ABSTRACT
Asexual spores are the main vehicle used by fungi to disperse to new niches. The Eurotiomycete Aspergillus nidulans is the main reference for the study of the genetic/molecular control of asexual development. In this species, Flb proteins control the expression of the master gene brlA, and thus, loss-of-function mutations in flb (upstream developmental activation [UDA]) genes block brlA transcription and, consequently, the production of conidiophores, the structures bearing asexual spores known as conidia. However, the aconidial phenotype of specific flb mutants, such as that of the ΔflbB strain, is reverted under salt-stress conditions. Previously, we generated a collection of second-site mutants of ΔflbB unable to conidiate on culture medium supplemented with NaH2PO4 (0.65 M). Here, we identified a Gly347Stop mutation within flpA as responsible for the FLIP57 phenotype and characterized the role of the putative cyclin FlpA and the remaining putative components of the C-terminal domain kinase-1 (CTDK-1) complex in A. nidulans and Aspergillus fumigatus. FlpA, Stk47, and FlpB are necessary (i) for timely germination, (ii) in the transition from metulae to phialides (the cells generating conidia) during conidiophore development, and (iii) for the development of sexual structures (cleistothecia) in A. nidulans. The three proteins are nuclear, and the nucleoplasmic localization of Stk47 depends on the activity of FlpA, which correlates with the retention of Stk47 by FlpA in pull-down assays. Overall, this work links the putative CTDK-1 complex of aspergilli with growth and developmental control. Identification of a mutation in flpA as inhibitor of conidiation in A. nidulans and functional characterization of FlpA, Stk47 and FlpB as putative members of the C-terminal domain kinase complex CTDK-1 in A. nidulans and A. fumigatus.IMPORTANCEAspergillus fumigatus has been included by the World Health Organization in the priority list of fungal pathogens because (i) it causes 90% of invasive aspergillosis cases, with a high mortality rate, and (ii) infections are becoming increasingly resistant to azole antifungals. A. nidulans is an opportunistic pathogen and a saprotroph which has served during the last 80 years as a reference system for filamentous fungi. Here, we characterized the role in morphogenesis and development of the putative transcriptional cyclin/kinase complex CTDK-1 in both aspergilli. The null mutants of the corresponding genes showed delayed germination, aberrant conidiophore development, and inhibition of cleistothecia production. While in higher eukaryotes this complex is formed only by a cyclin and a kinase, the fungal complex would incorporate a fungal-specific third component, FlpB, which would enable the interaction between the kinase (Stk47) and the cyclin (FlpA) and may be used as a target for antifungals.
ABSTRACT
The importance of the metabolic route of nitrogen in the fungus Penicillium rubens (strain PO212) is studied in relation to its biocontrol activity (BA). PO212 can resist a high concentration of chlorate anion and displays a classical nitrate-deficiency (nit-) phenotype resulting in poor colonial growth when nitrate is used as the main source of nitrogen. Analyses of genes implicated in nitrate assimilation evidenced the strong sequence conservation of PO212 and CH8 genome with penicillin producers such as reference strain P. rubens Wisconsin 54-1255, P2niaD18 and Pc3, however also revealed the presence of mutations. PO212 carries a mutation in the gene coding for zinc-binuclear cluster transcription factor NirA that specifically mediates the regulation of genes involved in nitrate assimilation. The nirA1 mutation causes an early stop of NirA factor, losing 66% of its sequence. The NirA1 mutant form is unable to mediate a nitrate-dependent regulation of nitrate and nitrite reductase coding genes. In this study, we study another isolate, CH8, with potential BA and nit- phenotype. A mutation in the nitrate permease coding gene crnA was found in CH8. An insertion of a guanine in the coding sequence cause a frameshift in CrnA with the loss of the last two transmembrane domains. Analysis of PO212 and CH8 isolates and complementation strains show the importance of NirA regulator in maintaining correct transcriptional levels of nitrate and nitrite reductases and suggest CrnA as the main nitrate transporter. the presence of alternative transporter for chlorate and the existence of a mechanism for preventing nitrite derived toxicity in Penicillum. BA of PO212 is partially altered when nirA1 mutation was complemented. This result and the finding of CH8, a novel biocontrol P. rubens strain with a nit- phenotype, suggest that nitrogen metabolism is a component of biocontrol capacity.
