Meryl A. Davis

Genetic Analysis of the Role of Peroxisomes in the Utilization of Acetate and Fatty Acids in Aspergillus Nidulans

Peroxisomes are organelles containing a diverse array of enzymes. In fungi they are important for carbon source utilization, pathogenesis, development, and secondary metabolism. We have studied Aspergillus nidulans peroxin (pex) mutants isolated by virtue of their inability to grow on butyrate or by the inactivation of specific pex genes. While all pex mutants are able to form colonies, those unable to import PTS1 proteins are partially defective in asexual and sexual development. The pex mutants are able to grow on acetate but are affected in growth on fatty acids, indicating a requirement for the peroxisomal localization of β-oxidation enzymes. However, mislocalization of malate synthase does not prevent growth on either fatty acids or acetate, showing that the glyoxylate cycle does not require peroxisomal localization. Proliferation of peroxisomes is dependent on fatty acids, but not on acetate, and on PexK (Pex11), expression of which is activated by the FarA transcription factor. Proliferation was greatly reduced in a farAΔ strain. A mutation affecting a mitochodrial ketoacyl-CoA thiolase and disruption of a mitochondrial hydroxy-acyl-CoA dehydrogenase gene prevented growth on short-chain but not long-chain fatty acids. Together with previous results, this is consistent with growth on even-numbered short-chain fatty acids requiring a mitochondrial as well as a peroxisomal β-oxidation pathway. The mitochondrial pathway is not required for growth on valerate or for long-chain fatty acid utilization.

Recent Advances in Nitrogen Regulation: a Comparison between Saccharomyces cerevisiae and Filamentous Fungi†

Myriad biological processes are dynamically regulated in response to environmental cues. Transcriptional gene regulation is of fundamental importance, and regulatory mechanisms ensure that genes are expressed at levels appropriate to the cellular state. Microorganisms undergo major shifts in their

Genetic manipulation of Aspergillus nidulans: meiotic progeny for genetic analysis and strain construction.

The multicellular microbial eukaryote Aspergillus nidulans is an excellent model for the study of a wide array of biological processes. Studies in this system contribute significantly to understanding fundamental biological principles and are relevant for biotechnology and industrial applications, as well as human, animal and plant fungal pathogenesis. A. nidulans is easily manipulated using classical and molecular genetics. Here, we describe the storage and handling of A. nidulans and procedures for genetic crossing, progeny analysis and growth testing. These procedures are used for Mendelian analysis of segregation of alleles to show whether a mutant phenotype segregates as a single gene and independent assortment of genes to determine the linkage relationship between genes. Meiotic crossing is used for construction of multiple mutant strains for genetic analysis. Genetic crossing and analysis of progeny can be undertaken in 2–3 weeks and growth testing takes 2–3 days.

AnCF, the CCAAT binding complex of Aspergillus nidulans, contains products of the hapB, hapC, and hapE genes and is required for activation by the pathway-specific regulatory gene amdR.

ABSTRACT CCAAT binding factors (CBFs) positively regulating the expression of the amdS gene (encoding acetamidase) and two penicillin biosynthesis genes (ipnA and aatA) have been previously found in Aspergillus nidulans. The factors were called AnCF and PENR1, respectively. Deletion of the hapCgene, encoding a protein with significant similarity to Hap3p ofSaccharomyces cerevisiae, eliminated both AnCF and PENR1 binding activities. We now report the isolation of the geneshapB and hapE, which encode proteins with central regions of high similarity to Hap2p and Hap5p of S. cerevisiae and to the CBF-B and CBF-C proteins of mammals. An additional fungus-specific domain present in HapE was revealed by comparisons with the homologs from S. cerevisiae,Neurospora crassa, and Schizosaccharomyces pombe. The HapB, HapC, and HapE proteins have been shown to be necessary and sufficient for the formation of a CCAAT binding complex in vitro. Strains with deletions of each of the hapB,hapC, and hapE genes have identical phenotypes of slow growth, poor conidiation, and reduced expression ofamdS. Furthermore, induction of amdS by omega amino acids, which is mediated by the AmdR pathway-specific activator, is abolished in the hap deletion mutants, as is growth on γ-aminobutyric acid as a sole nitrogen or carbon source. AmdR and AnCF bind to overlapping sites in the promoters of the amdSand gatA genes. It is known that AnCF can bind independently of AmdR. We suggest that AnCF binding is required for AmdR binding in vivo.

