Renata Natorff

At least four regulatory genes control sulphur metabolite repression in Aspergillus nidulans

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).

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 ΔmetR 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.

THE ASPERGILLUS NIDULANS SULPHUR REGULATORY GENE SCONB ENCODES A PROTEIN WITH WD40 REPEATS AND AN F-BOX

Abstract The Aspergillus nidulans gene sconB, one of the four identified genes controlling sulphur metabolite repression, was cloned and analysed. It encodes a polypeptide of 678 amino acids containing seven WD repeats characteristic of the large WD40 family of eukaryotic regulatory proteins. The SCONB protein has nuclear localisation signals and is very similar to the Neurospora crassa SCON2 and Saccharomyces cerevisiae Met30 proteins, both of which are involved in the regulation of sulphur metabolism. The N. crassa scon-2 gene complements the sconB2 mutation. All three proteins also contain a newly identified motif, the F-box, found in a number of eukaryotic regulatory proteins. This motif is responsible, at least in some cases, for ubiquitin-mediated proteolysis. The sconB transcript is derepressed under sulphur limitation conditions and partly repressed by high methionine.

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 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”.

Mutations affecting cysteine synthesis in Aspergillus nidulans : characterization and chromosome mapping

Selection and mapping of mutations affecting cysteine synthesis in Aspergillus nidulans was carried out. A new locus, cysE , is described, the mutants of which are deficient in in vivo conversion of O-acetylserine to cysteine, a step mediated by cysteine synthase. Three loci ( cysB, C and E ) were thus found to control this step in vivo , apparently without affecting the enzyme activity in vitro . By scoring for propargylglycine sensitivity of cys mutants, chromosomal map positions were obtained for all five cysteine loci ( A, B, C, D and E ).

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.

The Function of RNA Polymerase and dnaA in the Intitiation of Chromosome Replication in Escherichia coli and Salmonella Typhimurium

The replication of bacterial chromosome is regulated at the step of initiation (Maaloe and Hanawalt, 1961; Maaloe and Kjeldgaard, 1966; Helmstetter, Cooper, Pierucci and Revelas, 1968). Initiation occurs at a unique site on the chromosome termed the “replicator” in the original replicon hypothesis of Jacob, Brenner and Cuzin (1963). At present it is most frequently referred to as the “chromosomal origin” or the “origin of replication” (Louarn, Funderburgh and Bird, 1974; Bird, Chandler and Caro, 1976; Fujisawa and Eisenstark, 1973; Gyurasits and Wake, 1973).

Regulation of folate-dependent enzyme levels in Aspergillus nidulans: studies with regulatory mutants.

The synthesis of folate-dependent enzymes in Aspergillus nidulans appears to be regulated by intracellular pools of homocysteine and methionine. The results are consistent with the view that homocysteine acts as an inducer and methionine as a corepressor, but the molecular mechanism of the regulation is still unknown. Methionine-requiring mutants, metH2 and metD10, apparently allelic, show deregulation of folate-dependent enzymes. Most characteristic of the mutants is a repressed level of folylpolyglutamate synthetase. New mutations suppressing the metH2 lesion which render folate enzymes insensitive to methionine-mediated repression have been isolated. These mutations are likely to identify new regulatory genes in folate metabolism.

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.

Regulation of Folate Metabolizing Enzymes In Fungus Aspergillus nidulans

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.