Subject(s)
Biological Control Agents/metabolism , Gene Expression Regulation, Fungal , Nitrates/metabolism , Nitrogen/metabolism , Penicillium/genetics , Penicillium/metabolism , Anion Transport Proteins/genetics , Chlorates/pharmacology , Solanum lycopersicum/microbiology , Mutation , Nitrate Transporters , Nitrite Reductases/genetics , Penicillium/drug effects , Phenotype , Plant Diseases/microbiology , Transcription Factors/geneticsABSTRACT
Light is a major environmental stimulus that has a broad effect on organisms, triggering a cellular response that results in an optimal adaptation enhancing fitness and survival. In fungi, light affects growth, and causes diverse morphological changes such as those leading to reproduction. Light can also affect fungal metabolism, including the biosynthesis of natural products. In this study we show that in Aspergillus nidulans the effect of light on the production of the sterigmatocystin (ST) toxin depends on the glucose concentration. In cultures grown with 1% glucose and exposed to light, ST production was lower than when grown in the dark. This lower ST production coincided with an elevated rate of cellular damage with partial loss of nuclear integrity and vacuolated cytoplasm. However, in cultures grown with 2% glucose these effects were reversed and light enhanced ST production. Glucose abundance also affected the light-dependent subcellular localization of the VeA (velvet) protein, a key regulator necessary for normal light-dependent morphogenesis and secondary metabolism in Aspergilli and other fungal genera. The role of other VeA-associated proteins, particularly the blue-light-sensing proteins LreA and LreB (WC-1 and WC-2 orthologs), on conidiation could also be modified by the abundance of glucose. We also show that LreA and LreB, as well as the phytochrome FphA, modulate not only the synthesis of sterigmatocystin, but also the production of the antibiotic penicillin.
Subject(s)
Aspergillus nidulans/growth & development , Aspergillus nidulans/radiation effects , Glucose/metabolism , Morphogenesis , Sterigmatocystin/biosynthesis , Aspergillus nidulans/genetics , Aspergillus nidulans/metabolism , Fungal Proteins/genetics , Fungal Proteins/metabolism , Gene Expression Regulation, Fungal/radiation effects , Light , Morphogenesis/radiation effects , Penicillins/metabolism , Protein Transport/radiation effectsABSTRACT
The Aspergillus nidulans zinc finger transcription factor PacC is activated by proteolytic processing in response to ambient alkaline pH. The pH-regulated step is the transition of full-length PacC from a closed to an open, protease-accessible conformation. Here we show that in the absence of ambient pH signaling, the C-terminal negative-acting domain prevents the nuclear localization of full-length closed PacC. In contrast, the processed PacC form is almost exclusively nuclear at any ambient pH. In the presence of ambient pH signaling, the fraction of PacC that is in the open conformation but has not yet been processed localizes to the nucleus. Therefore, ambient alkaline pH leads to an increase in nuclear PacC by promoting the proteolytic elimination of the negative-acting domain to yield the processed form and by increasing the proportion of full-length protein that is in the open conformation. These findings explain why mutations resulting in commitment of PacC to processing irrespective of ambient pH lead to permanent PacC activation and alkalinity mimicry. A nuclear import signal that targets Escherichia coli beta-galactosidase to the nucleus has been located to the PacC zinc finger region. A mutation abolishing DNA binding does not prevent nuclear localization of the processed form, showing that PacC processing does not lead to nuclear localization by passive diffusion of the protein made possible by the reduction in size, followed by retention in the nucleus after DNA binding.
