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Guttation, the formation of exudation water, is widespread among plants and fungi, yet the underlying mechanisms remain largely unknown. We describe the conditions for inducing guttation in sporangiophores of the mucoracean fungus, Phycomyces blakesleeanus. Cultivation on peptone-enriched potato dextrose agar elicits vigorous guttation mainly below the apical growing zone, while sporangiophores raised on a glucose-mineral medium manifest only moderate guttation. Mycelia do not guttate irrespective of the employed media. The topology of guttation droplets allows identifying the non-growing part of the sporangiophore as a guttation zone, which responds to humidity and medium composition in ways that become relevant for turgor homeostasis and thus the sensor physiology of the growing zone. Apparently, the entire sporangiophore, rather than exclusively the growing zone, participates in signal reception and integration to generate a common growth output. Exogenous auxin applied to the growing zones elicits two correlated responses: (i) formation of guttation droplets in the growing and transition zones below the sporangium and (ii) a diminution of the growth rate. In sporangiophore populations, guttation-induction by exogenous control buffer occurs at low frequencies; the bias for guttation increases with increasing auxin concentration. Synthetic auxins and the transport inhibitor NPA suppress guttation completely, but leave growth rates largely unaffected. Mutants C2 carA and C148 carA madC display higher sensitivities for auxin-induced guttation compared to wild type. A working model for guttation includes aquaporins and mechanosensitive ion channels that we identified in Phycomyces by sequence domain searches. [ABSTRACT FROM AUTHOR]

The growing zone (GZ) of the unicellular coenocytic sporangiophore of Phycomyces blakesleeanus represents the site of stimulus reception (light, gravity, gas) and stimulus response, i.e., local modulations of the elongation growth, which may result, in dependence of the stimulus direction, in tropic bending. Until now, evidence for a possible participation of the columella in sensory reception is absent. We confirm with light microscopy earlier studies that show that the GZ and the columella are not separated by a membrane or cell wall, but rather form a spatial continuum that allows free exchange of cytoplasm and organelle transport. Evidence is presented that the columella is responsive to external stimuli. Columellae, from which spores and sporangial cell wall had been removed, respond to exogenous auxin with a local depolarization of the membrane potential and an increased growth rate of the GZ. In contrast, auxin applied to the GZ causes a decrease of the growth rate irrespective of the presence or absence of sporangia. The response pattern is specific and relevant for the sensory reception of Phycomyces, because the light-insensitive mutant C148carAmadC, which lacks the RAS-GAP protein MADC, displays abnormal IAA sensitivity and membrane depolarization. We argue that the traditional concept of the GZ as the only stimulus-sensitive zone should be abandoned in favor of a model in which GZ and columella operate as a single entity capable to orchestrate a multitude of stimulus inputs, including auxin, to modulate the membrane potential and elongation growth of the GZ. [ABSTRACT FROM AUTHOR]

Newly available genome sequences of two Mucoralean fungi, Phycomyces blakesleeanus and Mucor circinelloides, provide evidence for an ancient whole-genome duplication that contributed to the generation of expanded gene families. These fungi have robust responses to light that can be correlated with the expansion of gene networks involved in light sensing and signaling.

Plants and fungi use light and other signals to regulate development, growth, and metabolism. The fruiting bodies of the fungus Phycomyces blakesleeanus are single cells that react to environmental cues, including light, but the mechanisms are largely unknown [1]. The related fungus Mucor circinelloides is an opportunistic human pathogen that changes its mode of growth upon receipt of signals from the environment to facilitate pathogenesis [2]. Understanding how these organisms respond to environmental cues should provide insights into the mechanisms of sensory perception and signal transduction by a single eukaryotic cell, and their role in pathogenesis. We sequenced the genomes of P. blakesleeanus and M. circinelloides and show that they have been shaped by an extensive genome duplication or, most likely, a whole-genome duplication (WGD), which is rarely observed in fungi [3-6]. We show that the genome duplication has expanded gene families, including those involved in signal transduction, and that duplicated genes have specialized, as evidenced by differences in their regulation by light. The transcriptional response to light varies with the developmental stage and is still observed in a photoreceptor mutant of P. blakesleeanus. A phototropic mutant of P. blakesleeanus with a heterozygous mutation in the photoreceptor gene madA demonstrates that photosensor dosage is important for the magnitude of signal transduction. We conclude that the genome duplication provided the means to improve signal transduction for enhanced perception of environmental signals. Our results will help to understand the role of genome dynamics in the evolution of sensory perception in eukaryotes.

