Carbon Catabolite Repression Governs Diverse Physiological Processes and Development in Aspergillus nidulans.

Carbon catabolite repression (CCR) is a common phenomenon of microorganisms that enable efficient utilization of carbon nutrients, critical for the fitness of microorganisms in the wild and for pathogenic species to cause infection. In most filamentous fungal species, the conserved transcription factor CreA/Cre1 mediates CCR. Previous studies demonstrated a primary function for CreA/Cre1 in carbon metabolism; however, the phenotype of / mutants indicated broader roles.

Influence of the Chelation Process on the Stability of Organic Trace Mineral Supplements Used in Animal Nutrition.

The effect of the chelation process on the pH-dependent stability of organic trace minerals (OTMs) used as mineral supplements in animal nutrition was assessed using analytical techniques such as potentiometry, Fourier Transform Infrared Spectroscopy (FTIRS) and amino acid profiling. The aim was to understand the influence and relative importance of the manufacturing conditions on mineral chelation and the subsequent pH stability of OTMs. A selection of OTMs were assessed over a wide pH range to account for the typical environmental changes encountered in the gastrointestinal (GI) tract.

Quantitative assessment of copper proteinates used as animal feed additives using ATR-FTIR spectroscopy and powder X-ray diffraction (PXRD) analysis.

The aim of the present work was to establish a reliable analytical method to determine the degree of complexation in commercial metal proteinates used as feed additives in the solid state. Two complementary techniques were developed. Firstly, a quantitative attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopic method investigated modifications in vibrational absorption bands of the ligand on complex formation.

Synthesis of New Benzocyclotrimer Analogues: New Receptors for Tetramethylammonium Ion Recognition.

Using a [2 + 2 + 2] cycloaddition/Mitsunobu reaction sequence, a convenient synthesis to access new benzocyclotrimer analogues has been developed. The new receptors have the geometry and functionality capable of recognizing the tetramethylammonium ion in the gas phase and in solution.

Dual DNA binding and coactivator functions of Aspergillus nidulans TamA, a Zn(II)2Cys6 transcription factor.

Transcription factors containing DNA binding domains generally regulate transcription by direct interaction with DNA. For most transcription factors, including the fungal Zn(II)2Cys6 zinc binuclear cluster transcription factors, the DNA binding motif is essential for function. However, Aspergillus nidulans TamA and the related Saccharomyces cerevisiae Dal81p protein contain Zn(II)2Cys6 motifs shown to be dispensable for function.

Multiple nuclear localization signals mediate nuclear localization of the GATA transcription factor AreA.

The Aspergillus nidulans GATA transcription factor AreA activates transcription of nitrogen metabolic genes in response to nitrogen limitation and is known to accumulate in the nucleus during nitrogen starvation. Sequence analysis of AreA revealed multiple nuclear localization signals (NLSs), five putative classical NLSs conserved in fungal AreA orthologs but not in the Saccharomyces cerevisiae functional orthologs Gln3p and Gat1p, and one putative noncanonical RRX33RXR bipartite NLS within the DNA-binding domain. In order to identify the functional NLSs in AreA, we constructed areA mutants with mutations in individual putative NLSs or combinations of putative NLSs and strains expressing green fluorescent protein (GFP)-AreA NLS fusion genes.

HgrA is necessary and sufficient to drive hyphal growth in the dimorphic pathogen Penicillium marneffei.

Fungi produce multiple morphological forms as part of developmental programs or in response to changing, often stressful, environmental conditions. An opportunistic pathogen of humans, Penicillium marneffei displays multicellular hyphal growth and asexual development (conidiation) in the environment at 25°C and unicellular yeast growth in macrophages at 37°C. We characterized the transcription factor, hgrA, which contains a C(2)H(2) DNA binding domain closely related to that of the stress-response regulators Msn2/4 of Saccharomyces cerevisiae.

Reprogramming of carbon metabolism by the transcriptional activators AcuK and AcuM in Aspergillus nidulans.

The ability of fungi to use carbon sources metabolized via the TCA cycle requires gluconeogenesis. In Aspergillus nidulans the AcuK and AcuM transcription factors regulate the expression of the gluconeogenic genes acuF, encoding phosphoenolpyruvate carboxykinase, and acuG, encoding fructose-1,6-bisphosphatase. Expressed proteins containing the AcuK/AcuM N-terminal DNA-binding domains bind together in vitro to motifs containing repeats of CGG separated by seven bases (CCGN7CCG) and the functionality of these sequences was verified in vivo by acuF-lacZ reporter studies.

