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.

Cloning and characterization of the ethanol utilization regulon in Aspergillus nidulans.

In Aspergillus nidulans alcohol dehydrogenase (ADH) I and aldehyde dehydrogenase (AldDH) are co-inducible by acetaldehyde (Pateman et al., 1983; Sealy-Lewis and Lockington, 1984) and subject to carbon catabolite repression. The structural genes alcA and aldA are unlinked, but alcA is closely linked to the positive control gene alcR. We have obtained cDNA clones of alcA and aldA and genomic clones comprising alcA and alcR. The location of these genes in a genomic clone carrying a 13-kb insert was determined by subcloning and subsequent transformation of previously characterised point mutants. We have characterised at the physical level some large deletions encompassing both linked genes. We have shown that induction affects the level of RNA hybridisible with alcA and aldA probes. Mutations in the regulatory gene alcR, result in non-inducibility of RNA hybridisible with either probe. Thus the induction process is possibly at the level of transcription. Analogous experiments suggest that carbon catabolite repression of alcohol dehydrogenase I is equally at the level of transcription.

A physical map of the ribosomal DNA repeat unit of Aspergillus nidulans.

The ribosomal DNA repeat unit of Aspergillus nidulans has been cloned in pBR322 and a restriction map constructed. The genes coding for the 17S, 5.8S and 25S rRNAs are found in blocks separated by a 1.7 kb spacer region, with the 5.8S RNA gene lying between the genes for the two larger RNAs. The total length of the repeat unit is 7.7 kb. The 5S rRNA is not present in the repeat unit.

Cloning and characterization of the aldA gene of Aspergillus nidulans.

We have cloned and sequenced the aldA (encoding aldehyde dehydrogenase) gene of Aspergillus nidulans. The gene contains two introns which are similar in size and structure to other fungal introns. The amino acid sequence of aldehyde dehydrogenase (497 residues) shows a significant level of homology with analogous sequences in other organisms. Comparison of the primary structure of the active sites of the mammalian cytosolic and mitochondrial enzymes shows that the Aspergillus enzyme closely resembles the mammalian mitochondrial enzyme. Analysis of the 5' non-coding region of the aldA gene shows a TATA-like sequence located 90 bp upstream from the initiation codon. Two messenger-RNA start points are located 36 and 42 bp upstream from the start codon.

Comparison of the cis-acting control regions of two coordinately controlled genes involved in ethanol utilization in Aspergillus nidulans.

The alcA and aldA genes of Aspergillus nidulans are regulated in exactly the same manner, being subject to positive control by the product of the alcR gene. We report the complete nucleotide sequence of the alcA gene and its 5' non-coding region, preliminary localization of the region involved in the regulation of alcA expression, and a detailed comparison of this region to the 5' non-coding region of aldA (Pickett et al., 1987). The 5' flanking regions of the genes contain six similar sequence elements. Three of these elements are located upstream from the messenger RNA start points and one is related to a sequence element found in the region responsible for ethanol induction of the yeast ADH2 gene (Beier et al., 1985). The other homologous elements are located within the messenger RNA leader and may be associated with selection of messenger RNA start points. The amino acid sequence of alcohol dehydrogenase I (348 residues) shows a significant level of homology with analogous sequences in other organisms. Gene alcA contains introns which are similar in size and structure to other fungal introns. We discuss the positions of the introns in alcA of A. nidulans with particular reference to the conservation of intron position in and the evolutionary assembly of enzymes which possess NAD-binding domains.

Transformation by integration in Aspergillus nidulans.

DNA-mediated genetic transformation of Aspergillus nidulans has been achieved by incubating protoplasts from a strain of A. nidulans carrying a deletion in the acetamidase structural gene with DNA of derivatives of plasmid pBR322 containing the cloned structural gene for acetamidase [Hynes et al., Mol. Cell. Biol. 3 (1983) 1430-1439; p3SR2] in the presence of polyethylene glycol and CaCl2. The highest frequency obtained was 25 transformants per microgram of DNA. No enhancement of the transformation frequency was observed when DNAs of plasmids carrying either a fragment of the A. nidulans ribosomal repeat (p3SR2rr) or a fragment containing a possible A. nidulans mitochondrial origin of replication (p3SR2mo) in addition to the acetamidase gene were used. Both pBR322 and acetamidase gene sequences become integrated into the genome of A. nidulans in transformant strains. Integration events into the residual sequences adjacent to the deletion in the acetamidase gene, and probably (for p3SR2rr and p3SR2mo) into the ribosomal repeat unit are described.

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.

Isolation and characterization of a temperate bacteriophage from the ruminal anaerobe Selenomonas ruminantium.

A temperate bacteriophage was obtained from an isolate of the ruminal anaerobe Selenomonas ruminantium. Clear plaques that became turbid on further incubation occurred on a lawn of host bacteria. Cells picked from a turbid plaque produced healthy liquid cultures, but these often lysed on storage. Mid-log-phase liquid cultures incubated with the bacteriophage lysed and released infectious particles with a titer of up to 3 X 10(7) PFU/ml. A laboratory strain of S. ruminantium, HD-4, was also sensitive to this bacteriophage, which had an icosohedral head (diameter, 50 nm) and a flexible tail (length, 140 nm). The bacteriophage contained 30 kilobases of linear, double-stranded DNA, and a detailed restriction map was constructed. The lysogenic nature of infection was demonstrated by hybridization of bacteriophage DNA to specific restriction fragments of infected host genomic DNA and by identification of a bacteriophage genomic domain which may participate in integration of the bacteriophage DNA. Infection of S. ruminantium in vitro was demonstrated by two different methods of cell transformation with purified bacteriophage DNA.