A DNA region in the mitochondrial genome of the fungus Phycomyces blakesleeanus (Mucorales, Mucoromycota) was characterized in a population of wild-type strains. The region encodes a predicted protein similar to the reverse transcriptases encoded by mitochondrial retroplasmids of Neurospora species and other Sordariomycetes (Ascomycota), but is uncommon in other fungi. DNA sequences of this element, named mystique, are highly variable between the strains, having greater than 2.5% divergence, yet most of the nucleotide differences fall in codon positions that do not change the amino acid sequence. The high proportion of polymorphisms coupled to the rarity of nonsynonymous changes suggests that mystique is subject to counteracting forces of hypermutation and purifying selection. However, while evidence for negative selection may infer that the element provides a fitness benefit, some strains of P. blakesleeanus do not have the element and grow equivalently well as those strains with it. A mechanism to explain the variability between the mystique alleles is proposed, of error-prone replication through an RNA intermediate, reverse transcription and reintegration of the element into the mitochondrial genome. [ABSTRACT FROM AUTHOR]

The roles of fungal auxins in the regulation of elongation growth, photo-, and gravitropism are completely unknown. We analyzed the effects of exogenous IAA (indole-3-acetic acid), various synthetic auxins including 1-NAA (1-naphthaleneacetic acid) and 2,4-D (2,4-dichlorophenoxyacetic acid), and the auxin transport inhibitor NPA (N-1-naphtylphtalamic acid) on the growth rate and bending of the unicellular sporangiophore of the zygomycete fungus, Phycomyces blakesleeanus. Sporangiophores that were submerged in an aqueous buffer responded to IAA with a sustained enhancement of the growth rate, while 1-NAA, 2,4-D, and NPA elicited an inhibition. In contrast, sporangiophores kept in air responded to IAA with a 20 to 40% decrease of the growth rate, while 1-NAA and NPA elicited an enhancement. The unilateral and local application of IAA in the growing zone of the sporangiophore elicited in 30 min a moderate negative tropic bending in wild type C2 and mutant C148madC, which was, however, partially masked by a concomitant avoidance response caused by the aqueous buffer. Auxin transport-related genes ubiquitous in plants were found in a BLAST search of the Phycomyces genome. They included members of the AUX1 (auxin influx carrier protein 1), PILS (PIN-LIKES, auxin transport facilitator protein), and ABCB (plant ATP-binding cassette transporter B) families while members of the PIN family were absent. Our observations imply that IAA represents an intrinsic element of the sensory transduction of Phycomyces and that its mode of action must very likely differ in several respects from that operating in plants. [ABSTRACT FROM AUTHOR]

A considerable amount of research has been conducted to determine how cell walls are loosened to produce irreversible wall deformation and expansive growth in plant and algal cells. The same cannot be said about fungal cells. Almost nothing is known about how fungal cells loosen their walls to produce irreversible wall deformation and expansive growth. In this study, anoxia is used to chemically isolate the wall from the protoplasm of the sporangiophores of Phycomyces blakesleeanus. The experimental results provide direct evidence of the existence of chemistry within the fungal wall that is responsible for wall loosening, irreversible wall deformation and elongation growth. In addition, constant-tension extension experiments are conducted on frozen-thawed sporangiophore walls to obtain insight into the wall chemistry and wall loosening mechanism. It is found that a decrease in pH to 4.6 produces creep extension in the frozen-thawed sporangiophore wall that is similar, but not identical, to that found in frozen-thawed higher plant cell walls. Experimental results from frozen-thawed and boiled sporangiophore walls suggest that protein activity may be involved in the creep extension.

Ion channels are vital components of filamentous fungi signaling in communication with their environment. We exploited the ability of the apical region of growing sporangiophores of Phycomyces blakesleeanus to form membrane-enveloped cytoplasmic droplets (CDs), to examine ion currents in the filamentous fungi native plasma membrane. In hypoosmotic conditions, the dominant current in the CDs is ORIC, an osmotically activated, anionic, outwardly rectified, fast inactivating instantaneous current that we have previously characterized. Here, we examined the effect of ATP on ORIC. We show that CDs contain active mitochondria, and that respiration inhibition by azide accelerates ORIC inactivation. ATP, added intracellularly, reduced ORIC run-down and shifted the voltage dependence of inactivation toward depolarized potentials, in a manner that did not require hydrolysis. Notably, ATP led to slowing down of ORIC inactivation, as evidenced by an increased time constant of inactivation, τ in , and slower decline of τ in during prolonged recordings. Flavonoids (genistein and quercetin) had the effect on ORIC opposite to ATP, acting as current inhibitors, possibly by disrupting the stabilizing effect of ATP on ORIC. The integration of osmotic sensing with ATP dependence of the anionic current, typical of vertebrate cells, is described here for the first time in filamentous fungi., (© 2023. The Author(s).)

DASH (Drosophila, Arabidopsis, Synechocystis, Human)-type cryptochromes (cry-DASH) belong to a family of flavoproteins acting as repair enzymes for UV-B-induced DNA lesions (photolyases) or as UV-A/blue light photoreceptors (cryptochromes). They are present in plants, bacteria, various vertebrates, and fungi and were originally considered as sensory photoreceptors because of their incapability to repair cyclobutane pyrimidine dimer (CPD) lesions in duplex DNA. However, cry-DASH can repair CPDs in single-stranded DNA, but their role in DNA repair in vivo remains to be clarified. The genome of the fungus Phycomyces blakesleeanus contains a single gene for a protein of the cryptochrome/photolyase family (CPF) encoding a cry-DASH, cryA, despite its ability to photoreactivate. Here, we show that cryA expression is induced by blue light in a Mad complex-dependent manner. Moreover, we demonstrate that CryA is capable of binding flavin (FAD) and methenyltetrahydrofolate (MTHF), fully complements the Escherichia coli photolyase mutant and repairs in vitro CPD lesions in single-stranded and double-stranded DNA with the same efficiency. These results support a role for Phycomyces cry-DASH as a photolyase and suggest a similar role for cry-DASH in mucoromycotina fungi. [ABSTRACT FROM AUTHOR]

