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PubMed1979

Serine proteinases and their inhibitors in Phycomyces blakesleeanus.

Three serine proteinases of Phycomyces blakesleeanus were isolated and characterized. The molecular weights were determined to be 18,000, 22,000, and 60,000. The proteinases were solubilized by detergent or salt treatment. Two soluble proteins that specifically inhibit these proteinases were also isolated and characterized. Both these proteins formed 1:1 complexes with the serine proteinases. A molecular weight of 10,000 was estimated for both inhibitors. They were found to be present in excess in the cells. An acid proteinase of Phycomyces was able to take the inhibitor off a serine proteinase.inhibitor complex. This proteinase was partially purified. The proteinases and inhibitors of three mutant strains were partially purified and compared with a standard strain. These mutants exhibit abnormal growth responses of the sporangiophore to light. Mutant specific changes of the proteinases and their inhibitors were detected, but a connection to the behavioral responses could not be demonstrated.

Endopeptidases↗

PubMed1975

Characterization of the genome of Phycomyces blakesleeanus.

DNA of Phycomyces blakesleeanus was extracted from whole mycelia and from nuclear and mitochondrial organelle fractions obtained from sporangiophores. DNA from all three sources exhibits one symmetrical band at p equals 1.688 g/ml in CsCl buoyant density gradients. Reassociation data are consistent with kinetic division of the DNA into three components: very rapidly renaturing (fraction I), rapidly reassociating (fraction II) and slowly reassociating (fraction III) base sequences. These components comprise approximately 10%, 20% and 64% of total cell DNA. Kinetic fractions were prepared from total cell DNA and reassociated separately. The corrected rate constant is 11.3 M-1-S-1 for the rapidly reassociating component and 0.055 M-1-S-1 for the slowly reassociating component. Based on these data and the data from unfractionated total cell DNA, the genome size of Phycomyces is approximately 1.9-10-10 daltons, 6.7 times that of Escherichia coli.

Cell Nucleus↗

PubMed1981

Replacement of riboflavin by an analogue in the blue-light photoreceptor of Phycomyces.

Under suitable conditions, roseoflavin [7-methyl-8-dimethylamino-10-(1'-D-ribityl)isoalloxazine] replaces riboflavin to about 80% in the photoreceptor of Phycomyces. The substitute-bearing photoreceptor functions with an efficiency of about 0.1% of that of the normal receptor. The substitution is proven by (i) a decrease of the effective light flux by a factor of 4.7, expressed as a corresponding increase in threshold, and (ii) an increase of the effectiveness of 529-nm light relative to 380-nm light. It has also been shown that roseoflavin is taken up by the mycelium, translocated to the sporangiophore, and effectively phosphorylated by the riboflavin kinase of Phycomyces.

Journal Article↗

PubMed1973

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 beta-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".

Carotenoids↗

PubMed1974

Linear dichroism and orientation of the Phycomyces photopigment.

The greater sensitivity of a cylindrical Phycomyces sporangiophore to blue light polarized transversely rather than longitudinally is a consequence of the dichroism and orientation of the receptor pigment. The abilities of wild type and several carotene mutants to distinguish between the two directions of polarization are the same. The E-vector angle for maximum response relative to the transverse direction is 42 +/- 4 degrees at 280 nm, 7 degrees +/- 3 degrees at 456 nm, and 7 degrees +/- 8 degrees at 486 nm. The in vivo attenuation of polarized light at these wavelengths is very small. The polarized light effect in Phycomyces cannot arise from reflections at the cell surface or from differential attenuations due to internal screening or scattering.

Fungi↗

PubMed1975

Intracellular recordings from phycomyces.

Intracellular recordings from the giant sporangiophore of Phycomyces stage II were obtained. The mean transmembrane potential for 30 observations was -119.9 millivolts (negative inside), and it did not change either as a result of a light stimulus or during dark adaptation. Injected depolarizing and hyperpolarizing step currents and steady currents did not produce any avidence of spike activity. We conclude that light transduction and dark adaptation in Phycomyces are not based on alterations of the transmembrane potential.

Journal Article↗

PubMed1992

Growth rate fluctuations in phycomyces sporangiophores.

