The Aspergillus nidulans rcoA gene is required for veA-dependent sexual development

The Aspergillus nidulans rcoADelta mutant exhibits growth and developmental defects. We show that the rcoADelta mutant lacks cleistothecia and is self-sterile. In crosses with wild-type strains, rcoADelta nuclei do not contribute to the cleistothecial walls. Furthermore, sexual development resulting from veA overexpression is rcoA dependent, indicating that rcoA lies downstream of veA in the sexual development pathway.     

Isolation of DNA from Aspergillus nidulans.

A procedure for isolation of DNA from Aspergillus nidulans on a preparative scale is described. Mechanical disruption of lyophilized material in high-salt medium and treatment with proteinase K, followed by sedimentation of the lysate into saturated CsC1 solution yielded pure, highly polymerized DNA.     

Identification of a gene for alpha-tubulin in Aspergillus nidulans.

This paper demonstrates that revertants of temperature-sensitive benA (β-tubulin) mutations in Aspergillus nidulans can be used to identify proteins which interact with β-tubulin. Three benomyl-resistant benA (β-tubulin) mutants of Aspergillus nidulans, BEN 9, BEN 15 and BEN 19, were found to be temperature-sensitive (ts^?) for growth. Temperature sensitivity co-segregated with benomyl resistance among the progeny of outcrosses of BEN 9, 15 and 19 to a wild-type strain, FGSC#99, indicating that temperature sensitivity was caused by mutations in the benA gene in these strains. Eighteen revertants to ts⁺ were isolated by selection at the restrictive temperature. Four had back-mutations in the benA gene and fourteen carried extragenic suppressor mutations. Two of the back-mutated strains had β-tubulins which differed from the β-tubulins of their parental strains by one (1^?) or two (2^?) negative charges on two-dimensional gel electrophoresis. Although the β-tubulins of the extragenic suppressor strains were all electrophoretically identical to those of the parental strains, one of the suppressor strains, BEN 9R7, had an electrophoretic abnormality in α1-tubulin (1⁺). A heterozygous diploid between this strain and a strain with wild-type α1-tubulin was found to have both wild-type and mutant (1⁺) α1-tubulins. This experiment rules out post-translational modification as a possible cause of the α1-tubulin abnormality. Thus the suppressor mutation in BEN 9R7 must be in a structural gene for α1-tubulin. We propose that this gene be designated tubA to denote that it is a gene for α1-tubulin in A. nidulans.     

Identification of a gene for beta-tubulin in Aspergillus nidulans.

The tubulins of Aspergillus nidulans have been characterized in wild-type and ben A, B and C benomyl-resistant strains by two-dimensional gel electrophoresis, co-polymerization with porcine brain tubulin and peptide mapping. Four α-tubulins and at least four β-tubulins were resolved by two-dimensional gel electrophoresis of wild-type proteins. Eighteen of 26 benA mutants studied had electrophoretically abnormal β-tubulins. In these strains, one or more of the β-tubulins had either an altered isoelectric point or an altered electrophoretic mobility in the SDS gel dimension, or was diminished in amount. The a-tubulins were normal. Two-dimensional gels of protein extracts of a ben A/wild-type diploid strain demonstrated co-expression of the wild-type β-tubulins with the variant ben A tubulin. This experiment rules out post-translational modification as the source of the β-tubulin abnormalities in the benA mutants. We therefore conclude that benA must be a structural gene for β-tubulin. Due to the variety of abnormalities affecting β-tubulins in ben A mutants, and the absence of abnormalities affecting α-tubulins in any of the benomyl-resistant mutants, we also believe that the benomyl binding site must be located on the β-subunit of the tubulin dimer. The benA mutants of A. nidulans promise to be useful not only for characterizing the biochemical determinants of the benomyl binding site of tubulin but also for understanding the relationship between tubulin structure and function.     

Isolation of a sexual sporulation hormone from Aspergillus nidulans.

Psi factor is a substance produced by Aspergillus nidulans that induces premature sexual sporulation. Chromatographic analysis of psi-active extracts showed that psi activity resides in several different forms. Two of the forms, psiA1 and psiB1, have been isolated and have been shown to have closely similar compositions. The most abundant form, psiA1, reacts with alcohols in acidic solution by the addition of one entire molecule of the alcohol. This reaction, which is reversible, suggests that psiA1 may be a lactone whose ring is opened by alcohol addition. At high concentration, psiA1 is antagonistic to the response exhibited by the other forms of psi, but this antagonism is lost by the alcoholic derivatives. At least one unpurified psi species can be converted to psiA1 by acid catalysis. We suggest that psiA1 may be the metabolic precursor of at least some of the other more active psi components and that this conversion during Aspergillus development may be part of the process that triggers sexual sporulation.     

Transformation of Aspergillus niger by the amdS gene of Aspergillus nidulans.