The fungal genus Phycomyces ( Mucoromycotina , Mucorales ) has been revised by examining 96 strains, received from established collections or newly isolated from different environments. Morphology, sexuality, DNA sequences, and population structure clearly identify the genus and set it apart from other Mucorales . The size of the spores, the sexual interactions, the sequences of genes sexM and sexP that determine sexual identity, and the DNA for ribosomal RNAs validate the species Phycomyces blakesleeanus and Phycomyces nitens and the family Phycomycetaceae . Isolates from the same sample of biomass often differ in spore size, sex, DNA sequences, and restriction-length polymorphisms. The widest diversity was found in similar environments in three of the Canary Islands, implying a failure to disperse rapidly over the sea. All strains have apparently functional sexM and sexP genes and all but some strains of P. nitens complete the sexual cycle in the laboratory. The genetic diversity of P. blakesleeanus strains provides evidence for geographical clustering. Various sequence comparisons, including the newly isolated genes sexM and sexP of P. nitens and Blakeslea trispora , clarify phylogenetic relationships in the Mucorales and recommend the sex genes for the study of speciation. [ABSTRACT FROM AUTHOR]

Vanadium speciation in the fungus Phycomyces blakesleeanus was examined by X-ray absorption near-edge structure (XANES) spectroscopy, enabling assessment of oxidation states and related molecular symmetries of this transition element in the fungus. The exposure of P. blakesleeanus to two physiologically important vanadium species (V and V) resulted in the accumulation of this metal in central compartments of 24 h old mycelia, most probably in vacuoles. Tetrahedral V, octahedral V, and proposed intracellular complexes of V were detected simultaneously after addition of a physiologically relevant concentration of V to the mycelium. A substantial fraction of the externally added V remained mostly in its original form. However, observable variations in the pre-edge-peak intensities in the XANES spectra indicated intracellular complexation and corresponding changes in the molecular coordination symmetry. Vanadate complexation was confirmed by V NMR and Raman spectroscopy, and potential binding compounds including cell-wall constituents (chitosan and/or chitin), (poly)phosphates, DNA, and proteins are proposed. The evidenced vanadate complexation and reduction could also explain the resistance of P. blakesleeanus to high extracellular concentrations of vanadium. [ABSTRACT FROM AUTHOR]

The objective of this study was to analyze the response of Phycomyces blakesleeanus to glucose starvation and acetate growth stress. At the onset of the exponential growth phase, the fungus shows a high tolerance to both stresses, being higher for the glucose starvation. In both stresses we have found higher activities of catalase and glutathione peroxidase, and a decrease of the pools of D-erythroascorbate (D-erythroascorbate+D-erythroascorbate monoglucoside) and glutathione (GSH+GSSG), while the intracellular GSH/GSSG redox balance becomes more reducing. Gallic acid was not detected under both stresses. Glycogen breakdown and the high levels of trehalose seem to be part of the stress response. Both stress, under the conditions of this study, seem to lead to a qualitatively similar response in P. blakesleeanus, with regard to the behavior of antioxidant system, the content of secondary metabolites and the role of the reserve carbohydrates. [ABSTRACT FROM AUTHOR]

Regulation of cell growth is paramount to all living organisms. In plants, algae and fungi, regulation of expansive growth of cells is required for development and morphogenesis. Also, many sensory responses of stage IVb sporangiophores of Phycomyces blakesleeanus are produced by regulating elongation growth rate (growth responses) and differential elongation growth rate (tropic responses). Stiff mutant sporangiophores exhibit diminished tropic responses and are found to be defective in at least four genes; madD, madE, madF and madG. Prior experimental research suggests that the defective genes affect growth regulation, but this was not verified. All the growth of the single-celled stalk of the stage IVb sporangiophore occurs in a short region termed the growth zone. Prior experimental and theoretical research indicates that elongation growth rate of the stage IVb sporangiophore can be regulated by controlling the cell wall mechanical properties within the growth zone and the magnitude of the turgor pressure. A quantitative biophysical model for elongation growth rate is required to elucidate the relationship between wall mechanical properties and turgor pressure during growth regulation. In this study, it is hypothesized that the mechanical properties of the wall within the growth zone of stiff mutant sporangiophores are different compared to wild type. A biophysical equation for elongation growth rate is derived for fungal and plant cells with a growth zone. Two strains of stiff mutants are studied, C149 madD120 (-) and C216 geo- (-). Experimental results demonstrate that turgor pressure is larger but irreversible deformation rates of the wall within the growth zone and growth zone length are smaller for stiff mutant sporangiophores compared to wild type. These findings explain the diminished tropic responses of the stiff mutant sporangiophores and suggest that the defective genes affect the amount of wall-building material delivered to the inner cell wall.

