Nucleotide sequence encoding the biosynthetic dehydroquinase function of the penta-functional arom locus of Aspergillus nidulans.

The nucleotide sequence of a 1.9 Kb HindIII fragment of DNA derived from the arom locus of A.nidulans and encoding the biosynthetic dehydroquinase activity has been determined. The sequences encoding the biosynthetic and catabolic dehydroquinase enzymes of A.nidulans show no detectable homology, strongly suggesting convergent evolutionary pathways. The messenger RNA specified by the arom locus was detected as a 5.3 Kb RNA species.     

Organization of Enzymes in the Polyaromatic Synthetic Pathway: Separability in Bacteria

ULTRACENTRIFUGATION IN SUCROSE DENSITY GRADIENTS WAS EMPLOYED TO ESTIMATE THE MOLECULAR WEIGHTS AND TO DETERMINE POSSIBLE PHYSICAL AGGREGATION OF THE FIVE ENZYMES CATALYZING STEPS TWO TO SIX IN THE PRECHORISMIC ACID PORTION OF THE POLYAROMATIC SYNTHETIC PATHWAY IN SIX SPECIES OF BACTERIA: Escherichia coli, Salmonella typhimurium, Aerobacter aerogenes, Bacillus subtilis, Pseudomonas aeruginosa, and Streptomyces coelicolor. The five enzymes were not aggregated in extracts of any of the species examined, nor are the genes encoding these enzymes clustered in those bacterial species for which genetic evidence exists. (An initial examination of the blue-green alga Anabaena variabilis indicates nonaggregation in this species also.) This situation in bacteria is in marked contrast to that found in Neurospora crassa and other fungi in which the same five enzymes are associated as an aggregate encoded (at least in the case of N. crassa) by a cluster of five genes. In addition, also in contrast to N. crassa, no evidence was obtained for more than one kind of dehydroquinase activity in any of the bacteria examined. These comparative results are discussed in relation to the origin, evolution, and functional significance of the gene-enzyme relationships existing in the early steps of aromatic biosynthesis in bacteria and fungi.     

Genome mining of cyanide-degrading nitrilases from filamentous fungi

A variety of fungal species are known to degrade cyanide through the action of cyanide hydratases, a specialized subset of nitrilases which hydrolyze cyanide to formamide. In this paper, we report on two previously unknown and uncharacterized cyanide hydratases from Neurospora crassa and Aspergillus nidulans. Recombinant forms of four cyanide hydratases from N. crassa, A. nidulans, Gibberella zeae, and Gloeocercospora sorghi were prepared after their genes were cloned with N-terminal hexahistidine purification tags, expressed in Escherichia coli, and purified using immobilized metal affinity chromatography. These enzymes were compared according to their relative specific activity, pH activity profiles, thermal stability, and ability to remediate cyanide contaminated waste water from silver and copper electroplating baths. Although all four were similar, the N. crassa cyanide hydratase (CHT) has the greatest thermal stability and widest pH range of >50% activity. N. crassa also demonstrated the highest rate of cyanide degradation in the presence of both heavy metals. The CHT of A. nidulans has the highest reaction rate of the four fungal nitrilases evaluated in this work. These data will help determine optimization procedures for the possible use of these enzymes in the bioremediation of cyanide-containing waste. Similar to known plant pathogenic fungi, both N. crassa and A. nidulans were induced to express CHT by growth in the presence of KCN.     

Molecular genetics of fungal siderophore biosynthesis and uptake: the role of siderophores in iron uptake and storage

To acquire iron, all species have to overcome the problems of iron insolubility and toxicity. In response to low iron availability in the environment, most fungi excrete ferric iron-specific chelators--siderophores--to mobilize this metal. Siderophore-bound iron is subsequently utilized via the reductive iron assimilatory system or uptake of the siderophore-iron complex. Furthermore, most fungi possess intracellular siderophores as iron storage compounds. Molecular analysis of siderophore biosynthesis was initiated by pioneering studies on the basidiomycete Ustilago maydis, and has progressed recently by characterization of the relevant structural and regulatory genes in the ascomycetes Aspergillus nidulans and Neurospora crassa. In addition, significant advances in the understanding of utilization of siderophore-bound iron have been made recently in the yeasts Saccharomyces cerevisiae and Candida albicans as well as in the filamentous fungus A. nidulans. The present review summarizes molecular details of fungal siderophore biosynthesis and uptake, and the regulatory mechanisms involved in control of the corresponding genes.     

