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Carbon repression in aspergilli

George J.G Ruijter, Jaap Visser
DOI: http://dx.doi.org/10.1111/j.1574-6968.1997.tb12557.x 103-114 First published online: 1 June 1997


Many microorganisms prefer easily metabolizable carbon sources over alternative, less readily metabolized carbon sources. One of the mechanisms to achieve this is repression of the synthesis of enzymes related to catabolism of the alternative carbon sources, i.e. carbon repression. It is now clear that in Aspergillus nidulans and Aspergillus niger the repressor protein CREA plays a major role in carbon repression. CREA inhibits transcription of many target genes by binding to specific sequences in the promoter of these genes. Unfortunately there is little information on other components of the signalling pathway that triggers repression by CREA. In this review we summarize the current understanding of carbon repression in aspergilli.

  • Carbon repression
  • Aspergillus
  • Glucose
  • Hexokinase
  • Cyclic AMP

1 Introduction

aspergilli, like many other microorganisms, adjust their carbon catabolism to prevailing conditions. One of the regulatory mechanisms involved in this adaptation is carbon repression. Readily metabolizable carbohydrates repress the synthesis of enzymes related to catabolism of alternative carbon sources ensuring preferential utilization of the most favored carbon source present. From the viewpoint of cellular physiology this is beneficial for two reasons. Firstly, the energetically most favorable carbon source is used and secondly, no energy is wasted on the synthesis of other catabolic systems.

As has been suggested by other authors, e.g. [1], the expression ‘carbon repression’ is used here instead of the frequently used term ‘carbon catabolite repression’, because the latter implies that catabolites are involved in the repression mechanism, which is unclear at present. Repression may be produced by various carbon sources, but glucose is probably the most repressive. Most experimental studies in fact concern the effects of glucose and this is probably the reason why the phrase ‘glucose repression’ is also utilized. Glucose repression is a specific case of the general phenomenon of carbon repression.

Amongst lower eukaryotes, carbon repression has been studied most extensively in Saccharomyces cerevisiae and Aspergillus nidulans (for reviews see [14]). In this minireview we summarize the current knowledge on carbon repression in aspergilli. Various aspects of the role of the repressor protein CREA, which mediates carbon repression in A. nidulans and A. niger, will be discussed, since this is addressed in most of the relevant literature. In addition we will consider other elements possibly involved in carbon repression.

2 Systems subject to carbon repression

Studies on carbon repression in aspergilli are abundant. Usually the observation that the activity of an enzyme is low after growth on a mixture of an inducing plus a repressing carbon source compared to growth on inducer only is taken as an indication of carbon repression. Such an observation does not prove that the biosynthesis of the enzyme studied is actually repressed. The repression effect may be indirect and due to, for example, lack of inducer formation [5]. In fact, only for a limited number of systems, e.g. ethanol [68] and proline [9, 10] catabolism, it has been shown clearly that the relevant genes are repressed. These systems will be discussed in more detail in Section 3.4.

Systems that are regulated by carbon repression can be divided into three groups on the basis of their metabolic function. The first and main group comprises genes encoding enzymes committed to catabolism of less preferred carbon sources of which a selection is given in Table 1. Most enzyme systems involved in degradation of polysaccharides, such as cellulose, pectin, xylan and araban, appear to be repressed by glucose.

View this table:

Catabolic systems subject to carbon repression in aspergilli

PectinPectate lyaseA. nidulans[11, 12]
Celluloseβ-GlucosidaseA. nidulans[13]
XylanEndo-xylanaseA. nidulans[14, 15]
A. tubingensis[16]
β-XylosidaseA. nidulans[17]
ArabanArabinofuranosidases A, BA. niger[18, 19]
l-Arabinosel-Arabinose reductase, l-arabitol dehydrogenaseA. niger[18]
ProteinExtracellular proteases PEPA, PEPB, PEPFA. niger[20, 21]
GlycerolGlycerol kinase, FAD-dependent glycerol-3-phosphate dehydrogenaseA. nidulans[22]
EthanolEthanol dehydrogenase, aldehyde dehydrogenase, ALCRA. nidulans[68]
ProlineProline permease, proline oxidaseA. nidulans[9, 10]