Growth, photo- and gravitropism of sporangiophores of the zygomycete Phycomyces blakesleeanus occur within the apical growing zone, a cylindrical structure (diameter about 100 μm) that reaches about 1.5-2.5 mm below the tip and has growth rates up to 50 μm·min−1. To better understand morphogenesis and growth of the giant aerial hypha, we investigated with confocal microscopy and inhibitors the actin cytoskeleton and by in-vivo particle tracking the associated organelle movement. We found stage-1 sporangiophores (without sporangium) possess an actin cytoskeleton with polar zonation. (i) In the apex, abundant microfilaments without preferential orientation entangled numerous nuclei as well as a conspicious complex of some 200 lipid globules. Microfilament patches (≈ 1.6-μm diameter) are clustered in the tip and were found in the apical cortex, whereas short, curved microfilament bundles (≈ 2.3-μm long) prevailed in the subapex. (ii) In a transition zone downwards to the shaft, the microfilaments rearranged into a dense mat of longitudinal microfilaments that was parallel close to the periphery but more random towards the cell centre. Numerous microfilament patches were found near the cortex (≈ 10/100 μm2); their number decreased rapidly in the subcortex. In contrast, the short, curved microfilament bundles were found only in the subcortex. (iii) The basal shaft segment of the sporangiophore (with central vacuole) exhibited bidirectional particle movement over long distances (velocity ≈ 2 μm·s−1) along massive longitudinal, subcortical microfilament cables. The zonation of the cytoskeleton density correlated well with the local growth rates at the tip of the sporangiophore, and appears thus as a structural prerequisite for growth and bending. [ABSTRACT FROM AUTHOR]

Unicellular zygomycete fungus Phycomyces blakesleeanus exhibits a typical apical growth that depends on a complex interaction of different physiological processes. Sensitivity to the light, wind, barriers, touch and gravity of Phycomyces sporangiophore implicate the existence of the cross-talk between different signaling pathways in the same cell. Touch and gravity in Phycomyces sporangiophore seem to share some common elements of Ca2+ and H+ ion-based signal transduction systems. Apoplastic Ca2+ and H+ ionic luxes are important for establishing cell polarity in tip growing sporangiophore both in vertical and tilted position. Upon gravistimulation sporangiophores display asymmetric ionic distribution and intracellular reorganization leading to change in the growth pattern. [ABSTRACT FROM AUTHOR]

Using an integrated analysis of phenotypic and genotypic characterizations, a total of 18 isolates of 'bristle-like' fungal colonizers of the Kitora and Takamatsuzuka Tumuli, which had been provisionally identified as Phialocephala phycomyces, were here determined to be Kendrickiella phycomyces (Auersw.) K. Jacobs & M. J. Wingf. The 18 isolates consisted of 10 from stone surfaces or viscous gels (biofilms) of the stone chamber interior and adjacent small room, and air in the adjacent small room of the Kitora Tumulus, and 8 from viscous gels on the stone surfaces of the stone chamber interior, plant roots, and soil in the adjacent space or stone wall interspaces (interstices) in the stone chamber of the Takamatsuzuka Tumulus. Plaster and stone walls in both tumuli were recorded as novel substrates of this fungus. Our 18S sequence-based phylogeny indicated that K. phycomyces and species of the leotiomycetous anamorph genera Chaetomella, Pilidium, Sphaerographium, and Synchaetomella formed a monophyletic lineage distant from the core taxa of the Leotiomycetes (Pezizomycotina, Ascomycota). The relationship between the physicochemical characteristics of these isolates on GYC agar plates, i.e., soluble brownish pigments and dissolution of calcium carbonate (CaCO), and the biodeterioration of the plaster and plaster walls of both tumuli, are briefly discussed. [ABSTRACT FROM AUTHOR]

Regulation of cell growth is paramount to all living organisms. In plants, algae and fungi, regulation of expansive growth of cells is required for development and morphogenesis. Also, many sensory responses of stage IVb sporangiophores of Phycomyces blakesleeanus are produced by regulating elongation growth rate (growth responses) and differential elongation growth rate (tropic responses). "Stiff" mutant sporangiophores exhibit diminished tropic responses and are found to be defective in at least five genes; madD, E, F, G, and J. Prior experimental research suggests that the defective genes affect growth regulation, but this was not verified. All the growth of the single-celled stalk of the stage IVb sporangiophore occurs in a short region termed the "growth zone." Prior experimental and theoretical research indicates that elongation growth rate of the stage IVb sporangiophore can be regulated by controlling the cell wall mechanical properties within the growth zone and the magnitude of the turgor pressure. A quantitative biophysical model for elongation growth rate is required to elucidate the relationship between wall mechanical properties and turgor pressure during growth regulation. In this study, it is hypothesized that the mechanical properties of the wall within the growth zone of stiff mutant sporangiophores are different compared to wild type (WT). A biophysical equation for elongation growth rate is derived for fungal and plant cells with a growth zone. Two strains of stiff mutants are studied, C149 madD120 (-) and C216 geo- (-). Experimental results demonstrate that turgor pressure is larger but irreversible wall deformation rates within the growth zone and growth zone length are smaller for stiff mutant sporangiophores compared to WT. These findings can explain the diminished tropic responses of the stiff mutant sporangiophores. It is speculated that the defective genes affect the amount of wall-building material delivered to the inner cell wall. [ABSTRACT FROM AUTHOR]

