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Aspergillus Nidulans (aspergillus + nidulan)
Kinds of Aspergillus Nidulans Selected AbstractsDeletion of mdmB impairs mitochondrial distribution and morphology in Aspergillus nidulansCYTOSKELETON, Issue 2 2003Katrin V. Koch Abstract Mitochondria form a dynamic network of interconnected tubes in the cells of Saccharomyces cerevisiae or filamentous fungi such as Aspergillus nidulans,Neurospora crassa, or Podospora anserina. The dynamics depends on the separation of mitochondrial fragments, their movement throughout the cell, and their subsequent fusion with the other parts of the organelle. Interestingly, the microtubule network is required for the distribution in N. crassa and S. pombe, while S. cerevisiae and A. nidulans appear to use the actin cytoskeleton. We studied a homologue of S. cerevisiae Mdm10 in A. nidulans, and named it MdmB. The open reading frame is disrupted by two introns, one of which is conserved in mdm10 of P. anserina. The MdmB protein consists of 428 amino acids with a predicted molecular mass of 46.5 kDa. MdmB shares 26% identical amino acids to Mdm10 from S. cerevisiae, 35% to N. crassa, and 32% to the P. anserina homologue. A MdmB-GFP fusion protein co-localized evenly distributed along mitochondria. Extraction of the protein was only possible after treatment with a non-ionic and an ionic detergent (1% Triton X-100; 0.5% SDS) suggesting that MdmB was tightly bound to the mitochondrial membrane fraction. Deletion of the gene in A. nidulans affected mitochondrial morphology and distribution at 20°C but not at 37°C. mdmB deletion cells contained two populations of mitochondria at lower temperature, the normal tubular network plus some giant, non-motile mitochondria. Cell Motil. Cytoskeleton 55:114,124, 2003. © 2003 Wiley-Liss, Inc. [source] Aspergillus nidulans,-galactosidase of glycoside hydrolase family 36 catalyses the formation of ,-galacto-oligosaccharides by transglycosylationFEBS JOURNAL, Issue 17 2010Hiroyuki Nakai The ,-galactosidase from Aspergillus nidulans (AglC) belongs to a phylogenetic cluster containing eukaryotic ,-galactosidases and ,-galacto-oligosaccharide synthases of glycoside hydrolase family 36 (GH36). The recombinant AglC, produced in high yield (0.65 g·L,1 culture) as His-tag fusion in Escherichia coli, catalysed efficient transglycosylation with ,-(1,6) regioselectivity from 40 mm 4-nitrophenol ,- d -galactopyranoside, melibiose or raffinose, resulting in a 37,74% yield of 4-nitrophenol ,- d -Galp -(1,6)- d -Galp, ,- d -Galp -(1,6)-,- d -Galp -(1,6)- d -Glcp and ,- d -Galp -(1,6)-,- d -Galp -(1,6)- d -Glcp -(,1,,2)- d -Fruf (stachyose), respectively. Furthermore, among 10 monosaccharide acceptor candidates (400 mm) and the donor 4-nitrophenol ,- d -galactopyranoside (40 mm), ,-(1,6) linked galactodisaccharides were also obtained with galactose, glucose and mannose in high yields of 39,58%. AglC did not transglycosylate monosaccharides without the 6-hydroxymethyl group, i.e. xylose, l -arabinose, l -fucose and l -rhamnose, or with axial 3-OH, i.e. gulose, allose, altrose and l -rhamnose. Structural modelling using Thermotoga maritima GH36 ,-galactosidase as the template and superimposition of melibiose from the complex with human GH27 ,-galactosidase supported that recognition at subsite +1 in AglC presumably requires a hydrogen bond between 3-OH and Trp358 and a hydrophobic environment around the C-6 hydroxymethyl group. In addition, successful transglycosylation of eight of 10 disaccharides (400 mm), except xylobiose and arabinobiose, indicated broad specificity for interaction with the +2 subsite. AglC thus transferred ,-galactosyl to 6-OH of the terminal residue in the ,-linked melibiose, maltose, trehalose, sucrose and turanose in 6,46% yield and the ,-linked lactose, lactulose and cellobiose in 28,38% yield. The product structures were identified using NMR and ESI-MS and five of the 13 identified products were novel, i.e. ,- d -Galp -(1,6)- d -Manp; ,- d -Galp -(1,6)-,- d -Glcp -(1,4)- d -Glcp; ,- d -Galp -(1,6)-,- d -Galp -(1,4)- d -Fruf; ,- d -Galp -(1,6)- d -Glcp -(,1,,1)- d -Glcp; and ,- d -Galp -(1,6)-,- d -Glcp -(1,3)- d -Fruf. [source] Relationships between the ethanol utilization (alc) pathway and unrelated catabolic pathways in Aspergillus nidulansFEBS JOURNAL, Issue 17 2003Michel Flipphi The ethanol utilization pathway in Aspergillus nidulans is a model system, which has been thoroughly elucidated at the biochemical, genetic and molecular levels. Three main elements are involved: (a) high level expression of the positively autoregulated activator AlcR; (b) the strong promoters of the structural genes for alcohol dehydrogenase (alcA) and aldehyde dehydrogenase (aldA); and (c) powerful activation of AlcR by the physiological inducer, acetaldehyde, produced from growth substrates such as ethanol and l -threonine. We have previously characterized the chemical features of direct inducers of the alc regulon. These studies allowed us to predict which type of carbonyl compounds might induce the system. In this study we have determined that catabolism of different amino acids, such as l -valine, l -isoleucine, l -arginine and l -proline, produces aldehydes that are either not accumulated or fail to induce the alc system. On the other hand, catabolism of d -galacturonic acid and putrescine, during which aldehydes are transiently accumulated, gives rise to induction of the alc genes. We show that the formation of a direct inducer from carboxylic esters does not depend on alcA -encoded alcohol dehydrogenase I or on AlcR, and suggest that a cytochrome P450 might be responsible for the initial formation of a physiological aldehyde inducer. [source] 2-Methylisocitrate lyases from the bacterium Escherichia coli and the filamentous fungus Aspergillus nidulansFEBS JOURNAL, Issue 12 2001Characterization, comparison of both enzymes In Escherichia coli and Aspergillus nidulans, propionate is oxidized to pyruvate via the methylcitrate cycle. The last step of this cycle, the cleavage of 2-methylisocitrate to succinate and pyruvate is catalysed by 2-methylisocitrate lyase. The enzymes from both organisms were assayed with chemically synthesized threo -2-methylisocitrate; the erythro -diastereomer was not active. 2-Methylisocitrate lyase from E. coli corresponds to the PrpB protein of the prp operon involved in propionate oxidation. The purified enzyme has a molecular mass of approximately 32 kDa per subunit, which is lower than those of isocitrate lyases from bacterial sources (, 48 kDa). 2-Methylisocitrate lyase from A. nidulans shows an apparent molecular mass of 66 kDa per subunit, almost equal to that of isocitrate lyase of the same organism. Both 2-methylisocitrate lyases have a native homotetrameric structure as identified by size-exclusion chromatography. The enzymes show no measurable activity with isocitrate. Starting from 250 mm pyruvate, 150 mm succinate and 10 µm PrpB, the enzymatically active stereoisomer could be synthesized in 1% yield. As revealed by chiral HPLC, the product consisted of a single enantiomer. This isomer is cleaved by 2-methylisocitrate lyases from A. nidulans and E. coli. The PrpB protein reacted with stoichiometric amounts of 3-bromopyruvate whereby the activity was lost and one amino-acid residue per subunit became modified, most likely a cysteine as shown for isocitrate lyase of E. coli. PrpB exhibits 34% sequence identity with carboxyphosphoenolpyruvate phosphonomutase from Streptomyces hygroscopicus, in which the essential cysteine residue is conserved. [source] Evaluating low level sequence identitiesFEBS JOURNAL, Issue 2 2001AROM homologous?, Are Aspergillus QUTA A review published several years ago [Hawkins, A.R. & Lamb, H.K. (1995) Eur. J. Biochem. 