AreA controls nitrogen source utilisation during both growth programs of the dimorphic fungus Penicillium marneffei.

The opportunistic pathogen Penicillium marneffei displays a temperature-dependent dimorphic switching program with saprophytic hyphal growth at 25 °C and yeast growth at 37 °C. The areA gene of P. marneffei has been isolated and found to be required for the utilisation of nonpreferred nitrogen sources during both growth programs of P.

Role of carnitine acetyltransferases in acetyl coenzyme A metabolism in Aspergillus nidulans.

The flow of carbon metabolites between cellular compartments is an essential feature of fungal metabolism. During growth on ethanol, acetate, or fatty acids, acetyl units must enter the mitochondrion for metabolism via the tricarboxylic acid cycle, and acetyl coenzyme A (acetyl-CoA) in the cytoplasm is essential for the biosynthetic reactions and for protein acetylation. Acetyl-CoA is produced in the cytoplasm by acetyl-CoA synthetase during growth on acetate and ethanol while β-oxidation of fatty acids generates acetyl-CoA in peroxisomes.

Mechanisms of hydrolysis of phenyl- and benzyl 4-nitrophenyl-sulfamate esters.

The kinetics of hydrolysis at medium acid strength (pH interval 2-5) of a series of phenylsulfamate esters 1 have been studied and they have been found to react by an associative S(N)2(S) mechanism with water acting as a nucleophile attacking at sulfur, cleaving the S-O bond with simultaneous formation of a new S-O bond to the oxygen of a water molecule leading to sulfamic acid and phenol as products. In neutral to moderate alkaline solution (pH ≥ ~ 6-9) a dissociative (E1cB) route is followed that involves i) ionization of the amino group followed by ii) unimolecular expulsion of the leaving group from the ionized ester to give N-sulfonylamine [HN=SO(2)] as an intermediate. In more alkaline solution further ionization of the conjugate base of the ester occurs to give a dianionic species which expels the aryloxide leaving group to yield the novel N-sulfonylamine anion [(-)N=SO(2)]; in a final step, rapid attack of hydroxide ion or a water molecule on it leads again to sulfamic acid.

Ga(III) complexes as models for the M(III) site of purple acid phosphatase: ligand effects on the hydrolytic reactivity toward bis(2,4-dinitrophenyl) phosphate.

The effects of a series of Ga(III) complexes with tripodal ligands on the hydrolysis rate of the activated phosphate diester bis(2,4-dinitrophenyl)phosphate (BDNPP) have been investigated. In particular, the influence of the nature of the ligand donor sites on the reactivity of Ga(III) which represents a mimic of the Fe(III) ion in purple acid phosphatase has been evaluated. It has been shown that replacing neutral nitrogen donor atoms and carboxylate groups by phenolate groups enhanced the reactivity of the Ga complexes.

ATP-citrate lyase is required for production of cytosolic acetyl coenzyme A and development in Aspergillus nidulans.

Acetyl coenzyme A (CoA) is a central metabolite in carbon and energy metabolism and in the biosynthesis of cellular molecules. A source of cytoplasmic acetyl-CoA is essential for the production of fatty acids and sterols and for protein acetylation, including histone acetylation in the nucleus. In Saccharomyces cerevisiae and Candida albicans acetyl-CoA is produced from acetate by cytoplasmic acetyl-CoA synthetase, while in plants and animals acetyl-CoA is derived from citrate via ATP-citrate lyase.

Aspergillus nidulans contains six possible fatty acyl-CoA synthetases with FaaB being the major synthetase for fatty acid degradation.

Aspergillus nidulans can use a variety of fatty acids as sole carbon and energy sources via its peroxisomal and mitochondrial beta-oxidation pathways. Prior to channelling the fatty acids into beta-oxidation, they need to be activated to their acyl-CoA derivates. Analysis of the genome sequence identified a number of possible fatty acyl-CoA synthetases (FatA, FatB, FatC, FatD, FaaA and FaaB).

Deletion of the RING-finger peroxin 2 gene in Aspergillus nidulans does not affect meiotic development.

Peroxins are required for protein import into peroxisomes as well as for peroxisome biogenesis and proliferation. Loss-of-function mutations in genes for the RING-finger peroxins Pex2, Pex10 and Pex12 lead to a specific block in meiosis in the ascomycete Podospora anserina. However, loss of protein import into peroxisomes does not result in this meiotic defect.

Metabolic and developmental effects resulting from deletion of the citA gene encoding citrate synthase in Aspergillus nidulans.