A 'graduate student' in biology [ABSTRACT FROM AUTHOR]

Phycomyces carRA gene encodes a protein with two domains. Domain R is characterized by red carR mutants that accumulate lycopene. Domain A is characterized by white carA mutants that do not accumulate significant amounts of carotenoids. The carRA-encoded protein was identified as the lycopene cyclase and phytoene synthase enzyme by sequence homology with other proteins. However, no direct data showing the function of this protein have been reported so far. Different Mucor circinelloides mutants altered at the phytoene synthase, the lycopene cyclase or both activities were transformed with the Phycomyces carRA gene. Fully transcribed carRA mRNA molecules were detected by Northern assays in the transformants and the correct processing of the carRA messenger was verified by RT-PCR. These results showed that Phycomyces carRA gene was correctly expressed in Mucor. Carotenoids analysis in these transformants showed the presence of ß-carotene, absent in the untransformed strains, providing functional evidence that the Phycomyces carRA gene complements the M. circinelloides mutations. Co-transformation of the carRA cDNA in E. coli with different combinations of the carotenoid structural genes from Erwinia uredovora was also performed. Newly formed carotenoids were accumulated showing that the Phycomyces CarRA protein does contain lycopene cyclase and phytoene synthase activities. The heterologous expression of the carRA gene and the functional complementation of the mentioned activities are not very efficient in E. coli. However, the simultaneous presence of both carRA and carB gene products from Phycomyces increases the efficiency of these enzymes, presumably due to an interaction mechanism. [ABSTRACT FROM AUTHOR]

The Rhizopus oryzae species complex is a group of zygomycete fungi that are common, cosmopolitan saprotrophs. Some strains are used beneficially for production of Asian fermented foods but they can also act as opportunistic human pathogens. Although R. oryzae reportedly has a heterothallic (+/-) mating system, most strains have not been observed to undergo sexual reproduction and the genetic structure of its mating locus has not been characterized. Here we report on the mating behavior and genetic structure of the mating locus for 54 isolates of the R. oryzae complex. All 54 strains have a mating locus similar in overall organization to Phycomyces blakesleeanus and Mucor circinelloides (Mucoromycotina, Zygomycota). In all of these fungi, the minus (2) allele features the SexM high mobility group (HMG) gene flanked by an RNA helicase gene and a TP transporter gene (TPT). Within the R. oryzae complex, the plus (+) mating allele includes an inserted region that codes for a BTB/POZ domain gene and the SexP HMG gene. Phylogenetic analyses of multiple genes, including the mating loci (HMG, TPT, RNA helicase), ITS1-5.8S-ITS2 rDNA, RPB2, and LDH genes, identified two distinct groups of strains. These correspond to previously described sibling species R. oryzae sensu stricto and R. delemar. Within each species, discordant gene phylogenies among multiple loci suggest an outcrossing population structure. The hypothesis of random-mating is also supported by a 50:50 ratio of plus and minus mating types in both cryptic species. When crossed with tester strains of the opposite mating type, most isolates of R. delemar failed to produce zygospores, while isolates of R. oryzae produced sterile zygospores. In spite of the reluctance of most strains to mate in vitro, the conserved sex locus structure and evidence for outcrossing suggest that a normal sexual cycle occurs in both species. [ABSTRACT FROM AUTHOR]

Abstract: Light is an environmental signal that modulates many aspects of the biology of zygomycete fungi. Light regulation has been investigated in the zygomycetes Phycomyces blakesleeanus, Mucor circinelloides and Pilobolus crystallinus. Examples of light regulation include the phototropism of the fruiting bodies, the regulation of the development of reproductive structures, and the activation of the biosynthesis of β-carotene. In fungi blue light is perceived by proteins homologous to WC-1, a Neurospora crassa photoreceptor and Zn finger protein that interacts with WC-2 to form a photoresponsive transcription factor complex. Unlike ascomycete and basidiomycete fungi that usually have one wc-1 and one wc-2 gene, several studies have uncovered an unexpected multitude of genes similar to wc-1 and wc-2 in the genomes of several zygomycete fungi. Some of these genes are required for fungal photoresponses, but the function of many of them remains unknown. The presence of multiple wc-1 genes confirms previous suggestions of multiple blue-light photoreceptors in Phycomyces. [ABSTRACT FROM AUTHOR]