232, 7,18] proposed that genetic, biochemical and physiological data can override sequence comparison in the determination of homology in instances where structural information is unavailable. Their lead example was the hypothesis that the transcriptional activator protein for quinate catabolism in Aspergillus nidulans, QUTA, is derived from the pentafunctional AROM protein by a gene duplication followed by cleavage [Hawkins, A.R., Lamb, H.K., Moore, J.D. & Roberts, C.F. (1993) Gene136, 49,54]. We tested this hypothesis by a sensitive combination of position-specific log-odds scoring matrix methods. The position-specific log-odds scoring matrices were derived from a large number of 3-dehydroquinate synthase and 5- enolpyruvylshikimate-3-phosphate synthase domains that were proposed to be the domains from the AROM protein that gave rise to the transcriptional activator protein for quinate metabolism. We show that the degree and pattern of similarity between these position-specific log-odds scoring matrices and the transcriptional activator protein for quinate catabolism in A. nidulans is that expected for random sequences of the same composition. This level of similarity provides no support for the suggested gene duplication and cleavage. The lack of any trace of evidence for homology following a comprehensive sequence analysis indicates that the homology hypothesis is without foundation, underlining the necessity to accept only similarity of sequence and/or structure as evidence of evolutionary relatedness. Further, QUTA is homologous throughout its entire length to an extended family of fungal transcriptional regulatory proteins, rendering the hypothesized QUTA,AROM homology even more problematic. [source] Cloning and sequence analysis of cnaA gene encoding the catalytic subunit of calcineurin from Aspergillus oryzaeFEMS MICROBIOLOGY LETTERS, Issue 1 2001Praveen Rao Juvvadi Abstract Calcineurin has been implicated in ion-homeostasis, stress adaptation in yeast and for hyphal growth in filamentous fungi. Genomic DNA and cDNA encoding the catalytic subunit of calcineurin (cnaA) were isolated from Aspergillus oryzae. The cnaA open reading frame extended to 1727 bp and encoded a putative protein of 514 amino acids. Comparative analysis of the nucleotide sequence of cnaA genomic DNA and cDNA confirmed the presence of three introns and a highly conserved calmodulin binding domain. The deduced amino acid sequence was homologous to calcineurin A from Aspergillus nidulans (92%), Neurospora crassa (84%), human (67%), Saccharomyces cerevisiae (58%) and Schizosaccharomyces pombe (54%). Further, A. oryzae cnaA cDNA complemented S. cerevisiae calcineurin disruptant strain (,cmp1,cmp2), which was not viable in the presence of high concentrations of NaCl (1.2 M) and at alkaline pH 8.5. [source] pH Control of the production of recombinant glucose oxidase in Aspergillus nidulansJOURNAL OF APPLIED MICROBIOLOGY, Issue 2 2004R. Luque Abstract Aims:, Recombinant Aspergillus nidulans sVAL040, capable of synthesizing and secreting glucose oxidase derived from Aspergillus niger was used to study the influence of pH and carbon source on enzyme production. Methods and Results:, Glucose oxidase gene (goxC) was expressed under transcriptional regulation by using the promoter of A. nidulans xlnB gene (encoding an acidic xylanase). A maximum specific glucose oxidase activity of approx. 10 U mg,1 protein and a maximum volumetric productivity of 29·9 U l,1 h,1 were obtained at pH 5·5, after 80 h of growth by using xylose as inducer. Enzyme volumetric productivity increased when xylans were used instead of xylose; however, specific glucose oxidase activity did not differ significantly. Conclusions:, Specific GOX activity obtained at pH 5·5 are two to three times more than those previously described for goxC multicopy transformants of A. nidulans. Xylans were a more powerful inducer than xylose although fungal growth was lower when the polymers were used. Significance and Impact of the Study:, The obtained results by using xlnB promoter in A. nidulans could be useful in improving heterologous enzyme production by using genetic- and process-engineering strategies. [source] Reducing the cost of resistance; experimental evolution in the filamentous fungus Aspergillus nidulansJOURNAL OF EVOLUTIONARY BIOLOGY, Issue 4 2006S. E. SCHOUSTRA Abstract We have studied compensatory evolution in a fludioxonil resistant mutant of the filamentous fungus Aspergillus nidulans. In an evolution experiment lasting for 27 weeks (about 3000 cell cycles) 35 parallel strains of this mutant evolved in three different environmental conditions. Our results show a severe cost of resistance (56%) in the absence of fludioxonil and in all conditions the mutant strain was able to restore fitness without loss of