The <Emphasis Type="Italic">Aspergillus nidulans creC</Emphasis> 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.     

The WD40-repeat protein CreC interacts with and stabilizes the deubiquitinating enzyme CreB in vivo in Aspergillus nidulans

Genetic dissection of carbon catabolite repression in Aspergillus nidulans has identified two genes, creB and creC, which, when mutated, affect expression of many genes in both carbon catabolite repressing and derepressing conditions. The creB gene encodes a functional deubiquitinating enzyme and the creC gene encodes a protein that contains five WD40 repeat motifs, and a proline-rich region. These findings have allowed the in vivo molecular analysis of a cellular switch involving deubiquitination. We demonstrate that overexpression of the CreB deubiquitinating enzyme can partially compensate for a lack of the CreC WD40-repeat protein in the cell, but not vice versa and, thus, the CreB deubiquitinating enzyme acts downstream of the CreC WD40-repeat protein. We demonstrate using co-immunoprecipitation experiments that the CreB deubiquitinating enzyme and the CreC WD40-repeat protein interact in vivo in both carbon catabolite repressing and carbon catabolite derepressing conditions. Further, we show that the CreC WD40-repeat protein is required to prevent the proteolysis of the CreB deubiquitinating enzyme in the absence of carbon catabolite repression. This is the first case in which a regulatory deubiquitinating enzyme has been shown to interact with another protein that is required for the stability of the deubiquitinating enzyme.     

A carbon catabolite repression mutant of Saccharomyces cerevisiae with elevated hexokinase activity: Evidence for regulatory control of hexokinase PII synthesis

Summary Mutants were investigated that had elevated hexokinase activity and had been isolated previously as resistant to carbon catabolite repression (Zimmermann and Scheel 1977). They were allele tested with mutant strains of Lobo and Maitra (1977), which had defects in one or more of the genes coding for glucokinase and unspecific hexokinases. It was shown, that the mutation abolishing carbon catabolite repression had occured in a gene that was not allelic to any of the structural genes coding for hexokinases. This indicated that a regulatory defect was responsible for elevated hexokinase activity. This agreed with observations that hexokinase activities were like wild-type during growth on non-fermentable carbon sources in hex2 mutants. Recombination between the mutant allele hex2 and mutant alleles hxk1 and hxk2, coding for hexokinase PI and PII respectively, clearly demonstrated that only hexokinase PII was elevated in hex2 mutants. When hex2 mutant cells grown on YEP ethanol were shifted to YEP glucose media, hexokinase activity increased after 30min. This increase depended on de novo protein synthesis. hex2 mutants provide evidence, that carbon catabolite repression and synthesis of hexokinase PII are under common regulatory control.     

Extracellular arabinases in Aspergillus nidulans: the effect of different cre mutations on enzyme levels

The regulation of the syntheses of two arabinan-degrading extracellular enzymes and several intracellular l-arabinose catabolic enzymes was examined in wild-type and carbon catabolite derepressed mutants of Aspergillus nidulans. α-l-Arabinofuranosidase B, endoarabinase, l-arabinose reductase, l-arabitol dehydrogenase, xylitol dehydrogenase, and l-xylulose reductase were all inducible to varying degrees by l-arabinose and l-arabitol and subject to carbon catabolite repression by d-glucose. With the exception of l-xylulose reductase, all were clearly under the control of creA, a negative-acting wide domain regulatory gene mediating carbon catabolite repression. Measurements of intracellular enzyme activities and of intracellular concentrations of arabitol and xylitol in mycelia grown on d-glucose in the presence of inducer indicated that carbon catabolite repression diminishes, but does not prevent uptake of inducer. Mutations in creA resulted in an apparently, in some instances very marked, elevated inducibility, perhaps reflecting an element of “self” catabolite repression by the inducing substrate. creA mutations also resulted in carbon catabolite derepression to varying degrees. The regulatory effects of a mutation in creB and in creC, two genes whose roles are unclear, but likely to be indirect, were, when observable, more modest. As with previous data showing the effect of creA mutations on structural gene expression, there were striking instances of phenotypic variation amongst creA mutant alleles and this variation followed no discernible pattern, i.e. it was non-hierarchical. This further supports molecular data obtained elsewhere, indicating a direct role for creA in regulating structural gene expression, and extends the range of activities under creA control.     