The second group includes genes encoding gluconeogenic and glyoxalate cycle enzymes. These genes have not been studied extensively in aspergilli. Actually it is unclear whether gluconeogenic genes are repressed at all. No data are available for fructose-1,6-bisphosphate phosphatase and Kelly and Hynes have reported that phosphoenolpyruvate carboxykinase is probably inducible and only weakly regulated by carbon repression [5]. The glyoxalate cycle enzymes isocitrate lyase and malate synthase are induced by acetate and repressed by glucose or sucrose. Malate synthase mRNA is barely detectable in wild-type A. nidulans cultured on acetate plus sucrose, whereas sucrose does not decrease transcription in an A. nidulans mutant possessing a hypofunctional CREA repressor protein [23]. Using lacZ as a reporter gene Bowyer et al. [24] showed that A. nidulans isocitrate lyase is repressed 3-fold by sucrose when acetate is present as an inducer. In a creA1 mutant sucrose repressed isocitrate lyase 2-fold, thus a mutation in CREA resulted in partial derepression.

The third group of genes subject to carbon repression are related to secondary metabolism. The best characterized system to date is penicillin production by A. nidulans. Penicillin formation is controlled mainly by extracellular pH [25]. At relatively alkaline pH carbon control is not operative, but at acidic pH media containing sucrose or glucose result in a lower level of penicillin accumulation compared to media containing lactose. It has been shown that transcription of ipnA, encoding isopenicillin N-synthetase, is regulated by carbon repression [25, 26]. The physiological rationale for carbon control on penicillin biosynthesis is not completely clear. Possibly production of an antibiotic, which inhibits growth of competitive microorganisms, is more relevant when carbon is limited than under conditions with carbon sources amply available.

In S. cerevisiae, enzymes related to citric acid cycle and respiratory chain are also repressed by glucose [1, 2]. For aspergilli, being obligate aerobes, this would not be expected and this is confirmed by Raitt et al. [27] who have shown that, in contrast to the situation in S. cerevisiae, cytochrome c synthesis is not repressed by glucose in A. nidulans.

3 The repressor protein CREA

3.1 Isolation of creA mutants

The A. nidulans creA gene was discovered in experiments aimed at selection of suppressor mutations for areA loss-of-function mutations [28]. AREA is a wide-domain regulator for nitrogen metabolism. It is a transcription activator protein; in the absence of preferred nitrogen sources, ammonia or glutamine, it is required for expression of genes related to utilization of other nitrogen sources. Some of these alternative nitrogen sources, such as acetamide and proline, are controlled by nitrogen and carbon repression as they can serve both as a nitrogen and as a carbon source. Relief of either nitrogen or carbon repression permits utilization of these compounds, but growth on a combination of a repressing nitrogen source and a repressing carbon source completely blocks proline and acetamide catabolism. Therefore areA null mutants are unable to use proline as a nitrogen source in the presence of a repressing carbon source, such as glucose. Arst and Cove [28] made use of this phenotype to isolate mutations that interfere with carbon repression designated creAd. Hynes and Kelly [29] employed the same approach using a combination of acetamide and sucrose.

Other strategies have also been used to isolate creA mutants. One of these uses A. nidulans pdhA mutants, lacking a functional pyruvate dehydrogenase complex. A pdhA mutant is unable to make acetyl CoA from pyruvate and therefore cannot grow on carbon sources that are metabolized via glycolysis. It can, however, grow on acetate or ethanol as these C2 carbon sources are metabolized via acetyl CoA. A number of creA mutants were isolated as pseudorevertants of pdhA on media containing glucose and ethanol [30]. Normally, glucose represses ethanol catabolism, but repression is (partially) abolished in creA mutants.