Subject(s)
Aspergillus nidulans/metabolism , Cell Nucleus/metabolism , Fungal Proteins , Transcription Factors/metabolism , Amino Acids/chemistry , Blotting, Western , Cytoplasm/metabolism , Cytosol/metabolism , DNA/metabolism , DNA-Binding Proteins/metabolism , Green Fluorescent Proteins , Hydrogen-Ion Concentration , Luminescent Proteins/metabolism , Microscopy, Fluorescence , Models, Biological , Mutagenesis , Mutation , Plasmids/metabolism , Protein Conformation , Protein Structure, Tertiary , Recombinant Fusion Proteins/metabolism , Subcellular Fractions/metabolism , Transcription Factors/chemistry , Zinc FingersABSTRACT
Previous work has shown that zinc finger transcription factor PacC mediates the regulation of gene expression by ambient pH in the fungus Aspergillus nidulans. This regulation ensures that the syntheses of molecules functioning in the external environment, such as permeases, secreted enzymes, and exported metabolites, are tailored to the pH of the growth environment. A direct role for PacC in activating the expression of an alkaline-expressed gene has previously been demonstrated, but the mechanism by which alkaline ambient pH prevents the expression of any eukaryotic acid-expressed gene has never been reported. Here we show that a double PacC binding site in the promoter of the acid-expressed gabA gene, encoding gamma-aminobutyrate (GABA) permease, overlaps the binding site for the transcriptional activator IntA, which mediates omega-amino acid induction. Using bacterially expressed fusion proteins, we have shown that PacC competes with IntA for DNA binding in vitro at this site. Thus, PacC repression of GABA permease synthesis is direct and occurs by blocking induction. A swap of IntA sites between promoters for gabA and amdS, a gene not subject to pH regulation, makes gabA expression pH independent and amdS acid expressed.
Subject(s)
Aspergillus nidulans/genetics , DNA-Binding Proteins/metabolism , Fungal Proteins/metabolism , Hydrogen-Ion Concentration , Membrane Transport Proteins/genetics , Organic Anion Transporters , Repressor Proteins/metabolism , Transcription Factors/metabolism , gamma-Aminobutyric Acid/metabolism , Alkalies , Amidohydrolases/genetics , Base Sequence , Binding Sites , Binding, Competitive , Consensus Sequence , DNA-Binding Proteins/genetics , Fungal Proteins/genetics , Gene Expression Regulation, Fungal , Mutagenesis , Promoter Regions, Genetic , Protein Binding , Recombinant Fusion Proteins/metabolism , Repressor Proteins/genetics , Transcription Factors/geneticsABSTRACT
In response to alkaline ambient pH, the Aspergillus nidulans PacC transcription factor mediating pH regulation of gene expression is activated by proteolytic removal of a negative-acting C-terminal domain. We demonstrate interactions involving the approximately 150 C-terminal PacC residues and two regions located immediately downstream of the DNA binding domain. Our data indicate two full-length PacC conformations whose relative amounts depend upon ambient pH: one 'open' and accessible for processing, the other 'closed' and inaccessible. The location of essential determinants for proteolytic processing within the two more upstream interacting regions probably explains why the interactions prevent processing, whereas the direct involvement of the C-terminal region in processing-preventing interactions explains why C-terminal truncating mutations result in alkalinity mimicry and pH-independent processing. A mutant PacC deficient in pH signal response and consequent processing behaves as though locked in the 'closed' form. Single-residue substitutions, obtained as mutations bypassing the need for pH signal transduction, identify crucial residues in each of the three interactive regions and overcome the processing deficiency in the 'permanently closed' mutant.
Subject(s)
Fungal Proteins , Transcription Factors/metabolism , Amino Acid Sequence , Amino Acid Substitution , Aspergillus nidulans/genetics , Aspergillus nidulans/metabolism , Binding Sites/genetics , Endopeptidases/metabolism , Gene Expression Regulation , Genes, Fungal , Hydrogen-Ion Concentration , Models, Biological , Molecular Sequence Data , Point Mutation , Protein Conformation , Protein Processing, Post-Translational , Protein Structure, Tertiary , Sequence Deletion , Signal Transduction , Transcription Factors/chemistry , Transcription Factors/genetics , Two-Hybrid System Techniques , Zinc Fingers/geneticsABSTRACT
Completing the molecular analysis of the six pal genes of the ambient pH signal transduction pathway in Aspergillus nidulans, we report the characterization of palC and palH. The derived translation product of palH contains 760 amino acids with prediction of seven transmembrane domains in its N-terminal moiety. Remarkably, a palH frameshift mutant lacking just over half the PalH protein, including almost all of the long hydrophilic region C-terminal to the transmembrane domains, retains some PalH function. The palC-derived translation product contains 507 amino acids, and the null phenotype of a frameshift mutation indicates that at least one of the C-terminal 142 residues is essential for function. Uniquely among the A. nidulans pH-signalling pal genes, palC appears to have no Saccharomyces cerevisiae homologue, although it does have a Neurospora crassa expressed sequence tag homologue. In agreement with findings for the palA, palB and palI genes of this signalling pathway, levels of the palC and palH mRNAs do not appear to be pH regulated.