Glucose kinase of Streptomyces coelicolor A3(2): large-scale purification and biochemical analysis

Glucose kinase of Streptomyces coelicolor A3(2) is essential for glucose utilisation and is required for carbon catabolite repression (CCR) exerted through glucose and other carbon sources. The protein belongs to the ROK-family, which comprises bacterial sugar kinases and regulators. To better understand glucose kinase function, we have monitored the cellular activity and demonstrated that the choice of carbon sources did not significantly change the synthesis and activity of the enzyme. The DNA sequence of the Streptomyces lividans glucose kinase gene glkA was determined. The predicted gene product of 317 amino acids was found to be identical to S. coelicolor glucose kinase, suggesting a similar role for this protein in both organisms. A procedure was developed to produce pure histidine-tagged glucose kinase with a yield of approximately 10 mg/l culture. The protein was stable for several weeks and was used to raise polyclonal antibodies. Purified glucose kinase was used to explore protein-protein interaction by surface plasmon resonance. The experiments revealed the existence of a binding activity present in S. coelicolor cell extracts. This indicated that glucose kinase may interact with (an)other factor(s), most likely of protein nature. A possible cross-talk with proteins of the phosphotransferase system, which are involved in carbon catabolite repression in other bacteria, was investigated.     

Improvement of pristinamycin-producing Streptomyces pristinaespiralis by rational screening

Summary According to the biosynthetic pathway of pristinamycin, a rational selection procedure with u.v. mutation was performed to obtain a high pristinamycin-producing strain. Aminoacetic acid-resistant mutants (AA^r), valine hydroxamate-resistant mutants (VH^r), kitasamycin-resistant mutants (KTM^r) and 2-deoxy-D-glucose-resistant mutants (DOG^r) were selected, successively. A strain Streptomyces pristinaespiralis 12–55 with AA^r, Val^r, KTM^r, and DOG^r was obtained, and its production of pristinamycin reached 3000 u/ml which is 100 times higher than that of the parent strain S. pristinaespiralis ATCC 25486. It is inferred that S. pristinaespiralis 12–55 can alleviate catabolite repression caused by carbon sources, provide more acetic acid and valine for pristinamycin biosynthesis and increase its resistance to pristinamycin produced by itself, all of which are favorable for pristinamycin production. The subculture experiments indicated that the hereditary character of high productivity of S. pristinaespiralis 12–55 is stable. The pristinamycin production of S. pristinaespiralis 12–55 in a 15-l fermentor could reach 3010 u/ml after a 56 h batch fermentation.     

Reactivity in vegetative incompatibility of the HE?T-E protein of the fungus Podospora anserina is dependent on GTP-binding activity and a WD40 repeated domain

The het-e gene of the filamentous fungus Podospora anserina is involved in vegetative incompatibility. Co-expression of antagonistic alleles of the unlinked loci het-e and het-c triggers a cell death reaction that prevents the formation of viable heterokaryons between strains that contain incompatible combinations of het-c and het-e alleles. The het-e1 ^A gene encodes a polypeptide that contains a putative GTP-binding site and WD40 repeats. The role of these two domains in the reactivity of the HET-E protein in incompatibility was analyzed. An in vitro assay confirmed that the first domain is functional and can bind GTP and not ATP, suggesting that GTP-binding is essential for triggering the incompatibility reaction. The relationship between the number of WD40 repeats and the reactivity of the protein in incompatibility was investigated by estimating this number in different wild-type and mutant het-e alleles. It was deduced that reactive alleles contain a minimal number of ten WD40 repeats. These results demonstrate that the reactivity of the HET-E protein depends on two functional elements, a GTP-binding domain and several WD40 repeats. These motifs are present in separate polypeptides in trimeric G proteins, suggesting that HET-E polypeptides are also involved in signal transduction. Disruption of the het-e locus does not impair the phenotype of strains but DNA hybridization analyses revealed that het-e may belong to a multigenic family. Received: 20 January 1997 / Accepted: 8 June 1997     