The areA and pdhA mutants have enabled a classification of the effectiveness of several carbon sources to provoke carbon repression [28]. This classification is based on the extent to which various carbon sources allow utilization of proline in an areA background and ethanol in a pdhA background. d-Glucose, d-xylose, sucrose and acetate are strongly repressing, d-mannose, maltose, d-fructose, d-mannitol and d-galactose are intermediate and glycerol, melibiose, lactose, l-arabinose and ethanol are non or weakly repressing (derepressing) carbon sources. Besides the type, also the concentration of carbon source is relevant, since a relatively low concentration (0.05–0.1%) of glucose and sucrose decreases their ability to repress. It must be mentioned that this classification is valid for CREA mediated repression, but might be different for other repression mechanisms (see also Section 4).

The most severe A. nidulans creA mutation to date is creA30 [31]. The creA30 mutation is caused by a pericentric inversion of chromosome I resulting in a truncated creA gene. The mutant was isolated as a suppressor of frA on mannitol plus arabinose. A. nidulans frA mutants lack hexokinase and are unable to utilize fructose or its metabolic precursors, such as mannitol [32]. Although mannitol cannot be metabolized, it is still able to repress catabolism of arabinose in a frA background, but apparently not in a frA creA30 double mutant.

Recently A. niger creA mutants have been isolated (Ruijter et al., unpublished) using a strategy based on selection of pseudorevertants of areA1 on a medium containing 4-aminobutyric acid (GABA) and d-glucose. Most of the mutations also resulted in derepression of proline and alanine utilization in the presence of d-glucose and the mutants could be transformed back to the areA phenotype with the A. niger creA gene. Several catabolic systems were tested for derepression and it appeared that arabinofuranosidases, enzymes of l-arabinose catabolism (Ruijter et al., unpublished) and several extracellular proteases (Fraissinet-Tachet et al., unpublished) were derepressed. As an illustration, Fig. 1 clearly shows derepression of the extracellular proteases PEPA and PEPB in an A. niger creA2 mutant.

Figure 1

Derepression of extracellular proteases PEPA and PEPB in an A. niger creA mutant. Cells were grown overnight in complete medium, washed and transferred to minimal medium supplemented with the following carbon sources: glycerol (lane 1); no carbon source (lane 2); glucose (lane 3); elastin (lane 4); elastin+glucose (lane 5). After 6–8 h of incubation total RNA was isolated and subjected to Northern analysis. In wild-type A. niger elastin induces expression of both pepA and pepB (lane 4), whereas glycerol and the absence of a carbon source result in a low expression level (lanes 1 and 2). Glucose represses both proteases in wild-type A. niger (lanes 3 and 5). In a mutant carrying the creA2 allele the expresion level of both proteases is high, regardless of the presence or type of carbon source.

3.2 Properties of creA mutants

The various creA alleles display non-hierarchical heterogeneity. This means that various creA alleles are not just different in the extent to which they cause derepression of a certain target system, but that derepression also varies with different target systems. This is exemplified by studies on arabinase expression in A. nidulans by Van der Veen et al. (Table 2) [18]. The creA1 mutation resulted in elevated arabinofuranosidase B activity during growth on l-arabitol, whereas endo-arabinase was not affected. A strain carrying creA2 behaved exactly opposite. Together with the finding that creA mutations are recessive [28, 29] the allelic heterogeneity was explained by a direct and negative role for the creA product in carbon repression, which has been confirmed in several studies now (see Section 3.3Section 3.4).

View this table:

Activities of α-l-arabinofuranosidase and endo-arabinase in culture filtrates of A. nidulans wild-type (WG096) and strains carrying various creA mutations

EnzymeCarbon sourceStrains
α-l-Arabinofuranosidase Bl-Arabinose8.210.18.570.1147.3
  • Carbon sources were present at a concentration of 1% (w/v). Activities are expressed in mU/ml culture volume. Data were taken from [18].

