Identification of the ergC gene involved in polyene drug sensitivity in the Mucorales species Phycomyces blakesleeanus.

BACKGROUND: A strain of Phycomyces blakesleeanus (Mucorales, Mucoromycota) that was previously isolated after ultraviolet mutagenesis has altered responses to polyene antifungal drugs, sterol profiles, and phototropism of its sporangia. In this study, the genetic basis for these changes was sought. METHODS AND RESULTS: Two base pair substitutions were identified in the mutant within a P. blakelesleeanus gene that is homologous to others characterized from fungi, such as the Saccharomyces cerevisiae ERG3 gene, encoding sterol Δ5,6-desaturase. The polyene resistance and growth reduction phenotypes co-segregated with mutations in the gene in genetic crosses. The P. blakelesleeanus wild type ergC gene complemented a S. cerevisiae deletion strain of ERG3. CONCLUSIONS: This gene discovery may contribute towards better antifungal use in treating mucormycoses diseases caused by related species in the order Mucorales.

Tangent algorithm for photogravitropic balance in plants and Phycomyces blakesleeanus: Roles for EHB1 and NPH3 of Arabidopsis thaliana.

Plant organs that are exposed to continuous unilateral light reach in the steady-state a photogravitropic bending angle that results from the mutual antagonism between the photo- and gravitropic responses. To characterize the interaction between the two tropisms and their quantitative relationship we irradiated seedlings of Arabidopsis thaliana that were inclined at various angles and determined the fluence rates of unilateral blue light required to compensate the gravitropism of the inclined hypocotyls. We found the compensating fluence rates to increase with the tangent of the inclination angles (0° < γ < 90° or max. 120°) and decrease with the cotangent (90°< γ < 180° or max. 120°of the inclination angles. The tangent dependence became also evident from analysis of previous data obtained with Avena sativa and the phycomycete fungus, Phycomyces blakesleeanus. By using loss-of function mutant lines of Arabidopsis, we identified EHB1 (enhanced bending 1) as an essential element for the generation of the tangent and cotangent relationships. Because EHB1 possesses a C2-domain with two putative calcium binding sites, we propose that the ubiquitous calcium dependence of gravi- and phototropism is in part mediated by Ca2+-bound EHB1. Based on a yeast-two-hybrid analysis we found evidence that EHB1 does physically interact with the ARF-GAP protein AGD12. Both proteins were reported to affect gravi- and phototropism antagonistically. We further showed that only AGD12, but not EHB1, interacts with its corresponding ARF-protein. Evidence is provided that AGD12 is able to form homodimers as well as heterodimers with EHB1. On the basis of these data we present a model for a mechanism of early tropism events, in which Ca2+-activated EHB1 emerges as the central processor-like element that links the gravi- and phototropic transduction chains and that generates in coordination with NPH3 and AGD12 the tangent / cotangent algorithm governing photogravitropic equilibrium.

Light regulates a Phycomyces blakesleeanus gene family similar to the carotenogenic repressor gene of Mucor circinelloides.

The transcription of about 5-10 % of the genes in Phycomyces blakesleeanus is regulated by light. Among the most up-regulated, we have identified four genes, crgA-D, with similarity to crgA of Mucor circinelloides, a gene encoding a repressor of light-inducible carotenogenesis. The four proteins have the same structure with two RING RING Finger domains and a LON domain, suggesting that they could act as ubiquitin ligases, as their M. circinelloides homolog. The expression of these genes is induced by light with different thresholds as in other Mucoromycotina fungi like Blakeslea trispora and M. circinelloides. Only the P. blakesleeanus crgD gene could restore the wild type phenotype in a M. circinelloides null crgA mutant suggesting that P. blakesleeanus crgD is the functional homolog of crgA in M. circinelloides. Despite their sequence similarity it is possible that the P. blakesleeanus Crg proteins do not participate in the regulation of beta-carotene biosynthesis since none of the carotene-overproducing mutants of P. blakesleeanus had mutations in any of the crg genes. Our results provide further support of the differences in the regulation of the biosynthesis of beta-carotene in these two Mucoromycotina fungi.