Characterization of oleate-nonutilizing mutants of Aspergillus nidulans isolated by the 3-amino-1,2,4-triazole positive selection method

Conidia of Aspergillus nidulans were mutagenized with ultraviolet light and were incubated on a special selective medium containing the catalase inhibitor 3-amino-1,2,4-triazole. From approximately 5 × 10⁷  viable UV-irradiated conidia tested, 423 stable mutants resistant to 3-amino-1,2,4-triazole were recovered, of which 40 were unable to grow on minimal medium with oleic acid as the sole carbon source. These oleate-nonutilizing (Ole^–) mutants did not grow on medium with carbon sources requiring functional peroxisomes (oleate, butyrate, acetate, or ethanol), but grew well on medium with carbon sources supposedly not requiring such organelles (glucose, glycerol, l-glutamate, or l-proline). The Ole^– mutants carried mutations in one of five nuclear genes affecting acetate utilization: acuJ, acuH, acuE, acuL, and perA. The perA21 strain (DL21) carried a mutation in a gene that is not allelic with any of the known acu loci and displayed a phenotype resembling that described in the Pim^– (peroxisome import defective) mutants of Hansenula polymorpha. Hyphae of the perA21 mutant contained a few small peroxisomes with the bulk of peroxisomal enzymes remaining in the 20,000 ×g supernatant, but produced wild-type levels of penicillin. Received: 16 April 1997 / Accepted: 26 July 1997     

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.     

A novel WD40 protein,BnSWD1, is involved in salt stress in <Emphasis Type="Italic">Brassica napus</Emphasis>

Genes that are expressed early in specific response to high salinity conditions were isolated from rapeseed plant (Brassica napus L.) using an mRNA differential display method. Five PCR fragments (DD1–5) were isolated that were induced by, but showed different response kinetics to, 200 mM NaCl. Nucleotide sequence analysis and homology search revealed that the deduced amino sequences of three of the five cDNA fragments showed considerable similarity to those of β-mannosidase (DD1), tomato Pti-6 proteins (DD5), and the tobacco harpin-induced protein hin1 (DD4), respectively. In contrast, the remaining clones, DD3 and DD2, did not correspond to any substantial existing annotation. Using the DD3 fragment as a probe, we isolated a full-length cDNA clone from the cDNA library, which we termed BnSWD1 (Brassica napus salt responsive WD40 1). The predicted amino-acid sequence of BnSWD1 contains eight WD40 repeats and is conserved in all eukaryotes. Notably, the BnSWD1 gene is expressed at high levels under salt-stress conditions. Furthermore, we found that BnSWD1 was upregulated after treatment with abscisic acid, salicylic acid, and methyl jasmonate. Our study suggests that BnSWD1, which is a novel WD40 repeat-containing protein, has a function in salt-stress responses in plants, possibly via abscisic acid-dependent and/or -independent signaling pathways.     

