Aspergillus nidulans, as many other fungi, can utilize sulfate as a sulfur source through a highly energy consuming sulfate assimilation pathway producing sulfide which is then incorporated into cysteine. When cysteine is in excess this pathway is repressed by the hierarchical sulfur metabolite repression (SMR) system, in which the SCF ubiquitin ligase activated by cysteine brings about degradation of the MetR transcription factor specific for sulfate assimilation pathway genes. Mutational impairment of SMR causes a permanent derepression of the sulfate assimilation pathway and leads to a direct synthesis of homocysteine from sulfide as an alternative route of synthesis of sulfur amino acids. This derepression results in overproduction of sulfide and sulfur-containing organic compounds associated with a considerable dissipation of ATP and NADPH. Transcriptomic analysis of mutants dysregulated in SMR has revealed considerable remodelling of cellular metabolism manifested by changes in the levels of several hundred transcripts. Among the up-regulated genes the most remarkable are those encoding proteins normally involved in responses to various environmental stresses. Expression of genes encoding enzymes involved in homocysteine metabolism is independent of SMR and is activated by this amino acid, which prevents its accumulation above a toxic level.

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.

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.

Fungi are able to utilize inorganic sulfur. They possess a sulfate assimilation pathway leading to formation of sulfide. Then cysteine is formed from sulfide and O-acetylserine in reaction catalyzed by cysteine synthase. Cysteine is a precursor of methionine. Some fungi have an alternative pathway for cysteine synthesis. In this pathway homocysteine is the first sulfur-containing amino acid made and serves as a precursor of both cysteine and methionine. Aspergillus nidulans possesses both pathways but that involving cysteine synthase is the main one. The second pathway becomes functional when cysteine synthase is impaired or when sulfur metabolite repression (SMR) system is defective. In the first case there is a shortage of cysteine in the cell since the alternative pathway is less effective while in the second case both pathways are operating since the SMR is abolished leading to an overproduction of sulfurcontaining compounds. The SMR system is composed of a transcription factor specific for some sulfur metabolism-related genes and ubiquitin ligase which inactivates the transcription factor in the presence of an excess of cysteine so that de novo synthesis of cysteine is repressed. One can expect that such marked changes in sulfur amino acid pool (shortage vs. abundance) should have an impact on cellular metabolism, which might be manifested in a change in transcriptional and proteomic profile of the cell. Primary results with mutants impaired in sulfur metabolism and the wild type show that they show some features of the reaction to various forms of stress. There are also many cases of up and down regulation, which are more difficult to interpretation since they probably represent secondary reactions to the changes in sulfur metabolism. L22.2

Expression of the sulfur assimilation pathway in Aspergillus nidulans is under control of sulfur metabolite repression, which is composed of scon genes encoding subunits of ubiquitin ligase and the metR gene coding for a transcriptional activator. In this paper we report three dominant suppressors of methionine requirement isolated from a metB3 diploid strain. All three mutations lead to the substitution of phenylalanine 48 by serine or leucine in the conserved N-terminal region of the MetR protein. Strains carrying the dominant suppressor mutations exhibit increased activities of homocysteine synthase and sulfur assimilation enzymes as well as elevated levels of the corresponding transcripts. These changes are observed even under conditions of methionine repression, which suggests that the mutated MetR protein may be resistant to inactivation or degradation mediated by sulfur metabolite repression. We also found that a mutant impaired in sulfite reductase activity, known until now as sG8, has a frameshift which changes 41 C-terminal amino acids. Therefore, it is now designated metR18. This mutant has elevated levels of MetR-regulated transcripts and of activities of sulfur assimilation enzymes (except sulfite reductase), which can be repressed to the wild type level by exogenous methionine. Thus, metR18 and the three dominant suppressors represent new types of mutations affecting different parts of the A. nidulans MetR protein.

The interdependence of the sulfane sulfur metabolism and sulfur amino acid metabolism was studied in the fungus Aspergillus nidulans wild type strain and in mutants impaired in genes encoding enzymes involved in the synthesis of cysteine (a precursor of sulfane sulfur) or in regulatory genes of the sulfur metabolite repression system. It was found that a low concentration of cellular cysteine leads to elevation of two sulfane sulfurtransferases, rhodanase and cystathionine γ-lyase, while the level of 3-mercaptopyruvate sulfurtransferase remains largely unaffected. In spite of drastic differences in the levels of biosynthetic enzymes and of sulfur amino acids due to mutations or sulfur supplementation of cultures, the level of total sulfane sulfur is fairly stable. This stability confirms the crucial role of sulfane sulfur as a fine-tuning regulator of cellular metabolism.