Ustilago maydis KP6 killer toxin: structure, expression in Saccharomyces cerevisiae, and relationship to other cellular toxins.

There are a number of yeasts that secrete killer toxins, i.e., proteins lethal to sensitive cells of the same or related species. Ustilago maydis, a fungal pathogen of maize, also secretes killer toxins. The best characterized of the U. maydis killer toxins is the KP6 toxin, which consists of two small polypeptides that are not covalently linked. In this work, we show that both are encoded by one segment of the genome of a double-stranded RNA virus. They are synthesized as a preprotoxin that is processed in a manner very similar to that of the Saccharomyces cerevisiae k1 killer toxin, also encoded by a double-strand RNA virus. Active U. maydis KP6 toxin was secreted from S. cerevisiae transformants expressing the KP6 preprotoxin. The two secreted polypeptides were not glycosylated in U. maydis, but one was glycosylated in S. cerevisiae. Comparison of known and predicted cleavage sites among the five killer toxins of known sequence established a three-amino-acid specificity for a KEX2-like enzyme and predicted a new, undescribed processing enzyme in the secretory pathway in the fungi. The mature KP6 toxin polypeptides had hydrophobicity profiles similar to those of other known cellular toxins.     

Unique biosynthesis of dehydroquinic acid?

A search of the genomic sequences of the thermophilic microorganisms Aquifex aeolicus, Archaeoglobus fulgidus, Methanobacterium thermoautotrophicum, and Methanococcus jannaschii for the first seven enzymes (aroG, B, D, E, K, A, and C ) involved in the shikimic acid biosynthetic pathway reveal two key enzymes are missing. The first enzyme in the pathway, 3-deoxy-d-arabino-heptulosonic acid 7-phosphate synthase (aroG) and the second enzyme in the pathway, 5-dehydroquinic acid synthase (aroB) are "missing." The remaining five genes for the shikimate pathway in these organism are present and are similar to the corresponding Escherichia coli genes. The genomic sequences of the thermophiles Pyrococcus abyssi and Thermotoga maritima contain the aroG and aroB genes. Several fungi such as Aspergillus fumigatus, Aspergillus nidulans, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Pneumocystis carinii f. sp. carinii, and Neurospora crassa contain the gene aroM, a pentafunctional enzyme whose overall activity is equivalent to the combined catalytic activities of proteins expressed by aroB, D, E, K, and A genes. Two of these fungi also lack an aroG gene. A discussion of potential reasons for these missing enzymes is presented.     

Purification of the arom multienzyme aggregate from Euglena gracilis.

The arom multienzyme complex that catalyzes steps two through six in the prechorismate polyaromatic amino acid biosynthetic pathway has been purified up to 2000-fold from Euglena gracilis. The native arom aggregate has a molecular weight of approx. 249 000 based on a sedimentation coefficient of 9.5 and Stokes radius of 60 angstrom. A comparison between the arom aggregates of Neurospora crassa and Euglena gracilis and the possible phylogenetic relationships between the organisms are discussed.     

Deletion of mdmB impairs mitochondrial distribution and morphology in Aspergillus nidulans

Mitochondria form a dynamic network of interconnected tubes in the cells of Saccharomyces cerevisiae or filamentous fungi such as Aspergillus nidulans, Neurospora crassa, or Podospora anserina. The dynamics depends on the separation of mitochondrial fragments, their movement throughout the cell, and their subsequent fusion with the other parts of the organelle. Interestingly, the microtubule network is required for the distribution in N. crassa and S. pombe, while S. cerevisiae and A. nidulans appear to use the actin cytoskeleton. We studied a homologue of S. cerevisiae Mdm10 in A. nidulans, and named it MdmB. The open reading frame is disrupted by two introns, one of which is conserved in mdm10 of P. anserina. The MdmB protein consists of 428 amino acids with a predicted molecular mass of 46.5 kDa. MdmB shares 26% identical amino acids to Mdm10 from S. cerevisiae, 35% to N. crassa, and 32% to the P. anserina homologue. A MdmB-GFP fusion protein co-localized evenly distributed along mitochondria. Extraction of the protein was only possible after treatment with a non-ionic and an ionic detergent (1% Triton X-100; 0.5% SDS) suggesting that MdmB was tightly bound to the mitochondrial membrane fraction. Deletion of the gene in A. nidulans affected mitochondrial morphology and distribution at 20 degrees C but not at 37 degrees C. mdmB deletion cells contained two populations of mitochondria at lower temperature, the normal tubular network plus some giant, non-motile mitochondria.     

