RcoA has pleiotropic effects on Aspergillus nidulans cellular development

Aspergillus nidulans rcoA encodes a member of the WD repeat family of proteins. The RcoA protein shares sequence similarity with other members of this protein family, including the Saccharomyces cerevisiae Tup1p and Neurospora crassa RCO1. Tup1p is involved in negative regulation of an array of functions including carbon catabolite repression. RCO1 functions in regulating pleiotropic developmental processes, but not carbon catabolite repression. In A. nidulans, deletion of rcoA (DeltarcoA), a recessive mutation, resulted in gross defects in vegetative growth, asexual spore production and sterigmatocystin (ST) biosynthesis. Expression of the asexual and ST pathway-specific regulatory genes, brlA and aflR, respectively, but not the signal transduction genes (i.e. flbA, fluG or fadA) regulating brlA and aflR expression was delayed (brlA) or eliminated (aflR) in a DeltarcoA strain. Overexpression of aflR in a DeltarcoA strain could not rescue normal expression of downstream targets of AflR. CreA-dependent carbon catabolite repression of starch and ethanol utilization was only weakly affected in a DeltarcoA strain. The strong role of RcoA in development, vegetative growth and ST production, compared with a relatively weak role in carbon catabolite repression, is similar to the role of RCO1 in N. crassa.     

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.     

The Aspergillus nidulans xprF gene encodes a hexokinase-like protein involved in the regulation of extracellular proteases

The extracellular proteases of Aspergillus nidulans are produced in response to limitation of carbon, nitrogen, or sulfur, even in the absence of exogenous protein. Mutations in the A. nidulans xprF and xprG genes have been shown to result in elevated levels of extracellular protease in response to carbon limitation. The xprF gene was isolated and sequence analysis indicates that it encodes a 615-amino-acid protein, which represents a new type of fungal hexokinase or hexokinase-like protein. In addition to their catalytic role, hexokinases are thought to be involved in triggering carbon catabolite repression. Sequence analysis of the xprF1 and xprF2 alleles showed that both alleles contain nonsense mutations. No loss of glucose or fructose phosphorylating activity was detected in xprF1 or xprF2 mutants. There are two possible explanations for this observation: (1) the xprF gene may encode a minor hexokinase or (2) the xprF gene may encode a protein with no hexose phosphorylating activity. Genetic evidence suggests that the xprF and xprG genes are involved in the same regulatory pathway. Support for this hypothesis was provided by the identification of a new class of xprG(-) mutation that suppresses the xprF1 mutation and results in a protease-deficient phenotype.     

ADHII in Aspergillus nidulans is induced by carbon starvation stress

In Aspergillus nidulans there are three NAD(+)-dependent alcohol dehydrogenases (ADHs) that are capable of utilizing ethanol as a substrate. ADHI is the physiological enzyme of ethanol catabolism and ADHIII is induced under conditions of anaerobiosis. The physiological role of ADHII (structural gene alcB) is unknown. We have measured beta-galactosidase in a transformant with an alcB::lacZ fusion and have shown that alcB is maximally expressed under conditions of carbon starvation. The behavior of the alcB::lacZ transformant suggests a hierarchy of repressing carbon sources characteristic of repression by the general carbon catabolite repressor protein, CreA, but in a creA(d)30 background the transformant shows only partial derepression of beta-galactosidase on 1% glucose compared to the creA+ strain. Our results suggest that, in addition to carbon catabolite repression acting via CreA, a CreA-independent mechanism is involved in induction of alcB on carbon starvation.     

Glycolytic enzymes and intermediates in carbon catabolite repression mutants of Saccharomyces cerevisiae

Summary Glycolytic parameters were determined in recessive yeast mutants with partial defects in carbon catabolite repression. Specific activities of pyruvate kinase and pyruvate decarboxylase in glucose grown cells of all mutant and wild type stains were 4–5 times higher than in ethanol grown cells. Mutants of gene HEX1 had a reduced hexose phosphorylating activity on allmedia wheras those of gene HEX2 had elevated levels but only in glucose grown cells. Mutants of gene CAT80 were normal in this respect. All other glycolytic enzymes were normal in all mutants. This was also true for glycolytic intermediates. Only hexlmutants showed a reduced fermentation of repressing sugars. The three genes appear to be involved in catabolite repression of several but not of all repressible enzymes. Even though all three types of mutants show a limited overlap in their effects on certain enzymes, they still are distinctly different in their action spectra. Carbon catabolite repression apparently does not depend on the sole accumulation of glycolytic intermediales. The activity of the products of the three genes HEX1, HEX2 and CAT80 are required directly or indirectly for triggering carbon catabolite repression. Even a small segment of carbon catabolite repression is controlled by several genes with regulatory functions indicating that the entire regulatory circuit is highly complex.     

