Co-option of an extracellular protease for transcriptional control of nutrient degradation in the fungus Aspergillus nidulans

Abstract

Nutrient acquisition is essential for all organisms. Fungi regulate their metabolism according to environmental nutrient availability through elaborate transcription regulatory programs. In filamentous fungi, a highly conserved GATA transcription factor AreA and its co-repressor NmrA govern expression of genes involved in extracellular breakdown, uptake, and metabolism of nitrogen nutrients. Here, we show that the Aspergillus nidulans PnmB protease is a moonlighting protein with extracellular and intracellular functions for nitrogen acquisition and metabolism. PnmB serves not only as a secreted protease to degrade extracellular nutrients, but also as an intracellular protease to control the turnover of the co-repressor NmrA, accelerating AreA transcriptional activation upon nitrogen starvation. PnmB expression is controlled by AreA, which activates a positive feedback regulatory loop. Hence, we uncover a regulatory mechanism in the well-established controls determining the response to nitrogen starvation, revealing functional evolution of a protease gene for transcriptional regulation and extracellular nutrient breakdown.

Fig. 1. pnmB deletion and overexpression affects NmrA levels in vivo.

a Western blot analysis of NmrA^FLAG in wild type (WT) and xylP(p)-pnmB strains grown in liquid ANM media containing glucose and xylose concentrations of 1% glucose, 1%glucose with 0.5% xylose, 1% glucose with 1% xylose, 0.5% glucose with 1% xylose, and 1% xylose. The loading order is arranged in the order of increasing PnmB induction. The histone H3 protein (HhtA) was used as a loading control. b Western blot analysis showing reciprocal co-IP experiments between NmrA^FLAG and PnmB^HA using anti-FLAG or anti-HA antibodies. c Western blot analysis of NmrA^FLAG in wild type (WT) and pnmB∆ strains in a time-course experiment upon nitrogen starvation. Strains were grown in liquid ANM media with ammonium (NH₄) as the sole nitrogen source for 16 h (t = 0), then transferred to ANM nitrogen-free (-N) media for 0, 1, 3, 4, and 6 h. The histone H3 protein (HhtA) was used as a loading control. d A histogram showing normalized PolII ChIP-seq signal (see “Methods”) on the pnmB coding region under nitrogen-sufficient (NH₄), -limiting (Ala), and -starvation (-N) conditions in wild type. Black dots present the results from two independent experiments. e Western blot result of PnmB^HA and HhtA in wild type before (t = 0) and after 0.5, 1, 2, 3, and 4 h of nitrogen starvation. f Plot showing the ratio of mean colony diameters for three replicates after 2 days growth on glucose versus xylose in the presence of 10 mM ammonium tartrate (NH₄) and a toxic nitrogen source analog (10 mM thiourea; TU) for wild type (WT) and xylP(p)pnmB strains. Statistical significance was calculated using unpaired t test. Derepression is observed as inhibited colony diameter due to loss of protection against the toxic analog TU by ammonium^,. g Colony pictures for the growth test showed in f. Scale bar represents 5 mm.

Fig. 2. AreA directly binds to the pnmB promoter and activates pnmB expression, leading to downregulation of NmrA levels and, consequently, acceleration of the nitrogen-starvation response.

a GATA motif distribution across the pnmB promoter. Locations of the previously published AreA recognition motif HGATAR (red), GATA sequences (blue), and real-time PCR primers used in ChIP analysis (blue arrows) are shown. b ChIP followed by real-time PCR analysis against AreA over different GATA sequences at the pnmB promoter using primers marked in a under nitrogen-sufficient (NH₄) and -starvation (-N) conditions. Average fold over background from two independent experiments is presented (black dots for NH₄ condition and gray dots for -N condition). c A histogram showing PolII ChIP-seq signal on pnmB coding region in WT (black dots) and areA∆ (gray dots) grown under nitrogen-sufficient (NH₄), -limiting (Ala), and -starvation (-N) conditions from two independent experiments. d Western blot analysis of NmrA^FLAG in wild-type (WT) and areA∆ strains before and after 0.5, 1, 2, 3, 4, 5, and 6 h of nitrogen starvation. The histone H3 protein (HhtA) was used as a loading control. e PolII ChIP followed by real-time PCR analysis of AreA-dependent genes (mepA and ureA) and housekeeping genes upon nitrogen starvation. PolII binding is expressed as fold over background and a representative of three independent experiments are presented. See Supplementary Fig. 6 for the results of biological repeats.

Fig. 3. PnmB is expressed both as a secreted and an intracellular protein.

a Western blot analysis of PnmB^HA and HhtA from growth media (secreted proteins) and total protein extract (intracellular proteins) of strains expressing PnmB^HA from its native promoter or from the strong xylose-inducible xylP promoter. The xylP(p)prtA^HA overexpression strain was included as a control. For the PnmB^HA strain, mycelia and growth media were harvested after 4 h of nitrogen starvation, while the xylP(p)pnmB^HA and xylP(p)prtA^HA strains were induced with 1% xylose for 4 h. b Milk clearing test of WT, xylP(p)pnmB, and xylP(p)prtA. Strains were point-inoculated to ANM + NH₄ + 20% liquid milk plates with 1% glucose and xylose as carbon source, respectively, and incubated for 2 days. Milk clearing is observed as a halo surrounding the colony. Scale bar represents 5 mm.

Fig. 4. Distribution of PnmB BLASTP hits in the fungal kingdom.

Phylum-level distribution of PnmB BLASTP hits across eight phyla of the fungal kingdom.

Fig. 5. Regulatory model of AreA-NmrA-PnmB loop.

In nitrogen-sufficient conditions, NmrA levels are high, while AreA levels are low. NmrA represses AreA-mediated activation of pnmB and nitrogen metabolism genes. In nitrogen-starvation conditions, AreA expression and activity is increased while NmrA levels are decreased. pnmB expression is activated by AreA. The increased PnmB levels further accelerate the decrease of NmrA levels. AreA, PnmB, and NmrA form a positive regulatory loop to enhance AreA activity and nitrogen response rate. At the same time, PnmB was also secreted outside the cell to facilitate nutrient acquisition.