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  • 1
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: 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 (ΔrcoA), 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 ΔrcoA strain. Overexpression of aflR in a ΔrcoA 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 ΔrcoA 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.
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  • 2
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: DNA fragments containing genetic information for five secretion-related small GTPases of Aspergillus niger (srgA–E) were isolated and identified as members of different Rab/Ypt subfamilies. This isolation and the search for similar sequences in fungal genomic and EST databases showed that, in contrast to Saccharomyces cerevisiae, filamentous fungi also possess homologues of mammalian Rab2 GTPases. Multiple transcripts with unusually long 5′ and 3′ untranslated regions were found for all srg genes. Their level of expression was independent of the type of carbon source used for growth. Although the transcripts of srgA and srgB were abundant to the same extent throughout the cultivation, that of the other genes peaked during the early growth phase and then declined. Two genes, srgA and srgB, were characterized further. The protein encoded by srgA exhibited relatively low identity (58%) to its closest S. cerevisiae homologue SEC4, whereas the protein encoded by srgB showed 73% identity with S. cerevisiae YPT1. In contrast to other SEC4 homologues, srgA was unable to complement an S. cerevisiae sec4 mutant, and its disruption was not lethal in A. niger. SrgA mutants displayed a twofold increase in their hyphal diameter, unusual apical branching and strongly reduced protein secretion during growth on glucose.
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  • 3
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: The creA gene of A. nidulans encodes a wide-domain regulatory protein mediating carbon catabolite repression. Northern blot analysis of creA mRNA revealed a complex expression profile: the addition of monosaccharides to a carbon-starved culture of A. nidulans provoked a strong transient stimulation of creA transcript formation within a few minutes. In the case of repressing carbon sources, creA mRNA levels were subsequently downregulated, whereas the high creA mRNA levels were maintained in a creA mutant strain and in the presence of derepressing monosaccharides. A high creA transcript level is essential to achieve carbon catabolite repression and is dependent on glucose transport and, at least partially, on the creB gene product. Subsequent downregulation of creA mRNA levels, on the other hand, is typical of carbon catabolite repression and requires a functional CreA recognition site in the creA promoter (and thus involves autoregulation) and formation of glucose-6-phosphate. Despite the presence of continuing high transcript levels of creA in the presence of derepressing carbohydrates, EMSA demonstrated the presence of only low levels of a CreA–DNA complex in respective cell-free extracts. Upon transfer of carbon catabolite derepressed mycelia to catabolite-repressing conditions, a CreA–DNA complex is formed, and this process is dependent on de novo protein synthesis.
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  • 4
    Electronic Resource
    Electronic Resource
    Oxford, UK : Blackwell Publishing Ltd
    Molecular microbiology 21 (1996), S. 0 
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: The filamentous fungus Trichoderma reesei forms two specific, xylan-inducible xylanases encoded by xyn1 and xyn2 to degrade the β-1,4-d-xylan backbone of hemicelluloses. This enzyme system is formed in the presence of xylan, but not glucose. The molecular basis of the absence of xylanase I formation on glucose was the purpose of this study. Northern blotting of the xyn1 transcript as well as the use of the Escherichia coli hygromycin B phosphotransferase-encoding gene (hph) as a reporter consistently showed that the basal expression of xyn1 was affected by glucose, whereas its induction by xylan remained uninfluenced. The repression of basal xyn1 transcription is mediated by the carbon catabolite repressor protein Cre1, which in vivo binds to two of four consensus sites (5-SYG-GRG-3) in the xyn1 promoter, which occurred in the form of an inverted repeat. T. reesei strains, bearing a xynlv. hph reporter construct, in which four nucleotides from the middle of the inverted repeat had been removed, expressed hph on glucose at a level comparable to that observed during growth on a carbon catabolite derepressing carbon source. Northern analysis of xynl expression in a T. reesei mutant strain (RUT C-30), which contains a truncated, non-functional crel gene, also confirmed basal transcription of xyn1. In this strain, xyn1 transcription was still inducible by xylose or xylan to an even higher degree than in the wild-type strain, suggesting that induction overcomes glucose repression at the level of xynl expression. Based on these data, we postulate that basal transcription of xyn1 is repressed by glucose and mediated by an inverted repeat of the consensus motif for Cre1-mediated carbon catabolite repression.
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  • 5
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: We have investigated the effect of disruption of the bgl1-(β-glucosidase l-encoding) gene of Trichoderma reesei on the formation of other β-glucosidase activities and on the induction of cellulases. To this end the bgl1 locus was disrupted by insertion of the Aspergillus nidulans amdS (acetamidase-encoding) gene. The bgl1-disrupted strain did not produce the 75kDa extracellular β-glucosidase on cellulose or lactose, but still formed β-glucosidase activity on glucose, cellobiose, xylan or β-1,3-glucan, suggesting that the enzyme(s) exhibiting this β-glucosidase activity is (are) not encoded by bgl1. The cellulose-inducer sophorose induced the bgl1-encoded β-glucosidase, whereas the remaining β-glucosidase activity was induced by methyl-β-D-glucoside. The bgl1-gene product was mainly secreted into the medium, whereas the other β-glucosidase activity was mainly associated with the cells. A bgl1-multicopy strain formed higher amounts of cellulases than the parent strain. Nonsaturating concentrations of sophorose efficiently induced cellobiohydrolase I formation in the bgl1-multicopy strain, but less efficiently in the bgl1-disrupted strain. The multicopy strain and the parent strain were comparably efficient at saturating sophorose concentrations. The β-glucosidase inhibitor nojirimycin strongly inhibited induction in all strains. These data suggest that the bgl1-encoded β-glucosidase is not identical to the plasma-membrane-bound, constitutive, methyl-β-glucoside inducible β-glucosidase, but represents an extracellular cellulose-induced enzyme. Both enzymes contribute to rapid induction of cellulases by modifying the inducer sophorose.