Regulation of two alcohol dehydrogenases in Aspergillus nidulans.

In Aspergillus nidulans there are two alcohol dehydrogenases. In the presence of ethanol, alcohol dehydrogenase I (AHH I) is induced and alcohol dehydrogenase II (ADH II) is repressed. ADH I and ADH II have molecular weights of 39,000 and 36,000 respectively. At least ADH I is under the control of alcR, a transacting regulatory gene that is adjacent to alcA (the structural gene for ADH I, Pateman et al. 1983). Mutations in the alcR regulatory gene result in non inducibility of ADH I specific mRNA. Extreme alcA and alcR mutations result in derepressed levels of ADH II, and it is not clear whether alcR controls ADH II directly or through its control of ADH I synthesis. Both enzymes are subject to carbon catabolite repression. Induction of ADH I and ADH II operates at the level of synthesis or processing of mRNA.

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.

Analysis of mutations in the creA gene involved in carbon catabolite repression in Aspergillus nidulans.

The molecular nature of a number of creA mutant alleles has been determined. Three alleles analysed are missense mutations in the DNA binding domain and predicted to reduce but not abolish binding. Of the other four alleles, two result from frameshifts: one has a nonsense mutilation and the other has an inversion. All four alleles result in truncations of the protein after the zinc finger domain, such that the protein no longer contains at least the carboxy terminal 145 amino acids, so identifying a region required for repression. Transcriptional analysis of creA indicates that the transcript is autoregulated and analysis using 5' rapid amplification of cDNA ends indicates that transcriptional start points exist in clusters over a region of 200 bp located up to 595 bp 5' of the translational start point. The two major clusters have potential CREA-binding sites (SYGGRG) at appropriate positions to allow autoregulation. Autoregulation leads to the creA transcript being most abundant in carbon catabolite nonrepressing conditions, and this, together with the phenotypes of the mutant alleles, has led to the suggestion that CREA has effects under conditions generally not considered as carbon catabolite repressing, as well as in carbon catabolite repressing conditions.

Use of a unique gene sequence as a probe to enumerate a strain of Bacteroides ruminicola introduced into the rumen.

Cloned fragments of genomic DNA from the ruminal anaerobe Bacteroides ruminicola subsp. brevis B14 were isolated and used as hybridization probes to identify closely related bacterial species. One DNA fragment unique to strain B14 was tested to determine its sensitivity in detecting homologous sequences among total ruminal microbial DNA. In a DNA titration experiment, the probe was capable of detecting strain B14 sequences in vitro down to 0.1% of the total bacterial DNA present in a hybridization assay. There was no detectable signal for total ruminal bacterial DNA. The specificity of this DNA fragment was exploited to enumerate strain B14 in a fresh mixed suspension of ruminal bacteria in vitro and after inoculation of the strain into the rumen. In vitro strain B14 had a half-life of 9 h. However, following inoculation into the rumen there was a very rapid loss of the strain to below the detectable limit within 3 h. The half-life was less than 30 min. This loss was not due to ruminal dilution or to bacteriophage attack but was possibly the result of a specific bacteriocinlike activity present in the rumen and detectable in fresh ruminal fluid.

Alcohol dehydrogenase III in Aspergillus nidulans is anaerobically induced and post-transcriptionally regulated.

An alcohol dehydrogenase was shown to be induced in Aspergillus 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 the alcC 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.

Null alleles of creA, the regulator of carbon catabolite repression in Aspergillus nidulans.

CreA is the major regulatory protein involved in carbon catabolite repression in Aspergillus nidulans. Previously we have reported the molecular characterization of a number of in vivo selected mutant alleles and showed that they were unlikely to represent total loss of function alleles (Shroff et al., 1996) and that a deletion of the creA gene and surrounding DNA has an extremely severe effect on morphology under both carbon catabolite repressing and carbon catabolite nonrepressing conditions (Dowzer and Kelly, 1991). Here we present an analysis of in vivo selected creA mutations with an extreme morphological phenotype and show that some of these alleles would be predicted to result in no functional CreA. The most extreme of these alleles resulted in a truncation of the protein within the first zinc finger. Precise gene disruptions, leaving the flanking sequences intact, show essentially the same phenotype as this truncated allele. Thus, a strain containing a null allele is viable, and the leaky-lethal phenotype of previous deletion alleles (Dowzer and Kelly, 1991) must be due to the deletion of additional 3' genomic sequence. A strain containing an allele that results in a deletion of the final 80 amino acids shows reduced sensitivity to carbon catabolite repression for a number of systems, thus localizing a region of the protein involved in repression. Surprisingly, the phenotypically most extreme allele studied is not a null allele, but results in an amino acid substitution that would disrupt the zinc finger region and abolish binding to DNA. This is the only allele that produces a full-length protein, predicted to be nuclear localized, but which completely abolishes DNA binding. The phenotype may be more extreme than the null alleles due to the nuclear located CreA protein titrating interacting proteins.

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 (DeltarcoA), 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 DeltarcoA strain. Overexpression of aflR in a DeltarcoA 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 DeltarcoA 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.