The growth rate of the Phycomyces sporangiophore fluctuates under constant environmental conditions. These fluctuations underlie the well-characterized sensory responses to environmental changes. We compared growth fluctuations in sporangiophores of unstimulated wild type and behavioral mutants by use of maximum entropy spectral analysis, a mathematical technique that estimates the frequency and amplitude of oscillations in a time series. The mutants studied are believed to be altered near the input ("night-blind") or output ("stiff" and "hypertropic") of the photosensory transduction chain. The maximum entropy spectrum of wild type shows a sharp drop-off in spectral density above 0.3 millihertz, several minor peaks between 0.3 and 10 millihertz, and a broad maximum near 10 millihertz. Similar spectra were obtained for a night-blind mutant and a hypertropic mutant. In contrast, the spectra of three stiff mutants, defective in genes madD, madE, or madG, had distinctive peaks near 1.6 mHz and harmonics of this frequency. A madF stiff mutant, which is less stiff than madD, madE, and madG mutants, had a spectrum intermediate between wild type and the three other stiff mutants. Our results indicate that alterations in one or more steps associated with growth regulation output cause the Phycomyces sporangiophore to express a rhythmic growth rate.

Journal Article↗

PubMed1998

Mutants of phycomyces with decreased gallic acid content.

Most plants and some fungi accumulate phenols. Two hydroxybenzoic acids, gallic and protocatechuic acids, are abundant in the giant sporangiophores of the zygomycete Phycomyces blakesleeanus, much more so than in the basal mycelium or the culture medium. The actual concentrations vary with illumination, age of the culture, and composition of the medium. We devised a simple screening procedure to isolate hba mutants whose sporangiophores contained less gallic acid than the wild type. The most useful mutant had very low concentrations of hydroxybenzoic acids in the sporangiophores, but about the same as the wild type in the basal mycelium and the medium. The mutant was only slightly different from the wild type in growth and morphology. Mutant and wild-type sporangiophores grew away from ultraviolet C sources (260 nm) equally well. Contrary to previous conjectures, ultraviolet tropism does not depend on the ultraviolet absorption of gallic acid or other free hydroxybenzoic acids in the sporangiophore. Against expectations, phenols did not impair DNA extraction: sporangiophores produced better DNA preparations than basal mycelia and the hba mutant only slightly better than the wild type.

DNA, Fungal↗

PubMed1998

Functional characteristics of pyruvate transport in Phycomyces blakesleeanus.

A saturable and accumulative transport system for pyruvate has been detected in Phycomyces blakesleeanus NRRL 1555(-) mycelium. It was strongly inhibited by alpha-cyano-4-hydroxycinnamate. l-Lactate and acetate were competitive inhibitors of pyruvate transport. The initial pyruvate uptake velocity and accumulation ratio was dependent on the external pH. The Vmax of transport greatly decreased with increasing pH, whereas the affinity of the carrier for pyruvate was not affected. The pyruvate transport system mediated its homologous exchange, which was essentially pH independent, and efflux, which increased with increasing external pH. The uptake of pyruvate was energy dependent and was strongly inhibited by inhibitors of oxidative phosphorylation and of the formation of proton gradients. Glucose counteracted the inhibitory effect of the pyruvate transport produced by inhibitors of mitochondrial ATP synthesis. Our results are consistent with a pyruvate/proton cotransport in P. blakesleeanus probably driven by an electrochemical gradient of H+ generated by a plasma membrane H+-ATPase.

Acetates↗

PubMed2000

Statoliths in Phycomyces: characterization of octahedral protein crystals.

To identify the molecular mechanisms of gravitropism in the fungus Phycomyces blakesleeanus we determined several biochemical and physical parameters of paracrystalline protein bodies, so-called octahedral crystals. The crystals, which are present throughout the central vacuoles of the sporangiophore, function as statoliths (Schimek et al., 1999a,b). They possess an average volume of 9.96 microm(3) and a specific mass of 1.26 g cm(-3). SDS-PAGE of purified crystals shows three major proteins with relative molecular masses of 16, 46.5, and 55 kDa. These proteins are absent in gravitropism mutants which lack the crystals. Phototropism mutants (genotype mad) which are graviresponsive (class 1) and those which are defective in gravitropism (class 2) contain the crystals and the three associated proteins. Absorption spectra of isolated crystals and in situ absorption spectra of growing zones indicate the presence of chromophores, probably oxidized and reduced flavins. The flavin nature of the chromophores is also indicated by their fluorescence properties. It appears likely that the chromophores represent an essential part of the statoliths and thus the gravitropic transduction chain.

