The wide-domain carbon catabolite repressor CreA indirectly controls expression of the Aspergillus nidulans xlnB gene, encoding the acidic endo-beta-(1,4)-xylanase X(24)

The Aspergillus nidulans xlnB gene, which encodes the acidic endo-beta-(1,4)-xylanase X(24), is expressed when xylose is present as the sole carbon source and repressed in the presence of glucose. That the mutation creA(d)30 results in considerably elevated levels of xlnB mRNA indicates a role for the wide-domain repressor CreA in the repression of xlnB promoter (xlnBp) activity. Functional analyses of xlnBp::goxC reporter constructs show that none of the four CreA consensus target sites identified in xlnBp are functional in vivo. The CreA repressor is thus likely to exert carbon catabolite repression via an indirect mechanism rather than to influence xlnB expression by acting directly on xlnB.     

CreA-mediated repression in Aspergillus nidulans does not require transcriptional auto-regulation, regulated intracellular localisation or degradation of CreA.

The major regulatory protein in carbon repression in Aspergillus nidulans is CreA. Strains constitutively over-expressing creA show normal responses to carbon repression, indicating that auto-regulation of creA is not essential for CreA-mediated regulation. In these strains, high levels of CreA are present whether cells are grown in repressing or derepressing conditions, indicating large-scale degradation of CreA does not play a key role. CreA is located in the nucleus and cytoplasm in cells when grown in either repressing or derepressing conditions, and absence of CreB, CreD or AcrB does not affect either the localisation or amount of CreA. Therefore, CreA must require some modification or interaction to act as a repressor. Deletion analysis indicates that a region of CreA thought to be important for repression in Trichoderma reesei and Sclerotina sclerotiorum CreA homologues is not critical for function in Aspergillus nidulans.     

CreA-mediated repression in Aspergillus nidulans does not require transcriptional auto-regulation, regulated intracellular localisation or degradation of CreA

The major regulatory protein in carbon repression in Aspergillus nidulans is CreA. Strains constitutively over-expressing creA show normal responses to carbon repression, indicating that auto-regulation of creA is not essential for CreA-mediated regulation. In these strains, high levels of CreA are present whether cells are grown in repressing or derepressing conditions, indicating large-scale degradation of CreA does not play a key role. CreA is located in the nucleus and cytoplasm in cells when grown in either repressing or derepressing conditions, and absence of CreB, CreD or AcrB does not affect either the localisation or amount of CreA. Therefore, CreA must require some modification or interaction to act as a repressor. Deletion analysis indicates that a region of CreA thought to be important for repression in Trichoderma reesei and Sclerotina sclerotiorum CreA homologues is not critical for function in Aspergillus nidulans.     

Xylanase production in Aspergillus nidulans: induction and carbon catabolite repression

Abstract The induction of the synthesis of extracellular xylanases was investigated in the fungus Aspergillus nidulans using a number of compounds, including xylans of different origin, monosaccharides, xylooligosaccharides and xylose derivatives. Certain xylans (wheat arabinoxylan, oat spelt xylan, birchwood xylan and 4-O-methyl-D-glucurono-D-xylan) were found to be the most powerful inducers. Also, xylooligosaccharides such as xylobiose, xylotriose and xylotetraose served as inducers, their efficiency being directly related to their chain length. Xylose, on the contrary, was not a true inducer. Of the three endo-β-(1,4)-xylanases secreted by A. nidulans , that of 24 kDa was not under carbon catabolite repression, whereas the other two, of 22 and 34 kDa, were under glucose repression mediated by the creA gene product.     

Analysis of the creA gene, a regulator of carbon catabolite repression in Aspergillus nidulans.