the resistance. In several cases, the evolved strain reached a higher fitness than the original sensitive ancestor. Fitness compensation occurred in one, two or three discrete steps. Genetic analysis of crosses between different evolved strains and between evolved and ancestral strains revealed interaction between compensatory mutations and provided information on the number of loci involved in fitness compensation. In addition, we discuss the opportunities for the experimental study of evolutionary processes provided by the filamentous fungus A. nidulans. [source] A phylogenomic approach to reconstructing the diversification of serine proteases in fungiJOURNAL OF EVOLUTIONARY BIOLOGY, Issue 6 2004G. Hu Abstract Using a phylogenomic approach with 10 fungi of very different virulence and habitat, we determined that there was substantial diversification of subtilase-type proteases early in ascomycete history (with subsequent loss in many lineages) but with no comparable diversification of trypsins. Patterns of intron loss and the degree of divergence between paralogues demonstrated that the proliferation of proteinase K subtilases and subtilisin type subtilases seen in pathogenic ascomycetes (Metarhizium anisopliae, Magnaporthe grisea, Fusarium graminearum) occurred after the basidiomycete/ascomycete split but predated radiation of ascomycete lineages. This suggests that the early ascomycetes had a lifestyle that selected for multiple proteases, whereas the current disparity in gene numbers between ascomycete lineages results from retention of genes in at least some pathogens that have been lost in other lineages (yeasts, Aspergillus nidulans, Neurospora crassa). A similar prevailing trend towards lineage specific gene loss of trypsins in saprophytes and some pathogens suggests that their phylogenetic breadth will have been much wider in early fungi than currently. [source] Mononucleotide repeats represent an important source of polymorphic microsatellite markers in Aspergillus nidulansMOLECULAR ECOLOGY RESOURCES, Issue 2 2009JULIAN K. CHRISTIANS Abstract In fungi, microsatellites occur less frequently throughout the genome and tend to be less polymorphic compared with other organisms. Most studies that develop microsatellites for fungi focus on dinucleotide and trinucleotide repeats, and thus mononucleotide repeats, which are much more abundant in fungal genomes, may represent an overlooked resource. This study examined the relative probabilities of polymorphism in mononucleotide, dinucleotide and trinucleotide repeats in Aspergillus nidulans. As previously found, the probability of polymorphism increased with increasing number of repeating units. Dinucleotide and trinucleotide repeats had higher probabilities of polymorphism than mononucleotide repeats, but this was offset by the presence of numerous long mononucleotide repeats within the genome. Mononucleotide microsatellites with 20 or more repeating units have a probability of polymorphism similar to dinucleotide and trinucleotide microsatellites, and therefore, consideration of mononucleotide repeats will substantially increase the number of potential markers available. [source] FlbC is a putative nuclear C2H2 transcription factor regulating development in Aspergillus nidulansMOLECULAR MICROBIOLOGY, Issue 5 2010Nak-Jung Kwon Summary Asexual development (conidiation) in Aspergillus is governed by multiple regulators. Here, we characterize the upstream developmental activator FlbC in Aspergillus nidulans. flbC mRNA is detectable throughout the life cycle, at relatively high levels during vegetative growth, early asexual and late sexual developmental phases. The deletion of flbC causes a delay/reduction in conidiation, brlA and vosA expression, and conidial germination. While overexpression of flbC (OEflbC) does not elaborate conidiophores, it inhibits hyphal growth and activates expression of brlA, abaA and vosA, but not wetA. FlbC is conserved in filamentous Ascomycetes containing two C2H2 zinc fingers at the C-terminus and a putative activation domain at the N-terminus. FlbC localizes in the nuclei of both hyphae and developmental cells. Localization and expression of FlbC are not affected