Relationship between Phylogenetic Distribution and Genomic Features in Neurospora crassa

In the post-genome era, insufficient functional annotation of predicted genes greatly restricts the potential of mining genome data. We demonstrate that an evolutionary approach, which is independent of functional annotation, has great potential as a tool for genome analysis. We chose the genome of a model filamentous fungus Neurospora crassa as an example. Phylogenetic distribution of each predicted protein coding gene (PCG) in the N. crassa genome was used to classify genes into six mutually exclusive lineage specificity (LS) groups, i.e. Eukaryote/Prokaryote-core, Dikarya-core, Ascomycota-core, Pezizomycotina-specific, N. crassa-orphans and Others. Functional category analysis revealed that only ∼23% of PCGs in the two most highly lineage-specific grouping, Pezizomycotina-specific and N. crassa-orphans, have functional annotation. In contrast, ∼76% of PCGs in the remaining four LS groups have functional annotation. Analysis of chromosomal localization of N. crassa-orphan PCGs and genes encoding for secreted proteins showed enrichment in subtelomeric regions. The origin of N. crassa-orphans is not known. We found that 11% of N. crassa-orphans have paralogous N. crassa-orphan genes. Of the paralogous N. crassa-orphan gene pairs, 33% were tandemly located in the genome, implying a duplication origin of N. crassa-orphan PCGs in the past. LS grouping is thus a useful tool to explore and understand genome organization, evolution and gene function in fungi.     

A specific and sensitive fluorometric assay for tryptophan oxygenase

A spectrophotofluorometric assay was used to measure tryptophan oxygenase activity in several species. The fluorescent assay depends on the conversion of the product of the reaction, N-formyl-l-kynurenine, to anthranilate by means of the coupling enzymes kynurenine formamidase and kynureninase. These enzymes are easily obtained from l-tryptophan-induced N. crassa; and the product, anthranilate, is readily separated by organic extraction from other tryptophan catabolites and easily identified fluorometrically. With this assay, tryptophan oxygenase has been demonstrated in vitro for the first time in N. crassa.     

Homology-dependent inactivation of LTR retrotransposons in <Emphasis Type="Italic">Aspergillus fumigatus</Emphasis> and <Emphasis Type="Italic">A. nidulans</Emphasis> genomes

The repeat-induced point mutation mechanism (RIP) is the most intriguing among the known mechanisms of homology-dependent gene inactivation (silencing) because of its ability to produce irreversible mutations in repetitive DNA sequences. Discovered for the first time in Neurospora crassa, RIP is characterized by C:G to T:A transitions in duplicated sequences. The mechanisms and range of occurrence of RIP are still poorly understood. Mobile elements, including retrotransposons, are a common target for the processes that lead to homology-dependent silencing because of their ability to propagate themselves. Comparative analysis of LTR retrotransposons was performed throughout the genomes of two ascomycetes, Aspergillus fumigatus and A. nidulans. “De-RIP” retroelements were reconstructed on the basis of several copies. CpG, CpA, and TpG sites, which are potential targets for mutagenesis, were found at a much lower frequency in mobile elements than in structural genes. The dinucleotide targets of the two species are affected by RIP at different frequencies: mutagenesis occurs at both CpG and CpA sites in A. fumigatus and is confined to CpG dinucleotides in A. nidulans. This work provides a theoretical background for planning the experimental investigation of RIP inactivation in aspergilli.     

