Specific induction and carbon/nitrogen repression of arginine catabolism gene of Aspergillus nidulans--functional in vivo analysis of the otaA promoter

The arginine catabolism gene otaA encoding ornithine transaminase (OTAse) is specifically induced by arginine and is under the control of the broad-domain carbon and nitrogen repression systems. Arginine induction is mediated by a product of arcA gene coding for Zn(2)C(6) activator. We have identified a region responsible for arginine induction in the otaA promoter (AnUAS(arg)). Deletions within this region result in non-inducibility of OTAse by arginine, whether in an arcA(+) strain or in the presence of the arcA(d)47 gain of function allele. AnUAS(arg) is very similar to the Saccharomyces cerevisiae UAS(arg), a sequence bound by the Zn(2)C(6) activator (ArgRIIp), acting in a complex with two MADS-box proteins (McmIp and ArgRIp).We demonstrate here that two CREA in vitro binding sites in the otaA promoter are functional in vivo. CREA is directly involved in carbon repression of the otaA gene and it also reduces its basal level of expression. Although AREA binds to the otaA promoter in vitro, it probably does not participate in nitrogen metabolite repression of the gene in vivo. We show here that another putative negatively acting GATA factor AREB participates directly or indirectly in otaA nitrogen repression. We also demonstrate that the high levels of OTAse activity are an important factor in the suppression of proline auxotrophic mutations. This suppression can be achieved neither by growing of the proline auxotroph under carbon/nitrogen derepressing conditions nor by introducing of a creA(d) mutation.     

ADHII in Aspergillus nidulans is induced by carbon starvation stress

In Aspergillus nidulans there are three NAD(+)-dependent alcohol dehydrogenases (ADHs) that are capable of utilizing ethanol as a substrate. ADHI is the physiological enzyme of ethanol catabolism and ADHIII is induced under conditions of anaerobiosis. The physiological role of ADHII (structural gene alcB) is unknown. We have measured beta-galactosidase in a transformant with an alcB::lacZ fusion and have shown that alcB is maximally expressed under conditions of carbon starvation. The behavior of the alcB::lacZ transformant suggests a hierarchy of repressing carbon sources characteristic of repression by the general carbon catabolite repressor protein, CreA, but in a creA(d)30 background the transformant shows only partial derepression of beta-galactosidase on 1% glucose compared to the creA+ strain. Our results suggest that, in addition to carbon catabolite repression acting via CreA, a CreA-independent mechanism is involved in induction of alcB on carbon starvation.     

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.     

The integration of nitrogen and carbon catabolite repression in Aspergillus nidulans requires the GATA factor AreA and an additional positive-acting element, ADA.

The expression of the structural genes of the proline utilization cluster of Aspergillus nidulans is repressed efficiently only when both repressing carbon and nitrogen sources are present. Two hypotheses can account for this fact. One is a direct or indirect competition mechanism between the positive-acting AreA GATA factor, mediating nitrogen metabolite repression, and the negative-acting CreA protein, mediating carbon catabolite repression. The second is to propose that CreA prevents the binding or activity of another, as yet unidentified, positive-acting factor, here called ADA. We show the second possibility to be the correct one, and we localize the new positive cis-acting element within 290 bp of the prnD-prnB divergent promoter.     

The WD40-repeat protein CreC interacts with and stabilizes the deubiquitinating enzyme CreB in vivo in Aspergillus nidulans

Genetic dissection of carbon catabolite repression in Aspergillus nidulans has identified two genes, creB and creC, which, when mutated, affect expression of many genes in both carbon catabolite repressing and derepressing conditions. The creB gene encodes a functional deubiquitinating enzyme and the creC gene encodes a protein that contains five WD40 repeat motifs, and a proline-rich region. These findings have allowed the in vivo molecular analysis of a cellular switch involving deubiquitination. We demonstrate that overexpression of the CreB deubiquitinating enzyme can partially compensate for a lack of the CreC WD40-repeat protein in the cell, but not vice versa and, thus, the CreB deubiquitinating enzyme acts downstream of the CreC WD40-repeat protein. We demonstrate using co-immunoprecipitation experiments that the CreB deubiquitinating enzyme and the CreC WD40-repeat protein interact in vivo in both carbon catabolite repressing and carbon catabolite derepressing conditions. Further, we show that the CreC WD40-repeat protein is required to prevent the proteolysis of the CreB deubiquitinating enzyme in the absence of carbon catabolite repression. This is the first case in which a regulatory deubiquitinating enzyme has been shown to interact with another protein that is required for the stability of the deubiquitinating enzyme.     

Catabolite repression in Aspergillus nidulans; the role of glutamine synthetase.

A mutant (nit8) with a lowered activity of glutamine synthetase (GS) was isolated in Aspergillus nidulans. The levels of GS and of an arginine catabolic enzyme, ornithine transaminase (OTA) were assayed under a variety of growth conditions leading to repression, depression and induction of OTA in the wild type, nit8 and several regulatory mutants. The results obtained appear to exclude the possibility of involvement of GS in the regulation of arginine catabolism in A. nidulans.