by the absence of FlbB or FlbE, and vice versa. Importantly, overexpression of flbC causes growth inhibition and activation of abaA and vosA in the absence of brlA and abaA respectively. In vitro DNA-binding assay reveals that FlbC binds to the brlA, abaA and vosA, but not the wetA, promoters. In summary, FlbC is a putative nuclear transcription factor necessary for proper activation of conidiation, and its balanced activity is crucial for governing growth and development in A. nidulans. [source] Characterization of an acyl-CoA: carboxylate CoA-transferase from Aspergillus nidulans involved in propionyl-CoA detoxificationMOLECULAR MICROBIOLOGY, Issue 3 2008Christian B. Fleck Summary Filamentous fungi metabolize toxic propionyl-CoA via the methylcitrate cycle. Disruption of the methylcitrate synthase gene leads to an accumulation of propionyl-CoA and attenuates virulence of Aspergillus fumigatus. However, addition of acetate, but not ethanol, to propionate-containing medium strongly reduces the accumulation of propionyl-CoA and restores growth of the methylcitrate synthase mutant. Therefore, the existence of a CoA-transferase was postulated, which transfers the CoASH moiety from propionyl-CoA to acetate and, thereby, detoxifying the cell. In this study, we purified the responsible protein from Aspergillus nidulans and characterized its biochemical properties. The enzyme used succinyl-, propionyl- and acetyl-CoA as CoASH donors and the corresponding acids as acceptor molecules. Although the protein displayed high sequence similarity to acetyl-CoA hydrolases this activity was hardly detectable. We additionally identified and deleted the coding DNA sequence of the CoA-transferase. The mutant displayed weak phenotypes in the presence of propionate and behaved like the wild type when no propionate was present. However, when a double-deletion mutant defective in both methylcitrate synthase and CoA-transferase was constructed, the resulting strain was unable to grow on media containing acetate and propionate as sole carbon sources, which confirmed the in vivo activity of the CoA-transferase. [source] Developmental regulation of the glyoxylate cycle in the human pathogen Penicillium marneffeiMOLECULAR MICROBIOLOGY, Issue 6 2006David Cánovas Summary Penicillium marneffei is a thermally dimorphic opportunistic human pathogen with a saprophytic filamentous hyphal form at 25°C and a pathogenic unicellular yeast form at 37°C. During infection. P. marneffei yeast cells exist intracellularly in macrophages. To cope with nutrient deprivation during the infection process, a number of pathogens employ the glyoxylate cycle to utilize fatty acids as carbon sources. The genes which constitute this pathway have been implicated in pathogenesis. To investigate acetate and fatty acid utilization, the acuD gene encoding a key glyoxylate cycle enzyme (isocitrate lyase) was cloned. The acuD gene is regulated by both carbon source and temperature in P. marneffei, being strongly induced at 37°C even in the presence of a repressing carbon source such as glucose. When introduced into the non-pathogenic monomorphic fungus Aspergillus nidulans, the P. marneffei acuD promoter only responds to carbon source. Similarly, when the A. nidulans acuD promoter is introduced into P. marneffei it only responds to carbon source suggesting that P. marneffei possesses both cis elements and trans -acting factors to control acuD by temperature. The Zn(II)2Cys6 DNA binding motif transcriptional activator FacB was cloned and is responsible for carbon source-, but not temperature-, dependent induction of acuD. The expression of acuD at 37°C is induced by AbaA, a key regulator of morphogenesis in P. marneffei, but deletion of abaA does not completely eliminate temperature-dependent induction, suggesting that acuD and the glyoxylate cycle are regulated by a complex network of factors in P. marneffei which may contribute to its pathogenicity. [source] Requirement of spermidine for developmental transitions in Aspergillus nidulansMOLECULAR MICROBIOLOGY, Issue 3 2002Yuan Jin Summary Deletion of the spermidine synthase gene in the fungus Aspergillus nidulans results in a strain, ,spdA, which requires