Diverse Regulation of the CreA Carbon Catabolite Repressor in Aspergillus nidulans

Abstract

Carbon catabolite repression (CCR) is a process that selects the energetically most favorable carbon source in an environment. CCR represses the use of less favorable carbon sources when a better source is available. Glucose is the preferential carbon source for most microorganisms because it is rapidly metabolized, generating quick energy for growth. In the filamentous fungus Aspergillus nidulans, CCR is mediated by the transcription factor CreA, a C2H2 finger domain DNA-binding protein. The aim of this work was to investigate the regulation of CreA and characterize its functionally distinct protein domains. CreA depends in part on de novo protein synthesis and is regulated in part by ubiquitination. CreC, the scaffold protein in the CreB-CreC deubiquitination (DUB) complex, is essential for CreA function and stability. Deletion of select protein domains in CreA resulted in persistent nuclear localization and target gene repression. A region in CreA conserved between Aspergillus spp. and Trichoderma reesei was identified as essential for growth on various carbon, nitrogen, and lipid sources. In addition, a role of CreA in amino acid transport and nitrogen assimilation was observed. Taken together, these results indicate previously unidentified functions of this important transcription factor. These novel functions serve as a basis for additional research in fungal carbon metabolism with the potential aim to improve fungal industrial applications.

Keywords: Aspergillus nidulans; carbon catabolite repression; cellulases; protein domains; ubiquitination.

Figure 1

Schematic diagram of the CreA protein domains as proposed by Roy et al. (2008). The creA gene has 1251 base pairs and encodes a protein of 416 amino acids. CreA domains and corresponding sizes and gene locations are indicated. Above the diagram are the names used throughout this study of the strains with the different CreA regions deleted.

Figure 2

CreA does not require de novo protein synthesis and is regulated via partial degradation. (A) (Right) Western blot of immunoprecipitated CreA::GFP protein from whole-cell protein extracts of strains TN02a3 (wild-type) and CreA::GFP. Mycelia were grown from spores for 16 hr in glucose (Glu) and then transferred to Xylan for 6 hr before glucose was added for 30 min (X6h+G30m) in the absence or presence of cycloheximide (+CH). (Left) Coomassie-stained SDS-PAGE gel of whole-cell protein extracts before immunoprecipitation. CreA::GFP is indicated by a red arrow. (B) (Right) Western blot of immunoprecipitated CreA::GFP protein from whole-cell protein extracts. Mycelia were grown from spores for 16 hr in glucose (G) and then transferred to Xylan (X) for 6 hr before glucose was added for 30 min (X6h+G30m), 60 min (X6h+G60m), and 120 min (X6h+G120m). (Left) Coomassie-stained SDS-PAGE gel of whole-cell protein extracts before immunoprecipitation. CreA::GFP is indicated by a red arrow. (C) Expression of creA after 20 min in the presence of glucose, cellulose, and Xylan at 24 hr or after the addition of glucose for 1 hr, as determined by qRT-PCR. SD is shown for three technical replicates, and values were normalized by tubulin C (tubC) expression.

Figure 3

The DUB complex scaffold protein CreC is important for CreA function. (Top) Western blot of immunoprecipitated CreA::GFP protein from whole-cell protein extracts of different strains. Mycelia were grown from spores for 16 hr in glucose and then transferred to Xylan for 6 hr before glucose was added (Xylan + Gluc) for 30 and 60 min. (Bottom) Coomassie-stained SDS-PAGE gel of whole-cell protein extracts before immunoprecipitation. CreA::GFP is indicated by a red arrow.

Figure 4

CreA is regulated by ubiquitination. Western blot of immunoprecipitated CreA::GFP protein from whole-cell protein extracts. Mycelia were grown from spores for 16 hr in glucose and then transferred to Xylan for 6 hr before glucose was added for 30 min (X6h+G30m), 60 min (X6h+G60m), and 120 min (X6h+G120m). Membranes were incubated with anti-GFP antibody (right) or anti-ubiquitin antibody (center). (Left) Coomassie-stained SDS-PAGE gel of whole-cell protein extracts before immunoprecipitation. CreA::GFP is indicated by a red arrow and the ubiquitination smears by white braces.

Figure 5

Truncation of CreA results in it being unable to leave the nucleus and in reduced cellulase and hemicellulase gene expression. (A) Growth of CreA-truncated strains on MM containing 1% (w/v) glucose (gluc) or xylose (xyl) supplemented with different concentrations of 2DG and AA. (B) Expression of xlnA, eglA, and xlnR in the wild-type and CreA-truncated strains as determined by qRT-PCR. Strains were grown for 24 hr in fructose and then transferred to sugarcane bagasse for 6 hr before glucose was added to a final concentration of 2% (w/v) for 1 hr. Gene expression was normalized by tubulin C (tubC) expression. SD was calculated for three technical replicates (**P < 0.005, ***P < 0.001 in an equal-variance paired Student’s t-test).

Figure 6

The CreA-conserved region is important for mediating growth in the presence of different carbon, nitrogen, and lipid sources. (A) Deletion of the CreA-conserved region results in the spores being unable to germinate. Photographs were taken by microscopy in the absence (DIC, differential interference contrast) and presence (DAPI) of fluorescence of the wild-type CreA::GFP and CreAΔConsv::GFP strains when grown overnight in MM supplemented with 50 mM leucine (left) or 50 mM valine (middle) or 1% (w/v) cellulose (right). Nuclei were stained with Hoechst and viewed under the DAPI filter. (B) Strains were grown on agar plates containing 1% (w/v) various carbon sources, 1% (w/v) casamino acids, 1% (v/v) ethanol, and 50 mM of individual amino acids or (C) on plates containing 1% (v/v) tributyrin and 1% (w/v) milk powder supplemented with 0.05% (v/v) Triton X-100. (D) Halo-colony ratio of the wild-type and CreA-truncated strains when grown on plates containing 1% (w/v) milk powder supplemented with 0.05% (v/v) Triton X-100. The SD was measured between biological triplicates (***P < 0.001 in a one-tailed equal-variance Student’s t-test).

Figure 7

Binding of the wild-type and CreA-truncated strains to the xlnA promoter region. (A) Schematic diagram of the CreA-binding sites in the xlnA (encoding xylanase A) promoter region. Red arrows indicate the primer pair used in the ChIP–qRT-PCR. (B) Quantity of xlnA detected by ChIP–qRT-PCR on CreA binding site 3 in the wild-type and CreA-truncated strains when grown for 24 hr in fructose and then transferred to either glucose or sugarcane bagasse for 6 hr. All xlnA expression values were normalized by the quantity of tubC (β-tubulin) in each sample. SDs are shown for technical duplicates.

Figure 8

CreA is involved in amino acid transport and metabolism. (A) Growth of CreA::GFP and CreAΔConsv on solid medium supplemented with different carbon and nitrogen sources without and with transfer from liquid cultures (4 hr at 37°). (B) Amino acid uptake of the wild-type and CreAΔConsv strains during a 2-hr incubation in medium supplemented either with 50 mM leucine or valine. Concentrations of the amino acids were measured in the supernatants of biological triplicates and normalized by fungal dry weight (**P < 0.01, ***P < 0.001 in a paired equal-variance Student’s t-test). (C) Halo-colony ratio of the growth of wild-type (CreA::GFP) and CreA-truncated strains on plates containing 1% glucose and 1% milk (control) or in the presence of 50 mM NaNO₃ or 1% casamino acids (CA). SD was calculated for biological triplicates (*P < 0.01, **P < 0.001, ***P < 0.0001 in a paired equal-variance Student’s t-test).