Fusarium sambucinum astA gene expressed during potato infection is a functional orthologue of Aspergillus nidulans astA.

Sulfate assimilation plays a vital role in prototrophic organisms. Orthologues of the alternative sulfate transporter (AstA) gene from Aspergillus nidulans were identified in the fungal plant pathogens Fusarium sambucinum and Fusarium graminearum. By physiological and biochemical analyses, the AstA orthologues were determined to be able to uptake sulfate from the environment. Similarly to astA in A. nidulans, the FsastA gene was found to be regulated by sulfur metabolite repression (SMR) in a sulfur-dependent manner. In contrast, the FgastA transcript was undetectable, however, when the FgastA gene was expressed heterologously in A. nidulans, the translated FgAstA protein acted as a sulfate transporter. Interestingly, F. sambucinum astA expression was remarkably augmented in infected potato tubers, despite the presence abundant sulfate and was found not to be correlated with plant resistance.

The Aspergillus nidulans metZ gene encodes a transcription factor involved in regulation of sulfur metabolism in this fungus and other Eurotiales.

In Aspergillus nidulans, expression of sulfur metabolism genes is activated by the MetR transcription factor containing a basic region and leucine zipper domain (bZIP). Here we identified and characterized MetZ, a new transcriptional regulator in A. nidulans and other Eurotiales. It contains a bZIP domain similar to the corresponding region in MetR and this similarity suggests that MetZ could potentially complement the MetR deficiency. The metR and metZ genes are interrupted by unusually long introns. Transcription of metZ, unlike that of metR, is controlled by the sulfur metabolite repression system (SMR) dependent on the MetR protein. Overexpression of metZ from a MetR-independent promoter in a ΔmetR background activates transcription of genes encoding sulfate permease, homocysteine synthase and methionine permease, partially complementing the phenotype of the ΔmetR mutation. Thus, MetZ appears to be a second transcription factor involved in regulation of sulfur metabolism genes.

Regulatory mutations affecting sulfur metabolism induce environmental stress response in Aspergillus nidulans.

Mutations in the cysB, sconB and sconC genes affect sulfur metabolism in Aspergillus nidulans in different ways. The cysB mutation blocks synthesis of cysteine by the main pathway and leads to a shortage of this amino acid. The sconB and sconC mutations affect subunits of the SCF ubiquitin ligase complex, which inactivates the MetR transcription factor in the presence of an excess of cysteine. In effect, both cysB and scon mutations lead to permanent derepression of MetR-dependent genes. We compared transcriptomes of these three mutants with that of a wild type strain finding altered expression of a few hundred genes belonging to various functional categories. Besides those involved in sulfur metabolism, many up-regulated genes are related to stress responses including heat shock and osmotic stress. However, only the scon strains are more resistant to exogenous stress agents than the wild type strain while cysB is more sensitive. The two-component signal transduction system is a functional category, which is most enriched among genes up-regulated in the cysB, sconB and sconC mutants. A large group of up-regulated genes are involved in carbohydrate and energy metabolism, including genes coding for enzymes of trehalose and glycerol synthesis. The altered expression of these genes is accompanied by changes in sugar and polyol accumulation in conidia of the mutants. Genes encoding enzymes of the glyoxylate bypass and the GABA shunt are also up-regulated along with genes coding for enzymes of alcohol fermentation. Among the down-regulated genes the most numerous are those encoding membrane proteins and enzymes involved in secondary metabolism, including the penicillin biosynthesis cluster.

Cohesin Irr1/Scc3 is likely to influence transcription in Saccharomyces cerevisiae via interaction with Mediator complex.

The evolutionarily conserved proteins forming sister chromatid cohesion complex are also involved in the regulation of gene transcription. The participation of SA2p (mammalian ortholog of yeast Irr1p, associated with the core of the complex) in the regulation of transcription is already described. Here we analyzed microarray profiles of gene expression of a Saccharomyces cerevisiae irr1-1/IRR1 heterozygous diploid strain. We report that expression of 33 genes is affected by the presence of the mutated Irr1-1p and identify those genes. This supports the suggested role of Irr1p in the regulation of transcription. We also indicate that Irr1p may interact with elements of transcriptional coactivator Mediator.

Aspergillus nidulans genes encoding reverse transsulfuration enzymes belong to homocysteine regulon.

Homocysteine is an intermediate in methionine synthesis in Aspergillus nidulans, but it can also be converted to cysteine by the reverse transsulfuration pathway involving cystathionine beta-synthase (CBS) and cystathionine gamma-lyase (CGL). Because homocysteine is toxic to the cell at high concentrations, this pathway also functions as a means of removal of its excess. We found that the transcription of the mecA and mecB genes encoding CBS and CGL was upregulated by excess of homocysteine as well as by shortage of cysteine. Homocysteine induced transcription of both genes when added to the growth medium or overproduced in a regulatory mutant. The derepressing effect of cysteine shortage was observed in some mutants and in the wild-type strain during sulfur starvation. An increase in the level of mecA or mecB transcript roughly parallel with the elevation of the respective enzyme activity. On the basis of the mode of mecA and mecB regulation by homocysteine, these genes may be classified in a group of genes upregulated directly or indirectly by this amino acid. We call this group of genes the "homocysteine regulon".