Motor-driven motility of fungal nuclear pores organizes chromosomes and fosters nucleocytoplasmic transport

Exchange between the nucleus and the cytoplasm is controlled by nuclear pore complexes (NPCs). In animals, NPCs are anchored by the nuclear lamina, which ensures their even distribution and proper organization of chromosomes. Fungi do not possess a lamina and how they arrange their chromosomes and NPCs is unknown. Here, we show that motor-driven motility of NPCs organizes the fungal nucleus. In Ustilago maydis, Aspergillus nidulans, and Saccharomyces cerevisiae fluorescently labeled NPCs showed ATP-dependent movements at ～1.0 μm/s. In S. cerevisiae and U. maydis, NPC motility prevented NPCs from clustering. In budding yeast, NPC motility required F-actin, whereas in U. maydis, microtubules, kinesin-1, and dynein drove pore movements. In the latter, pore clustering resulted in chromatin organization defects and led to a significant reduction in both import and export of GFP reporter proteins. This suggests that fungi constantly rearrange their NPCs and corresponding chromosomes to ensure efficient nuclear transport and thereby overcome the need for a structural lamina.     

Neurospora crassa ve-1 affects asexual conidiation

The velvet factor of the homothallic fungus Aspergillus nidulans promotes sexual fruiting body formation. The encoding veA gene is conserved among fungi, including the ascomycete Neurospora crassa. There, the orthologous ve-1 gene encodes a deduced protein with high similarity to A. nidulans VeA. Cross-complementation experiments suggest that both the promoter and the coding sequence of N. crassa ve-1 are functional to complement the phenotype of an A. nidulans deletion mutant. Moreover, ve-1 expression in the heterologous host A. nidulans results in development of reproductive structures in a light-dependent manner, promoting sexual development in the darkness while stimulating asexual sporulation under illumination. Deletion of the N. crassa ve-1 locus by homologous gene replacement causes formation of shortened aerial hyphae accompanied by a significant increase in asexual conidiation, which is not light-dependent. Our data suggest that the conserved velvet proteins of A. nidulans and N. crassa exhibit both similar and different functions to influence development of these two ascomycetes.     

A comparative genomic analysis of the calcium signaling machinery in Neurospora crassa, Magnaporthe grisea, and Saccharomyces cerevisiae

A large number of Ca2+ -signaling proteins have been previously identified and characterized in Saccharomyces cerevisiae but relatively few have been discovered in filamentous fungi. In this study, a detailed, comparative genomic analysis of Ca2+ -signaling proteins in Neurospora crassa, Magnaporthe grisea, and S. cerevisiae has been made. Our BLAST analysis identified 48, 42, and 40 Ca2+ -signaling proteins in N. crassa, M. grisea, and S. cerevisiae, respectively. In N. crassa, M. grisea, and S. cerevisiae, 79, 100, and 13% of these proteins, respectively, were previously unknown. For N. crassa, M. grisea, and S. cerevisiae, respectively, we have identified: three Ca2+ -permeable channels in each species; 9, 12, and 5 Ca2+/cation-ATPases; eight, six, and four Ca2+ -exchangers; four, four, and two phospholipase C's; one calmodulin in each species; and 23, 21, and 29 Ca2+/calmodulin-regulated proteins. Homologs of a number of key proteins involved in the release of Ca2+ from intracellular stores, and in the sensing of extracellular Ca2+, in animal and plant cells, were not identified. The greater complexity of the Ca2+ -signaling machinery in N. crassa and M. grisea over that in S. cerevisiae probably reflects their more complex cellular organization and behavior, and the greater range of external signals which filamentous fungi have to respond to in their natural habitats. To complement the data presented in this paper, a comprehensive web-based database resource (http://www.fungalcell.org/fdf/) of all Ca2+ -signaling proteins identified in N. crassa, M. grisea, and S. cerevisiae has been provided.     