An interesting observation from the results of Van der Veen et al. is the increased inducibility of arabinases in creA mutants (Table 2) [18]. Arabinases are induced by l-arabinose or l-arabitol and are subject to repression by d-glucose. One would expect to find derepression of the enzymes in media containing inducing plus repressing carbon source, but the effects of creA mutations on arabinase expression were most manifest in the presence of just inducer. This might be explained in two ways. Perhaps l-arabinose or l-arabitol not only act as an inducer, but also repress arabinase synthesis via CREA. An alternative explanation would be that CREA has some repressor activity even in the absence of a repressing carbon source [7]. Similar effects have been observed in other systems, such as ethanol dehydrogenase [7, 8], glycerol kinase [22], endo-xylanase [15] and pectate lyase [11]. Interestingly, recent molecular studies employ in vitro binding studies of CREA to promoter fragments [6, 7, 25] and demonstrate that CREA does bind to promoter sites in the absence of activation by a repressing carbon source, although it must be stated that the concentrations of both CREA and target DNA are probably far from physiological in these experiments.

Besides derepression of certain target systems, creA mutants have a few other properties worth mentioning. A distinct characteristic of severe creA mutants is compact colony morphology and reduced sporulation on solid media [29, 30]. During submerged growth the biomass level of creA mutants may be lower than obtained for wild-type strains. These effects may be related to recent findings of Van der Veen et al. [33]. Upon examination of glycolysis and polyol metabolism in A. nidulans wild-type and in a mutant carrying creA30 after growth on d-glucose, it was observed that hexokinase and mannitol-1-phosphate dehydrogenase activity were increased in the creA30 mutant, whereas phosphofructokinase, pyruvate kinase and citrate synthase activity were decreased. These alterations in enzyme activities resulted in concentration changes of glycolytic metabolites. In addition it was found that the creA30 strain accumulated much higher polyol levels in the medium. It is presently not clear whether the observed changes in primary metabolism are a direct or an indirect effect of creA30, but they emphasize the pleiotropic effects of creA mutations and suggest a broader function for CREA than just its role as a mediator of carbon repression.

3.3 The CREA protein

The A. nidulans creA gene has been cloned and characterized by Dowzer and Kelly [34, 35]. These authors have made use of the fact that creA mutants are sensitive to allylalcohol in the presence of a repressing carbon source. Under the same conditions wild-type A. nidulans is resistant due to tight repression of alcohol dehydrogenase, which converts allylalcohol into the toxic acrolein. This difference in phenotype was used to clone creA by complementation. A mutant carrying creA204 was transformed with a plasmid library and colonies were selected on 1% sucrose plus 2.5 mM allylalcohol. From one such colony a plasmid could be rescued that carried the creA gene.

A. nidulans creA encodes a protein of 415 amino acids containing several features characteristic for DNA binding proteins, such as zinc fingers, an alanine rich region and frequently appearing SPXX and TPXX motifs (Fig. 2) [35]. The CREA protein has two zinc finger structures of the Cys2His2 type very similar to the zinc fingers of MIG1, a repressor protein in the main glucose repression pathway of S. cerevisiae[36].

Figure 2

Schematic representation of A. nidulans CREA. Features in the amino acid sequence that are probably important for CREA function are indicated by blocks and described at the top. Mutations in several creA alleles are given at the bottom.

Recently, Shroff et al. [37] have sequenced a number of creA alleles. Three of the alleles analyzed have missense mutations in the zinc finger domain (Fig. 2). These alleles result in partial derepression of the target systems analyzed and most probably encode CREA proteins with a reduced binding affinity for their recognition sites in target promoters. Four other mutations result in truncations of CREA between the zinc finger domain and the C-terminus of the protein (Fig. 2). The residual length of these CREA proteins varies from 130 amino acid residues in creA30 to 281 amino acid residues in creA221. Derepression of alcohol dehydrogenase is strongest in creA30 suggesting a correlation between residual length of the truncated CREA and its phenotype, but with these four alleles it is not yet possible to identify a domain specifically involved in repression.