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 β-synthase (CBS) and cystathionine γ-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”.

Sulfate uptake, the first step of sulfate assimilation in all organisms, is a highly endoergic, ATP requiring process. It is under tight control at the transcriptional level and is additionally modulated by posttranslational modifications, which are not yet fully characterized. Sulfate anion is taken up into the cell by specific transporters, named sulfate permeases, located in the cell membrane. Bacterial sulfate permeases differ significantly from the eukaryotic transporters in their evolutionary origins, structure and subunit composition. This review focuses on the diversity and regulation of sulfate permeases in various groups of organisms.

Glycosylphosphatidylinositol (GPI) anchoring is the main mechanism allowing proper localization of secretory proteins in cell membranes. We have isolated an Aspergillus nidulans homolog of the human PIG-P gene, which encodes a subunit of acetylglucosaminyltransferase (GPI-GnT)-an enzyme involved in the synthesis of GPI anchors. A. nidulans pigP mutants have significantly decreased GPI synthesis. On solid media they show strong growth retardation (the "button" phenotype) while in liquid minimal media they show overall good growth but with hyperbranched and bulbous hyphae with impaired septation. Furthermore, the pigP strains, in contrast to the wild-type, abundantly secrete a 33-kDa alkaline serine protease (ALP) into the liquid medium.

The identification and annotation of protein-coding genes is one of the primary goals of whole-genome sequencing projects, and the accuracy of predicting the primary protein products of gene expression is vital to the interpretation of the available data and the design of downstream functional applications. Nevertheless, the comprehensive annotation of eukaryotic genomes remains a considerable challenge. Many genomes submitted to public databases, including those of major model organisms, contain significant numbers of wrong and incomplete gene predictions. We present a community-based reannotation of the Aspergillus nidulans genome with the primary goal of increasing the number and quality of protein functional assignments through the careful review of experts in the field of fungal biology.

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.

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.

Schizosaccharomyces pombe, in contrast to Saccharomyces cerevisiae and Aspergillus nidulans, lacks cystathionine beta-synthase and cystathionine gamma-lyase, two enzymes in the pathway from methionine to cysteine. As a consequence, methionine cannot serve as an efficient sulphur source for the fungus and does not bring about repression of sulphur assimilation, which is under control of the cysteine-mediated sulphur metabolite repression system. This system operates at the transcriptional level, as was shown for the homocysteine synthase encoding gene. Our results corroborate the growing evidence that cysteine is the major low-molecular-weight effector in the regulation of sulphur metabolism in bacteria, fungi and plants.

Mutations in four genes: sconA (formerly suA25meth, mapA25), sconB (formerly mapBl), sconC and sconD, the last two identified in this work, relieve a group of sulphur amino acid biosynthetic enzymes from methionine-mediated sulphur metabolite repression. Exogenous methionine has no effect on sulphate assimilation in the mutant strains, whereas in the wild type it causes almost complete elimination of sulphate incorporation. In both mutant and wild-type strains methionine is efficiently taken up and metabolized to S-adenosylmethionine, homocysteine and other compounds. scon mutants also show elevated levels of folate-metabolizing enzymes which results from the large pool of homocysteine found in these strains. The folate enzymes apear to be inducible by homocysteine and repressible by methionine (or Sadenosylmethionine).

Summary The regulation of methionine synthase. serine hydroxymethyltransferase. methylenetetrahydrofolate oxidorelluctase and dihydrofolate reductase in Aspergillus nidulans have been found to be controlled by endogenous pools methionine and homocysteine. Mutants impaired in sulfur metaholite repression. which have highly elevated pools of these amino acids. also show elevated levels of folate metabolizing enzymes mentioned above. This effect was found to he the result of the accumulation of endogenous homocysteine. which induces folate enzymes. High concentration (up to 5 mM) or methionine in growth medium leads to repression of these enzymes. It appears. therefore. that the levels of folate metaholizing enzymes arc determined by the ratio of cellular levels or methionine and homocysteine.