Heather M. Sealy-Lewis

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.

A Possible Regulatory Gene for the Molybdenum-Containing Cofactor in Aspergillus nidulans

Aspergillus nidulans has three molybdoenzymes, nitrate reductase, purine hydroxylase I and purine hydroxylase II. These three enzymes share a molybdenum-containing cofactor whose synthesis requires the integrity of five loci, designated cnxABC, cnxE, cnxF, cnxG and cnxH. Here we report the existence of a sixth locus, designated cnxJ, which might be involved inthe regulation of cofactor levels. When grown in the presence, but not in the absence, of tungstate or methylammonium, strains carrying cnxJ1 or cnxJ2 have reduced molybdoenzyme levels as judged both from growth properties and enzyme determinations. A new cryosensitive cnxC- allele is also reported. Its phenotype at 37 degrees C (but not 25 degrees C) shows some similarities to that of the two cnxJ- alleles. A structural role for the cnxC (or cnxABC) product in the cofactor is tentatively suggested.

An NADP+-dependent glycerol dehydrogenase in Aspergillus nidulans is inducible by D-galacturonate

SummaryIn Aspergillus nidulans there is an NADP+-dependent glycerol dehydrogenase that is specifically induced on transfer to D-galacturonate medium. In contrast to the previously characterised constitutive NADP+-dependent glycerol dehydrogenase it has a much broader substrate specificity, having activity as an ethanol dehydrogenase, and is subject to carbon-catabolite repression. In addition to the two NADP+-dependent glycerol dehydrogenases, alcohol dehydrogenase I and II are also present on transfer to D-galacturonate medium, and have weak activity as glycerol dehydrogenases.

Isolation of mutants deficient in acetyl-CoA synthetase and a possible regulator of acetate induction in Aspergillus niger

Acetate-non-utilizing mutants in Aspergillus niger were selected by resistance to 1.2% propionate in the presence of 0.1% glucose. Mutants showing normal morphology fell into two complementation groups. One class of mutant lacked acetyl-CoA synthetase but had high levels of isocitrate lyase, while the second class showed reduced levels of both acetyl-CoA synthetase and isocitrate lyase compared to the wild-type strain. By analogy with mutants selected by resistance to 1.2% propionate in Aspergillus nidulans, the properties of the mutants in A. niger suggest that the mutations are either in the structural gene for acetyl-CoA synthetase (acuA) or in a possible regulatory gene of acetate induction (acuB). A third class of mutant in a different complementation group was obtained which had abnormal morphology (yellow mycelium and few conidia); the specific lesion in these mutants has not been determined.

A new selection method for isolating mutants defective in acetate utilisation inAspergillus nidulans

Propionate medium is normally toxic for the growth ofAspergillus nidulans. Spontaneous mutations relieving the toxicity to propionate, which arose on propionate medium, have been shown to be mutations in acetate metabolism. Oneacu− mutant is allelic withacuA (the structural gene for acetyl-CoA synthetase), another withacuB (the regulatory gene involved in the induction of enzymes concerned with acetate metabolism, including acetyl-CoA synthetase), and a third mutants,acuO, represents a newacu− locus that maps on likage group V.

Chromosomal mapping and gene disruption of the ADHIII gene in aspergillus nidulans.

SummaryThere are at least three alcohol dehydrogenases in Aspergillus nidulans. ADHIII has no obvious physiological function. We describe here the cloning of the ADHIII gene (alcC), its mapping on linkage group VII by “reverse genetics”, and the properties of multicopy transformants tested for their ability to grow on a range of alcohols (butan-1-ol being the best substrate tested for growth). We were unable to detect any obvious alteration in phenotype of a strain carrying a disrupted copy of the ADHIII gene.

The identification of mutations in Aspergillus nidulans that lead to increased levels of ADHII

SummaryThere are at least three alcohol dehydrogenases in Aspergillus nidulans. ADHII has been observed in polyacrylamide gels stained for ADH activity but, unlike ADHI and ADHIII, no physiological function has been attributed to it. This paper describes mutations that have been isolated from strains carrying a deletion in the structural gene for ADHI (alcA) and its adjacent positively-acting regulatory gene (alcR) that restore some ability to utilise ethanol as a carbon source. The mutations map at three loci, and all show elevated levels of the ADHII staining band. An assay for ADHII has been developed. The growth on ethanol has been shown to be dependent on the previously identified aldehyde dehydrogenase (structural gene, aldA). Two of the mutations, alcD and alcE, represent newly discovered mutations affecting ethanol utilisation while the third mutation is in amdA, a previously described trans-acting regulatory protein.

The cloning and sequencing of thealcB gene, coding for alcohol dehydrogenase II, inAspergillus nidulans

Alcohol dehydrogenase II (ADH II, structural genealcB) was purified from a strain H1035,biA1; alcE1; alc500 alcD1, which produces 100-times more ADH II activity than thealcAalcR deletion strain (alc500). Antibodies were raised against this ADH, and were used to screen a cDNA library in γgt11. We have isolated the gene for an ADH which is over-expressed in H1035, and which we believe to be thealcB gene; cDNA and genomic clones were sequenced. The sequence contains three introns and encodes a protein of 367 amino acids. This protein shows a clear level of identity to a range of alcohol dehydrogenases, but is no more closely related to the ADH I and ADH III previously described inA. nidulans than to the ADHs ofS. pombe andS. cerevisiae. The significance of consensus sequences found in the 5′ region of the gene is discussed in relation to the regulation of the gene.

Substrate specificity of nine NAD+-dependent alcohol dehydrogenases in Aspergillus nidulans

In Aspergillus nidulans three alcohol dehydrogenases (ADHs) have been described. ADHI is induced by ethanol and is the physiological enzyme of ethanol utilization, ADHII has not been attributed a function but is repressed by ethanol. The ALCR regulatory protein acts positively to induce ADHI, and negatively in its control of ADHII. ADHIII is specifically induced by anaerobic stress. We have characterized the substrate specificity of these three enzymes by looking at their staining profile on polyacrylamide gels with a range of alcohols. In addition to these enzymes we have observed six other NAD(+)-dependent ADHs, two of which, propan-2-ol dehydrogenase and pentan-2-ol dehydrogenase, share similar control with ADHII. The inducibility of these enzymes with some alcohols has also been investigated. The profile of ADHs with NADP+ as an electron acceptor is also reported.

In Aspergillus nidulans the Suppressors suaA and suaC Code for Release Factors eRF1 and eRF3 and suaD Codes for a Glutamine tRNA

In Aspergillus nidulans, after extensive mutagenesis, a collection of mutants was obtained and four suppressor loci were identified genetically that could suppress mutations in putative chain termination mutations in different genes. Suppressor mutations in suaB and suaD have a similar restricted spectrum of suppression and suaB111 was previously shown to be an alteration in the anticodon of a gln tRNA. We have shown that like suaB, a suaD suppressor has a mutation in the anticodon of another gln tRNA allowing suppression of UAG mutations. Mutations in suaA and suaC had a broad spectrum of suppression. Four suaA mutations result in alterations in the coding region of the eukaryotic release factor, eRF1, and another suaA mutation has a mutation in the upstream region of eRF1 that prevents splicing of the first intron within the 5′UTR. Epitope tagging of eRF1 in this mutant results in 20% of the level of eRF1 compared to the wild-type. Two mutations in suaC result in alterations in the eukaryotic release factor, eRF3. This is the first description in Aspergillus nidulans of an alteration in eRF3 leading to suppression of chain termination mutations.