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PubMed1988

Structural organization of the TRP1 gene of Phycomyces blakesleeanus: implications for evolutionary gene fusion in fungi.

The complete nucleotide (nt) sequence of the cloned TRP1 gene from Phycomyces blakesleeanus is reported. The gene encodes a trifunctional polypeptide that represents a sequential fusion of glutamine amidotransferase (TrpG), indoleglycerolphosphate synthetase (TrpC), and phosphoribosylanthranilate isomerase (TrpF) activities from the amino- to the carboxy terminus. This genetic organization is characteristic of filamentous fungi in general. The transcription start sites and the polyadenylation sites of the gene have been mapped. The two predominant TRP1 transcripts have a short leader of 13 and 18 nt, while minor species with significantly longer leaders are also detectable. Approximately 50 bp and 70 bp upstream from the major transcription start points, sequences that match the canonical eukaryotic TATA and CAAT boxes, respectively, are found. Two major polyadenylation signals are located approximately 50 and 70 nt downstream from the translational stop. Three closely clustered minor polyadenylation sites map to roughly 120-150 bp 3' to the termination codon. The TRP1 gene is the first and only Phycomyces gene that has been cloned and sequenced. The information regarding the promoter and terminator of a Phycomyces gene derived from these studies should benefit strategies aimed at gene manipulations in this organism.

Amino Acid Sequence↗
PubMed1996

Genomic organization of the fungus Phycomyces.

The fungus Phycomyces blakesleeanus has a relatively small genome, 30 megabases (Mb), with a low guanine and cytosine (G + C) content, 35%; the coding sequences cloned to date all have a G + C content of about 50%. In order to investigate the organization of the genome of this fungus, we have cloned and sequenced 251 DNA fragments. One hundred and twenty-six clones were obtained by digestion with MspI (target sequence 5'-CCGG-3') and 125 random clones were obtained by sonication. The average length of sequence obtained was about 200 base pairs (bp) and the total length was about 50 kilobases (kb). The G + C content is not homogeneous throughout the genome: sequences obtained after digestion with MspI have an average of 5% more G + C content than the random fragments, and are enriched in coding sequences. Fourteen MspI fragments show similarities to known proteins and 21 encode ribosomal RNA (rRNA). By contrast, only three of the random fragments are similar to known proteins and only one to a rRNA. We conclude that the Phycomyces genome is composed of G + C-rich genes surrounded by G + C-poor areas. Two clones have similarities to the transposase of the transposon Tc1 from Caenorhabditis elegans. This result suggests the presence of a high copy number of a Tc1-like transposable element in the Phycomyces genome. Another clone was similar to the transposon Tx1 from Xenopus laevis. A novel repetitive nt sequence has been characterized; about 5% of the total genome is a repetition of any of two consensus sequences of 31 bp named PrA1 and PrA2.

Amino Acid Sequence↗
PubMed1999

Protein crystals in Phycomyces sporangiophores are involved in graviperception.

The sporangiophores of the zygomycete fungus Phycomyces blakesleeanus contain octahedral crystals with diameters of up to 5 micrometers in their vacuole. The crystals are associated with the intracellular membrane system. In tilted or horizontally placed sporangiophores, the crystals sediment to the respective lower face of the vacuole with a velocity of up to 100 micrometers per minute. The sedimentation is completed within about 2 minutes, well within the latency period for the negative gravitropic response of Phycomyces. Crystal-lacking mutant strains display a smaller maximal bending angle and a reduced gravitropic bending rate in comparison to the wild type. We therefore conclude that the crystals serve as statoliths for gravitropism in Phycomyces.

Crystallization↗
PubMed1996

Two NAD+-isocitrate dehydrogenase forms in Phycomyces blakesleeanus. Induction in response to acetate growth and characterization, kinetics, and regulation of both enzyme forms.