Subject(s)
Aspergillus nidulans/genetics , Aspergillus nidulans/metabolism , Fungal Proteins/genetics , Fungal Proteins/metabolism , Signal Transduction , Amino Acid Sequence , Base Sequence , Cloning, Molecular , Frameshift Mutation , Hydrogen-Ion Concentration , Molecular Sequence Data , Sequence Homology, Amino Acid , Transcription, GeneticABSTRACT
We describe the cloning, sequence and expression of gabA, encoding the gamma-amino-n-butyrate (GABA) permease of the fungus Aspergillus nidulans. Sequence changes were determined for three up-promoter (gabI ) and six gabA loss-of-function mutations. The predicted protein contains 517 residues and shows 30.3% overall identity with a putative GABA permease of Arabidopsis thaliana, 29.6% identity with the yeast choline transporter and 23.4% identity with the yeast UGA4 GABA permease. Structural predictions favour 11-12 transmembrane domains. Comparison of the genomic and cDNA sequences shows the presence of 19 introns, an unusually large number of introns for, we believe, any fungal gene. In agreement with the wealth of genetic data available, transcript level analyses demonstrate that gabA is subject to carbon catabolite and nitrogen metabolite repression, omega-amino acid induction and regulation in response to ambient pH (being acid-expressed). In agreement with this, we report consensus binding sites 5' to the coding region, six each for CreA and AREA and one for PacC, the transcription factors mediating carbon catabolite and nitrogen metabolite repression and response to ambient pH respectively.
Subject(s)
Aspergillus nidulans/genetics , Membrane Transport Proteins/genetics , Organic Anion Transporters , Amino Acid Sequence , Base Sequence , Cloning, Molecular , Exons , Fungal Proteins/genetics , Fungal Proteins/metabolism , Gene Expression Regulation, Fungal , Membrane Transport Proteins/metabolism , Molecular Sequence Data , Mutation , Promoter Regions, Genetic , Sequence Analysis , Sequence Homology, Amino Acid , Transcription, GeneticABSTRACT
The Aspergillus nidulans transcription factor PacC, which mediates pH regulation, is proteolytically processed to a functional form in response to ambient alkaline pH. The full-length PacC form is unstable in the presence of an operational pH signal transduction pathway, due to processing to the relatively stable short functional form. We have characterized and used an extensive collection of pacC mutations, including a novel class of "neutrality-mimicking" pacC mutations having aspects of both acidity- and alkalinity-mimicking phenotypes, to investigate a number of important features of PacC processing. Analysis of mutant proteins lacking the major translation initiation residue or truncated at various distances from the C terminus showed that PacC processing does not remove N-terminal residues, indicated that processing yields slightly heterogeneous products, and delimited the most upstream processing site to residues approximately 252 to 254. Faithful processing of three mutant proteins having deletions of a region including the predicted processing site(s) and of a fourth having 55 frameshifted residues following residue 238 indicated that specificity determinants reside at sequences or structural features located upstream of residue 235. Thus, the PacC protease cuts a peptide bond(s) remote from these determinants, possibly thereby resembling type I endonucleases. Downstream of the cleavage site, residues 407 to 678 are not essential for processing, but truncation at or before residue 333 largely prevents it. Ambient pH apparently regulates the accessibility of PacC to proteolytic processing. Alkalinity-mimicking mutations L259R, L266F, and L340S favor the protease-accessible conformation, whereas a protein with residues 465 to 540 deleted retains a protease-inaccessible conformation, leading to acidity mimicry. Finally, not only does processing constitute a crucial form of modulation for PacC, but there is evidence for its conservation during fungal evolution. Transgenic expression of a truncated PacC protein, which was processed in a pH-independent manner, showed that appropriate processing can occur in Saccharomyces cerevisiae.