Mystique of Phycomyces blakesleeanus is a peculiar mitochondrial genetic element that is highly variable in DNA sequence while subjected to strong negative selection.

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.

A Ras GTPase associated protein is involved in the phototropic and circadian photobiology responses in fungi.

Light is an environmental signal perceived by most eukaryotic organisms and that can have major impacts on their growth and development. The MadC protein in the fungus Phycomyces blakesleeanus (Mucoromycotina) has been postulated to form part of the photosensory input for phototropism of the fruiting body sporangiophores, but the madC gene has remained unidentified since the 1960s when madC mutants were first isolated. In this study the madC gene was identified by positional cloning. All madC mutant strains contain loss-of-function point mutations within a gene predicted to encode a GTPase activating protein (GAP) for Ras. The madC gene complements the Saccharomyces cerevisiae Ras-GAP ira1 mutant and the encoded MadC protein interacts with P. blakesleeanus Ras homologs in yeast two-hybrid assays, indicating that MadC is a regulator of Ras signaling. Deletion of the homolog in the filamentous ascomycete Neurospora crassa affects the circadian clock output, yielding a pattern of asexual conidiation similar to a ras-1 mutant that is used in circadian studies in N. crassa. Thus, MadC is unlikely to be a photosensor, yet is a fundamental link in the photoresponses from blue light perceived by the conserved White Collar complex with Ras signaling in two distantly-related filamentous fungal species.

The inhibition of mating in Phycomyces blakesleeanus by light is dependent on the MadA-MadB complex that acts in a sex-specific manner.

Light is an environmental signal that influences reproduction in the Mucoromycotina fungi, as it does in many other species of fungi. Mating in Phycomyces blakesleeanus is inhibited by light, but the molecular mechanisms for this inhibition are uncharacterized. In this analysis, the role of the light-sensing MadA-MadB complex in mating was tested. The MadA-MadB complex is homologous to the Neurospora crassa White Collar complex. Three genes required for cell type determination in the sex locus or pheromone biosynthesis are transcriptionally-regulated by light and are controlled by MadA and MadB. This regulation acts through the plus partner, indicating that the inhibitory effect of light on mating is executed through only one of the two sexes. These results are an example whereby the mating types of fungi have acquired sex-specific properties beyond their role in conferring cell-type identity, and provide insight into how sex-determining chromosomal regions can expand the traits they control.

Metabolite profiling of Phycomyces blakesleeanus carotene mutants reveals global changes across intermediary metabolism.

The filamentous fungus Phycomyces blakesleeanus provides a renewable biosource of industrial high-value compounds such as carotenes, other isoprenoids (ubiquinone and sterols), organic acids and fatty acids. Several Phycomyces mutants involved in the formation of ß-carotene are available. For example, the carA mutants have a leaky mutation in the phytoene synthase and produce significantly lower amounts of carotenes, while the carB and carR mutants produce phytoene and lycopene, respectively, due to a null mutation in the genes encoding the phytoene dehydrogenase and lycopene cyclase, respectively. The carS mutants are mutated in the gene encoding the oxygenase responsible for the conversion of ß-carotene into apocarotenoids and, as a result, ß-carotene accumulates. In order to ascertain further the biochemical changes arising in these potential industrial strains, a metabolite profiling workflow was implemented for Phycomyces. GC-MS and ultra-performance liquid chromatography-photodiode array platforms enabled the identification of over 100 metabolites in 11 carA, carB, carR and carS mutant strains and their wild-type comparator. All mutant strains possessed decreased TCA cycle intermediates, galactose, alanine and ribitol, while dodecanol and valine showed a general increase. As predicted, other terpenoid levels were affected in the carB, carR and carS mutants but not in the carA mutants. The global changes across intermediary metabolism of the mutants suggest that complex metabolic networks exist between intermediary and secondary metabolism or that other mutations beyond the carotene pathway may exist in these mutants. These data show the utility of the methodology in metabolically phenotyping Phycomyces strains with potential industrial exploitation.

