The role of the GATA transcription factor AreB in regulation of nitrogen and carbon metabolism in Aspergillus nidulans

Abstract

In Aspergillus nidulans, nitrogen and carbon metabolism are under the control of wide-domain regulatory systems, including nitrogen metabolite repression, carbon catabolite repression and the nutrient starvation response. Transcriptomic analysis of the wild type strain grown under different combinations of carbon and nitrogen regimes was performed, to identify differentially regulated genes. Carbon metabolism predominates as the most important regulatory signal but for many genes, both carbon and nitrogen metabolisms coordinate regulation. To identify mechanisms coordinating nitrogen and carbon metabolism, we tested the role of AreB, previously identified as a regulator of genes involved in nitrogen metabolism. Deletion of areB has significant phenotypic effects on the utilization of specific carbon sources, confirming its role in the regulation of carbon metabolism. AreB was shown to regulate the expression of areA, tamA, creA, xprG and cpcA regulatory genes suggesting areB has a range of indirect, regulatory effects. Different isoforms of AreB are produced as a result of differential splicing and use of two promoters which are differentially regulated by carbon and nitrogen conditions. These isoforms are likely to be functionally distinct and thus contributing to the modulation of AreB activity.

Keywords: AreA; AreB; CreA; Nitrogen metabolite repression; RNA-Seq; carbon catabolite repression.

Figure 1.

Nitrogen and carbon regulated genes in A. nidulans wild type strain. A. Effect of ammonium or glucose on the wild type strain transcriptome under carbon or nitrogen repressing/derepressing conditions, respectively. Expression of genes under two different carbon/nitrogen conditions was compared. Number of up or down regulated genes is shown. CR—carbon repression; CD—carbon derepression; NR—nitrogen repression; ND—nitrogen derepression. For detailed lists of differentially expressed genes see Table S3A–D (Supporting Information). B. Expression profile codes for groups of genes regulated by nitrogen and/or carbon source in the wild type strain. Expressions decreased (↓), increased (↑) or not changed (−) in the presence of ammonium or glucose, respectively. GU vs. GNH₄–effect of ammonium under carbon repressing conditions (glucose as a carbon source); FU vs. FNH₄–effect of ammonium under carbon derepressing conditions (fructose as a carbon source); FU vs. GU—effect of glucose under nitrogen derepressing conditions (urea as a nitrogen source); FNH₄ vs. GNH₄–effect of glucose under nitrogen repressing conditions (ammonium as a nitrogen source). Number of genes in each group is shown. For detailed lists of genes in each group see Table S4 (Supporting Information).

Figure 2.

Effect of ammonium and glucose in the wild type strain grown under different carbon or nitrogen conditions, respectively. (A), Number of genes induced or repressed by ammonium under carbon derepressing (yellow), repressing (pink) or independently on carbon conditions (orange). (B), Number of genes induced or repressed by glucose under nitrogen derepressing (yellow), repressing (pink) or independently on nitrogen conditions (orange)

Figure 3.

Deletion of areBΔ influences the growth on several carbon sources. Growth of areBΔ and the wild type strain on glucose, fructose and some other selected carbon sources. Amino acids (in green); tricarboxylic acids from TCA (Krebs) cycle (in blue); compounds utilised by areBΔ as a carbon source better than by the wild type (in red). For detailed lists of carbon sources utilised less effectively by areBΔ see Fig. S3 (Supporting Information).

Figure 4.

Deletion of areB influences the expression of the wide-domain regulatory genes areA, tamA, creA, xprG and cpcA. (A), Quantitative transcriptional analysis of areA, tamA, creA, xprG and cpcA in areB deletion strain. Relative expression in areBΔ mutant in comparison with the control wild type strain was calculated by RT-qPCR analysis. *—P-value < 0.1; **—P-value < 0.05; FC—fold change areBΔ/wt. (B), Number of GATA sites in potential promoter regions of areA, tamA, creA, xprG and cpcA. GATA pair was defined as two sites at a distance of less than 30 bp. Potential promoter region was defined as 1 kb upstream of ATG for tamA, creA and xprG or 1.5 kb for areA and cpcA that comprise much longer 5' UTR. C. Deletion of areB decreases the expression of the CreA, the carbon catabolite repressor. Sensitivity of the wild type, areBΔ and creA1 strains was tested on minimal medium with 10 mM sodium nitrate and 1% glucose (MM) or with other carbon/nitrogen sources, as described in Materials and Methods.

Figure 5.

Transcriptional analysis of the areB gene under different carbon and nitrogen regimes. (A), Structure of areB gene and its three transcripts (based on Conlon et al. 2001). Differential RT-qPCR analysis of areB transcripts was performed using primers specific for each mRNA. Introns are marked in red. Amplified fragments, specific for α, β or γ areB mRNA are marked with blue arrows. Left primer for areBγ mRNA amplification is complementary to exon—exon junction in the spliced transcript. (B), Comparison of expression levels of three areB transcripts in the wild type strain grown under specific carbon/nitrogen repressing and/or derepressing conditions. The level of areBβ mRNA was arbitrarily set to 1. *- P-value < 0.1; **- P-value < 0.05. (C), Comparison of expression levels of each specific areB transcript, in the wild type strain grown under different carbon/nitrogen repressing and/or derepressing conditions. The level of each mRNA under nitrogen and carbon de-repressing conditions (FU) was arbitrarily set to 1. *- P-value < 0.1; **- P-value < 0.05. NS—difference not significant.