Multiple fungal enzymes possess cysteine synthase activity in vitro.

We present evidence that there are at least three Aspergillus nidulans enzymes which catalyze in vitro the reaction of O-acetylserine (OAS) with sulfide forming cysteine. This activity is shared by cysteine synthase (CS) encoded by the cysB gene, homocysteine synthase encoded by cysD and by at least one more enzyme. Moreover, arginine, histidine or proline starvation leads to derepression of CS activity even in the cysB,cysD double mutant strains, while neither cysB nor cysD gene transcription is derepressed by amino acid starvation. Using a cpcA mutant, we show that starvation-inducible CS activity is under control of cross-pathway regulation. We identify CysF as a putative CS in A. nidulans. However, cysF gene transcription is not elevated by amino acid starvation. Therefore, it seems that there exists yet another enzyme, thus far unidentified, which possesses CS activity. Using mutants impaired during various steps of cysteine synthesis we prove that the cysB-encoded enzyme is the only CS of physiological importance in the studied fungus. Similar results were obtained with Schizosaccharomyces pombe mutant strains impaired in cysteine synthesis, indicating that the presence of multiple enzymes with in vitro CS activity may be a common feature of many fungal species.

Sulfate transport in Aspergillus nidulans: a novel gene encoding alternative sulfate transporter.

In Aspergillus nidulans sulfate is taken up by sulfate permease encoded by the sB gene. A unique tight auxotrophic mutant with an impaired promoter region of the sulfate permease gene, sB1(pr), was isolated. Three suppressor genes were cloned by complementation of this mutation. One of them, described here, is the astA gene (alternative sulfate transporter) derived from a genomic library of the Japanese A. nidulans IAM 2006 strain. In the reference strain of Glasgow origin the astA gene was found to be a pseudogene having several nucleotide deletions in ORF. The gene encodes a novel type of sulfate transporter which is distinct from other known sulfate permeases forming the SulP family. The putative ASTA protein belongs to an extensive and poorly characterized Dal5 allantoate permease family of fungal organic anion transporters. We have shown that ASTA is a physiological sulfate transporter. We also report cloning and characterization of the sB gene in this work. Both genes, sB and astA, are regulated at the transcriptional level by sulfur metabolite repression (SMR).

Two Aspergillus nidulans genes encoding methylenetetrahydrofolate reductases are up-regulated by homocysteine.

Methylenetetrahydrofolate reductase (MTHFR) catalyzes the reduction of 5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate, a co-substrate in the synthesis of methionine from homocysteine. We have cloned and characterized two Aspergillus nidulans genes encoding MTHFRs: metA and metF. Mutations in either gene result in methionine requirement; the metA-encoded enzyme is responsible for only 10-15% of total MTHFR activity. These two enzymes belong to different classes of MTHFRs. Mutations in metA but not in the metF gene are suppressed by mutations resulting in enhancement of homocysteine synthesis. The expression of both genes is up-regulated by homocysteine.

The Aspergillus nidulans metR gene encodes a bZIP protein which activates transcription of sulphur metabolism genes.

The identification, isolation and characterization of a new Aspergillus nidulans positive-acting gene metR, which encodes a transcriptional activator of sulphur metabolism, is reported. metR mutants are tight auxotrophs requiring methionine or homocysteine for growth. Mutations in the metR gene are epistatic to mutations in the negative-acting sulphur regulatory scon genes. The metR coding sequence is interrupted by a single intron of 492 bp which is unusually long for fungi. Aspergillus nidulans METR is a member of bZIP family of DNA-binding proteins. The bZIP domains of METR and the Neurospora crassa CYS3 transcriptional activator of sulphur genes are highly similar. Although Neurospora cys-3 gene does not substitute for the metR function, a chimeric metR gene with a cys-3 bZIP domain is able to transform the DeltametR mutant to methionine prototrophy. This indicates that METR recognizes the same regulatory sequence as CYS3. The metR gene is not essential, as deletion mutants are viable and have similar phenotype as point mutants. In contrast to the Neurospora cys-3, transcription of the metR gene was found to be regulated neither by METR protein nor by sulphur source. Transcription of metR gene is derepressed in the sconB2 mutant. Transcription of genes encoding sulphate permease, homocysteine synthase, cysteine synthase, ATP-sulphurylase, and sulphur controller--sconB is strongly regulated by the metR gene product and depends on the character of the metR mutation and sulphur supplementation.