Crystallization↗

PubMed2002

Specific and reversible inactivation of Phycomyces blakesleeanus isocitrate lyase by ascorbate-iron: role of two redox-active cysteines.

Phycomyces blakesleeanus isocitrate lyase (EC 4.1.3.1) is in vivo reversibly inactivated by hydrogen peroxide. The purified enzyme showed reversible inactivation by an ascorbate plus Fe(2+) system under aerobic conditions. Inactivation requires hydrogen peroxide; was prevented by catalase, EDTA, Mg(2+), isocitrate, GSH, DTT, or cysteine; and was reversed by thiols. The ascorbate served as a source of hydrogen peroxide and also reduced the Fe(3+) ions produced in a "site-specific" Fenton reaction. Two redox-active cysteine residues per enzyme subunit are targets of oxidative modification; one of them is located at the catalytic site and the other at the metal regulatory site. The oxidized enzyme showed covalent and conformational changes that led to inactivation, decreased thermal stability, and also increased inactivation by trypsin. These results represent an example of redox regulation of an enzymatic activity, which may play a role as a sensor of redox cellular status.

Antioxidants↗

PubMed2001

Topology of an intracellular transduction chain (phototropism of Phycomyces): 1. Joint review of functional, temporal, and spatial aspects.

Two light-induced growth reactions in a unicellular cylindrical sporangiophore of Phycomyces blakesleeanus-vertical growth acceleration under symmetrical irradiation (photomecism) and directional growth under unilateral irradiation (phototropism)-share common input light perception as well as common output growth mechanism but have strongly divergent dynamics and other distinctive features. This divergence culminates in the phototropic paradoxes the main of which states that photomecism shows total adaptation, while phototropism does not adapt. The basis for this contradiction is that the phototropic transduction chain, unlike that of photomecism, faces a spatially non-uniform stimulus and processes a series of spatial patterns (light and absorption profiles, adaptation profile, etc.). The only way to resolve the paradoxes and correlate features of both responses within a single transduction chain is to assume non-local signal transduction, e.g. a cross-talk between different azimuthal locations within the cylindrical cell. On the other hand, to establish the presence of an appropriate cross-talk is equivalent of gaining insight into the topology of the transduction chain. This series of two papers contains a review reconsidering the entire field from this viewpoint (Paper 1) and a mathematical model of pattern transduction which unifies features of phototropism and resolves the paradoxes (Paper 2). At the same time, this is the first "proof of concept" for the "activity/pooling (a/p) networks"-a specific mathematical apparatus designed to analyse systemic properties and control in metabolic pathways.

Absorption↗

PubMed1991

Action spectra of the light-growth response in three behavioral mutants of Phycomyces.

Null-point action spectra of the light-growth response were measured for three mutants of Phycomyces blakesleeanus (Burgeff) and compared with the action spectrum of the wild type (WT). The action spectrum for L150, a recently isolated "night-blind" mutant, differs from the WT spectrum. The L150 action spectrum has a depression near 450 nm and small alterations in its long-wavelength cutoff, the same spectral regions where its photogravitropism action spectrum is altered. This indicates that the affected gene product influences both phototropism and the light-growth response. For L85, a "hypertropic" (madH) mutant, the light-growth-response action spectrum is very similar to that of WT even though the photogravitropism action spectrum of L85 has been shown previously to be altered in the near-UV region. The affected gene product in this mutant appears to affect phototropic transduction but not light-growth-response transduction. The action spectrum of C110, a "stiff" (madE) mutant, differs significantly from the WT spectrum near 500 nm, the same spectral region where sporangiophores of madE mutants have been shown to have small alterations in second-derivative absorption spectra. This indicates that the madE gene product may be physically associated with a photoreceptor complex, as predicted by system-analysis studies.

