone and 14 alpha-hydroxytestosterone. The testosterone and androstenedione transformation products have not been reported previously for this organism. We also report for the first time the preparation of P. blakesleeanus cell-free extracts which transformed progesterone reasonably efficiently and faithfully in vitro, although the proportions of each product varied from one extract to another.     

OCTAHEDRAL CRYSTALS IN PHYCOMYCES. II

"Phycomyces blakesleeanus" sporangiophores contain octahedral crystals throughout their cytoplasm and vacuole. More octahedral crystals were found in the wild-type strain G5 (+) than in the β-carotene-deficient mutant C5 (-), and much more than in the mutant C141 (-), which is sensitive to only high light intensity. In the wild type, the number of crystals per sporangiophore increased until the sporangiophore reached stage IV, and then decreased. Stage I contained the most crystals per unit volume. Cultures grown in darkness had the maximum number of crystals. Under high light intensity, there was an overall reduction of crystals. The crystals are regular octahedrons. The crystals were isolated from the sporangiophores by a method of sucrose density-gradient centrifugation. They contain nearly 95% protein, are stable in organic solvents, but can be solubilized in buffer solution above pH 9.5 and below 2.5. The crystals weakly fluoresce with an emission peak at 540 nm, which is affected by irradiation with white light. Absorption spectra of freshly prepared crystals show absorption maxima around 265–285 nm, 350–380 nm, and 450–470 nm. These absorption peaks for the crystals are close to those of the phototropic and light-growth action spectra. These data suggest that the crystals may contain a flavoprotein which may be the photoreceptor pigment of "Phycomyces".