CcpA-mediated repression of Clostridium difficile toxin gene expression

The presence of glucose or other rapidly metabolizable carbon sources in the bacterial growth medium strongly represses Clostridium difficile toxin synthesis independently of strain origin. In Gram-positive bacteria, carbon catabolite repression (CCR) is generally regarded as a regulatory mechanism that responds to carbohydrate availability. In the C. difficile genome all elements involved in CCR are present. To elucidate in vivo the role of CCR in C. difficile toxin synthesis, we used the ClosTron gene knockout system to construct mutants of strain JIR8094 that were unable to produce the major components of the CCR signal transduction pathway: the phosphotransferase system (PTS) proteins (Enzyme I and HPr), the HPr kinase/phosphorylase (HprK/P) and the catabolite control protein A, CcpA. Inactivation of the ptsI, ptsH and ccpA genes resulted in derepression of toxin gene expression in the presence of glucose, whereas repression of toxin production was still observed in the hprK mutant, indicating that uptake of glucose is required for repression but that phosphorylation of HPr by HprK is not. C. difficile CcpA was found to bind to the regulatory regions of the tcdA and tcdB genes but not through a consensus cre site motif. Moreover in vivo and in vitro results confirmed that HPr-Ser45-P does not stimulate CcpA-dependent binding to DNA targets. However, fructose-1,6-biphosphate (FBP) alone did increase CcpA binding affinity in the absence of HPr-Ser45-P. These results showed that CcpA represses toxin expression in response to PTS sugar availability, thus linking carbon source utilization to virulence gene expression in C. difficile.     

Corynebacterium diphtheriae: a PTS view to the genome

We have surveyed the publicly available genome sequence of Corynebacterium diphtheriae (www.sanger.ac.uk) to identify components of the phosphotransferase system (PTS), which plays a central role in carbon metabolism in many bacteria. Three gene loci were found to contain putative pts genes. These comprise: (i) the genes of the general phosphotransferases enzyme I (ptsI) and HPr (ptsH), a fructose-specific enzyme IIABC permease (fruA), and a fructose 1-phosphate kinase (fruK); (ii) a gene that encodes an enzyme IIAB of the fructose/mannitol family, and a novel HPr-like gene, ptsF, that encodes an HPr domain fused to a domain of unknown function; (iii) and a gene for a glucose-specific enzyme IIBCA (ptsG). A search for genes that may be putative PTS-targets or that may operate in general carbon regulation revealed a possible regulatory gene encoding an antiterminator protein downstream from ptsG. Furthermore, genes were detected encoding glycerol kinase, glucose kinase, and a homologue of the global activator of carbon catabolite repression in Escherichia coli, CAP. The possible significance of these observations in carbon metabolism and the novel features of the detected genes are discussed.     

The genes gmdA, encoding an amidase, and bzuA, encoding a cytochrome P450, are required for benzamide utilization in Aspergillus nidulans

Two unlinked loci, gmdA and bzuA, have previously been identified as being required for the utilization of benzamide as the sole nitrogen source by Aspergillus nidulans. We have cloned each of these genes via direct complementation. The gmdA gene encodes a predicted product belonging to the amidase signature sequence family that displays similarity to AmdS from A. nidulans. However, identity is significantly higher to the amdS gene from Aspergillus niger. The bzuA gene encodes a protein belonging to the cytochrome P450 superfamily and is orthologous to the benzoate para-hydroxylase-encoding gene bphA of A. niger. The bzuA1 mutation prevents the use of benzoate as a carbon source and intracellular accumulation of benzoate results in growth inhibition on benzamide. Northern blot analysis has shown that gmdA expression is subject solely to AreA-dependent nitrogen metabolite repression while bzuA is strongly benzoate inducible and subject to CreA-mediated carbon catabolite repression and a probable inactivation of benzoate induction by glucose. Fluorescence microscopy of a fusion of the N-terminal end of BzuA to green fluorescent protein revealed that this protein localizes to the endoplasmic reticulum.     

Genetic and biochemical evidence for hexokinase PII as a key enzyme involved in carbon catabolite repression in yeast

Summary Mutants with reduced hexokinase activity previously isolated as resistant to carbon catabolite repression of invertase and maltase (Zimmermann and Scheel, 1977) were allele tested with mutant strains of Lobo and Maitra (1977) which had defects in one or several of the genes coding for glucokinase and the two unspecific hexokinases. It could be demonstrated, that the mutation abolishing carbon catabolite repression had occurred in a gene allelic to the structural gene of hexokinase PII. Moreover, the defective mutant allele for hexokinase PII isolated by Lobo and Maitra (1977) was also defective in carbon catabolite repression. Neither glucokinase nor hexokinase PI showed any effect on this regulatory system. Biochemical analysis in crude extracts also showed altered kinetic properties of hexokinases in the hex1 mutants. The results directly support the hypothesis previously put forward, that one of the hexokinases is not only active as a catalytic, but also as a regulatory protein.     

A role for creD, a carbon catabolite repression gene from Aspergillus nidulans, in ubiquitination

In Aspergillus nidulans, it is known that creB encodes a deubiquitinating enzyme that forms a complex with the WD40 motif containing protein encoded by creC, that mutations in these genes lead to altered carbon source utilization and that the creD34 mutation suppresses the phenotypic effects of mutations in creC and creB. Therefore, creD was characterized in order to dissect the regulatory network that involves the CreB-CreC deubiquitination complex. CreD contains arrestin domains and PY motifs and is highly similar to the Rod1p and Rog3p proteins from Saccharomyces cerevisiae. An additional gene was identified in the A. nidulans genome that also encodes an arrestin and PY motif-containing protein, which we have designated apyA, and thus two similar proteins also exist in A. nidulans. In S. cerevisiae, Rod1p and Rog3p interact with the ubiquitin ligase Rsp5p, and so the A. nidulans homologue of Rsp5p was identified, and the gene encoding this HECT ubiquitin ligase was designated hulA. CreD and ApyA were tested for protein-protein interactions with HulA via the bacterial two-hybrid system, and ApyA showed strong interaction, and CreD showed weak interaction, with HulA in this system.