Robin A. Lockington

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.

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.

Genetic diversity in ruminal isolates ofSelenomonas ruminantium

Diversity in the ruminal bacterial speciesSelenomonas ruminantium has been investigated by DNA fingerprinting, DNA-DNA hybridization, plasmid analysis, bacteriophage sensitivity, and monoclonal antibody-based immunoassay. Twenty different isolates from the sheep rumen were initially classified morphologically and by carbon source utilization. DNA fingerprint analyses and quantitative genomic DNA hybridizations showed that limited grouping of these isolates was possible, with the largest group comprising four isolates, and two other groups comprising two isolates each. The remaining isolates were unique. Plasmids in four different size classes, 2.5, 3.7, 6.5 and 12.0 kbp, were identified, but these did not appear in all isolates. There was no apparent relationship between DNA fingerprint pattern and plasmid content. Only three isolates were sensitive to theS. ruminantium-specific temperate bacteriophage S-1. These data indicate that substantial genetic diversity exists within the ruminal speciesS. ruminantium, but that at least one strain may represent up to 20% of isolates.

A second component of the SltA-dependent cation tolerance pathway in Aspergillus nidulans

Highlights • SltB is a novel component of the cation stress responsive pathway.• Loss of SltB function results in sensitivity to elevated extracellular concentrations of cations and to alkalinity.• SltB is involved in signaling to transcription factor SltA.• SltA regulates expression of sltB.• The Slt pathway is unique to fungi from the pezizomycotina subphylum.

The Spt-Ada-Gcn5 Acetyltransferase (SAGA) complex in Aspergillus nidulans.

A mutation screen in Aspergillus nidulans uncovered mutations in the acdX gene that led to altered repression by acetate, but not by glucose. AcdX of A. nidulans is highly conserved with Spt8p of Saccharomyces cerevisiae, and since Spt8p is a component of the Spt-Ada-Gcn5 Acetyltransferase (SAGA) complex, the SAGA complex may have a role in acetate repression in A. nidulans. We used a bioinformatic approach to identify genes encoding most members of the SAGA complex in A. nidulans, and a proteomic analysis to confirm that most protein components identified indeed exist as a complex in A. nidulans. No apparent compositional differences were detected in mycelia cultured in acetate compared to glucose medium. The methods used revealed apparent differences between Yeast and A. nidulans in the deubiquitination (DUB) module of the complex, which in S. cerevisiae consists of Sgf11p, Sus1p, and Ubp8p. Although a convincing homologue of S. cerevisiae Ubp8p was identified in the A. nidulans genome, there were no apparent homologues for Sus1p and Sgf11p. In addition, when the SAGA complex was purified from A. nidulans, members of the DUB module were not co-purified with the complex, indicating that functional homologues of Sus1p and Sgf11p were not part of the complex. Thus, deubiquitination of H2B-Ub in stress conditions is likely to be regulated differently in A. nidulans compared to S. cerevisiae.

SAGA complex components and acetate repression in Aspergillus nidulans.

Alongside the well-established carbon catabolite repression by glucose and other sugars, acetate causes repression in Aspergillus nidulans. Mutations in creA, encoding the transcriptional repressor involved in glucose repression, also affect acetate repression, but mutations in creB or creC, encoding components of a deubiquitination system, do not. To understand the effects of acetate, we used a mutational screen that was similar to screens that uncovered mutations in creA, creB, and creC, except that glucose was replaced by acetate to identify mutations that were affected for repression by acetate but not by glucose. We uncovered mutations in acdX, homologous to the yeast SAGA component gene SPT8, which in growth tests showed derepression for acetate repression but not for glucose repression. We also made mutations in sptC, homologous to the yeast SAGA component gene SPT3, which showed a similar phenotype. We found that acetate repression is complex, and analysis of facA mutations (lacking acetyl CoA synthetase) indicates that acetate metabolism is required for repression of some systems (proline metabolism) but not for others (acetamide metabolism). Although plate tests indicated that acdX- and sptC-null mutations led to derepressed alcohol dehydrogenase activity, reverse-transcription quantitative real-time polymerase chain reaction showed no derepression of alcA or aldA but rather elevated induced levels. Our results indicate that acetate repression is due to repression via CreA together with metabolic changes rather than due to an independent regulatory control mechanism.