Dowzer and Kelly attempted to inactivate creA in A. nidulans by gene disruption, but only succeeded to do so in a heterokaryon or in a diploid strain [35]. Deletion of creA was not possible in a haploid strain. Haploid conidiospores obtained from heterokaryons and carrying a disrupted creA were able to germinate, but were unable to grow further [35]. Thus, disruption of creA in A. nidulans appeared to be lethal. This is in line with the finding that none of creA mutations isolated by classical genetic techniques were complete loss-of-function mutations and with the pleiotropic effects of severe creA mutations. The lethality of a creA null allele can be explained in two ways. Either complete derepression of particular or maybe all systems under CREA control is lethal or CREA has some positive function in addition to its negative role in carbon repression [35].

The A. niger creA [38] and the Trichoderma reesei homologue cre1 [39, 40] have now also been cloned. Interestingly, the T. reesei mutant rutC30, which is well-known for its overproduction of cellulolytic enzymes, is actually a cre1 mutant [40]. rutC30 contains a truncated CRE1 with only one zinc finger. Introduction of intact cre1 into rutC30 abolished overproduction of cellulases. A. niger CREA is a 427 amino acid protein which has 82% overall identity with A. nidulans CREA. Near the C-terminus of the proteins a stretch of approximately 40 amino acids is completely conserved and was found to be similar to a part of another S. cerevisiae protein thought to be involved in carbon repression, RGR1 [38]. However, Strauss et al. [39] concluded on the basis of an extensive database search that this domain is a general feature of a particular class of eukaryotic DNA-binding proteins and not specific for RGR1. This hypothesis is substantiated by the recent finding that RGR1 is in fact a component of the RNA polymerase complex and plays a more general role in transcription regulation [41].

In A. nidulans the level of creA mRNA is higher in mycelium cultured on 1% glycerol or 1%l-arabinose than in mycelium grown on 1% glucose [34, 37]. Similar results have been obtained with T. reesei[40]. This result suggests autoregulation, i.e. CREA represses its own synthesis to some extent. Two recent observations by Shroff et al. [37] are in agreement with this notion. Firstly, in strains carrying creA mutations expression of creA is always at the higher level, regardless of the growth conditions. Secondly, the promoter of creA contains a number of putative CREA binding sites. Repression of creA by glucose is unexpected, since the presence of glucose is a situation that requires CREA for repression of many target systems. Possibly, these observations are related to the proposed activator function of CREA [35, 37, 40]. Such an explanation assumes that this activator function requires more CREA under non-repressing conditions than under repressing conditions.

3.4 Mechanism of repression by CREA

Already 20 years ago the group of Arst postulated, on the basis of genetic data, that the creA gene product most likely was a repressor protein [42], but it is only a few years since this was confirmed by molecular studies. CREA binds to specific short sequences in the promoter of target systems and this binding prevents transcription of these target genes. This has been rigorously shown for the A. nidulans alc and prn systems, involved in catabolism of ethanol and proline respectively and will be discussed in the following paragraphs. An important tool in these studies has been a fusion protein of the Schistosoma japonicum glutathione S-transferase and the N-terminus of CREA, comprising the zinc fingers and the alanine-rich region (GST::CREA). The GST::CREA fusion protein was purified and used in in vitro binding assays.