Expansion of Signal Transduction Pathways in Fungi by Extensive Genome Duplication.

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.

Fungal cryptochrome with DNA repair activity reveals an early stage in cryptochrome evolution.

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.

Diversity, ecology, and evolution in Phycomyces.

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.

Deciphering the uniqueness of Mucoromycotina cell walls by combining biochemical and phylogenomic approaches.

Most fungi from the Mucoromycotina lineage occur in ecosystems as saprobes, although some species are phytopathogens or may induce human mycosis. Mucoromycotina represent early diverging models that are most valuable for understanding fungal evolution. Here we reveal the uniqueness of the cell wall structure of the Mucoromycotina Rhizopus oryzae and Phycomyces blakesleeanus compared with the better characterized cell wall of the ascomycete Neurospora crassa. We have analysed the corresponding polysaccharide biosynthetic and modifying pathways, and highlight their evolutionary features and higher complexity in terms of gene copy numbers compared with species from other lineages. This work uncovers the presence in Mucoromycotina of abundant fucose-based polysaccharides similar to algal fucoidans. These unexpected polymers are associated with unusually low amounts of glucans and a higher proportion of chitin compared with N. crassa. The specific structural features are supported by the identification of genes potentially involved in the corresponding metabolic pathways. Phylogenomic analyses of genes encoding carbohydrate synthases, polysaccharide modifying enzymes and enzymes involved in nucleotide-sugar formation provide evidence for duplication events during evolution of cell wall metabolism in fungi. Altogether, the data highlight the specificity of Mucoromycotina cell walls and pave the way for a finer understanding of their metabolism.

Sex determination directs uniparental mitochondrial inheritance in Phycomyces.

Uniparental inheritance (UPI) of mitochondria is common among eukaryotes. The underlying molecular basis by which the sexes of the parents control this non-Mendelian pattern of inheritance is yet to be fully understood. Two major factors have complicated the understanding of the role of sex-specific genes in the UPI phenomenon: in many cases (i) fusion occurs between cells of unequal size or (ii) mating requires a large region of the genome or chromosome that includes genes unrelated to sex determination. The fungus Phycomyces blakesleeanus is a member of the Mucoromycotina and has a simple mating type locus encoding only one high-mobility group (HMG) domain protein, and mating occurs by fusion of isogamous cells, thus providing a model system without the limitations mentioned above. Analysis of more than 250 progeny from a series of genetic crosses between wild-type strains of Phycomyces revealed a correlation between the individual genes in the mating type locus and UPI of mitochondria. Inheritance is from the plus (+) sex type and is associated with degradation of the mtDNA from the minus (-) parent. These findings suggest that UPI can be directly controlled by genes that determine sex identity, independent of cell size or the complexity of the genetic composition of a sex chromosome.

The Uve1 endonuclease is regulated by the white collar complex to protect cryptococcus neoformans from UV damage.

The pathogenic fungus Cryptococcus neoformans uses the Bwc1-Bwc2 photoreceptor complex to regulate mating in response to light, virulence and ultraviolet radiation tolerance. How the complex controls these functions is unclear. Here, we identify and characterize a gene in Cryptococcus, UVE1, whose mutation leads to a UV hypersensitive phenotype. The homologous gene in fission yeast Schizosaccharomyces pombe encodes an apurinic/apyrimidinic endonuclease acting in the UVDE-dependent excision repair (UVER) pathway. C. neoformans UVE1 complements a S. pombe uvde knockout strain. UVE1 is photoregulated in a Bwc1-dependent manner in Cryptococcus, and in Neurospora crassa and Phycomyces blakesleeanus that are species that represent two other major lineages in the fungi. Overexpression of UVE1 in bwc1 mutants rescues their UV sensitivity phenotype and gel mobility shift experiments show binding of Bwc2 to the UVE1 promoter, indicating that UVE1 is a direct downstream target for the Bwc1-Bwc2 complex. Uve1-GFP fusions localize to the mitochondria. Repair of UV-induced damage to the mitochondria is delayed in the uve1 mutant strain. Thus, in C. neoformans UVE1 is a key gene regulated in response to light that is responsible for tolerance to UV stress for protection of the mitochondrial genome.