Genes, Fungal↗

PubMed1976

Regulation of carotene synthesis in Phycomyces.

Three independent mutations of Phycomyces blakesleeanus resulting in overaccumulation of beta-carotene are recessive and belong to the same complementation group. The corresponding gene has been named carS. Evidence is presented that gene carS is not the same as gene carA, previously defined by mutations blocking carotene production. Vitamin A increases carotenogenesis in wild types and in carS mutants to about the same extent. Intersexual heterokaryosis increases carotenogenesis most prominently in carS genetic backgrounds (up to 300 times the production of the wild type in the same conditions). Vitamin A, intersexual heterokaryosis and carS mutations are thought to stimulate carotenogenesis through different mechanisms. It is suggested that the carS gene product participates in end-product regulation of the pathway.

Carotenoids↗

PubMed1993

Chitin synthetase mutants of Phycomyces blakesleeanus.

Mutants resistant to nikkomycin, an inhibitor of chitin biosynthesis, were isolated after exposure of wild-type spores of the fungus Phycomyces blakesleeanus to N-methyl-N'-nitro-N-nitrosoguanidine. Genetic analysis revealed that nikkomycin resistance was due to mutations in a single gene, chsA. Mutants and wild type grew equally well in the absence of nikkomycin. In contrast to the wild type, whose spore germination and mycelial growth were inhibited by 5 microM nikkomycin, chsA mutants grew reasonably well in the presence of 50 microM nikkomycin. Chitin synthesis in vivo was much less affected by the drug in the mutants than in the wild type. Resistance was not due to impaired uptake or detoxification of the drug. Analysis of the kinetics of chitin synthesis in vitro showed that the mutants had a decreased Ka for the allosteric activator, N-acetylglucosamine, and gross alterations in nikkomycin inhibition kinetics. These results indicate that chsA is the structural gene for chitin synthetase, or at least for the polypeptide that bears the catalytic and allosteric sites.

Aminoglycosides↗

PubMed1994

Isolation of the facA (acetyl-CoA synthetase) gene of Phycomyces blakesleeanus.

A 5.6 kb DNA fragment from the fungus Phycomyces blakesleeanus has been cloned and sequenced. The fragment contains a gene that probably codes for the enzyme acetyl-coenzyme A synthetase (facA). The amino acid sequence deduced for the P. blakesleeanus protein is highly homologous to those of acetyl-coA-synthetases from other organisms. When placed under the control of a constitutive promoter from Aspergillus nidulans, the cloned gene complemented a facA- mutation of this organism. In P. blakesleeanus, the expression of facA is induced by acetate.

Acetate-CoA Ligase↗

PubMed1990

A new gene (madI) involved in the phototropic response of Phycomyces.

Only eight genes are known to be involved in the phototropic response of Phycomyces (madA-H). Mutants affected in these genes have played a major role in the analysis of photosensory transduction processes in this system. A set of new mutants isolated by Alvarez et al. (1989) that are unable to bend towards dim unilateral blue light were studied by complementation and recombination. Two of these mutants have mutations in madE, one has a mutation in madF and one is a double madE madF mutant. The three remaining mutants tested did not complement each other and showed positive complementation with strains carrying mutations in the genes madA, madB, and madC, indicating that they carried mutations in a new gene designated madI. Recombination analysis showed that madI is unlinked to madA, madB and madC.

Genes, Fungal↗

PubMed1995

PkpA, a novel Phycomyces blakesleeanus serine/threonine protein kinase.

This work reports the cloning and sequencing of pkpA, a gene of the filamentous fungus Phycomyces blakesleeanus, whose expression seems to be coupled to vegetative growth. This gene encodes a putative serine/threonine-specific protein kinase, whose sequence is related to that of the yeast protein STE20, involved in pheromone-response pathways, and to a number of MAPK kinase proteins. However, detailed analysis of the kinase sequence suggests that PkpA is a novel serine/threonine protein kinase that probably participates as an intermediate in an intracellular system controlling nuclear proliferation in P. blakesleeanus.

Amino Acid Sequence↗