Joan M. Kelly

Cloning of the creA gene from Aspergillus nidulans: a gene involved in carbon catabolite repression.

SummaryThe creA gene from A. nidulans has been cloned by complementation of a non-revertable mutant allele using a genomic library and marker rescue techniques. The rescued sequence was subcloned and a 2.3 kb fragment identified which complements several creA mutant alleles. Northern analyses showed that creA encodes a transcript of approximately 1.8 kb in length and that the levels of this transcript varied by up to two fold depending on the carbon source. Transformants containing more than two extra copies of creA grew as wildtype on a range of carbon sources, but there was evidence for tighter carbon catabolite repression.

The Aspergillus niger carbon catabolite repressor encoding gene, creA

In order to undertake a comparative analysis of carbon catabolite repression in two Aspergillus species, the creA gene has been isolated from A. niger by cross hybridization, using the cloned A. nidulans gene. The A. niger gene has been shown to be functional in A. nidulans by heterologous complementation of the creA204 mutation of A. nidulans. Overall, the genes show 90% sequence similarity (82% identity) at the amino acid (aa) level. There were some striking similarities between the aa sequences encoded by the two fungal creA genes and two genes involved in carbon catabolite repression in Saccharomyces cerevisiae. The zinc-finger regions showed 96% similarity (84% identity) with the zinc-finger region of the MIG1 gene of S. cerevisiae. The CREA protein contains a stretch of 42 aa that is identical in A. niger and A. nidulans, and these show 81% similarity (33% identity) with a region of the S. cerevisiae RGR1 gene.

Notes An inversion truncating the creA gene of Aspergillus niduians results in carbon catabolite derepression

The creAd‐30 mutation leading to carbon catabolite derepression in Aspergillus nidulans is a pericentric inversion, having one breakpoint within the creA gene on the left arm of chromosome I and the other breakpoint between binG and yA on the right arm. The left‐arm breakpoint alters the creA transcript. The likelihood that the inversion truncates creA centrally strengthens a previous proposal that derepression is the phenotype of loss‐of‐function mutations in creA.

Carbon catabolite repression in Aspergillus nidulans involves deubiquitination

The best studied role of ubiquitination is to mark proteins for destruction by the proteasome but, in addition, it has recently been shown to promote macromolecular assembly and function, and alter protein function, thus playing a regulatory role distinct from protein degradation. Deubiquinating enzymes, the ubiquitin‐processing proteases (ubps) and the ubiquitin carboxy‐terminal hydrolases (uchs), remove ubiquitin from ubiquitinated substrates. We show here that the creB gene involved in carbon catabolite repression in Aspergillus nidulans encodes a functional member of the novel subfamily of the ubp family defined by the human homologue UBH1, thus implicating ubiquitination in the process of carbon catabolite repression. Members of the novel subfamily of ubps that include CreB are widespread amongst eukaryotes, with homologues present in mammals, nematodes, Drosophila and Arabidopsis, but mutations in the genes have only been identified in A. nidulans. From phenotypes of the A. nidulans mutants it is probable that this subfamily is involved in complex regulatory pathways. Mutations in the gene encoding the WD40 repeat protein CreC result in an identical phenotype, implicating both genes in this pathway.

RcoA has pleiotropic effects on Aspergillus nidulans cellular development.

Aspergillus nidulans rcoA encodes a member of the WD repeat family of proteins. The RcoA protein shares sequence similarity with other members of this protein family, including the Saccharomyces cerevisiae Tup1p and Neurospora crassa RCO1. Tup1p is involved in negative regulation of an array of functions including carbon catabolite repression. RCO1 functions in regulating pleiotropic developmental processes, but not carbon catabolite repression. In A. nidulans, deletion of rcoA (ΔrcoA), a recessive mutation, resulted in gross defects in vegetative growth, asexual spore production and sterigmatocystin (ST) biosynthesis. Expression of the asexual and ST pathway‐specific regulatory genes, brlA and aflR, respectively, but not the signal transduction genes (i.e. flbA, fluG or fadA) regulating brlA and aflR expression was delayed (brlA) or eliminated (aflR) in a ΔrcoA strain. Overexpression of aflR in a ΔrcoA strain could not rescue normal expression of downstream targets of AflR. CreA‐dependent carbon catabolite repression of starch and ethanol utilization was only weakly affected in a ΔrcoA strain. The strong role of RcoA in development, vegetative growth and ST production, compared with a relatively weak role in carbon catabolite repression, is similar to the role of RCO1 in N. crassa.

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 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 ex‐periments 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.

Alcohol dehydrogenase III inAspergillus nidulans is anaerobically induced and post-transcriptionally regulated

SummaryAn alcohol dehydrogenase was shown to be induced inAspergillus nidulans by periods of anaerobic stress. This alcohol dehydrogenase was shown to correspond to the previously described cryptic enzyme, alcohol dehydrogenase III (McKnight et al. 1985), by analysis of a mutation in the structural gene of alcohol dehydrogenase III,alcC, created by gene disruption. Survival tests on agar plates showed that this enzyme is required for long-term survival under anaerobic conditions. Northern blot analysis and gene fusion studies showed that the expression of thealcC gene is regulated at both the transcriptional and translational levels. Thus there are mechanisms in this filamentous fungus allowing survival under anaerobic stress that are similar to those described in higher plants.

Pyruvate decarboxylase and anaerobic survival in Aspergillus nidulans

The presence of pyruvate decarboxylase activity has been demonstrated in Aspergillus nidulans, and a gene encoding a pyruvate decarboxylase has been isolated from this organism and physically characterized. The isolation of the pdcA gene in A. nidulans confirms the existence of the alcoholic fermentation pathway in this fungus, despite it being an obligate aerobic organism. Southern analysis showed that it is most probably a single copy gene. Several potential binding sites for a GATAR-binding protein were identified in the sequence just prior to the start point of transcription, and mutant alleles of the GATAR-binding protein-encoding gene, areA, affected pdcA mRNA levels in a manner that suggested that it influences pdcA expression in nitrogen repressing conditions. Other previously reported cases of AREA action are in nitrogen-limiting conditions. Interestingly, the production of ethanol was affected in a similar way by the same areA alleles, suggesting that changes in pdcA mRNA level are reflected in the changes in the level of ethanol production. The experiments presented here confirm that PDC levels are a major determinant of ethanol production under these conditions.

Disruption of Trichoderma reesei cre2, encoding an ubiquitin C-terminal hydrolase, results in increased cellulase activity

BackgroundThe filamentous fungus Trichoderma reesei (Hypocrea jecorina) is an important source of cellulases for use in the textile and alternative fuel industries. To fully understand the regulation of cellulase production in T. reesei, the role of a gene known to be involved in carbon regulation in Aspergillus nidulans, but unstudied in T. reesei, was investigated.ResultsThe T. reesei orthologue of the A. nidulans creB gene, designated cre2, was identified and shown to be functional through heterologous complementation of a creB mutation in A. nidulans. A T. reesei strain was constructed using gene disruption techniques that contained a disrupted cre2 gene. This strain, JKTR2-6, exhibited phenotypes similar to the A. nidulans creB mutant strain both in carbon catabolite repressing, and in carbon catabolite derepressing conditions. Importantly, the disruption also led to elevated cellulase levels.ConclusionsThese results demonstrate that cre2 is involved in cellulase expression. Since the disruption of cre2 increases the amount of cellulase activity, without severe morphological affects, targeting creB orthologues for disruption in other industrially useful filamentous fungi, such as Aspergillus oryzae, Trichoderma harzianum or Aspergillus niger may also lead to elevated hydrolytic enzyme activity in these species.