Reorganization of plasma membrane lipid domains during conidial germination

Neurospora crassa, a filamentous fungus, in the unicellular conidial stage has ideal features to study sphingolipid (SL)-enriched domains, which are implicated in fundamental cellular processes ranging from antifungal resistance to apoptosis. Several changes in lipid metabolism and in the membrane composition of N. crassa occur during spore germination. However, the biophysical impact of those changes is unknown. Thus, a biophysical study of N. crassa plasma membrane, particularly SL-enriched domains, and their dynamics along conidial germination is prompted.Two N. crassa strains, wild-type (WT) and slime, which is devoid of cell wall, were studied. Conidial growth of N. crassa WT from a dormancy state to an exponential phase was accompanied by membrane reorganization, namely an increase of membrane fluidity, occurring faster in a supplemented medium than in Vogel's minimal medium. Gel-like domains, likely enriched in SLs, were found in both N. crassa strains, but were particularly compact, rigid and abundant in the case of slime cells, even more than in budding yeast Saccharomyces cerevisiae. In N. crassa, our results suggest that the melting of SL-enriched domains occurs near growth temperature (30 °C) for WT, but at higher temperatures for slime. Regarding biophysical properties strongly affected by ergosterol, the plasma membrane of slime conidia lays in between those of N. crassa WT and S. cerevisiae cells. The differences in biophysical properties found in this work, and the relationships established between membrane lipid composition and dynamics, give new insights about the plasma membrane organization and structure of N. crassa strains during conidial growth.     

Evidence for Transceptor Function of Cellodextrin Transporters in Neurospora crassa

Neurospora crassa colonizes burnt grasslands and metabolizes both cellulose and hemicellulose from plant cell walls. When switched from a favored carbon source to cellulose, N. crassa dramatically up-regulates expression and secretion of genes encoding lignocellulolytic enzymes. However, the means by which N. crassa and other filamentous fungi sense the presence of cellulose in the environment remains unclear. Previously, we have shown that a N. crassa mutant carrying deletions of three β-glucosidase enzymes (Δ3βG) lacks β-glucosidase activity, but efficiently induces cellulase gene expression and cellulolytic activity in the presence of cellobiose as the sole carbon source. These observations indicate that cellobiose, or a modified version of cellobiose, functions as an inducer of lignocellulolytic gene expression and activity in N. crassa. Here, we show that in N. crassa, two cellodextrin transporters, CDT-1 and CDT-2, contribute to cellulose sensing. A N. crassa mutant carrying deletions for both transporters is unable to induce cellulase gene expression in response to crystalline cellulose. Furthermore, a mutant lacking genes encoding both the β-glucosidase enzymes and cellodextrin transporters (Δ3βGΔ2T) does not induce cellulase gene expression in response to cellobiose. Point mutations that severely reduce cellobiose transport by either CDT-1 or CDT-2 when expressed individually do not greatly impact cellobiose induction of cellulase gene expression. These data suggest that the N. crassa cellodextrin transporters act as “transceptors” with dual functions - cellodextrin transport and receptor signaling that results in downstream activation of cellulolytic gene expression. Similar mechanisms of transceptor activity likely occur in related ascomycetes used for industrial cellulase production.     

Human-Like Eukaryotic Translation Initiation Factor 3 from Neurospora crassa

Eukaryotic translation initiation factor 3 (eIF3) is a key regulator of translation initiation, but its in vivo assembly and molecular functions remain unclear. Here we show that eIF3 from Neurospora crassa is structurally and compositionally similar to human eIF3. N. crassa eIF3 forms a stable 12-subunit complex linked genetically and biochemically to the 13^th subunit, eIF3j, which in humans modulates mRNA start codon selection. Based on N. crassa genetic analysis, most subunits in eIF3 are essential. Subunits that can be deleted (e, h, k and l) map to the right side of the eIF3 complex, suggesting that they may coordinately regulate eIF3 function. Consistent with this model, subunits eIF3k and eIF3l are incorporated into the eIF3 complex as a pair, and their insertion depends on the presence of subunit eIF3h, a key regulator of vertebrate development. Comparisons to other eIF3 complexes suggest that eIF3 assembles around an eIF3a and eIF3c dimer, which may explain the coordinated regulation of human eIF3 levels. Taken together, these results show that Neurospora crassa eIF3 provides a tractable system for probing the structure and function of human-like eIF3 in the context of living cells.     