The complete nucleotide sequence derived from a genomic clone and two cDNA clones of the creA gene of Aspergillus nidulans is presented. The gene contains no introns. The derived polypeptide of 415 amino acids contains two zinc fingers of the C2H2 class, frequent S(T)PXX motifs, and an alanine-rich region indicative of a DNA-binding repressor protein. The amino acid sequence of the zinc finger region has 84% similarity to the zinc finger region of Mig1, a protein involved in carbon catabolite repression in yeast cells, and it is related both to the mammalian Egr1 and Egr2 proteins and to the Wilms' tumor protein. A deletion removing the creA gene was obtained, by using in vitro techniques, in both a heterokaryon and a diploid strain but was unobtainable in a pure haploid condition. Evidence is presented suggesting that the phenotype of such a deletion, when not complemented by another creA allele, is leaky lethality allowing limited germination of the spore but not colony formation. This phenotype is far more extreme than that of any of the in vivo-generated mutations, and thus either the gene product may have an activator activity as well as a repressor function or some residual repressor function may be required for full viability.     

Carbon catabolite repression of the Aspergillus nidulans xlnA gene

Expression of the Aspergillus nidulans 22 kDa endoxylanase gene, xlnA , is controlled by at least three mechanisms: specific induction by xylan or xylose; carbon catabolite repression (CCR); and regulation by ambient pH. Deletion analysis of xlnA upstream sequences has identified two positively acting regions: one that mediates specific induction by xylose; and another that mediates the influence of ambient pH and contains two PacC consensus binding sites. The extreme derepressed mutation creA^d 30 results in considerable, although not total, loss of xlnA glucose repressibility, indicating a major role for CreA in its CCR. Three consensus CreA binding sites are present upstream of the structural gene. Point mutational analysis using reporter constructs has identified a single site, xlnA .C1, that is responsible for direct CreA repression in vivo . Using the creA^d 30 derepressed mutant background, our results indicate the existence of indirect repression by CreA.     

A role for creD, a carbon catabolite repression gene from Aspergillus nidulans, in ubiquitination

In Aspergillus nidulans, it is known that creB encodes a deubiquitinating enzyme that forms a complex with the WD40 motif containing protein encoded by creC, that mutations in these genes lead to altered carbon source utilization and that the creD34 mutation suppresses the phenotypic effects of mutations in creC and creB. Therefore, creD was characterized in order to dissect the regulatory network that involves the CreB-CreC deubiquitination complex. CreD contains arrestin domains and PY motifs and is highly similar to the Rod1p and Rog3p proteins from Saccharomyces cerevisiae. An additional gene was identified in the A. nidulans genome that also encodes an arrestin and PY motif-containing protein, which we have designated apyA, and thus two similar proteins also exist in A. nidulans. In S. cerevisiae, Rod1p and Rog3p interact with the ubiquitin ligase Rsp5p, and so the A. nidulans homologue of Rsp5p was identified, and the gene encoding this HECT ubiquitin ligase was designated hulA. CreD and ApyA were tested for protein-protein interactions with HulA via the bacterial two-hybrid system, and ApyA showed strong interaction, and CreD showed weak interaction, with HulA in this system.     

The Aspergillus nidulans creC gene involved in carbon catabolite repression encodes a WD40 repeat protein

Expression of many microbial genes required for the utilisation of less favoured carbon sources is carbon catabolite repressed in the presence of a preferred carbon source such as D-glucose. In Aspergillus nidulans, creC mutants show derepression in the presence of D-glucose of some, but not all, systems normally subject to carbon catabolite repression. These mutants also fail to grow on some carbon sources, and show minor morphological impairment and altered sensitivity to toxic compounds including molybdate and acriflavin. The pleiotropic nature of the phenotype suggests a role for the creC gene product in the carbon regulatory cascade. The creC gene was cloned and found to encode a protein which contains five WD40 motifs. The sequence changes in three mutant alleles were found to lead to production of truncated proteins which lack one or more of the WD40 repeats. The similarity of the phenotypes conferred by these alleles implies that these alleles represent loss of function alleles. Deletion analysis also showed that at least the most C-terminal WD40 motif is required for function. The CreC protein is highly conserved relative to the Schizosaccharomyces pombe protein Yde3 – whose function is unknown – and human and mouse DMR-N9, which may be associated with myotonic dystrophy. Received: 1 July 1999 / Accepted: 31 January 2000