Fungal small nuclear ribonucleoproteins share properties with plant and vertebrate U-snRNPs.

snRNAs with properties closely related to those of the major vertebrate U-snRNAs are present in the fungi Aspergillus nidulans, Neurospora crassa and Schizosaccharomyces pombe. These RNAs possess a tri-methyl guanosine cap structure and a subset cross-hybridizes with human U1 and U2 clones. In the form of snRNPs, snRNAs from these fungi as well as from Saccharomyces cerevisiae and pea plants are immunoprecipitated by human and anti-Sm or anti-(U1)RNP autoimmune antibodies. On micro-injection into the cytoplasm of Xenopus oocytes, the snRNAs are packaged into ribonucleoprotein particles and migrate into the nucleus. The results demonstrate a hitherto unsuspected degree of evolutionary conservation in snRNA structure, snRNP protein structure, and sites of RNA-protein interaction within snRNPs.     

Characterization of Neurospora crassa catabolic dehydroquinase purified from N. crassa and Escherichia coli.

1. Neurospora crassa catabolic dehydroquinase has been purified from N. crassa and Escherichia coli. 2. Protein-sequence and gel-electrophoretic data show that apparently pure, homogeneous native dehydroquinase is a mixture of intact and proteinase-cleaved enzyme monomers. 3. Protein-sequence data and steady-state kinetics show that the catabolic dehydroquinase gene of N. crassa is expressed with fidelity in E. coli.     

Purification and isolation of the arom aggregates of Schizosaccharomyces pombe

The five enzymes that catalyzing steps two through six in the prechorismate polyaromatic amino acid biosynthetic pathway are physically associated and have been purified up to 400-fold from Schizosaccharomyces pombe. The native arom aggregate has a molecular weight of approx. 140,000-145,000 based on gel filtration, glycerol-density-gradient centrifugation, and polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate. Similarities between the S. pombe arom aggregate and that of Neurospora crassa and Euglena gracilis are discussed.     

The a and b loci of Ustilago maydis hybridize with DNA sequences from other smut fungi.

The smut fungi are obligately parasitic during the sexual phase of their life cycle, and the mating-type genes of these fungi play key roles in both sexual development and pathogenicity. Among species of smut fungi it is common to find a bipolar mating system in which one locus with two alternate alleles is believed to control cell fusion and establishment of the infectious cell type. Alternatively, several species have a tetrapolar mating system in which two different genetic loci, one of which has multiple alleles, control fusion and subsequent development of the infection hyphae. Cloned sequences from the a and b mating-type loci of the tetrapolar smut fungus Ustilago maydis were used as hybridization probes to DNAs from 23 different fungal strains, including smut fungi with both tetrapolar and bipolar mating systems. In general, all of the smut fungi hybridized with the mating-type genes from U. maydis, suggesting conservation of the sequences involved in mating interactions. A selection of DNAs from other ascomycete and basidiomycete fungi failed to hybridize with the U. maydis mating-type sequences. Exceptions to this finding include hybridization of DNA from the a1 idiomorph of U. maydis to DNA from one strain of U. violacea and hybridization of both a idiomorphs to DNA from Saccharomyces cerevisiae.     

Temperature-Sensitive Pleiotropic Revertants from a Mutant in the AROM Gene Cluster of NEUROSPORA CRASSA

Genetical and biochemical studies have been performed with revertants induced in a polyaromatic mutant (No. 58) in the arom gene cluster of Neurospora crassa. In addition to complete and partial revertants able to grow on minimal at both 25 degrees and 35 degrees , temperature-sensitive revertants capable of growth on minimal at 25 degrees but not at 35 degrees have been recovered. One of these revertants has been shown to lack biosynthetic dehydroquinase activity at both temperatures (utilizing the inducible catabolic isozyme for growth at 25 degrees ), to have dehydroshikimate reductase activity only at 25 degrees , and to form an arom aggregate having a molecular weight approximately one-half that of wild type. These results are interpreted as indicating that pleiotropic mutants in the arom gene cluster can result from missense mutations, as well as from nonsense mutations as indicated in previous studies.