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.

FIG. 1.

Growth of regulatory gene mutants on fatty acids as sole carbon sources. The following carbon sources were added to minimal medium with 10 mM ammonium chloride as the nitrogen source: glucose (1%); acetate (50 mM); butyrate and valerate (10 mM); hexanoate (5 mM), propionate, Tween 20, and Tween 80 (0.1%); lauric, myristic, palmitic, heptadecanoic, stearic, oleate, elaidic, and erucic acids (2.5 mM). Growth was for 2 to 3 days at 37°C.

FIG. 2.

Structures of farA and farB genes and comparisons with orthologs in other fungi. (A) The farA gene, showing intron positions and the sequences coding for the indicated domains. farAΔ was generated by replacing sequences from +225 to +2670 (relative to the start codon) with the A. nidulans pyrG gene followed by gene replacement. (B) Structure of the farB gene, showing intron positions and the sequences coding for the indicated domains. farBΔ was generated by replacing sequences from −227 to +2368 (relative to the start codon) with the A. nidulans pyrG gene followed by gene replacement. (C) Comparison of the Zn₂-Cys₆ binuclear cluster domains of the FarA and FarB proteins in filamentous fungi and the orthologs in hemiascomycetes compared to A. nidulans FarA (Yeast group). Identical residues present in at least 60% of the sequences are indicated by black boxes, whereas gray shading represents similar residues. Overall comparisons of the proteins, database accession numbers, and intron positions in the genes are presented in the Fig. S1 to S3 in the supplemental material. (D) Unrooted neighbor joining distance tree showing the relationships of the FarA, FarB, and yeast proteins to each other. Sequences were aligned using ClustalW (70) and distance matrices were calculated (19), which were used to create the tree in NJPlot (52). Species abbreviations: A.n, Aspergillus nidulans; A.f; Aspergillus fumigatus; S.n, Stagonospora nodorum; F.g, Fusarium graminearium; N.h, Nectria hematococca; M.g, Magnaporthe grisea; N.c, Neurospora crassa; C.g, Chaetomium globosum; B.c, Botrytis cineria; C.a, Candida albicans; D.h, Debaryomyces hansenii; Y.l, Yarrowia lipolytica.

FIG. 3.

Effects of regulatory gene mutations on expression of an acuJ-lacZ reporter. (A) A fragment of the acuJ 5′ region was inserted upstream of a minimal promoter (gpd mini) driving lacZ expression. The black bars represent CCGAGG sequences present in this sequence. (B to H) Mycelia of strains of the indicated genotypes were grown for 16 to 18 h in 1% glucose-10 mM ammonium tartrate medium and then transferred to minimal medium with the indicated carbon sources together with 10 mM ammonium chloride for 6 h or for the indicated times (D and E) before harvesting. Mycelia were extracted and assayed for β-galactosidase as previously described (13). The specific activity is expressed in Miller units per minute per milligram of protein. The bars represent standard errors. The concentrations of carbon sources were 10 mM for proline, acetate, butyrate, valerate, and oleate, 5 mM for hexanoate, 0.1% for propionate and Tween 20, and 2.5 mM for myristic, palmitic, heptadecanoic, elaidic, stearic, and erucic acids.

FIG. 4.

Analysis of the effects of regulatory gene mutations on expression of various genes. RNAs were extracted from strains of the indicated genotypes grown for 16 h in 1% glucose-10 mM ammonium tartrate medium and then transferred to medium with the indicated carbon sources for 6 h. Abbreviations: pro, 10 mM l-proline; ace, 10 mM acetate; but, 10 mM n-butyrate; ole, 10 mM oleate; Tw20, 0.1% Tween 20. The results represent three different Northern blot membranes probed multiple times with sequences corresponding to the indicated genes with their proposed functions. Probes were made by labeling either gel-purified restriction fragments or PCR products generated using primers based on genome sequences. H3 represents an EcoRI fragment of the histone H3 clone (17), and rRNA refers to the large rRNA species observed by ethidium bromide staining.

FIG. 5.

EMSA analysis of DNA binding by expressed MBP fusion proteins. (A) Summary of the key DNA binding studies of cutinase transcription factors in N. hematococca (43, 44). Binding was detected to the probe Pal2 but not Pal2M1 or Pal2M2. Methylation protection studies showed the protection of the G residues, indicated by asterisks. (B) Probes used in EMSAs in this study. Complementary pairs of oligonucleotides were designed based on sequences from the 5′ regions of the genes acuJ, echA, acuE, and pexK, with Spe1/Xba1- or BamH1/BglΙΙ-compatible ends added (in lowercase letters). The database designation of these genes is given in Table S1 in the supplemental material. The conserved core CCGAGG sequence is indicated in bold, and only one strand is shown. Probe J3-4 is identical to J1-2 except for the modification of the core CCGAGG to CCcgGG. (C and D) EMSA of extracts (20 μg of protein added to binding reaction mixtures) to the indicated probes together with no-extract controls. Free probe is designated as F. Binding is represented by the decreased mobility of the probe in the presence of protein, compared to the free probe. (E) Specific competition of binding of FarB and FarA to probe J7-8. Binding was performed with MBP-FarA and MBP-FarB extracts (20 μg of protein) in the presence of increasing amounts of unlabeled competitor DNA. Competitors were the unlabeled probes J1-2 and J3-4, increasing from 10 to 100 to 500 times the concentration of labeled probe.

FIG. 6.

Positions of FacB binding sites and CCTCGG motifs in the 5′ regions of genes involved in acetate utilization in A. nidulans. FacB binding sites are based on those previously proposed (67, 74) or by sequence inspection in the case of acuJ. Vertical lines represent 100-bp intervals upstream of the start codon for each gene.

FIG. 7.

Structure of the scfA gene. (A) The scfA gene, showing intron positions and the sequences coding for the indicated domains. scfAΔ was generated by replacing sequences from −295 to +1470 (relative to the start codon) with the A. nidulans pyrG gene followed by gene replacement. (B) Comparison of the Zn₂-Cys₆ binuclear cluster domains of scfA with proposed orthologs in N. crassa and A. fumigatus. Identical residues present in at least 60% of the sequences are indicated by black boxes, whereas gray shading represents similar residues. Overall comparisons of the proteins and database accession numbers are presented in Fig. S5 in the supplemental material.