Subject(s)
Aspergillus nidulans/metabolism , Fungal Proteins/metabolism , Transcription Factors/metabolism , Amino Acid Sequence , Aspergillus nidulans/genetics , Binding Sites , Fungal Proteins/chemistry , Fungal Proteins/genetics , Hydrogen-Ion Concentration , Molecular Sequence Data , Mutation , Plasmids/genetics , Protein Processing, Post-Translational , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Signal Transduction , Transcription Factors/chemistry , Transcription Factors/genetics , Transformation, GeneticABSTRACT
The zinc finger regions of the Aspergillus nidulans PacC transcription factor, mediating regulation of gene expression by ambient pH, and the Saccharomyces cerevisiae Rim1p transcription factor, mediating control of meiosis and invasiveness, are homologous and both transcription factors undergo proteolytic processing of the C-terminus for conversion to the functional form. In both cases, functioning of a signal transduction pathway involving several gene products is a necessary prerequisite for processing. We now show that the Aspergillus PalI pH signal transduction component is homologous to the Saccharomyces Rim9p meiotic signal transduction component throughout a region containing four hydrophobic, putative membrane-spanning segments. This suggests that PalI might be a membrane sensor for ambient pH. Deletion of the palI gene established that the less extreme phenotype of palI mutations compared with mutations in the other five genes of the pH signalling pathway is a general feature of palI mutations.
Subject(s)
Aspergillus nidulans/genetics , Fungal Proteins/genetics , Fungal Proteins/metabolism , Meiosis , Membrane Proteins , Saccharomyces cerevisiae Proteins , Saccharomyces cerevisiae/genetics , Signal Transduction , Amino Acid Sequence , Aspergillus nidulans/metabolism , Base Sequence , Blotting, Northern , Cloning, Molecular , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Deoxyribonucleases, Type II Site-Specific/genetics , Deoxyribonucleases, Type II Site-Specific/metabolism , Hydrogen-Ion Concentration , Molecular Sequence Data , Mutation , Repressor Proteins , Saccharomyces cerevisiae/metabolism , Sequence Homology, Amino Acid , Transcription Factors/genetics , Transcription Factors/metabolismABSTRACT
pmaA, an Aspergillus nidulans gene encoding a P-ATPase, has been cloned by heterologous hybridization with the yeast PMA1 gene. The putative 990-residue PmaA polypeptide shows 50% identity to Saccharomyces cerevisiae and Neurospora crassa plasma membrane H(+)-ATPases and weak (19-26%) identity to other yeast P-type cation-translocating ATPases. PmaA contains all catalytic domains characterizing H(+)-ATPases. pmaA transcript levels are not regulated by PacC, the transcription factor mediating pH regulation, and were not significantly affected by an extreme creAd mutation resulting in carbon catabolite derepression. Deletion of pmaA causes lethality, but a single copy of the gene is sufficient to support normal growth rate in pmaA hemizygous diploids, even under acidic growth conditions. As compared to other fungal H(+)-ATPases, PmaA presents three insertions, 39, 7, and 16 residues long, in the conserved central region of the protein. Two of these insertions are predicted to be located in extracellular loops and might be of diagnostic value for the identification of Aspergillus species. Their absence from most mammalian P-type ATPases may have implications for antifungal therapy.