Expression of genes from the prn cluster is regulated by PRNA, the transcription activator mediating proline induction, and by AREA and CREA. The prnB and prnD genes, encoding proline permease and proline oxidase respectively, are transcribed divergently and the intergenic region is crucial for regulation of expression. Expression of prnA is constitutive, it is not regulated by CREA, but it has been shown by Cubero and Scazzocchio [10] that carbon repression of the prnBD genes is effected by binding of CREA to the prnBD intergenic region. They identified seven GST::CREA binding sites in the prnBD intergenic region by gel retardation and DNase I footprinting experiments. Two of the sites were separated by only one base, and contained previously isolated mutations that result in specific derepression of the prn cluster [9, 10]. These cis-acting mutations prevented in vitro binding of GST::CREA and demonstrate that the pair of CREA binding sites is active in vivo [10].

Whereas carbon repression of proline catabolism is only at the level of prnBD, it has been shown by the group of Felenbok that in the case of the ethanol regulon, CREA operates at two levels [6, 7]. It represses both alcR, the transcription activator for alc genes and the structural genes alcA, encoding alcohol dehydrogenase I and aldA, encoding aldehyde dehydrogenase. Gel retardation and DNase I footprinting experiments revealed four GST::CREA binding sites in the alcR promoter and two sites in the alcA promoter [6]. The alcR promoter was subsequently modified in two different ways. Firstly, part of the alcR promoter, containing one of the CREA binding sites was deleted [6] and secondly, two guanidines in the same CREA site were substituted by adenines [7]. In both cases alcR was derepressed proving that this CREA site was functional. In transformants containing either of these two altered alcR constructs and in transformants in which alcR was expressed constitutively using the promoter of glyceraldehyde-3-phosphate dehydrogenase, alcA was only partly derepressed, indicating that CREA also repressed alcA directly [7]. This was confirmed with a creA30 mutant, in which alcR, alcA and aldA were almost completely derepressed. Thus, in ethanol catabolism a double-lock mechanism operates with CREA repressing both alcR, preventing stimulation of transcription of the structural alc genes, and alcA and aldA themselves.

The binding sites of ALCR and CREA in the alcR promoter overlap and it was found that the two proteins actually compete for binding to the promoter [7]. These results have been important in formulating a model for regulation of the alc system, which might be valid in general for catabolic systems [7, 8]. In the absence of ethanol, ALCR is not active in stimulating alc expression and CREA will repress alc, in particular in the presence of glucose, but probably also in the presence of weak repressing carbon sources and maybe even in the absence of a carbon source. When both ethanol and glucose are present there will be a smooth transition between repression and induction. Repression by CREA is relieved upon consumption of the glucose due to a decrease in the affinity of CREA for its binding site and CREA will then be replaced by ALCR resulting in induction of alc genes.

The DNase I footprinting experiments have permitted definition of a consensus binding site for CREA, 5′-SYGGRG-3′[6, 10]. Not all possible sequences included in this consensus are always functional, i.e. the binding affinity for certain variants depends on the sequences outside the hexanucleotide [10]. In addition, it was found that CREA also binds to non-consensus sites in the A. nidulans ipnA promoter, but that this binding is dependent on the presence of an adjacent CREA binding site [43]. The possibility that CREA binds as a dimer is substantiated by the presence of two proximate sites in the alcR, alcA and prnBD promoters [43]. However, no dimerization motif could be found in CREA and it might be that binding of a CREA dimer is due to the use of the GST::CREA fusion protein, since GST is a dimeric protein. It has been shown that the GST moiety introduces significant bias in determination of specific DNA targets in the case of ALCR (Lenouvel et al., unpublished).

4 Other elements involved in carbon repression

CREA is the only regulatory protein for which mediation of carbon repression has been demonstrated in aspergilli. Although it is very likely that other proteins are involved, there is surprisingly little information on this.