Aspergillus niger grows poorly on acetamide as a nitrogen or carbon source and lacks sequences detectably homologous to the amdS gene encoding the acetamidase of Aspergillus nidulans. We have taken advantage of these observations to develop a transformation system for A. niger using the amdS gene as a dominant heterologous marker for selecting transformants on the basis of acetamide utilization. Transformants varied in their ability to grow on amide media and the number of integrated copies of the amdS plasmid ranged from 1 or 2 to greater than 100. Southern analysis of transformants revealed that the multiple copies were integrated into the chromosome in tandem arrays. This result indicates that transformation of A. niger is more similar to mammalian cells than to yeast. Analysis of enzyme activity levels and RNA levels showed that most of the copies of amdS were expressed. Mitotic stabilities of transformants were found to be high. A transformant containing greater than 100 copies of the amdS gene was impaired in omega-amino acid utilization, a result that has also been found in A. nidulans. Since, in A. nidulans, omega-amino acids induce acetamidase via a characterizied regulatory gene (amdR/intA) this observation implies that titration of an analogous A. niger regulatory gene product by multiple amdS copies has occurred. Additional evidence suggested that the amdS gene is regulated in A. niger. It has also been shown that an unselected plasmid can be co-transformed with the amdS plasmid into A. niger.     

The tamA gene of Aspergillus nidulans contains a putative zinc cluster motif which is not required for gene function.

Expression of many nitrogen catabolic enzymes is controlled by nitrogen metabolite repression in Aspergillus nidulans. Although the phenotypes of tamA mutants have implicated this gene in nitrogen regulation, its function is unknown. We have cloned the tamA gene by complementation of a new tamA allele. The tamA sequence shares significant homology with the UGA35/DAL81/DURL gene of Saccharomyces cerevisiae. In vitro mutagenesis of sequences encoding a putative zinc cluster DNA binding domain indicated that this motif is not required for in vivo TamA function.     

Characterization of phiA, a gene essential for phialide development in Aspergillus nidulans

We have previously identified genes and proteins involved in the fungal response to the Streptomyces-produced antibiotics, bafilomycin B1 and concanamycin A, known inhibitors of V-ATPases. Using mRNA differential display we identified an Aspergillus nidulans gene with 30-fold up-regulated expression in the presence of bafilomycin. This gene, here denoted phiA, and its gene product, were further characterized by targeted gene disruption and immunohistochemistry. Phenotypically, the phiA mutation resulted in reduced growth and severely reduced sporulation. The abnormality could be traced to the phialides, which divided several times instead of forming a single flask-shaped cell. The importance of phiA for phialide and conidium development was supported by immunohistochemistry experiments that showed the protein to be mainly present in these two cell types. Attempts to relate phiA to inhibition of V-ATPases did not result in unambiguous conclusions, but suggest the possibility that changed expression of phiA is correlated with growth arrest caused by inhibited V-ATPases.     

Cloning an Aspergillus nidulans developmental gene by transformation.

We have developed a transformation system for Aspergillus nidulans giving a frequency of transformation high enough to screen a gene bank from which we were able to isolate and clone the A. nidulans developmental gene brlA by visual selection. The vector contains the selective marker argB+, and with it a frequency of transformation of 500 stable transformants/micrograms plasmid DNA can regularly be achieved. The evidence suggests that transformation is by integration but spontaneous excision of integrated plasmids is apparently frequent enough to allow the recovery of transforming plasmids in Escherichia coli.     

Aspergillus nidulans verA is required for production of the mycotoxin sterigmatocystin.

Aspergillus nidulans produces the carcinogenic mycotoxin sterigmatocystin (ST), the next-to-last precursor in the aflatoxin (AF) biosynthetic pathway found in the closely related fungi Aspergillus flavus and Aspergillus parasiticus. We identified and characterized an A. nidulans gene, verA, that is required for converting the AF precursor versicolorin A to ST. verA is closely related to several polyketide biosynthetic genes involved in polyketide production in Streptomyces spp. and exhibits extended sequence similarity to A. parasiticus ver-1, a gene proposed to encode an enzyme involved in converting versicolorin A to ST. By performing a sequence analysis of the region 3' to verA, we identified two additional open reading frames, designated ORF1 and ORF2. ORF2 is closely related to a number of cytochrome P-450 monooxygenases, while ORF1 shares identity with the gamma subunit of translation elongation factor 1. Given that several steps in the ST-AF pathway may require monooxygenase activity and that AF biosynthetic genes are clustered in A. flavus and A. parasiticus, we suggest that verA may be part of a cluster of genes required for ST biosynthesis. We disrupted the verA coding region by inserting the A. nidulans argB gene into the center of the coding region and transformed an A. nidulans argB2 mutant to arginine prototrophy. Seven transformants that produced DNA patterns indicative of a verA disruption event were grown under ST-inducing conditions, and all of the transformants produced versicolorin A but negligible amounts of ST (200-fold to almost 1,000-fold less than the wild type), confirming the hypothesis that verA encodes an enzyme necessary for converting versicolorin A to ST.     

Isolation and characterization of the positively acting regulatory gene QUTA from Aspergillus nidulans.