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  • 6
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: Lactose is the only soluble carbon source which can be used economically for the production of cellulases or heterologous proteins under cellulase expression signals by Hypocrea jecorina (=Trichoderma reesei). Towards an understanding of lactose metabolism and its role in cellulase formation, we have cloned and characterized the gal1 (galactokinase) gene of H. jecorina, which catalyses the first step in d-galactose catabolism. It exhibits a calculated Mr of 57 kDa, and shows moderate identity (about 40%) to its putative homologues of Saccharomyces cerevisiae and Kluyveromyces lactis. Gal1 is a member of the GHMP family, shows conservation of a Gly/Ser rich region involved in ATP binding and of amino acids (Arg 51, Glu 57, Asp 60, Asp 214, Tyr 270) responsible for galactose binding. A single transcript was formed constitutively during the rapid growth phase on all carbon sources investigated and accumulated to about twice this level during growth on d-galactose, l-arabinose and their corresponding polyols. Deletion of gal1 reduces growth on d-galactose but does only slightly affect growth on lactose. This is the result of the operation of a second pathway for d-galactose catabolism, which involves galactitol as an intermediate, and whose transient concentration is strongly enhanced in the delta-gal1 strain. In this pathway, galactitol is catabolised by the lad1-encoded l-arabinitol-4-dehydrogenase, because a gal1/lad1 double delta-mutant failed to grow on d-galactose. In the delta-gal1 strain, induction of the Leloir pathway gene gal7 (encoding galactose-1-phosphate uridylyltransferase) by d-galactose, but not by l-arabinose, is impaired. Induction of cellulase gene expression by lactose is also impaired in a gal1 deleted strain, whereas their induction by sophorose (the putative cellulose-derived inducer) was shown to be normal, thus demonstrating that galactokinase is a key enzyme for cellulase induction during growth on lactose, and that induction by lactose and sophorose involves different mechanisms.
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  • 7
    ISSN: 1546-1696
    Source: Nature Archives 1869 - 2009
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: [Auszug] As a first step to exploit the potential of Trichoderma reesei to produce hemicellulases, we have purified two endo-β-1,4-xylanases (1,4-β-D-xylan xylanohydrolase, EC 3.2.1.8) and cloned their genes. The enzymes were isolated from culture filtrates of T. reesei C 30 grown on xylan as a ...
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  • 8
    ISSN: 1574-6968
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology
    Notes: Carbon catabolite repression by the CreA-transcriptional repressor is widespread in filamentous fungi, but the mechanism by which glucose triggers carbon catabolite repression is still poorly understood. We investigated the hypothesis that the growth rate on glucose may control CreA-dependent carbon catabolite repression by using glucose-limited chemostat cultures and the intracellular β-galactosidase activity of Aspergillus nidulans, which is repressed by glucose, as a model system. Chemostat cultures at four different dilution rates (D=0.095, 0.068, 0.045 and 0.015 h−1) showed that formation of β-galactosidase activity is repressed at the two highest Ds, but increasingly derepressed at the lower Ds, the activity at 0.015 h−1 equalling that in derepressed batch cultures. Chemostat cultures with the carbon catabolite derepressed A. nidulans mutant strain creAΔ4 revealed a dilution-rate independent constant β-galactosidase activity of the same range as that found in the wild-type strain at D=0.015 h−1. Two other enzymes – isocitrate lyase, which is almost absent on glucose due to a CreA-independent mechanism; and galactokinase, which is formed constitutively and independent of CreA – were measured as controls. They were formed at constant activity at each dilution rate, both in the wild-type strain as well as in the carbon catabolite derepressed mutant strain. We conclude that the growth rate on glucose is a determinant of carbon catabolite repression in A. nidulans, and that below a certain growth rate carbon catabolite derepression occurs.
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  • 9
    ISSN: 1574-6968
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology
    Notes: Expression of the endochitinase encoding ech42 gene of the mycoparasite Trichoderma atroviride is subject to control by several environmental signals, including derepression by carbon starvation. In order to identify promoter areas involved in control by this condition, we prepared fusions of several mutant forms of the ech42 promoter to the Aspergillus niger goxA gene as a reporter. Removal of a 130-bp fragment comprising a binding site for the carbon catabolite repressor Cre1, an AGGGG element and three separate binding sites identical and highly similar, respectively, to those for the Aspergillus nidulans regulator of conidiation BrlA resulted in a three-fold increase in derepression under carbon starvation. A truncation of the promoter to 196 bp, which removed all of the observed DNA binding motifs, resulted in five-fold derepression. In vitro protein–DNA binding analyses showed that only the BrlA-like sites, but neither the AGGGG element nor the Cre1 binding site, bound proteins from cell-free extracts from carbon-starved mycelia of T. atroviride. Thus this study identifies a new regulator of chitinase gene expression in Trichoderma, a BrlA-like binding motif.
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  • 10
    Electronic Resource
    Electronic Resource
    Oxford, UK : Blackwell Publishing Ltd
    FEMS microbiology letters 12 (1981), S. 0 
    ISSN: 1574-6968
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology
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