Subject(s)
Aspergillus nidulans/genetics , Genes, Fungal , Proton-Translocating ATPases/genetics , Amino Acid Sequence , Aspergillus nidulans/enzymology , Base Sequence , Blotting, Northern , Cloning, Molecular , Gene Deletion , Hydrogen-Ion Concentration , Molecular Sequence Data , Phylogeny , Proton Pumps , Proton-Translocating ATPases/chemistry , Proton-Translocating ATPases/metabolism , Sequence Alignment , Sequence Analysis, DNA , Transcription, GeneticABSTRACT
The three zinc fingers of PacC, the transcription factor mediating pH regulation in Aspergillus nidulans, are necessary and sufficient to recognise specifically the target ipnA2 site. Missing nucleoside footprints confirmed the core target (double-stranded) hexanucleotide 5'-GCCAAG-3'. Any base substitution resulted in substantial or complete loss of binding, excepting A5 (partially replaceable by G). A T preceding the hexanucleotide enhanced binding. Interference footprinting indicates that the four Gs and A4 participate in specific contacts and that five pyrimidines are essential for binding. The size of the target sequence and the amino acid sequence of finger 1 suggested that its probe helix would not participate in base-specific contacts. Using site-directed mutagenesis and analogy to GLI, we propose that finger 1 crucially interacts with finger 2, a pair of conserved Trp residues in the Cys knuckles contacting hydrophobically. Finger 2 would also participate in extensive base contacts with the 5' moiety of the hexanucleotide. The specificity mutation Lys159Gln shows that finger 3 binds the 3' moiety of the hexanucleotide. Replacement of residues in positions +3 (His128Asn) and +2 (Gln155Lys) of the reading helices of fingers 2 and 3, respectively, prevented binding. Our biochemical and molecular data plus modelling using previously determined zinc finger-DNA complexes, predict specific contacts of fingers 2 and 3 to ipnA2. Our data indicate compact organisation of the PacC-ipnA2 complex (with nearly every base involved in specific contacts), illustrate the binding versatility of zinc finger domains and should facilitate analysis of other PacC family members, including Saccharomyces cerevisiae RIM1.
Subject(s)
Aspergillus nidulans/chemistry , DNA/metabolism , Fungal Proteins , Transcription Factors/chemistry , Transcription Factors/metabolism , Zinc Fingers , Amino Acid Sequence , Base Composition , Binding Sites , DNA Footprinting , Models, Molecular , Molecular Sequence Data , Mutagenesis, Site-Directed , Oligonucleotides , Protein Conformation , Transcription Factors/geneticsABSTRACT
The isopenicillin N synthase (ipnA) gene, encoding a key penicillin biosynthetic enzyme in Aspergillus nidulans, represents a prototype of an alkaline-expressed gene. ipnA is under ambient pH regulation, and its promoter (ipnAp) contains binding sites for the zinc-finger transcription factor PacC. We show here that three of these sites, denoted ipnA2, ipnA3, and ipnA4AB, are efficiently recognized by the protein in an isolated sequence context. Single, double, and triple inactivation of these sites in any possible combination reduced promoter activity under alkaline conditions but had no effect under acidic conditions (under which promoter activity was low), as measured by the expression of wild-type and mutant ipnAp::lacZ fusion genes integrated in single copy into a common chromosomal location. This establishes a physiological role for these PacC binding sites and demonstrates a direct role for PacC in ambient pH regulation of ipnA gene expression. In addition, this confirms our previous proposal that PacC is an activator for alkaline-expressed genes. Notably, our experiments show that ipnA2, the highest affinity site for PacC in the ipnAp, contributes relatively modestly to PacC-mediated activation. By contrast, the lower affinity sites ipnA3 and ipnA4AB contribute more substantially to regulation by ambient pH. Inactivation of these three binding sites reduced promoter activity under alkaline conditions to that observed under acidic conditions, showing that these three PacC sites at ipnAp are sufficient to account for its activation by alkaline ambient pH.
Subject(s)
Aspergillus nidulans/metabolism , Fungal Proteins , Oxidoreductases/genetics , Promoter Regions, Genetic , Transcription Factors/metabolism , Zinc Fingers , Binding Sites , Hydrogen-Ion Concentration , Mutagenesis, Site-Directed , Sequence Deletion , Transcription Factors/geneticsABSTRACT
Extremes of pH are an occupational hazard for many microorganisms. In addition to efficient pH homeostasis, survival effectively requires a regulatory system tailoring the syntheses of molecules functioning beyond the cell boundaries (permeases, secreted enzymes, and exported metabolites) to the pH of the growth environment. Our previous work established that the zinc finger PacC transcription factor mediates such pH regulation in the fungus Aspergillus nidulans in response to a signal provided by the products of the six pal genes at alkaline ambient pH. In the presence of this signal, PacC becomes functional, activating transcription of genes expressed at alkaline pH and preventing transcription of genes expressed at acidic pH. Here we detect two forms of PacC in extracts, both forming specific retardation complexes with a PacC-binding site. Under acidic growth conditions or in acidity-mimicking pal mutants (defective in ambient pH signal transduction), the full-length form of PacC predominates. Under alkaline growth conditions or in alkalinity-mimicking pacCc mutants (independent of the ambient pH signal), a proteolysed version containing the amino-terminal approximately 40% of the protein predominates. This specifically cleaved shorter version is clearly functional, both as an activator for alkaline-expressed genes and as a repressor for acid-expressed genes, but the full-length form of PacC must be inactive. Thus, PacC proteolysis is an essential and pH-sensitive step in the regulation of gene expression by ambient pH. Carboxy-terminal truncations, resulting in a gain-of-function (pacCc) phenotype, bypass the requirement for the pal signal transduction pathway for conversion of the full-length to the proteolyzed functional form.