Nuclear Accumulation of the GATA Factor AreA in Response to Complete Nitrogen Starvation by Regulation of Nuclear Export

ABSTRACT Both the availability and the quality of nutrients affect cellular functions by controlling gene activity. AreA, a member of the GATA family of transcription factors, globally activates expression of genes involved in nitrogen source utilization in Aspergillus nidulans. The quality of the nitrogen source determines the level and activation capacity of AreA through controls at the level of areA mRNA stability and by interaction of AreA with the corepressor NmrA. The availability of potential nitrogen sources also affects the activation capacity of AreA. We show that the complete absence of a nitrogen source results in an enhanced level of AreA-dependent gene expression and that this response is independent of mechanisms regulating AreA activity in response to nitrogen source quality. During nitrogen starvation AreA accumulates in the nucleus, but the presence of a potential nitrogen source or carbon starvation prevents this accumulation. Furthermore, accumulated AreA is rapidly lost from the nuclei of nitrogen-starved cells when a nitrogen source is supplied or when a carbon source is absent, and this accompanies arrest of the AreA-dependent nitrogen starvation response on regulated gene expression. By the generation of a leptomycin B-sensitive mutant, we have been able to show that nuclear exit occurs via the CrmA exportin. We conclude that sensing mechanisms discriminate between starvation and the presence of potential nutrients that can signal to the AreA transcription factor. Nitrogen source availability, but not quality, affects nuclear accumulation by regulating nuclear exit of AreA, providing a rapid response to changes in the supply of nutrients.

Regulatory Genes Controlling Fatty Acid Catabolism and Peroxisomal Functions in the Filamentous Fungus Aspergillus nidulans

ABSTRACT The catabolism of fatty acids is important in the lifestyle of many fungi, including plant and animal pathogens. This has been investigated in Aspergillus nidulans, which can grow on acetate and fatty acids as sources of carbon, resulting in the production of acetyl coenzyme A (CoA). Acetyl-CoA is metabolized via the glyoxalate bypass, located in peroxisomes, enabling gluconeogenesis. Acetate induction of enzymes specific for acetate utilization as well as glyoxalate bypass enzymes is via the Zn2-Cys6 binuclear cluster activator FacB. However, enzymes of the glyoxalate bypass as well as fatty acid beta-oxidation and peroxisomal proteins are also inducible by fatty acids. We have isolated mutants that cannot grow on fatty acids. Two of the corresponding genes, farA and farB, encode two highly conserved families of related Zn2-Cys6 binuclear proteins present in filamentous ascomycetes, including plant pathogens. A single ortholog is found in the yeasts Candida albicans, Debaryomyces hansenii, and Yarrowia lipolytica, but not in the Ashbya, Kluyveromyces, Saccharomyces lineage. Northern blot analysis has shown that deletion of the farA gene eliminates induction of a number of genes by both short- and long-chain fatty acids, while deletion of the farB gene eliminates short-chain induction. An identical core 6-bp in vitro binding site for each protein has been identified in genes encoding glyoxalate bypass, beta-oxidation, and peroxisomal functions. This sequence is overrepresented in the 5′ region of genes predicted to be fatty acid induced in other filamentous ascomycetes, C. albicans, D. hansenii, and Y. lipolytica, but not in the corresponding genes in Saccharomyces cerevisiae.

The hapC gene of Aspergillus nidulans is involved in the expression of CCAAT-containing promoters