Citrate synthase is a central activity in carbon metabolism. It is required for the tricarboxylic acid (TCA) cycle, respiration, and the glyoxylate cycle. In Saccharomyces cerevisiae and Arabidopsis thaliana, there are mitochondrial and peroxisomal isoforms encoded by separate genes, while in Aspergillus nidulans, a single gene, citA, encodes a protein with predicted mitochondrial and peroxisomal targeting sequences (PTS).

The RFX protein RfxA is an essential regulator of growth and morphogenesis in Penicillium marneffei.

Fungi are small eukaryotes capable of undergoing multiple complex developmental programs. The opportunistic human pathogen Penicillium marneffei is a dimorphic fungus, displaying vegetative (proliferative) multicellular hyphal growth at 25 degrees C and unicellular yeast growth at 37 degrees C. P.

AoxA is a major peroxisomal long chain fatty acyl-CoA oxidase required for beta-oxidation in A. nidulans.

Filamentous fungi can use a variety of fatty acids (FA) as sole carbon and energy sources. Aspergillus nidulans has been shown to possess both peroxisomal and mitochondrial beta-oxidation pathways. In these studies, the major peroxisomal long chain fatty acyl coenzyme A oxidase AoxA was identified.

Deletion and overexpression of the Aspergillus nidulans GATA factor AreB reveals unexpected pleiotropy.

The Aspergillus nidulans transcription factor AreA is a key regulator of nitrogen metabolic gene expression. AreA contains a C-terminal GATA zinc finger DNA-binding domain and activates expression of genes necessary for nitrogen acquisition. Previous studies identified AreB as a potential negative regulator of nitrogen catabolism showing similarity with Penicillium chrysogenum NreB and Neurospora crassa ASD4.

Anomalously slow rates of reduction of iron(III) polypyridine complexes by triphenyl verdazyl radicals in acetonitrile.

The kinetics and mechanisms of the electron transfer reactions of [Fe(N-N)(3)](ClO(4))(3) [N-N = 2,2'-bipyridine, 1,10-phenanthroline] and [Fe(terpy)(2)](ClO(4))(3) [terpy = 2,2':6,2''-terpyridine] with a range of triphenyl verdazyl radicals have been investigated in acetonitrile at 25 degrees C and I = 0.05 mol dm(-3) (C(4)H(9))(4)NPF(6) using stopped-flow spectrophotometry. It was found that all three iron(III) complexes underwent extensive dissociation in acetonitrile and it was necessary to carry out the reactions in the presence of an excess of the relevant ligand.

Inducer-dependent nuclear localization of a Zn(II)(2)Cys(6) transcriptional activator, AmyR, in Aspergillus nidulans.

AmyR is a Zn(II)(2)Cys(6) transcriptional activator that regulates expression of the amylolytic genes in Aspergillus species. Subcellular localization studies of GFP-fused AmyR in A. nidulans revealed that the fusion protein preferentially localized to the nucleus in response to isomaltose, the physiological inducer of the amylolytic genes.

Contribution of peroxisomes to penicillin biosynthesis in Aspergillus nidulans.

Peroxisomal localization of the third enzyme of the penicillin biosynthesis pathway of Aspergillus nidulans, acyl-coenzyme A:IPN acyltransferase (IAT), is mediated by its atypical peroxisomal targeting signal 1 (PTS1). However, mislocalization of IAT by deletion of either its PTS1 or of genes encoding proteins involved in peroxisome formation or transport does not completely abolish penicillin biosynthesis. This is in contrast to the effects of IAT mislocalization in Penicillium chrysogenum.

Identification of the novel penicillin biosynthesis gene aatB of Aspergillus nidulans and its putative evolutionary relationship to this fungal secondary metabolism gene cluster.

The final step of penicillin biosynthesis in the filamentous fungus Aspergillus nidulans is catalysed by isopenicillin N acyltransferase encoded by the aatA gene. Because there is no bacterial homologue, its evolutionary origin remained obscure. As shown here,disruption of aatA still enabled penicillin production.

Sumoylation in Aspergillus nidulans: sumO inactivation, overexpression and live-cell imaging.

Sumoylation, the reversible covalent attachment of small ubiquitin-like modifier (SUMO) peptides has emerged as an important regulator of target protein function. In Saccharomyces cerevisiae, but not in Schizosaccharyomes pombe, deletion of the gene encoding SUMO peptides is lethal. We have characterized the SUMO-encoding gene, sumO, in the filamentous fungus Aspergillus nidulans.