Abstract: Carotene biosynthesis in Phycomyces is photoinducible and carried out by phytoene dehydrogenase (encoded by carB) and a bifunctional enzyme possessing lycopene cyclase and phytoene synthase activities (carRA). A light pulse followed by periods of darkness produced similar biphasic responses in the expression of the carB and carRA genes, indicating their coordinated regulation. Specific binding complexes were formed between the carB–carRA intergenic region and protein extracts from wild type mycelia grown in the dark or 8min after irradiation. These two conditions correspond to the points at which the expression of both genes is minimal, suggesting that these binding complexes are involved in the down-regulation of photocarotenogenesis in Phycomyces. Protein extracts from carotene mutants failed to form the dark retardation complex, suggesting a role of these genes in the regulation of photocarotenogenesis. In contrast, protein extracts from phototropic mutants formed dark retardation complexes identical to that of the wild type. [ABSTRACT FROM AUTHOR]

Background: The biosynthesis of leucine is a biochemical pathway common to prokaryotes, plants and fungi, but absent from humans and animals. The pathway is a proposed target for antimicrobial therapy. Methodology/Principal Findings: Here we identified the leuA gene encoding α-isopropylmalate synthase in the zygomycete fungus Phycomyces blakesleeanus using a genetic mapping approach with crosses between wild type and leucine auxotrophic strains. To confirm the function of the gene, Phycomyces leuA was used to complement the auxotrophic phenotype exhibited by mutation of the leu3+ gene of the ascomycete fungus Schizosaccharomyces pombe. Phylogenetic analysis revealed that the leuA gene in Phycomyces, other zygomycetes, and the chytrids is more closely related to homologs in plants and photosynthetic bacteria than ascomycetes or basidiomycetes, and suggests that the Dikarya have acquired the gene more recently. Conclusions/Significance: The identification of leuA in Phycomyces adds to the growing body of evidence that some primary metabolic pathways or parts of them have arisen multiple times during the evolution of fungi, probably through horizontal gene transfer events. [ABSTRACT FROM AUTHOR]

The article presents a study on the parasexual cycle of the haploid, multinucleated fungus, Phycomyces. The unstable characteristics of heterokaryons in Phycomyces include high-growth rate sectoring and genetic recombination or chromosomal re-assortments. The author reveals that he has created a situation leading to anastomosis in Phycomyces.

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Abstract: The sporangiophore of the zygomycete Phycomyces blakesleeanus displays negative gravitropism that manifests with a latency of some 5–15min after stimulation. Sporangiophores possess an apical organelle that consists of some 200 lipid globules (ρ =0.79gcm−3) held together in a spherical complex of a dense mesh of F-actin. The complex of lipid globules meets basic physical criteria for a function as gravisusceptor that operates by buoyancy. In addition to the lipid globules also vacuolar protein crystals that sediment upon reorientation contribute to gravitropic bending. The negative gravitropism of Phycomyces can be modulated by tonic irradiation with near-UV, blue and also by red light which indicates the presence of a red-light receptor or a red-light absorbing intermediate of the blue-light receptor. Exogenous Ca2+ at concentrations above 10−6 M enhances gravitropism when the vacuolar crystals are lacking. The findings are compatible with a model in which statoliths exert their function not by pressure but rather by contact and subsequent exchange of binding or redox molecules with the gravireceptor (contact model, (Eibel, P., Schimek, C., Fries, V., et al. Statoliths in it Phycomyces: characterization of octahedral protein crystals. Fungal Gen. Biol. 29, 211–220, 2000; Fries, V., Krockert, T., Grolig, F. et al. Statoliths in Phycomyces: spectrofluorometric characterization of octahedral protein crystals. J. Plant Physiol. 159, 39–47, 2002; Limbach, C., Hauslage, J., Schäfer, C., et al. How to activate a plant gravireceptor. Early mechanisms of gravity sensing studied in Characean rhizoids during parabolic flights. Plant Physiol. 139, 1030–1040, 2005). [Copyright &y& Elsevier]

The Zygomycetes Phycomyces blakesleeanus and Blakeslea trispora are actual or potential sources of β-carotene, ergosterol, ubiquinone, edible oil, and other compounds. By feeding [14C]acetyl-CoA, L-[14C]leucine, or R-[14C]mevalonate in the presence of excess unlabeled glucose, we found that ubiquinone (the terpenoid moiety), β-carotene, and triacylglycerols were made from separate pools of all their common intermediates; the pools for ubiquinone and ergosterol were indistinguishable. Fatty acids were not labeled from mevalonate, showing the absence in these fungi of a shunt pathway that would recycle carbon from mevalonate and its products back to central metabolism. The overproduction of carotene in a Phycomyces mutant and in sexually mated cultures of Blakeslea modified the relative use of labeled and unlabeled carbon sources in the production of carotene, but not of the other compounds. We concluded that carotene, ubiquinone, and triacylglycerols are synthesized in separate subcellular compartments, while sterols and ubiquinone are synthesized in the same compartments or in compartments that exchange precursors. Carotene biosynthesis was regulated specifically and not by flow diversion in a branched pathway. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