Two forms of NAD+-isocitrate dehydrogenase, named ICDH-1 and ICDH-2, have been identified and purified in Phycomyces blakesleeanus NRRL-1555(-). These enzymes forms may be separated by chromatography on DEAE-Sephacel. ICDH-2 induction was a response to the adaptation of Phycomyes growth on acetate as the carbon source. Both enzyme forms were octamers of 388 + or - 30 kDa with apparently identical subunits of 40.5 +/- 5 kDa, but they were distinguishable by their electrophoretic mobilities on polyacrylamide gel electrophoresis. Isoelectric pH values were 5.28 and 4.96 for ICDH-1 and ICDH-2, respectively. ICDH-2 was more stable to urea denaturation than ICDH-1. At pH 7.6, ICDH-1 showed a markedly sigmoidal kinetic behavior with respect to isocitrate. However, ICDH-1 and ICDH-2 showed hyperbolic kinetics with respect to NAD+. THe tribasic form of isocitrate (I3-) and its magnesium complex (MI-) are the true substrates for both enzyme forms. Kinetic data obtained with Mg2+ as a divalent cation for both enzyme forms are compatible with the kinetic mechanism proposed by Cohen and Colman (1974) [Eur. J. Biochem. 47, 35-45] but assuming some degree of interaction between binding sites for the active form of isocitrate. This report describes for the first time the existence of two forms of NAD+-isocitrate dehydrogenase in filamentous fungi. From the changes in activity levels for each form, during adaptation of Phycomyces to growth on acetate and taking into account the kinetic and regulatory properties of both enzyme forms, we discuss the role of ICDH-1 and ICDH-2 in the control of isocitrate flux in Phycomyces.

Acetates↗
PubMed2006

The Phycomyces madA gene encodes a blue-light photoreceptor for phototropism and other light responses.

Phycomyces blakesleeanus is a filamentous zygomycete fungus that produces striking elongated single cells that extend up to 10 cm into the air, with each such sporangiophore supporting a sphere containing the spores for dispersal. This organism has served as a model for the detection of environmental signals as diverse as light, chemicals, touch, wind, gravity, and adjacent objects. In particular, sporangiophore growth is regulated by light, and it exhibits phototropism by bending toward near-UV and blue wavelengths and away from far-UV wavelengths in a manner that is physiologically similar to plant phototropic responses. The Phycomyces madA mutants were first isolated more than 40 years ago, and they exhibit reduced sensitivity to light. Here, we identify two (duplicated) homologs in the White Collar 1 family of blue-light photoreceptors in Phycomyces. We describe that the madA mutant strains contain point mutations in one of these genes and that these mutations cosegregate with a defect in phototropism after genetic crosses. Thus, the phototropic responses of fungi through madA and plants through phototropin rely on diverse proteins; however, these proteins share a conserved flavin-binding domain for photon detection.

Base Sequence↗
PubMed1996

Purification and characterization of an extracellular aspartate protease from Phycomyces blakesleeanus.

An acid protease has been found in the culture broth of Phycomyces blakesleeanus growing under standard conditions. It has been induced up to 70-fold with several complex growth media and the enzyme has been purified to homogeneity and characterized. The molecular mass of the native enzyme was estimated by gel filtration to be 40 kDa. The acid protease of Phycomyces migrated as a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, corresponding to a molecular mass of 35 kDa. The glycoprotein nature for the acid protease was deduced from its binding to a concanavalin A-Sepharose 4B column. The carbohydrate moiety is composed of mannose and rhamnose. Its amino acid composition was determined, and its isoelectric point was estimated to be 4.2, the optimum pH was 2.5 to 3, and the optimum temperature was 70 degrees C, using hemoglobin as a substrate. The enzyme showed thermal stability between 37 and 50 degrees C. The thermodynamic parameters for hemoglobin hydrolysis and thermal inactivation were calculated. With Lys-Pro-Ile-Glu-Phe-Phe(4-N02)-Arg-Leu as the substrate, the Km, kcat, and Vmax values were 8.78 microM, 1.25 s(-1), and 2.12 mumol min(-1) mg(-1), respectively. The protease was insensitive to phenylmethylsulfonyl fluoride, O-phenanthroline, N-ethylmaleimide, iodoacetamide, ethylenediaminetetraacetate, [ethyl-enebis(oxyethylenenitrilo)]tetraacetic acid, and trypsin inhibitor. However, pepstatin A established a strong competitive inhibition against it, with a K(i) value of 1.33 nM. The data suggest that this protease has properties of an aspartate-type proteinase.

Amino Acid Sequence↗
PubMed1996

(-) mating type-specific mutants of Phycomyces defective in sex pheromone biosynthesis.