Tip growth of <Emphasis Type="Italic">Neurospora crassa</Emphasis> upon resource shortage: Disturbances of the coordination of elongation,branching, and septation

The characteristics of elongation, branching, septation, and nuclear morphology in hyphal tips (of ~400 μm in length) of the mycelial fungus Neurospora crassa isolated from the mycelium and cultivated for several hours have been investigated using intracellular fluorescent markers. The newly formed branches had the following characteristic features: (1) the predefined orientation was conserved, whereas the diameter decreased (from 10–20 to 6.5 ± 0.4 μm), as did the elongation rate (from 24 ± 1 to 6.7 ± 0.5 μm/min); (2) a disturbed branching pattern with abnormally large internodal distances (up to 1471 μm) and developmental arrest of part of the buds of lateral branches; and (3) a conserved septation pattern and a relatively constant length of hyphal segments (68 ± 2 μm). The size of the nucleus-free zone at the tip (5–33 μm) and the distance between the first septum and the growth point (210 ± 15 μm) in the daughter branches of the isolated fragments were almost the same as in hyphae connected to the mycelium, whereas the average distance between the growth point and the first lateral branch (492 ± 127 μm) and the variability of this parameter were higher in the isolated fragments. The morphology of the nuclei and the size of the nucleus-free zone near the growth point did not differ from those reported for normal vegetative hyphae of N. crassa. The experimental model developed may be used for the elucidation of details of molecular genetic mechanisms that underlie the regulation of interactions between the intracellular structures that provide tip growth of the hyphae in N. crassa.     

Regulation of vectorial supply of vesicles to the hyphal tip determines thigmotropism in Neurospora crassa

Thigmotropism is the ability of an organism to respond to a topographical stimulus by altering its axis of growth. The thigmotropic response of the model fungus Neurospora crassa was quantified using microfabricated glass slides with ridges of defined height. We show that the polarity machinery at the hyphal tip plays a role in the thigmotropic response of N. crassa. Deletion of N. crassa genes encoding the formin, BNI-1, and the Rho-GTPase, CDC-42, an activator of BNI-1 in yeast, CDC-24, its guanine nucleotide exchange factor (GEF), and BEM-1, a scaffold protein in the same pathway, were all shown to significantly decrease the thigmotropic response. In contrast, deletion of genes encoding the cell end-marker protein, TEA-1, and KIP-1, the kinesin responsible for the localisation of TEA-1, significantly increased the thigmotropic response. These results suggest a mechanism of thigmotropism involving vesicle delivery to the hyphal tip via the actin cytoskeleton and microtubules. Neurospora crassa thigmotropic response differed subtly from that of Candida albicans where the stretch-activated calcium channel, Mid1, has been linked with thigmotropic behaviour. The MID-1 deficient mutant of N. crassa (Δmid-1) and the effects of calcium depletion were examined here but no change in the thigmotropic response was observed. However, SPRAY, a putative calcium channel protein, was shown to be required for N. crassa thigmotropism. We propose that the thigmotropic response is a result of changes in the polarity machinery at the hyphal tip which are thought to be downstream effects of calcium signalling pathways triggered by mechanical stress at the tip.     

Structure and function of a mating-type gene from the homothallic species Neurospora africana

The homothallic Neurospora species, N. africana, contains sequences that hybridize to the A but not to a mating-type sequences of the heterothallic species N. crassa. In this study, the N. africana mating-type gene, mt A-1, was cloned, sequenced and its function analyzed in N. crassa. Although N. africana does not mate in a heterothallic manner, its mt A-1 gene functions as a mating activator in N. crassa. In addition, the N. africana mt A-1 gene confers mating type-associated vegetative incompatibility in N. crassa. DNA sequence analysis shows that the N. africana mt A-1 open reading frame (ORF) is 93% identical to that of N. crassa mt A-1. The mt A-1 ORF of N. africana contains no stop codons and was detected as a cDNA which is processed in a similar manner to mt A-1 of N. crassa. By DNA blot and orthogonal field agarose gel electrophoretic analysis, it is shown that the composition and location of the mating-type locus and the organization of the mating-type chromosome of N. africana are similar to that of N. crassa.     

Biotechnological production of ethanol from renewable resources by Neurospora crassa: an alternative to conventional yeast fermentations?