Quantitation of the Effects of Disruption of Catabolite (De)Repression Genes on the Cell Cycle Behavior of Saccharomyces cerevisiae

We have studied the effect of disrupting catabolite (de)repression genes SNF1, SNF4, and MIG1 on the cell cycle behavior of the CEN.PK122 wild type (WT) strain of Saccharomyces cerevisiae by flow cytometry in glucose-limited chemostat cultures or batch growth in the presence of different carbon sources. Through a combination of flow cytometry of propidium iodide–stained cells and mathematical modeling we showed that the deletion of the SNF4 gene provoked a decrease in the length of G1 with respect to the WT strain along with a smaller difference in the cell cycle length of parent and daughter cells. snf1 and mig1 mutants exhibited slightly shorter G1 respect to the WT. Additionally, in the mig1 mutant the cell cycle length of parent and daughter cells was slightly altered. The results obtained are in agreement with the view that the SNF4 gene is involved in the regulation of cell cycle in yeast. Received: 28 May 1998 / Accepted: 20 July 1998     

ScoC Mediates Catabolite Repression of Sporulation in <Emphasis Type="Italic">Bacillus subtilis</Emphasis>

Sporulation in Bacillus subtilis can be triggered by carbon catabolite limitation. Conversely, carbon source excess can repress the production of extracellular enzymes, motility, and sporulation. Recent studies have implicated a pH-sensing mechanism, involving AbrB, the TCA cycle, Spo0K, and Ï^H in controlling the catabolite repression of sporulation gene expression. In an accompanying paper, we demonstrate that the AbrB-dependent pH-sensing mechanism may not be the only means by which carbon catabolites affect sporulation. In the studies reported here, we have examined the molecular basis underlying the catabolite repression phenotype of mutations in the hpr (scoC), rpoD (crsA47), and spo0A (rvtA11) loci. Loss of function mutations in hpr (scoC) restored sporulation gene expression and sporulation in the presence of excess catabolite(s), suggesting that Hpr (ScoC) has a pivotal role in mediating catabolite repression. Moreover, hpr gene expression increased substantially in the presence of excess catabolite(s), further supporting the involvement of Hpr (ScoC) in the carbon catabolite response system. We suggest that alterations in the phosphorelay response to catabolites may be one mechanism by which catabolite-resistant mutants such as crsA and rvtA are able to sporulate in the presence of excess glucoseReceived: 12 November 2002 / Accepted: 13 December 2002     

New genes involved in carbon catabolite repression and derepression in the yeast Saccharomyces cerevisiae.

A mutation causing resistance to carbon catabolite repression in gene HEX2, mutant allele hex2-3, causes an extreme sensitivity to maltose when in combination with the genes necessary for maltose metabolism. This provided a convenient system for the selective isolation of mutations in genes specifically required for maltose metabolism and other genes involved in general carbon catabolite repression. In addition to reversion of the hex2-3 allele, mutations in three other genes were detected. These genes were called CAT1, CAT3, and MUR1 and in a mutated form abolished maltose inhibition caused by mutant allele hex2-3. Mutant alleles cat1 and cat3 also restored normal repression in the presence of the hex2-3 allele. Segregants having only mutant alleles cat1 or cat3 were obtained by tetrad analysis. These segregants could not grow on nonfermentable carbon sources. Mutant alleles of gene CAT1 were allelic to a mutant allele cat1-1 previously isolated (Zimmermann et al., Mol. Gen. Genet. 151:95-103). Such mutants prevented derepression not only of the maltose catabolizing system, the selected property, but also of glyoxylate shunt and gluconeogenic enzymes. However, respiratory activities and invertase formation were not affected under derepressing conditions. cat3 mutants had the same phenotypic properties as cat1 mutants. This showed that carbon metabolism in yeast cells is under a very complex and ramified control of repressing and derepressing genes, which are interdependent.     

Arabidopsis sucrose synthase 2 and 3 modulate metabolic homeostasis and direct carbon towards starch synthesis in developing seeds