We have isolated the first mating type-specific mutants in mucoraceous fungi. Both mutants in Phycomyces blakesleeanus appear to be defective in the same gene. The gene, present in both mating types, is necessary only in cultures of the (-) mating type. The gene codes for an enzyme in sex pheromone biosynthesis. The pheromone precursor made by the mutants is detectable only in cross-feeding experiments. The biological and solubility properties of the precursor suggest the precursor is 4-dihydrotrisporin, a metabolite of beta-carotene. Separate studies with beta-carotene-deficient mutants and Compound-P, a new chemically synthesized precursor of the pheromones, imply the constitutive level of enzymes for pheromone biosynthesis in Phycomyces is extremely low. In comparison, the level of enzymes for pheromone conversion to trisporic acid is higher. The mating type-specific mutants also catalyze the conversion of (+) pheromone to trisporic acid. This finding was unexpected because literature models predicted this reaction was catalyzed by the same enzyme which catalyzed the conversion of 4-dihydrotrisporin to (-) pheromone-a reaction missing in the (-) mating type-specific mutants. Thus, we propose a revised model for trisporic acid biosynthesis.

Crosses, Genetic↗
PubMed1999

Blue light signaling chains in Phycomyces: phototransduction of carotenogenesis and morphogenesis involves distinct protein kinase/phosphatase elements.

Carotenogenesis and morphogenesis represent two of the several responses sensitive to blue light which characterize the lower eukaryote Phycomyces blakesleeanus. Speculating that reversible phosphorylation may be an intracellular event beyond the photoperception step, we resorted to the use of first-choice inhibitors of protein phosphatases and protein kinases. The mycelial beta-carotene content of dark-grown cultures was induced by all agents administered, while the morphogenic output showed the typical trend effected by light only with one of the protein kinase inhibitors. Our data provide convincing evidence that protein phosphorylation plays a regulatory role in photocarotenogenesis and photomorphogenesis of Phycomyces. According to the model we propose, the putative signaling elements involved are anticipated to have a repressive function in the dark so that the responses are maintained in the "off" mode until the moment photon information has to flow through the regulatory circuit.

Culture Media↗
PubMed1989

System analysis of Phycomyces light-growth response in single and double night-blind mutants.

The sum-of-sinusoids method of nonlinear system identification has been applied to the light-growth response of the Phycomyces sporangiophore. Experiments were performed on the Phycomyces tracking machine with the wild-type strain with single and double mutants affected in genes madA, madB, and madC. The sum-of-sinusoids test stimuli were applied to the logarithm of the light intensity. The log-mean intensity level was 10(-1) W m-2 and the wavelength was 477 nm. The system identification results are in the form of first- and second-order frequency kernels, which are related to temporal kernels that appear in the Wiener functional series. The first-order kernels agree well with those obtained previously by the white noise method. In particular, the madA madB and madB madC double mutants show very weak responses. With the superior precision of the sum-of-sinusoids methods, we have achieved sufficient resolution to measure and analyze their second-order kernels. The first- and second-order frequency kernels were interpreted by system analysis methods involving a nonlinear parametric model. In addition a nonparametric hypothesis concerning interactions of gene products was tested. Results from the interaction tests confirm the earlier conclusion that the madB and madC gene products interact. In addition, with the enhanced precision and with the extension to nonlinear analysis, we have found evidence of interaction of the madA gene product with the madB and madC gene products. Thus all three genes appear to have mutual interactions, presumably because of their close physical association in a photoreceptor complex.

Cells, Cultured↗
PubMed1994

Isolation and characterization of phototropism mutants of Phycomyces insensitive to ultraviolet light.

Phototropism mutants of the zygomycete fungus Phycomyces blakesleeanus were isolated on the basis of their loss of responsivity to UV light. Four of these mutants had retained a partial sensitivity to near-UV and to blue light. Gravitropism and the avoidance response were unaffected in these mutants. One mutant, A909, had lost most of its sensitivity to near-UV and blue light while the sensitivity to far-UV light was only slightly affected. Additionally, the gravitropic and the avoidance responses were significantly reduced in A909. A complementation analysis of the five strains of Phycomyces with known phototropism mutants indicated that strains A896, A897, and A898 were defective in the madA gene, and that A905 was affected in the madC gene. In strain A909 the input, as well as the output, of the transduction chain is affected.

Carotenoids↗
PubMed1986

System analysis of Phycomyces light-growth response with Gaussian white-noise test stimuli.