A new genetic linkage map of the zygomycete fungus Phycomyces blakesleeanus.

Phycomyces blakesleeanus is a member of the subphylum Mucoromycotina. A genetic map was constructed from 121 progeny of a cross between two wild type isolates of P. blakesleeanus with 134 markers. The markers were mostly PCR-RFLPs. Markers were located on 46 scaffolds of the genome sequence, covering more than 97% of the genome. Analysis of the alleles in the progeny revealed nine or 12 linkage groups, depending on the log of the odds (LOD) score, across 1583.4 cM at LOD 5. The linkage groups were overlaid on previous mapping data from crosses between mutants, aided by new identification of the mutations in primary metabolism mutant strains. The molecular marker map, the phenotype map and the genome sequence are overall congruent, with some exceptions. The new genetic map provides a genome-wide estimate for recombination, with the average of 33.2 kb per cM. This frequency is one piece of evidence for meiosis during zygospore development in Mucoromycotina species. At the same time as meiosis, transmission of non-recombinant chromosomes is also evident in the mating process in Phycomyces. The new map provides scaffold ordering for the genome sequence and a platform upon which to identify the genes in mutants that are affected in traits of interest, such as carotene biosynthesis, phototropism or gravitropism, using positional cloning.

A gene for carotene cleavage required for pheromone biosynthesis and carotene regulation in the fungus Phycomyces blakesleeanus.

Mating and sexual development in fungi are controlled by molecular mechanisms that are specific for each fungal group. Mating in Phycomyces blakesleeanus and other Mucorales requires pheromones derived from ß-carotene. Phycomyces mutants in gene carS accumulate large amounts of ß-carotene but do not enter the sexual process. We show that carS encodes a ß-carotene-cleaving oxygenase that catalyzes the first step in the biosynthesis of a variety of apocarotenoids, including those that act as pheromones. Therefore carS mutants cannot stimulate their sexual partners, although they respond to them. CarS catalyzes the biosynthesis of a ß-ring-containing apocarotenoid that inhibits the activity of the carotenogenic enzyme complex in vegetative cells and provides a feedback regulation for the ß-carotene pathway. The carS gene product is a keystone in carotenogenesis and in sexual reproduction.

Cleavage oxygenases for the biosynthesis of trisporoids and other apocarotenoids in Phycomyces.

Mixed cultures of strains of opposite sex of the Mucorales produce trisporic acids and other compounds arising from cleavage of ß-carotene, some of which act as signals in the mating process. The genome of Phycomyces blakesleeanus contains five sequences akin to those of verified carotenoid cleavage oxygenases. All five are transcribed, three of them have the sequence traits that are considered essential for activity, and we have discovered the reactions catalysed by the products of two of them, genes carS and acaA. The transcripts of carS became more abundant in the course of mating, and its expression in ß-carotene-producing Escherichia coli cells led to the formation of ß-apo-12'-carotenal, a C25 cleavage product of ß-carotene. Joint expression of both genes in the same in vivo system resulted in the production of ß-apo-13-carotenone, a C18 fragment. In vitro, AcaA cleaved ß-apo-12'-carotenal into ß-apo-13-carotenone and was active on other apocarotenoid substrates. According to these and other results, the first reactions in the apocarotenoid pathway of Phycomyces are the cleavage of ß-carotene at its C11'-C12' double bond by CarS and the cleavage of the resulting C25-fragment at its C13-14 double bond by AcaA.

Functional analysis of the Phycomyces carRA gene encoding the enzymes phytoene synthase and lycopene cyclase.

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.

Splitting of beta-carotene in the sexual interaction of Phycomyces.

Two new 7-carbon compounds, 1 and 2, have been found in the culture medium of Phycomyces blakesleeanus. A genetic test showed that they derive from beta-carotene. These new molecules represent the missing link that proves that beta-carotene is split into fragments of 18, 15 and 7 carbon fragments, each head of a separate family of apocarotenoids.