An F-Box/WD40 repeat-containing protein important for Dictyostelium cell-type proportioning, slug behaviour, and culmination

FbxA is a novel member of a family of proteins that contain an F-box and WD40 repeats and that target specific proteins for degradation via proteasomes. In fruiting bodies formed from cells where the fbxA gene is disrupted (fbxA(-) cells), the spore mass fails to fully ascend the stalk. In addition, fbxA(-) slugs continue to migrate under environmental conditions where the parental strain immediately forms fruiting bodies. Consistent with this latter behaviour, the development of fbxA(-) cells is hypersensitive to ammonia, the signaling molecule that regulates the transition from the slug stage to terminal differentiation. The slug comprises an anterior prestalk region and a posterior prespore region and the fbxA mRNA is highly enriched in the prestalk cells. The prestalk zone of the slug is further subdivided into an anterior pstA region and a posterior pstO region. In fbxA(-) slugs the pstO region is reduced in size and the prespore region is proportionately expanded. Our results indicate that FbxA is part of a regulatory pathway that controls cell fate decisions and spatial patterning via regulated protein degradation.     

Regulation of the acuF Gene, Encoding Phosphoenolpyruvate Carboxykinase in the Filamentous Fungus Aspergillus nidulans

Phosphoenolpyruvate carboxykinase (PEPCK) is a key enzyme required for gluconeogenesis when microorganisms grow on carbon sources metabolized via the tricarboxylic acid (TCA) cycle. Aspergillus nidulans acuF mutants isolated by their inability to use acetate as a carbon source specifically lack PEPCK. The acuF gene has been cloned and shown to encode a protein with high similarity to PEPCK from bacteria, plants, and fungi. The regulation of acuF expression has been studied by Northern blotting and by the construction of lacZ fusion reporters. Induction by acetate is abolished in mutants unable to metabolize acetate via the TCA cycle, and induction by amino acids metabolized via 2-oxoglutarate is lost in mutants unable to form 2-oxoglutarate. Induction by acetate and proline is not additive, consistent with a single mechanism of induction. Malate and succinate result in induction, and it is proposed that PEPCK is controlled by a novel mechanism of induction by a TCA cycle intermediate or derivative, thereby allowing gluconeogenesis to occur during growth on any carbon source metabolized via the TCA cycle. It has been shown that the facB gene, which mediates acetate induction of enzymes specifically required for acetate utilization, is not directly involved in PEPCK induction. This is in contrast to Saccharomyces cerevisiae, where Cat8p and Sip4p, homologs of FacB, regulate PEPCK as well as the expression of other genes necessary for growth on nonfermentable carbon sources in response to the carbon source present. This difference in the control of gluconeogenesis reflects the ability of A. nidulans and other filamentous fungi to use a wide variety of carbon sources in comparison with S. cerevisiae. The acuF gene was also found to be subject to activation by the CCAAT binding protein AnCF, a protein homologous to the S. cerevisiae Hap complex and the mammalian NFY complex.     

Identification and complementation of abnormal hyphal branch mutants ahbA1 and ahbB1 in Aspergillus nidulans

Branching generates new axes of polar growth in filamentous fungi and is critical for development, reproduction, and pathogenicity. To investigate branching we screened an Aspergillus nidulans temperature-sensitive mutant collection for abnormal hyphal branch (ahb) mutants. We identified two mutants, ahbA1, which showed reduced branching relative to wild type at restrictive temperature, and ahbB1, which showed increased branching relative to wild type at restrictive temperature. Both mutants also showed abnormal conidiophore development at restrictive temperature. The ahbA1 hypobranching mutant showed defects in nuclear division and hydroxyurea resistance. Complementation and sequencing showed that ahbA1 is a previously identified allele of the cell cycle regulator nimX. The ahbB1 hyperbranching mutant had an increased number of nuclei, was osmotically remedial and Calcofluor resistant. The ahbB gene is predicted to encode a novel protein that has homologues exclusively in filamentous fungi. The C-terminal domain of the predicted AhbB protein showed homology with the heme-binding domain of a cytochrome P450 protein and sequencing of the ahbB1 mutant allele showed that the lesion lies just before this putative heme-binding domain. The ahbB1 mutant showed increased sensitivity to the ergosterol biosynthesis inhibitor imidazole. Our results suggest a link between nuclear division and branching and a possible role for membrane synthesis in branching.     