Subject(s)
Aspergillus nidulans/genetics , Aspergillus nidulans/metabolism , Fungal Proteins , Gene Expression Regulation, Fungal , Genes, Fungal , Hydrogen-Ion Concentration , Transcription Factors/metabolism , Aspergillus nidulans/growth & development , Base Sequence , Binding Sites , Blotting, Western , Consensus Sequence , Endopeptidases/metabolism , Genotype , Mutagenesis, Site-Directed , Protein Biosynthesis , Recombinant Fusion Proteins/metabolism , Transcription Factors/genetics , Transcription, Genetic , Zinc FingersABSTRACT
Transcription of the Aspergillus nidulans ipnA gene is under carbon regulation. Loss-of-function mutations in creB or creC do not cause full derepression of ipnA transcript levels in sucrose-grown mycelia and do not elevate repressed penicillin levels, indicating that neither of these genes plays a major regulatory role in penicillin biosynthesis. However, these mutations reduce external pH acidification, accelerate sucrose degradation and result in extracellular accumulation of resulting D-glucose and D-fructose. These effects would explain the partial elevation of carbon-repressed ipnA transcript levels observed in strains carrying creB- or creC- mutations.
Subject(s)
Aspergillus nidulans/metabolism , Gene Expression Regulation, Bacterial , Genes, Bacterial/genetics , Oxidoreductases/genetics , Penicillins/biosynthesis , Aspergillus nidulans/drug effects , Aspergillus nidulans/genetics , Enzyme Repression , Genes, Regulator/genetics , Hydrogen-Ion Concentration , Mutation , Oxidoreductases/biosynthesis , RNA, Messenger/analysis , Sucrose/metabolism , Sucrose/pharmacologyABSTRACT
The pH regulation of gene expression in Aspergillus nidulans is mediated by pacC, whose 678 residue-derived protein contains three putative Cys2His2 zinc fingers. Ten pacCc mutations mimicking growth at alkaline pH remove between 100 and 214 C-terminal residues, including a highly acidic region containing an acidic glutamine repeat. Nine pacC+/- mutations mimicking acidic growth conditions remove between 299 and 505 C-terminal residues. Deletion of the entire pacC coding region mimics acidity but leads additionally to poor growth and conidiation. A PacC fusion protein binds DNA with the core consensus GCCARG. At alkaline ambient pH, PacC activates transcription of alkaline-expressed genes (including pacC itself) and represses transcription of acid-expressed genes. pacCc mutations obviate the need for pH signal transduction.
Subject(s)
Acid-Base Equilibrium , Aspergillus nidulans/genetics , DNA-Binding Proteins/metabolism , Fungal Proteins/genetics , Transcription Factors/physiology , Amino Acid Sequence , Base Sequence , Binding Sites , Cloning, Molecular , Epistasis, Genetic , Gene Expression Regulation, Fungal , Molecular Sequence Data , Mutagenesis, Site-Directed , Promoter Regions, Genetic , RNA, Messenger/genetics , Sequence Alignment , Sequence Homology, Amino Acid , Structure-Activity Relationship , Zinc FingersABSTRACT
The two zinc-fingers of the Aspergillus nidulans repressor CreA recognize the consensus hexanucleotide 5'-SYGGRG-3'. We have determined all the CreA binding sites in a approximately 2 kb region upstream the ipnA gene. Our analysis shows that (i) CreA binds to certain consensus sites in a context-dependent manner; (ii) five non-consensus 6-bp sequences are also recognized by CreA; this non-canonical binding correlates with the presence of a second, neighbouring CreA binding site, suggesting that recognition of two linked sites stabilizes CreA binding. Our results suggest that the binding possibilities of CreA might be more complex than originally envisaged.