In addition to creA a few other cre mutations were isolated by Hynes and Kelly [29, 44]. Mutants carrying creB or creC showed derepression of a number of enzymes including alcohol dehydrogenase, acetamidase and α-glucosidase, but mutations in creB and creC also caused other defects such as difficulties to grow on certain carbon sources, e.g. l-proline, d-quinate and d-glucuronate, and relatively low levels of particular catabolic enzymes, such as β-galactosidase and d-quinate dehydrogenase [29]. Mutations in creB and creC have subsequently also been shown to cause derepression of β-glucosidase [13] and arabinases [18]. creB and creC were recessive to the wild-type allele in diploids and exhibited hierarchical heterogeneity [3, 29], in contrast to the different creA alleles. Therefore the role of creB and creC is likely to be indirect. Their products may have a function in the signal transduction pathway for carbon repression upstream of CREA and/or interact with CREA. The pleiotropic effects of mutations in creB and creC are difficult to explain and may indicate a more general role in carbon metabolism.

Kelly and Hynes [44] isolated a suppressor of the effects of creC27 on acetamidase expression, designated creD34 and an interesting property of this mutation is that it not only suppresses creC27, but also creA204 and creB15. This mutation suggests some interaction between the products of the four cre genes, but the role of creB, creC and creD in carbon repression is at present unclear.

In S. cerevisiae a large number of proteins involved in carbon repression have been identified. As such this yeast serves as a model organism, although it might be that the mechanism of carbon repression in aspergilli is very different. For example, S. cerevisiae hexokinase PII is involved in glucose repression [2]. In mutants lacking hexokinase PII a number of catabolic systems are relieved from glucose repression. In contrast, the absence of hexokinase activity in an A. nidulans frA mutant did not interfere with glucose repression of the enzymes involved in alcohol and l-arabinose catabolism [32]. This suggests that, unlike the situation in yeast, the A. nidulans hexokinase is not involved in glucose repression.

Glucose represses genes encoding respiratory proteins, such as cyc1, in S. cerevisiae[1, 2]. This repression is mediated by the HAP2/HAP3/HAP4 complex. In the presence of glucose hap4 is repressed five-fold and the reduced HAP4 level then results in decreased expression of cyc1. The HAP2/HAP3/HAP4 complex binds to CCAAT sequences in the promoters of their target genes. Recently, a CCAAT binding factor was found in A. nidulans. Gel retardation experiments showed binding of a protein (or a protein complex) to a CCAAT motif in the A. nidulans amdS promoter [45]. This CCAAT element is known to be required for derepression of amdS. Subsequently, it was shown by Bonnefoy et al. [46] that in S. cerevisiae the HAP2/HAP3/HAP4 complex is able to recognize the amdS CCAAT sequence and regulates expression of a reporter gene from this sequence. Thus, CCAAT elements, which have also been observed in promoters of other Aspergillus genes, appear to regulate expression in response to carbon availability. Possibly, the CCAAT binding factor found in A. nidulans resembles the yeast HAP2/HAP3/HAP4 complex.

Up to now there is little evidence for CREA-independent mechanisms for carbon repression in aspergilli. One such case is expression of genes related to penicillin biosynthesis in A. nidulans. At acidic pH sucrose and glucose repress A. nidulans ipnA encoding isopenicillin N synthetase [26]. Several observations suggest a carbon repression mechanism for ipnA which is independent of CREA. Firstly, in strains carrying mutations in either creA, creB or creC the ipnA transcript level is only moderately increased under repressing conditions [26, 47], although this might be explained by allele specific effects. Secondly, the region in the ipnA promoter, that has been shown to be involved in carbon regulation, contains only one CREA site and deletion of this binding site did not affect carbon regulation of ipnA expression [25]. Finally, the classification of repressing and derepressing carbon sources as described earlier does not hold for regulation of ipnA expression. Glycerol, which is considered a derepressing carbon source, strongly represses ipnA, whereas acetate acts in an opposite manner [25]. These observations suggest the existence of an alternative carbon repression mechanism of which the components still remain to be identified.

5 Is cyclic AMP involved?

In S. cerevisiae the addition of glucose to cells growing on non-fermentable carbon sources or to stationary phase cells results in a transient increase in the level of cyclic AMP (cAMP). This temporary increase in cAMP concentration is believed to signal the presence of easily fermentable carbon sources in order to change metabolism accordingly [48]. The cAMP signal does not play a role in the main glucose repression pathway of S. cerevisiae.