DIRS-1 is a retroelement from the slime mold Dictyostelium discoideum. Until recently only two related retrotransposons had been described: PAT from the nematode Panagrellus redivivus and Prt1 from the zygomycete fungus Phycomyces blakesleeanus. Analyses of the reverse transcriptase sequences encoded by these three elements suggested that they were closely related to each other and more distantly related to the Ty3/gypsy Long Terminal Repeat (LTR) retroelements. They have several unusual structural features that distinguish them from typical LTR elements. For instance, they each encode a tyrosine recombinase (YR), but not a DDE-type integrase or an aspartic protease. Although the DIRS-1-related elements are bordered by terminal repeats these differ from typical LTRs in a number of ways. In DIRS-1, for example, the terminal repeats are inverted (complementary), non-identical in sequence, and the outer edges of the terminal sequences are repeated (adjacent to each other) in the internal region. PAT has so-called “split” direct repeats in which the unrelated terminal sequences appear as direct repeats adjacent to each other in the internal region. The only repetition displayed by Prt1 is the presence of short inverted terminal repeats, but the sequenced copy of this element is believed to be a truncated version of an element with a structure resembling DIRS-1. The unusual structure of the terminal repeats of the DIRS1-like elements appears to be related to their replication via free circular intermediates. Site-specific recombination is believed to integrate the circle without creating duplications of the target sites. In recognition of these important distinctions it is proposed that the retrotransposons that encode tyrosine recombinases be called the tyrosine recombinase (or YR) retrotransposons. Recently a large number of additional YR retrotransposons have been described, including elements from fungi (zygomycetes and basidiomycetes), plants (green algae) and a wide range of animals including nematodes, insects, sea urchins, fish and amphibia, while remnants of elements related to DIRS-1 occur in the human genome. The complete set of YR retrotransposons can be divided into two major groups, the DIRS elements and the Ngaro elements, the two groups forming distinct clades on phylogenetic trees based on alignments of RT/RH and recombinase sequences, and also having some structural distinctions. A third group of transposable elements, which we call Cryptons, also carry tyrosine recombinases. These elements do not encode a reverse transcriptase and so are believed to be DNA transposons not retrotransposons. They have been detected in several pathogenic fungi, including the basidiomycete Cryptococcus neoformans, and the ascomycetes Coccidioides posadasii and Histoplasma capsulatum. Sequence comparisons suggest that the Crypton YRs are related to those of the YR retrotransposons. We suggest that the YR retrotransposons arose from the combination of a Crypton-like YR DNA transposon and the RT/RH encoding sequence of a retrotransposon. Copyright © 2005 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

The single-celled fungus Phycomyces blakesleeanus forms vertically oriented sporangiophores from hyphae, which display a negative gravitropic response. Longitudinal growth and gravitropic bending of these sporangiophores were measured with noninvasive H+ and Ca2+ ion-selective microelectrode measurements. The directions of H+ and Ca2+ fluxes, recorded at different locations of sporangiophores, were opposite when the sporangiophores were kept in vertical position. Ca2+ fluxes were in most experiments positive (efflux), while H+ fluxes were negative (influx). The direction of ion fluxes depended on developmental stage of sporangiophores and changed with gravistimulation. [ABSTRACT FROM AUTHOR]

The effect of anoxia and the respiratory chain inhibitors azide and cyanide on the polyphosphate content of Phycomyces was studied by in vivo 31P NMR spectroscopy. Anoxia was manifested by a decrease of core polyphosphates (PPi) and increase of intracellular inorganic phosphate (Pi) signal. Normalized changes in PPi/Pi ratio between control and nitrogen-purged mycelia suggest that the sensitivity to anoxia differs with growth phases. Azide acts in the same way as anoxia, by decreasing the PPi/Pi ratio, while cyanide causes an increase of the PPi/Pi ratio. [ABSTRACT FROM AUTHOR]

Abstract: Two forms of acetyl-CoA synthetase (ACS1 and ACS2) have been detected in Phycomyces blakesleeanus. ACS1, encoded by the gene facA, was induced by acetate and repressed by glucose at the transcriptional level. ACS2, not encoded by the gene facA, was detected as a response to carbon starvation both in the wild type and in an facA − mutant. Both enzymes were purified and characterized. They can use acetate and propionate as substrates. ACS2 is a much more stable enzyme than ACS1. After 60 min incubation at 55 °C, ACS2 retained 50% of its activity whereas ACS1 only retained 3%. The optimum temperature was 50 °C for ACS2 and 30 °C for ACS1. [Copyright &y& Elsevier]

Presents a study on molecular cloning of a gene encoding endo-β-D-1,4-glucanase PCE1 from Phycomyces nitens. Previous cloning of three genes from Rhizpus oryzae as the first cellulase genes from the subdivision Zygomycota; Methods use to clone an endoglucanase gene, designated a pce1 gene; Indication that the optimum pH for the CMCase activity of PCE1 was 6.0 and the optimum temperature was 50°C, the lowest among the family 45 endoglucanases.