Subject(s)
Aspergillus nidulans/metabolism , DNA, Fungal/metabolism , Fungal Proteins/metabolism , Genes, Fungal , Repressor Proteins/metabolism , Zinc Fingers , Aspergillus nidulans/genetics , Base Composition , Base Sequence , Binding Sites , Consensus Sequence , DNA, Fungal/chemistry , Molecular Sequence Data , Oligodeoxyribonucleotides/metabolism , Recombinant Fusion Proteins/metabolismABSTRACT
Transcription of the ipnA gene encoding isopenicillin N synthetase, an enzyme of secondary metabolism, is under the control of the pH regulatory system in the fungus Aspergillus nidulans. External alkaline pH or mutations in pacC, the wide domain regulatory gene which mediates pH regulation, override carbon regulation of ipnA transcript levels, resulting in elevation of the levels of this message in sucrose broth. Strains carrying these mutations, which mimic growth at alkaline pH, produce higher levels of penicillins when grown in sucrose broth compared with the wild type strain grown under carbon derepressing conditions. ipnA transcription is regulated by carbon (C) source, but extreme mutations in creA (the regulatory gene mediating carbon catabolite repression) only slightly increase repressed transcript levels. Precise deletion of the only in vitro CreA binding site present in a region of the ipnA promoter involved in carbon regulation has no effect on ipnA expression. The levels of ipnA transcript in broths with acetate or glycerol as principal C sources are inconsistent with direct or indirect creA-mediated transcriptional control of the gene. We conclude that a second, creA-independent mechanism of carbon repression controls expression of this gene. All derepressing C sources tested result in alkalinization of the growth media. In contrast, all repressing C sources result in external acidification. Neither acidic external pH nor pal mutations, mimicking the effects of growth at acid pH, prevent carbon derepression, providing strong support for independent regulatory mechanisms, one mediating carbon regulation (via thus far unidentified genes) and another mediating pH regulation (through the pacC-encoded transcriptional regulator). External pH measurements suggest that these two independent forms of regulation normally act in concert. We propose that external alkalinity represents a physiological signal which triggers penicillin biosynthesis.
Subject(s)
Aspergillus nidulans/enzymology , Oxidoreductases/genetics , Penicillins/biosynthesis , Acetates/metabolism , Aspergillus nidulans/genetics , Base Sequence , Carbon/metabolism , DNA, Fungal , Fungal Proteins/metabolism , Gene Expression Regulation, Enzymologic , Genes, Fungal , Genes, Regulator , Glycerol/metabolism , Hydrogen-Ion Concentration , Kinetics , Molecular Sequence Data , Mutation , Oxidoreductases/metabolism , Promoter Regions, Genetic , Repressor Proteins/metabolism , Transcription, GeneticABSTRACT
Aspergillus nidulans synthesizes penicillins as secondary metabolites when grown under certain culture conditions. Broths containing carbon (C) sources that give rise to carbon catabolite repression (CCR) support a much lower antibiotic yield than broths with non-repressing C sources. Steady-state levels of the isopenicillin N synthetase (IPNS) gene transcript are considerably reduced in mycelia grown with repressing C sources and are depressed in mycelia grown with sugars which do not cause CCR, indicating that penicillin biosynthesis is regulated by CCR through transcriptional control of structural genes. CCR is sufficient to explain the temporal window of expression of the IPNS gene during the growth cycle since (i) the transcript becomes derepressed as soon as the concentration of a repressing C source drops to non-repressing levels and (ii) derepressing C sources sustain derepressed IPNS transcription at all tested moments of the growth cycle. Several tested hypofunctional mutations in creA (the negatively acting regulatory gene which mediates CCR in A. nidulans) do not cause full derepression of IPNS transcript in the presence of a repressing C source. The slight degree of IPNS derepression caused by some creAd (derepressed) alleles parallels the strength of the mutation (as determined by the morphological effect they elicit).