Genome organization and evolution of a eukaryotic nicotinate co-inducible pathway

Abstract

In Aspergillus nidulans a regulon including 11 hxn genes (hxnS, T, R, P, Y, Z, X, W, V, M and N) is inducible by a nicotinate metabolic derivative, repressible by ammonium and under stringent control of the nitrogen-state-sensitive GATA factor AreA and the specific transcription factor HxnR. This is the first report in a eukaryote of the genomic organization of a possibly complete pathway of nicotinate utilization. In A. nidulans the regulon is organized in three distinct clusters, this organization is variable in the Ascomycota. In some Pezizomycotina species all 11 genes map in a single cluster; in others they map in two clusters. This variable organization sheds light on cluster evolution. Instances of gene duplication followed by or simultaneous with integration in the cluster, partial or total cluster loss, and horizontal gene transfer of several genes (including an example of whole cluster re-acquisition in Aspergillus of section Flavi) were detected, together with the incorporation in some clusters of genes not found in the A. nidulans co-regulated regulon, which underlie both the plasticity and the reticulate character of metabolic cluster evolution. This study provides a comprehensive phylogeny of six members of the cluster across representatives of all Ascomycota classes.

Keywords: Aspergillus nidulans; ascomycetes; eukaryotic nicotinate utilization; gene cluster co-regulation; gene cluster evolution; horizontal gene transmission.

Figure 1.

Expanded clusters in Eurotiomycetes uncover new hxn genes. Comparison of the organization of known [11] and putative novel hxn genes in three species: A. nidulans, A. terreus and Cyphellophora europaea. Each orthologous gene is symbolized by a thick arrow of a different colour, which also indicates relative orientation. Colour-coded double-headed arrows connect the five new putative C. europaea hxn genes to orthologues in the A. nidulans genome. Dashed lines connect similarly arranged cluster segments in the three species. For A. nidulans, a double vertical line indicates separation of clusters in different chromosomes (super-scaffold BN001306 for chromosome VI, BN001301 for chromosome I). For A. terreus, a single vertical line separates two distinct contigs (Contig AAJN01000215 for the nine-gene cluster, AAJN01000156 for the two-gene cluster). In C. europaea, the 11-gene cluster is contained in contig AOBU01000059.

Figure 2.

HxnR-dependent co-induction by 6-NA and ammonium repression of genes in clusters 2/VI and 3/I. All genes in clusters 2/VI and 3/I (a) and the cognate cluster-flanking genes (b) were tested together with hxnS (in cluster VI/1), which was included as a positive control of expression. The relative mRNA levels were measured by RT-qPCR and data were processed according to the relative standard curve method [24] with the γ-actin transcript (actA/AN6542) as reference. Mycelia were grown on 10 mM acetamide as sole N-source for 8 h at 37°C. They were either kept on the same medium for a further 2 h (non-induced, NI) or induced with 1 mM 6-NA (as the sodium salt, I) or induced as above together with 5 mM of l-(+)di-ammonium-tartrate (induced-repressed, IR), also for 2 h. Strains used were hxnR⁺ (FGSC A26), hxnRΔ (HZS.136) and hxnR^c7 (FGSC A872) (electronic supplementary material, table S1). Standard deviations of three independent experiments are shown. Primers are listed in electronic supplementary material, table S2.

Figure 3.

The GATA factor AreA is essential for expression of all hxn genes with the exception of hxnN. Relative mRNA levels in strains of areA⁺ (FGSC A26), a generally de-repressed areA mutant (xprD1, HZS.216) and an areA-null mutant (areA600, CS3095) were determined (electronic supplementary material, table S1). Non-induced conditions (NI): Strains were grown on MM media with 5 mM l-(+)di-ammonium-tartrate as sole N-source for 8 h, then the mycelia were transferred to MM with 10 mM acetamide for further 2 h. Induced conditions (I): as above but transferred to 10 mM nicotinic acid as sole N-source. Induced-repressed (IR) conditions: transferred to 10 mM nicotinic acid and 5 mM l-(+)di-ammonium-tartrate for further 2 h. N-starvation conditions (St): transferred to nitrogen source-free medium. RT-qPCR data were processed according to the standard curve method [24] with the γ-actin transcript (actA/AN6542) as reference. Standard deviations based on three biological replicates are shown. Primers are listed in electronic supplementary material, table S2.