Improving extracellular production of food-use enzymes from Aspergillus nidulans

Filamentous fungi, and particularly those of the genus Aspergillus, are major producers of enzymatic activities that have important applications in the food and beverage industries. Prior to the availability of transformation systems improvement of industrial production strains was largely restricted to the strategy of mutagenesis, screening and selection. Aspergillus nidulans is a genetically amenable filamentous fungus the ease of handling and analysis of which has led to its use as a model system for the investigation of eukaryotic gene regulation. Although not used industrially it is able to produce a wide variety of extracellular enzymatic activities. As a consequence of half a century of study a considerable resource of characterised mutants has been generated in conjunction with extensive genetic and molecular information on various gene regulatory systems in this micro-organism. Investigation of xylanase gene regulation in A. nidulans as a model for the production of food-use extracellular enzymes suggests strategies by which production of these enzymes in industrially useful species may be improved.     

cAMP and ras signalling independently control spore germination in the filamentous fungus Aspergillus nidulans

The role of cAMP signalling during germination of asexual spores (conidia) of the filamentous fungus Aspergillus nidulans was investigated. A. nidulans strains defective for adenylate cyclase (CyaA) or for the functionally overlapping cAMP-dependent protein kinase (PkaA) and newly characterized SchA protein kinase, homologous to Saccharomyces cerevisiae Sch9, show altered trehalose mobilization and kinetics of germ tube outgrowth, in addition to other defects in colony formation. cAMP-dependent trehalose breakdown is triggered by the addition of a carbon source independently of further catabolism, suggesting that cAMP signalling controls early events of conidial germination in response to carbon source sensing. Additional results suggest that cAMP has targets other than PkaA and SchA and that PkaA retains activity in the absence of cAMP. Conversely, PkaA regulates cAMP levels in A. nidulans because these are elevated by approximately 250-fold in a strain that lacks PkaA. Furthermore, analysis of mutant strains impaired in both adenylate cyclase and RasA GTPase previously implicated in the control of A. nidulans spore germination suggested that RasA and cAMP signalling proceed independently during germination in A. nidulans.     

Regulatory genes controlling fatty acid catabolism and peroxisomal functions in the filamentous fungus Aspergillus nidulans

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.     

A novel F-box protein involved in sexual development and pathogenesis in Gibberella zeae

Gibberella zeae is an ascomyceteous fungus that causes serious diseases in cereal crops. Severe epidemics require strains that are virulent and that can reproduce sexually. We characterized an insertional mutant (designated ZH436) with a pleiotropic defect in both traits, and identified a novel F-box protein gene encoding FBP1 (F-box protein 1) that is similar to fungal F-box proteins including Saccharomyces cerevisiae Grr1, a well-characterized component of the Skp1-Cullin-F-box protein (SCF(Grr1)) E3 ligase complex required for protein degradation. FBP1 also can bind both S. cerevisiae Skp1 protein, the other component of the SCF(Grr1) complex, and its G. zeae sequence homologue SKP1. Two putative protein interacting domains in FBP1 are essential for in vivo function. FBP1 and ScGRR1 are not so interchangeable between S. cerevisiae and G. zeae, but FBP1 can partially complement several defects of a yeast grr1 deletion mutant. Functional analyses confirmed that FBP1 is required for several phenotypes including both sexual development and virulence in G. zeae; the phenotype of DeltaFBP1 strains is different from those of null mutants for F-box proteins in other filamentous fungi as well as from S. cerevisiae grr1Delta strains. Thus, FBP1 is a versatile F-box protein that presumably participates in the formation of the SCF(FBP1) complex that probably controls the ubiquitin-mediated degradation of proteins involved in sexual reproduction and virulence important for disease development by G. zeae.