The 5′ regulatory region of theamdS gene ofAspergillus nidulans, which encodes an acetamidase required for growth on acetamide as a carbon and nitrogen source, contains a CCAAT sequence which is required for setting the basal level ofamdS expression. Mobility shift studies have identified a factor inA. nidulans nuclear extracts which binds to this CCAAT sequence. InSaccharomyces cerevisiae theHAP3 gene encodes one component of a multisubunit complex that binds CCAAT sequences. A search of the EMBL and SwissProt databases has revealed anA. nidulans sequence with significant homology to theHAP3 gene adjacent to the previously cloned regulatory geneamdR. Sequencing of the remainder of this region has confirmed the presence of a gene, designatedhapC, with extensive homology toHAP3. The predicted amino acid sequence of HapC shows extensive identity to HAP3 in the central conserved domain, but shows little conservation in the flanking sequences. A haploid carrying ahapC deletion has been created and is viable, but grows poorly on all media tested. This null mutant grows especially slowly on acetamide as a sole carbon and nitrogen source, indicating thathapC plays a role inamdS expression. In agreement with this notion, it has been shown that thehapC deletion results in reduced levels of expression of anamdS::lacZ reporter gene and this effect is particularly evident under conditions of carbon limitation. Nuclear extracts prepared from thehapC deletion mutant show no CCAAT binding activity to theamdS orgatA promoters, indicating thathapC may encode a component of the complex binding at this sequence.

The acetate regulatory gene facB of Aspergillus nidulans encodes a Zn(II)2Cys6 transcriptional activator.

Abstract Genetic studies have indicated that the facB gene of Aspergillus nidulans is a major regulatory gene involved in acetamide and acetate utilisation. Sequencing of the facB gene revealed that it encodes a protein that contains an N-terminal GAL4-like Zn(II)2Cys6 (or C6 zinc) binuclear cluster for DNA binding, leucine zipper-like heptad repeat motifs and central and C-terminal acidic α-helical regions, consistent with a function as a DNA-binding transcriptional activator. The Zn(II)2Cys6 cluster shows strong similarity with those of the Saccharomyces cerevisiae carbon metabolism regulatory proteins CAT8 and SIP4. A significant level of similarity with CAT8 is found throughout the length of the protein, suggesting at least partial functional homology. The facB genes of Aspergillus oryzae and Aspergillus niger were also sequenced and found to be highly conserved. Deletion of the facB gene confirmed that it is required for growth on acetate as a sole carbon source. Functional dissection using deletion and fusion constructs and in vitro mutagenesis indicated that the Zn(II)2Cys6 cluster and the C-terminal end of the protein are required for function.

FacB, the Aspergillus nidulans activator of acetate utilization genes, binds dissimilar DNA sequences

The facB gene is required for acetate induction of acetamidase (amdS) and the acetate utilization enzymes acetyl‐CoA synthase (facA), isocitrate lyase (acuD) and malate synthase (acuE) in Aspergillus nidulans. The facB gene encodes a transcriptional activator with a GAL4‐type Zn(II)2Cys6 zinc binuclear cluster DNA‐binding domain which is shown to be required for DNA binding. In vitro DNA‐binding sites for FacB in the 5′ regions of the amdS, facA, acuD and acuE genes have been identified. Mutations in amdS FacB DNA‐binding sites affected expression of an amdS–lacZ reporter in vivo and altered the affinity of in vitro DNA binding. This study shows that the FacB Zn(II)2Cys6 cluster binds to dissimilar sites which show similarity in form but not sequence with DNA‐binding sites of other Zn(II)2Cys6 proteins. Sequences with homology to FacB sites are found in the 5′ regions of genes regulated by the closely related yeast Zn(II)2Cys6 protein CAT8.

An amdS-lacZ fusion for studying gene regulation in Aspergillus

A translational fusion has been constructed between the amdS gene of Aspergillus nidulans and the lacZ gene of Escherichia coli. Sequencing across the fusion junction confirmed the generation of an in-frame fusion at amino acid 34 of amdS and a novel protein has been detected in transformants carrying the fusion plasmid. Transformants of A. nidulans and Aspergillus niger carrying the fusion plasmid were obtained by co-transformation with a second selectable plasmid. These transformants were readily identified on media containing XGal. The intensity of the reaction on XGal media was indicative of the number of copies of the fusion plasmid carried by the transformants. The growth of highly expressing strains of A. nidulans was inhibited on XGal media. The fusion plasmid was used to develop a two-step gene replacement strategy in which the resident amdS gene was replaced with the fusion gene free of vector sequences. Plate tests and in vitro assays of the beta-galactosidase enzyme confirmed that expression of the fusion gene was regulated by amdS flanking sequences and trans-acting regulatory genes.