Figure 4.

AreA and putative HxnR-binding sites are extant in the 11 genes of the hxn regulon. (a) Sequence logo of the DNA-binding motif of the HxnR transcription factor generated by the ‘DNA-binding site predictor for Cys2His2 Zinc Finger Proteins' application (http://zf.princeton.edu/) [33]. (b) Distribution of 5′HGATAR AreA-binding sites (orange boxes) [32] and putative canonical 5′GHGGGG HxnR-binding sites (dark green lozenges) in hxn gene promoters and also in the promoter of the hxB gene. The latter encodes a trans-sulphurylase necessary for the activity of the MOCO cofactor in enzymes of the xanthine oxidoreductase group (including HxnS and HxA). UaY-binding sites on the hxB promoter are marked by blue coloured ovals [34]. Sequences conforming to the consensus 5′GHGGGG sequence are present in all HxnR-regulated genes, except hxnN. Nevertheless, figure 2 shows clearly that hxnN is under the control of HxnR. Thus, the physiological binding sites may have a more relaxed consensus sequence. We propose 5′GNGGDG motif as a non-canonical consensus binding site that can be found in hxnN as well as in other hxn promoters. Light green lozenges indicate the location of the more relaxed consensus 5′GNGGDG motif. Note that the hxnT/hxnS, hxnP/hxnY and hxnX/hxnW gene couples share bi-directional promoters.

Figure 5.

Genomic arrangement of the hxn gene clusters. (a) Selected species from Aspergillaceae including section Nidulantes, section Nigri, section Terrei, section Aspergillus, section Flavi and Penicillium (b) selected species from other Eurotiomycetes compared with H. repandus (Leotiomycetes, Helotiales). Orthologues found in different species are indicated by arrows of the same colour as in figure 1. A single vertical line symbolizes physical separation of genes on different contigs.

Figure 6.

Summary of proposed hxn HGT events between fungal taxa. F: Aspergillus section Flavi; U: Aspergillus section Usti; P: Penicillia; T: early diverging species of Talaromyces; 1: HGT of pfdB gene found between Penicillia and species of Talaromyces and between species of Talaromyces and Aspergillus section Flavi; 2: HGTs of hxnM gene from Xylonomycetes (Symbiotaphrina) to Dothideomycetes; from the common ancestor of Fusaria (Ascomycota, Sordariomycetes) to Basidiomycota (the common ancestor of the Panellus stipticus and Mycena galopus belonging to Agaricomycetes) and from Penicillia (Ascomycota, Eurotiomycetes) to Aspergillus section Usti (Ascomycota, Eurotiomycetes) (see detailed phylogeny in electronic supplementary material, figure S9); 3: HGT of hxnS gene from Aspergillus section Usti to Penicillium section Citrina; red asterisk: transfer of the whole hxn cluster composed of nine hxn genes and including the pfdB gene from Penicillia to species of Talaromyces and from Talaromyces to Aspergillus section Flavi. Lines connecting taxa mark confirmed HGTs.

Figure 7.

HGT from Talaromyces to Aspergillus section Flavi is supported by the phylogenies of four different proteins based on Eurotiomycetes data set (see electronic supplementary material, figures S6, S7, S9 and S10 for the complete phylogenies). Cyan: Talaromyces; blue: Penicillium; red: Aspergillus section Flavi; purple: Aspergillus section Polypaecilum; light brown: Aspergillus section Tannerorum; green: Aspergillus section Terrei; grey: Aspergillus section Cremei; black: Aspergillus section Flavipedes. When hxnM paralogues are extant in the genome, the cluster-related protein is called HxnM1, while the cluster-non-related paralogues are numbered consecutively. Otherwise, when no paralogues are extant in the genome, the cluster-related protein is referred to as HxnM.