Microbial production of ethanol might be a potential route to replace oil and chemical feedstocks. Bioethanol is by far the most common biofuel in use worldwide. Lignocellulosic biomass is the most promising renewable resource for fuel bioethanol production. Bioconversion of lignocellulosics to ethanol consists of four major unit operations: pretreatment, hydrolysis, fermentation, and product separation/distillation. Conventional bioethanol processes for lignocellulosics apply commercial fungal cellulase enzymes for biomass hydrolysis, followed by yeast fermentation of resulting glucose to ethanol. The fungus Neurospora crassa has been used extensively for genetic, biochemical, and molecular studies as a model organism. However, the strain's potential in biotechnological applications has not been widely investigated and discussed. The fungus N. crassa has the ability to synthesize and secrete all three enzyme types involved in cellulose hydrolysis as well as various enzymes for hemicellulose degradation. In addition, N. crassa has been reported to convert to ethanol hexose and pentose sugars, cellulose polymers, and agro-industrial residues. The combination of these characteristics makes N. crassa a promising alternative candidate for biotechnological production of ethanol from renewable resources. This review consists of an overview of the ethanol process from lignocellulosic biomass, followed by cellulases and hemicellulases production, ethanol fermentations of sugars and lignocellulosics, and industrial application potential of N. crassa.     

Involvement of protein kinase C in the response of Neurospora crassa to blue light

As a first step towards understanding the process of blue light perception, and the signal transduction mechanisms involved, in Neurospora crassa we have used a pharmacological approach to screen a wide range of second messengers and chemical compounds known to interfere with the activity of well-known signal transducing molecules in vivo. We tested the influence of these compounds on the induction of the al-3 gene, a key step in light-induced carotenoid biosynthesis. This approach has implicated protein kinase C (PKC) as a component of the light transduction machinery. The conclusion is based on the effects of specific inhibitors (calphostin C and chelerythrine chloride) and activators of PKC (1,2-dihexanoyl-sn-glycerol). During vegetative growth PKC may be responsible for desensitization to light because inhibitors of the enzyme cause an increase in the total amount of mRNA transcribed after illumination. PKC is therefore proposed here to be an important regulator of transduction of the blue light signal, and may act through modification of the protein White Collar-1, which we show to be a substrate for PKC in N. crassa.     

Osmotic Effects on the Polyamine Pathway of Neurospora crassa

Davis, R. H., and Ristow, J. L. 1995. Osmotic effects on the polyamine pathway of Neurospora crassa. Experimental Mycology 19, 314-319. In bacteria, mammals, and certain plants, the induction of the polyamine synthetic enzyme, ornithine decarboxylase (ODC), and the accumulation of its product, putrescine, follows osmotic manipulations of cells. In at least some of these cases, this response is indispensable for survival. We wished to determine whether the polyamine pathway of Neurospora crassa was regulated in response to hyper- or hypoosmotic conditions. Unlike ODC of most other classes of organisms, the N. crassa enzyme and the accumulation of putrescine appears to be relatively indifferent to these conditions, either during sudden transitions or in steady-state. We conclude that other mechanisms of osmotic adjustment or tolerance have evolved in N. crassa and perhaps other fungi that obviate the need for putrescine accumulation.     

The protease problem in Neurospora. Structural modification of the arom multienzyme system during its extraction and isolation.