The level of cAMP in A. nidulans appears to be higher when glucose is present at high concentration than after depletion of the glucose [49] and this compares to the situation in yeast. It is, however, not known what the cAMP levels would be in cells growing on weakly repressing carbon sources or upon a pulse of glucose to such cells. In a few studies the effect of exogenous addition of cAMP or its analog dibutyryl-cAMP on carbon repression of various systems was tested. Addition of 20 mM cAMP did not relieve β-glucosidase synthesis from repression by glucose in A. nidulans[13]. Arst and Bailey found that cAMP, dibutyryl-cAMP or inhibitors of phosphodiesterase, the enzyme catalyzing degradation of cAMP, did not affect carbon repression in A. nidulans areA or pdhA mutants [42]. However, these authors suspected that cAMP and dibutyryl-cAMP did not enter the cells, since these components could neither supplement adenine auxotrophy nor serve as nitrogen source. For a few other aspergilli addition of cAMP did have effects on the synthesis of extracellular enzymes. Addition of 0.1 mM dibutyryl-cAMP, but not cAMP itself, could partially relieve repression of xylanase and β-xylosidase by glucose [50]. However, in this case synthesis of these enzymes was also stimulated by dibutyryl-cAMP in the absence of glucose and the effect might therefore be independent from carbon repression. Whereas these studies are concerned with relief from carbon repression, an opposite effect was found in the case of Aspergillus awamori glucoamylase [51]. Addition of 4 mM cAMP to mycelium growing on starch resulted in a two-fold decrease in the glucoamylase mRNA level. This is in fact the only study where transcription was investigated. In the other cases mentioned and in a number of other investigations which are not cited here, it is unclear whether the effect of cAMP, if there is any at all, is on expression or on another level, such as protein modification.

Notably, sequences similar to cAMP responsive elements as observed in promoters of higher eukaryotic genes, are also found in fungal promoters, e.g. of arabinases of A. niger[19]. It remains to be shown whether these elements are functional. Furthermore, we have recently cloned A. niger pkaC, encoding the catalytic subunit of protein kinase A (Bencina et al., in press). In the course of these investigations we found that both moderate overexpression of pkaC, probably resulting in uncontrolled PKAC activity, and addition of 5 mM cAMP have severe effects on carbon metabolism. Thus, cAMP does play a role in regulation of carbon metabolism by protein phosphorylation, but this signalling pathway might be distinct from carbon repression via CREA.

6 Concluding remarks

Presently, the mechanism of repression by CREA is understood in great detail. However, we are completely unaware of the signal transduction pathway for carbon repression in aspergilli. The presence of a favored carbon source, such as glucose, must somehow be signalled to CREA to activate it, subsequently resulting in repression. Uptake of sugars appears to be required for this signalling, since the screening procedures that produced the creA mutants also resulted in selection of sugar uptake mutants [30]. However, hexokinase does not seem to be involved in carbon repression in A. nidulans[32], suggesting that the signalling pathway is distinct from the main glucose repression pathway in yeast. In addition, no mutations resulting in permanent repression, such as in the SNF1 protein kinase of S. cerevisiae, have been described for aspergilli, but this might be due to biased selection procedures.

The simplest model would be activation of CREA by binding of a sugar to it. However, the observation that different sugars are capable of eliciting carbon repression is perhaps the best evidence that components other than CREA are required, since it is difficult to imagine that CREA would be able to bind so many different sugars.


G.R. is financially supported by the Dutch Ministry of Economic Affairs, the Ministry of Education, Culture and Science, The Ministry of Agriculture, Nature Management and Fishery within the framework of an industrially relevant research programme of the Netherlands Association of Biotechnology Centres (ABON). The authors thank Drs. M.A. Peñalva and F. Lenouvel for critical reading of the manuscript.


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