Two genes encoding sucrose synthase (SUS), namely SUS2 (At5g49190) and SUS3 (At4g02280), are strongly and differentially expressed in Arabidopsis seed. Detailed biochemical analysis was carried out in developing seeds 9–21 days after flowering (DAF) of wild type and two knockouts. SUS2 and SUS3 are not redundant genes since single knockouts show a phenotype in developing seeds. The mutants had 30–50% less SUS activity and therefore accumulated 40% more sucrose and 50% less fructose at 15 DAF. This did not affect the hexose-P pool, but led to 30–70% less starch in embryo and seed coat. Lipids were 55% higher in both mutants at 9–15 DAF. It seems that sucrolysis via SUS is not required for oil or protein synthesis but rather for channeling carbon toward ADP-glucose and starch in seeds. Metabolite profiling with GC–TOF revealed specific downstream changes in primary metabolism as a consequence of signaling or regulatory fine-tuning. While sucrose increased, hexoses and specific amino acids decreased reciprocally. There was a developmental shift regarding an earlier timing of dry weight accumulation, germinative maturity, oil deposition, sugar levels, transient starch buildup, and protein storage. Nevertheless, final seed size and composition were unaltered due to an earlier cessation of growth, thus giving rise to an apparent silent phenotype of mature mutant seeds. We conclude that SUS is important for metabolite homeostasis and timing of seed development, and propose that an altered sucrose/hexose ratio can modify carbon partitioning and the pattern of storage compounds in Arabidopsis.     

Nitrogen metabolite repression in Aspergillus nidulans

Summary In Aspergillus nidulans, mutations, designated areA^r, can result in the inability to utilise a wide variety of nitrogen sources including amino acids, purines, amides, nitrate, and nitrite, whilst not affecting growth on ammonium. Other allelic areA mutations, designated areA^d, lead to derepression of one or more activities which are ammonium repressible in wild type (areA⁺) strains, whilst not affecting their inducibility. Various areA mutations exhibit a wide variety of phenotypes: areA^r alleles can be temperature sensitive on some nitrogen sources while not on others, and different alleles can be temperature sensitive for utilisation of different nitrogen sources. areA^d alleles can be derepressed for one ammonium-repressible activity, be normally repressible for another, and lead to abnormally low levels for a third. Once again each areA^d allele has its own highly specific phenotype. The inability of areA^r strains to utilise most nitrogen sources is paralleled by low activities of certain ammonium-repressible enzymes. areA^r mutations appear to be epistatic to some but not all regulatory mutations leading to constitutive synthesis of inducible enzymes and also epistatic to gdhA mutations which lead both to loss of NADP-linked glutamate dehydrogenase and to derepression of ammonium-repressible activities. areA^r mutations do not interfere with repair of a large number of auxotrophies in double mutants. Furthermore, although areA^r mutations prevent utilisation of L-arginine, L-ornithine, and L--amino-n-butyrate as nitrogen sources, they do not prevent the metabolism of these compounds necessary for repairing auxotrophies for proline and isoleucine in the appropriate double mutants. Utilisation of acetamide and most amino acids as carbon or carbon and nitrogen sources is unaffected by areA^r mutations, and areA^r strains are able to utilise acetamide and L-proline (but not other amino acids) as nitrogen sources in the presence of non-catabolite-repressing carbon sources such as L-arabinose, glycerol, melibiose, and lactose. Suppressor mutations, designated creA^d, probably leading to loss of carbon catabolite repression, allow utilisation of acetamide and proline as nitrogen sources in areA^r double mutants in the presence of carbon catabolite-repressing carbon sources. creA^d mutations allow ethanol to serve as a source of acetate for pyruvate dehydrogenaseless (pdhA) strains in the presence of carbon catabolite-repressing carbon sources, whereas pdhA single mutants respond to ethanol as sole carbon source only in the presence of non-carbon catabolite-repressing carbon sources. Specific suppressor mutations, designated amd ^d and prn ^d, allow utilisation of acetamide or proline, respectively, in areA^r double mutants.The areA locus can be interpreted as specifying a protein which is capable of (and in most cases essential for) allowing the synthesis of a number of enzymes of nitrogen metabolism but which cannot function in the presence of ammonium (i.e., as specifying a positive regulatory element which mediates ammonium repression) although the possibility that the areA product also plays a negative regulatory role cannot at present be ruled out.