The light-growth response of the Phycomyces sporangiophore is a transient change of elongation rate in response to changes in ambient blue-light intensity. The white-noise method of nonlinear system identification (Wiener-Lee-Schetzen theory) has been applied to this response, and the results have been interpreted by system analysis methods in the frequency domain. Experiments were performed on the Phycomyces tracking machine. Gaussian white-noise stimulus patterns were applied to the logarithm of the light intensity. The log-mean intensity of the broad-band blue illumination was 0.1 Wm-2 and the standard deviation of the Gaussian white-noise was 0.58 decades. The results, in the form of temporal functions called Wiener kernels, represent the input-output relation of the light-growth response system. The transfer function, which was obtained as the Fourier transform of the first-order kernel, was analyzed in the frequency domain in terms of a dynamic model that consisted of a first-order high-pass filter, two second-order low-pass filters, a delay element, and a gain factor. Parameters in the model (cutoff frequencies, damping coefficients, latency, and gain constant) were evaluated by nonlinear least-squares methods applied to the complex-valued transfer function. Analysis of the second-order kernel in the frequency domain suggests that the residual nonlinearity of the system lies close to the input.

Light↗
PubMed1985

A new allele with abnormal cyclic-AMP phosphodiesterase activity in Phycomyces.

A mutant of the fungus Phycomyces blakesleeanus (Burgeff), C21 (madA7) that was isolated for its abnormal phototropism (Bergman et al. 1973) carries a secondary mutation pde-1 which is unlinked to the madA gene. The pde-1 allele causes the loss of about 80% of the cAMP phosphodiesterase activity. This allele is not essential for the photoreactions of the mycelium or the sporangiophore, and the bulk activity of the phosphodiesterase appears to play no role in the phototransduction pathway of Phycomyces.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1985

Electrophoretic analysis of proteins from night-blind mutants of Phycomyces.

Using two-dimensional gel electrophoresis, we have analyzed proteins from a plasma membrane-enriched fraction from Phycomyces sporangiophores. Specifically, we have compared gels for night-blind mutants and a wild-type strain to find proteins involved in the early steps of the sensory transduction chain for phototropism. In the gels for a mutant affected in the gene madA, a protein spot [51 kilodaltons (kdal) and pI 6.35] appears that is absent from the wild-type and the other mad mutants. Mutants affect in either of two madB alleles lack a protein spot (57 kdal and pI 6.6) that is present in the wild-type and all other mad strains; this spot probably represents the madB gene product. In some madC mutants, two spots (59 kdal, pI 6.5, with a covalently linked flavin; and 50 kdal, pI 6.4) are absent; however, in other madC strains, one or both of these spots are present. These four protein spots that are altered in madA, madB, and madC mutants may represent component of the photoreceptor complex responsible for phototropism in Phycomyces.

Electrophoresis, Polyacrylamide Gel↗
PubMed1999

Heterologous expression of the Phycomyces blakesleeanus phytoene dehydrogenase gene (carB) in Mucor circinelloides.

A phytoene dehydrogenase-deficient mutant of Mucor circinelloides accumulating only phytoene was transformed with the gene encoding the corresponding enzyme (carB gene) of Phycomyces blakesleeanus. Carotenoids derived from phytoene were detected in the transformants showing that the P. blakesleeanus carB gene complements the M. circinelloides carB mutation. These newly formed carotenoids accumulated in low quantities, indicating that functional complementation was poor. carB mRNA molecules correctly transcribed were detected in the transformants, but they represented a small proportion of the total population of carB-derived mRNAs, mostly constituted by truncated transcripts and by transcripts longer than the transcript that is functional in Phycomyces. These results showed that the P. blakesleeanus carB gene was expressed in M. circinelloides and suggested that the poor complementation observed was owing, at least in part, to the lack of specificity in the recognition of the transcription initiation and termination signals of the P. blakesleeanus carB gene by the M. circinelloides transcriptional machinery.

Carotenoids↗
PubMed2004

The gene for the heat-shock protein HSP100 is induced by blue light and heat-shock in the fungus Phycomyces blakesleeanus.