The positively acting regulator gene QUTA from Aspergillus nidulans has been identified and located within a cluster of quinic acid utilisation (QUT) genes isolated within a recombinant phage lambda (lambda Q1). The DNA sequence of the QUTA gene reveals a single uninterrupted reading frame coding for a protein of mw 90.416 Kd. The QUTA protein sequence has a protein motif in the form of a putative "DNA finger" that shows strong homology to other such motifs in the GAL4, PPR1, ARGRII, LAC9 and QA1F regulatory gene products of S. cerevisiae, K. lactis and N. crassa. The data presented confirm the view deduced by genetical analysis that the QUTA gene of A. nidulans encodes a protein capable of interacting with QUT specific DNA sequences.     

Two Delta9-stearic acid desaturases are required for Aspergillus nidulans growth and development

Unsaturated fatty acids are important constituents of all cell membranes and are required for normal growth. In the filamentous fungus Aspergillus nidulans, unsaturated fatty acids and their derivatives also influence asexual (conidial) and sexual (ascospore) sporulation processes. To investigate the relationship between fatty acid metabolism and fungal development, we disrupted the A. nidulans sdeA and sdeB genes, both encoding Delta9-stearic acid desaturases responsible for the conversion of palmitic acid (16:0) and stearic acid (18:0) to palmitoleic acid (16:1) and oleic acid (18:1). The effects of sdeA deletion on development were profound, such that growth, conidial and ascospore production were all reduced at 22 and 37 degrees C. Total fatty acid content was increased over 3-fold in the DeltasdeA strain, reflected in up-regulation of the expression of the fasA gene encoding the alpha chain of the fatty acid synthase, compared to wild type. Stearic acid accumulated approximately 3-fold compared to wild type in the DeltasdeA strain, while unsaturated fatty acid production was decreased. In contrast, disruption of sdeB reduced fungal growth and conidiation at 22 degrees C, but did not affect these processes at 37 degrees C compared to wild type. Interestingly, ascospore production was increased at 37 degrees C for DeltasdeB compared to wild type. Total fatty acid content was not increased in this strain, although stearic acid accumulated 2-fold compared to wild type, and unsaturated fatty acid production was decreased. Combining the DeltasdeA and DeltasdeB alleles created a synthetic lethal strain requiring the addition of oleic acid to the medium for a modicum of growth. Taken together, our results suggest a role for sdeA in growth and development at all temperatures, while sdeB is involved in growth and development at lower temperatures.     

The molecular cloning and identification of a gene product specifically required for nuclear movement in Aspergillus nidulans

A temperature-sensitive mutation in the nudC gene (nudC3) of Aspergillus nidulans specifically prevents the microtubule-based movement of nuclei in this organism at the restrictive temperature. The mutation does not affect short term growth, nuclear division, or the movement of other subcellular organelles. Immunofluorescence analysis of cells blocked at the restrictive temperature, using antitubulin antibodies, shows that the inability of nuclei to move under these conditions is not related to an inability of a particular class of microtubule to form. The inability to move nuclei in this mutant is also shown to be independent of both mitosis and the number of nuclei in the cell as a double mutant carrying both nudC3 and a cell cycle-specific mutation blocks with a single immotile nucleus at the restrictive temperature. The molecular cloning of the nudC gene and sequence analysis reveal that it encodes a previously unidentified protein of 22 kd. Affinity-purified antisera reactive to the nudC protein cross reacts to a single protein of 22 kD in Aspergillus protein extracts. This purified sera failed to reveal a subcellular location for the nudC protein at the level of indirect immunofluorescence. The data presented suggest that the 22-kD nudC gene product functions by interacting between microtubules and nuclei and/or is involved in the generation of force used to move nuclei during interphase.     

The extremely conserved pyroA gene of Aspergillus nidulans is required for pyridoxine synthesis and is required indirectly for resistance to photosensitizers.

Numerous disparate studies in plants, filamentous fungi, yeast, Archaea, and bacteria have identified one of the most highly conserved proteins (SNZ family) for which no function was previously defined. Members have been implicated in the stress response of plants and yeast and resistance to singlet oxygen toxicity in the plant pathogen Cercospora. However, it is found in some anaerobic bacteria and is absent in some aerobic bacteria. We have cloned the Aspergillus nidulans homologue (pyroA) of this highly conserved gene and define this gene family as encoding an enzyme specifically required for pyridoxine biosynthesis. This realization has enabled us to define a second pathway for pyridoxine biosynthesis. Some bacteria utilize the pdx pyridoxine biosynthetic pathway defined in Escherichia coli and others utilize the pyroA pathway. However, Eukarya and Archaea exclusively use the pyroA pathway. We also found that pyridoxine is destroyed in the presence of singlet oxygen, helping to explain the connection to singlet oxygen sensitivity defined in Cercospora. These data bring clarity to the previously confusing data on this gene family. However, a new conundrum now exists; why have highly related bacteria evolved with different pathways for pyridoxine biosynthesis?