The “aromatic complex” or “arom aggregate” of Neurospora crassa catalyzes five consecutive reactions in the central pathway leading to the biosynthesis of the aromatic amino acids. Previously, this multienzyme system was shown variously to have a molecular weight of 230,000 to 300,000 and to contain up to four subunits. Recently, a protease and a corresponding specific inhibitor have been isolated from N. crassa and, as described in this report, a new method for isolating the multienzyme system has been developed. We have made the following observations: (a) Detergent (sodium dodecyl sulfate) gel electrophorograms of the “complex” isolated by two different methods are not comparable. In an earlier method, which involved more manipulations and time, the detergent gel banding patterns showed four polypeptides with molecular weights totaling about 300,000. With the new purification procedure, there are two major bands: the first with an apparent molecular weight of about 150,000 and the second with a molecular weight of 50,000. (b) When the freshly purified multienzyme system is incubated at 25 °C, four new bands appear within 30 h and a fifth is visible after 40 h. (c) The formation of these new bands is prevented for up to 40 h by the addition of phenylmethanesulfonylfluoride or a purified preparation of the specific N. crassa protease inhibitor, (d) The multienzyme system appears to remain intact, as shown by standard polyacrylamide gel electrophoresis, even after it has suffered several proteolytic clips. These results demonstrate that the purified complex is contaminated with a small but influential quantity of the inhibitable N. crassa protease and show that this protease is capable of creating an artificial subunit structure in the multienzyme system. Based on these observations, we hypothesize that the arom enzyme system is a five-component multifunctional enzyme.     

What's in the genome of a filamentous fungus? Analysis of the Neurospora genome sequence

The German Neurospora Genome Project has assembled sequences from ordered cosmid and BAC clones of linkage groups II and V of the genome of Neurospora crassa in 13 and 12 contigs, respectively. Including additional sequences located on other linkage groups a total of 12 Mb were subjected to a manual gene extraction and annotation process. The genome comprises a small number of repetitive elements, a low degree of segmental duplications and very few paralogous genes. The analysis of the 3218 identified open reading frames provides a first overview of the protein equipment of a filamentous fungus. Significantly, N.crassa possesses a large variety of metabolic enzymes including a substantial number of enzymes involved in the degradation of complex substrates as well as secondary metabolism. While several of these enzymes are specific for filamentous fungi many are shared exclusively with prokaryotes.     

Characterization of the cit-1 gene from Neurospora crassa encoding the mitochondrial form of citrate synthase

We have isolated the cDNA and corresponding genomic DNA encoding citrate synthase in Neurospora crassa. Analysis of the protein coding region of this gene, named cit-1, indicates that it specifies the mitochondrial form of citrate synthase. The predicted protein has 469 amino acids and a molecular mass of 52002 Da. The gene is interrupted by four introns. Hybridization experiments show that a cit-1 probe binds to two different fragments of genomic DNA, which are located on different chromosomes. Neurospora crassa may have two isoforms of citrate synthase, one in the mitochondria and the other in microbodies.     

The Saccharomyces cerevisiae PRM1 Homolog in Neurospora crassa Is Involved in Vegetative and Sexual Cell Fusion Events but Also Has Postfertilization Functions

Cell–cell fusion is essential for a variety of developmental steps in many eukaryotic organisms, during both fertilization and vegetative cell growth. Although the molecular mechanisms associated with intracellular membrane fusion are well characterized, the molecular mechanisms of plasma membrane merger between cells are poorly understood. In the filamentous fungus Neurospora crassa, cell fusion events occur during both vegetative and sexual stages of its life cycle, thus making it an attractive model for studying the molecular basis of cell fusion during vegetative growth vs. sexual reproduction. In the unicellular yeast Saccharomyces cerevisiae, one of the few proteins implicated in plasma membrane merger during mating is Prm1p; prm1Δ mutants show an ~50% reduction in mating cell fusion. Here we report on the role of the PRM1 homolog in N. crassa. N. crassa strains with deletions of a Prm1-like gene (Prm1) showed an ~50% reduction in both vegetative and sexual cell fusion events, suggesting that PRM1 is part of the general cell fusion machinery. However, unlike S. cerevisiae, N. crassa strains carrying a Prm1 deletion exhibited complete sterility as either a male or female mating partner, a phenotype that was not complemented in a heterokaryon with wild type (WT). Crosses with ΔPrm1 strains were blocked early in sexual development, well before development of ascogenous hyphae. The ΔPrm1 sexual defect in N. crassa was not suppressed by mutations in Sad-1, which is required for meiotic silencing of unpaired DNA (MSUD). However, mutations in Sad-1 increased the number of progeny obtained in crosses with a ΔPrm1 (Prm1-gfp) complemented strain. These data indicate multiple roles for PRM1 during sexual development.