We cloned and sequenced the Phycomyces hspA gene. The hspA gene product is a 901-amino-acid protein member of the clpB/HSP100 family. HSP100 proteins are ATPases involved in high-temperature tolerance, proteolysis, and protein disaggregation. Phycomyces HSP100 is composed of a domain presumably involved in protein-protein interactions and two ATP-binding domains. The hspA promoter contains three heat-shock elements that are presumably involved in the activation of hspA after heat-shock. In addition, four short sequences are present in the hspA promoter and in the promoter of the photoinducible genes carB and carRA; and these are candidates as binding sites for light-regulated transcription factors. Blue light can increase transcription of the hspA gene 10-fold, with a threshold of 1 J/m2. The threshold for hspA photoactivation is 10(4) times higher than the thresholds for blue-light regulation of sporangiophore development and photocarotenogenesis, which suggests that there are differences in the photosensory systems for gene photoactivation and mycelial photoresponses. A heat-shock of 30 min at 34 degrees C or 42 degrees C increased hspA gene activity 160-fold. The differences in maximum hspA gene transcription by blue light and heatshock suggest the presence of different regulatory mechanisms.

Adenosine Triphosphatases↗
PubMed2003

Gravity susception by buoyancy: floating lipid globules in sporangiophores of Phycomyces.

To elucidate the mechanisms of gravity susception that operate in the sporangiophore of Phycomyces blakesleeanus, we characterized the function and topography of a large apical complex of lipid globules. Stage-1 sporangiophores (without sporangium) possess a roughly spherical complex of 100-200 large lipid globules whose center is localized 110 microm below the apex. The complex of lipid globules (CLG) is rather stable and is kept in place by positioning forces that resist centrifugal accelerations of up to 150 g. The lipid globules possess an average diameter of 2 to 2.5 microm and a density of 0.791 g cm(-3), which is below that of typical plant oleosomes. The potential energy which is generated by the buoyancy of a CLG of 100 globules is in the order of 10(-17) to 10(-16) J, which is 4 to 5 orders of magnitude above thermal noise. The formation of lipid globules can be suppressed by raising stage-1 sporangiophores for 24 hs at 5 degrees C. Sporangiophores with a reduced number of lipid globules display gravitropic bending angles that are 3 to 4 times smaller than those of sporangiophores with the normal number of lipid globules. The results suggest that the lipid globules function as gravisusceptors of Phycomyces and that buoyancy is the physical principle for their mode of action. The globules contain beta-carotene and two distinct fluorescing pigments that are, however, dispensable for graviperception.

Gravitropism↗
PubMed1999

Light-induced absorbance changes in Phycomyces: evidence for cryptochrome-associated flavosemiquinones.

Light-induced absorbance changes (LIACs), which are associated with early photochemical events of blue-light transduction, were detected in growing zones of Phycomyces sporangiophores. The novel LIACs meet all the essential requirements for a spectrophotometric photoreceptor assay which was previously unavailable for blue-light receptors (cryptochromes). In-vivo absorption spectra of growing zones were derived from reflection spectra which were measured with a novel rapid-scan spectrophotometer. To detect photoreceptor-associated absorbance changes white mutants were employed which lack the interfering bulk pigment beta-carotene. Blue and white light, not however red light, induced in these strains absorbance changes near 460-490 and 600-620 nm. The LIACs were absent in light-insensitive mutants with defects in the genes madA, madB and madC. Because these genes affect photosensory adaptation and the blue-light receptor itself, the novel in-vivo LIACs must be associated with photochemical events which occur early in the transduction chain. The spectral characteristics of the LIACs are in accordance with a blue- and red-light absorbing flavosemiquinone which is generated upon light absorption by an oxidized flavin receptor. It is proposed that the flavosemiquinone functions itself as photoreceptor which mediates several red-light responses of Phycomyces.

Cryptochromes↗
PubMed2001

Genes for mevalonate biosynthesis in Phycomyces.

Terpenoids or isoprenoids constitute a vast family of organic compounds that includes sterols and carotenoids. The terpenoids in many organisms share early steps in their biosynthesis, including the synthesis of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) and its conversion to mevalonate. We have cloned and characterised the genes hmgS for HMG-CoA synthase and hmgR for HMG-CoA reductase from the Zygomycete Phycomyces blakesleeanus. Single copies of these genes are present in the Phycomyces genome. The predicted product of hmgS is largely hydrophilic and that of hmgR has eight putative transmembrane segments and a large hydrophilic domain. The hydrophilic domain suffices for catalytic activity, as shown by expressing it in Escherichia coli. Several features in the promoter of hmgS and in HMG-CoA reductase resemble motifs known to be involved in sterol-mediated regulation and sterol sensing. Carotene-overproducing mutants contain more hmgS mRNA than the wild type, possibly in response to an increased demand for HMG-CoA.

Amino Acid Sequence↗