We cloned and sequenced thePhycomyces hspAgene. ThehspAgene 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.PhycomycesHSP100 is composed of a domain presumably involved in protein-protein interactions and two ATP-binding domains. ThehspApromoter contains three heat-shock elements that are presumably involved in the activation ofhspAafter heat-shock. In addition, four short sequences are present in thehspApromoter and in the promoter of the photoinducible genescarBandcarRA; and these are candidates as binding sites for light-regulated transcription factors. Blue light can increase transcription of thehspAgene 10-fold, with a threshold of 1 J/m2. The threshold forhspAphotoactivation is 104 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°C or 42°C increasedhspAgene activity 160-fold. The differences in maximumhspAgene transcription by blue light and heat-shock suggest the presence of different regulatory mechanisms. [ABSTRACT FROM AUTHOR]

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 ×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 × g while the albino strain C2 carAgeo-3 (without crystals) has a threshold of about 2 × 10-1 × g. The threshold for the aiming error angle is ill defined and is between 10-2 and 10-1 × g. The threshold for the polar angle of the wild type NRRL1555 (with crystals) is near 8 × 10-2 × g. [ABSTRACT FROM AUTHOR]

Introduction of plasmids in Phycomyces blakesleeanus caused extensive changes in the exogenous DNA and in the resident genome. Plasmids with a bacterial gene for geneticin resistance under a Phycomyces promoter were either injected into immature sporangia or incubated with spheroplasts. An improved method produced about one viable spheroplast per cell. Colonies resistant to geneticin were rare and only about 0.1% of their spores grew in the presence of geneticin. The transformation frequency was very low, ⩽1 transformed colony per million spheroplasts or per μg DNA. Few nuclei in the transformants contained exogenous DNA, as shown by a selective procedure that sampled single nuclei from heterokaryons. The exogenous DNA was not integrated into the genome and no stable transformants were obtained. The plasmids were replicated in the recipient cells, but their DNA sequences were modified by deletions and rearrangements and the transformed phenotype was eventually lost. The spores developed in injected sporangia were often inviable; a genetic test showed that spore death was caused by impaired nuclear proliferation and induction of lethal mutations. About one-fourth of the viable spores from injected sporangia formed abnormal colonies with obvious changes in shape, texture, or color. The abnormalities that could be investigated were due to dominant mutations. The results indicate that incoming DNA is not only attacked, but signals a situation of stress that leads to increased mutation and nuclear and cellular death. [Copyright &y& Elsevier]

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 µm 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 µm 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 supressed by raising stage-1 sporangiophores for 24 hs at 5°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 ß-carotene and two distinct fluorescing pigments that are, however, dispensible for graviperception. [ABSTRACT FROM AUTHOR]

The effects of the Ca2+/H+ exchanger A23187 and the K+/H+ exchanger nigericin, the electrogenic membrane-potential depleters valinomycin and CCCP, and the calcium channel blockers ruthenium red, nifedipine, and nitrendipine on the apical growth of Phycomyces blakesleeanus were analyzed. While all of the compounds inhibited the growth of germlings in liquid medium, the Ca2+ channel blockers were the least effective. Chitin synthesis in vivo was also sensitive to the inhibitors; here again, the calcium channel blockers were less efficient, and their effect occurred after a lag phase, in contrast to the electroneutral ionophores whose effects were immediate. The ionophores rapidly inhibited protein secretion, and reduced the number of secretory vesicles and chitosomes in the hyphal apex of P. blakesleeanus. The results suggest that not only tip-to-base calcium gradients but also transmembrane ionic gradients and membrane potential have a role in the apical growth of P. blakesleeanus. They are probably involved in the formation, migration, and/or fusion with the plasmalemma of secretory vesicles and chitosomes. [ABSTRACT FROM AUTHOR]

An understanding of the relationship between the two components of helical growth (rotation rate and elongation rate) is fundamental to understanding the biophysical and molecular mechanism(s) of cell wall extension in algal cells, fungal cells, and plant stems and roots. Helical growth occurs throughout development of the sporangiophores of Phycomyces blakesleeanus. Previous studies within the growth zone of stage-IVb sporangiophores have reported conflicting conclusions. An implicit assumption in the previous studies [E.S. Castle (1937) J Cell Comp Physiol 9:477–489; R. Cohen and M. Delbruck (1958) J Cell Comp Physiol 52:361–388; J.K.E. Ortega et al. (1974) Plant Physiol 53:485–490] was that the relationship between rotation rate and elongation rate was independent of the magnitude of the elongation rate. In the present study, for stage-IVb sporangiophores growing at a steady rate, it is shown that the ratio of rotation rate and elongation rate decreases as the elongation rate increases. Previously proposed biophysical and molecular mechanisms cannot account for the observed behavior. The previously postulated fibril-reorientation mechanism [J.K.E. Ortega and R.I. Gamow (1974) J Theor Biol 47:317–332; J.K.E. Ortega et al. (1974) Plant Physiol 53:485–490] is modified to accommodate this new finding. Other experiments were conducted to determine how the ratio of rotation rate and elongation rate behaves during a pressure response (a transient decrease in elongation rate produced by a large step-up in turgor pressure using the pressure probe). Results of these experiments indicate that this ratio increases during the pressure response. [ABSTRACT FROM AUTHOR]