Functional characterisation of the non-essential protein kinases and phosphatases regulating Aspergillus nidulans hydrolytic enzyme production

Abstract

Background: Despite recent advances in the understanding of lignocellulolytic enzyme regulation, less is known about how different carbon sources are sensed and the signaling cascades that result in the adaptation of cellular metabolism and hydrolase secretion. Therefore, the role played by non-essential protein kinases (NPK) and phosphatases (NPP) in the sensing of carbon and/or energetic status was investigated in the model filamentous fungus Aspergillus nidulans.

Results: Eleven NPKs and seven NPPs were identified as being involved in cellulase, and in some cases also hemicellulase, production in A. nidulans. The regulation of CreA-mediated carbon catabolite repression (CCR) in the parental strain was determined by fluorescence microscopy, utilising a CreA::GFP fusion protein. The sensing of phosphorylated glucose, via the RAS signalling pathway induced CreA repression, while carbon starvation resulted in derepression. Growth on cellulose represented carbon starvation and derepressing conditions. The involvement of the identified NPKs in the regulation of cellulose-induced responses and CreA derepression was assessed by genome-wide transcriptomics (GEO accession 47810). CreA::GFP localisation and the restoration of endocellulase activity via the introduction of the ∆creA mutation, was assessed in the NPK-deficient backgrounds. The absence of either the schA or snfA kinase dramatically reduced cellulose-induced transcriptional responses, including the expression of hydrolytic enzymes and transporters. The mechanism by which these two NPKs controlled gene transcription was identified, as the NPK-deficient mutants were not able to unlock CreA-mediated carbon catabolite repression under derepressing conditions, such as carbon starvation or growth on cellulose.

Conclusions: Collectively, this study identified multiple kinases and phosphatases involved in the sensing of carbon and/or energetic status, while demonstrating the overlapping, synergistic roles of schA and snfA in the regulation of CreA derepression and hydrolytic enzyme production in A. nidulans. The importance of a carbon starvation-induced signal for CreA derepression, permitting transcriptional activator binding, appeared paramount for hydrolase secretion.

Figure 1

The AVICEL-specific reduction in growth of eleven NPKs. The dry weight of the NPKs when grown in complete media (A) and the total protein content of the NPKs when grown directly on minimal media plus AVICEL as a sole carbon source (B). One-tailed t-test (* P <0.05, ** P < 0.01, *** P < 0.001).

Figure 2

The identified NPKs mutants demonstrated a reduction in the transcription, and capacity of, hydrolytic enzymes. The parental (WT), NPKs mutant strains were grown in MM plus 1% fructose overnight and then transferred to AVICEL or xylan as a sole carbon source for an additional 5 or 3 days, respectively. The endocellulase activity of the 5 day AVICEL culture (A). The relative expression of the major endoglucanase genes, eglA and eglB, in the mycelia isolated from the cultures used to determine endocellulase activity (B). The endoxylanase activity of the xylan cultures (C).

Figure 3

The seven NPPs identified as being required for growth on AVICEL. The dry weight of the NPPs when grown in MM plus 2% glucose (A) and the total protein content of the NPPs when grown directly on MM plus AVICEL as a sole carbon source (B). The parental (WT) and NPPs mutant strains were grown in MM plus 1% fructose overnight and then transferred to AVICEL as a sole carbon source for an additional 5 days. The endocellulase activity of the AVICEL culture (C) and the relative expression of two endoglucanase genes, eglA and eglB, in the mycelia isolated from the same cultures (D).

Figure 4

Protein kinase A was hyperactivated upon growth on AVICEL or carbon starvation. The parental (WT), ∆pkaC and ∆cyaA strain were grown in minimal media plus 1% fructose overnight (o/n) and then transferred to various alternative carbon sources for 8 h. The PKA activity from the respective cultures, and the internal cAMP (positive) and water (negative) controls, were determine via densitometry. One-tailed t-test (* P <0.05, ** P < 0.01, *** P < 0.001).

Figure 5

CreA::GFP localisation alters under repressing and derepressing condition. Under repressing conditions (glucose 16 h), CreA::GFP constantly localised to the nucleus, while under derepressing conditions (AVICEL 16 h), CreA::GFP was absent from the nucleus. The brightfield (DIC), GFP and the DAPI (for Hoechst nuclear staining) channels are presented. Bar = 5 μm.

Figure 6

The identification of the genes modulated by schA and snfA upon growth on AVICEL. The venn diagrams for the differentially up (A,B) or down (C,D) regulated genes (p < 0.01) in the parental (WT), ∆schA and ∆snfA strains post 8 (A,C) and 24 h (B,D) growth on AVICEL as a sole carbon source.

Figure 7

The contribution of schA and snfA to AVICEL-induced gene induction. The distribution of different functional categories and CAZy enzymes, including glycoside hydrolases (GH), among the genes that demonstrated elevated transcription, in the parental strain, post 24 h growth on AVICEL, and whether this induction was dependent on the action of schA and/or snfA. Note; the near absence of genes independent of schA or snfA (blue), the substantial contribution of snfA represented by the sum of snfA dependent (purple) and snfA plus schA dependent (red) genes, while the majority of schA dependent genes overlapped with snfA and only a limited number of genes were specifically schA dependent responses (green).

Figure 8

Cellulase and hemicellulase transcription is reduced in absence of schA or snfA. The log2 fold change in the expression of a selection of cellulases (A) and hemicellulases (B) in the parental (WT), ∆schA and ∆snfA strains post transfer from complete media to minimal media plus AVICEL as a sole carbon source for 24 h. Gene IDs and glycoside hydrolase families are presented.

Figure 9

A schematic for the mechanisms by which NPKs regulate CreA repression and hydrolytic enzyme production. Upon growth on cellulose as a sole carbon source, a drop in phosphorylated glucose and RAS-mediated PKA activation, results in the loss of the positive signal for CreA nuclear localisation and repression. Subsequently, the drop in intracellular ATP and an alteration in TOR signalling, results in SchA and SnfA controlling CreA re-localisation and hydrolytic enzyme production. Bold arrows represent experimentally proven, while dotted arrow represent hypothetical, connections.