spermidine for growth and accumulates putrescine as the sole polyamine. Vegetative growth but not sporulation or sterigmatocystin production is observed when ,spdA is grown on media supplemented with 0.05,0.10 mM exogenous spermidine. Supplementation of ,spdA with , 0.10 mM spermidine restores sterigmatocystin production and , 0.50 mM spermidine produces a phenotype with denser asexual spore production and decreased radial hyphal growth compared with the wild type. ,spdA spores germinate in unsupplemented media but germ tube growth ceases after 8 h upon which time the spores swell to approximately three times their normal diameter. Hyphal growth is resumed upon addition of 1.0 mM spermidine. Suppression of a G protein signalling pathway could not force asexual sporulation and sterigmatocystin production in ,spdA strains grown in media lacking spermidine but could force both processes in ,spdA strains supplemented with 0.05 mM spermidine. These results show that increasing levels of spermidine are required for the transitions from (i) germ tube to hyphal growth and (ii) hyphal growth to tissue differentiation and secondary metabolism. Suppression of G protein signalling can over-ride the spermidine requirement for the latter but not the former transition. [source] Dynamic distribution of BIMGPP1 in living hyphae of Aspergillus indicates a novel role in septum formationMOLECULAR MICROBIOLOGY, Issue 5 2002H. Fox Summary Mutation of bimG, the major protein phosphatase 1 gene in Aspergillus nidulans, causes multiple cell cycle and hyphal growth defects that are associated with overphosphorylation of subcellular components. We have used functional translational fusions with the green fluorescent protein (GFP) to show that BIMG has at least four discrete locations within growing hyphae. Three of these locations, the hyphal tip, the spindle pole body and the nucleus, correlate with previously known requirements for bimGPP1 in mitosis and hyphal growth and are highly dynamic. BIMG-GFP in the hyphal tip seemed to be associated with the plasma membrane and formed a collar of fluorescence within the apical dome. The distribution of nuclear BIMG-GFP varied depending on nutritional conditions; on poor medium, it concentrated more in the nucleolus than in the nucleoplasm, whereas on rich medium, it was more evenly distributed between the two nuclear regions. The association of BIMG-GFP with developing septa was transient, and we present evidence that BIMG phosphatase plays a direct role in septum formation, distinct from its role in mitosis. We conclude that, by being physically present at several sites, the BIMG phosphatase has roles in multiple cellular processes. [source] SEPH, a Cdc7p orthologue from Aspergillus nidulans, functions upstream of actin ring formation during cytokinesisMOLECULAR MICROBIOLOGY, Issue 1 2001Kenneth S. Bruno In the filamentous fungus, Aspergillus nidulans, multiple rounds of nuclear division occur before cytokinesis, allowing an unambiguous identification of genes required specifically for cytokinesis. As in animal cells, both an intact microtubule cytoskeleton and progression through mitosis are required for actin ring formation and contraction. The sepH gene from A. nidulans was discovered in a screen for temperature-sensitive cytokinesis mutants. Sequence analysis showed that SEPH is 42% identical to the serine,threonine kinase Cdc7p from fission yeast. Signalling through the Septation Initiation Network (SIN), which includes Cdc7p and the GTPase Spg1p, is emerging as a primary regulatory pathway used by fission yeast to control cytokinesis. A similar group of proteins comprise the Mitotic Exit Network (MEN) in budding yeast. This is the first direct evidence for the existence of a functional SIN,MEN pathway outside budding and fission yeast. In addition to SEPH, potential homologues were also identified in other fungi and plants but not in animal cells. Deletion of sepH resulted in a viable strain that failed to septate at any temperature. Interestingly, quantitative analysis of the actin cytoskeleton revealed that sepH is required for construction of the actin ring. Therefore, SEPH is distinct from its counterpart in fission yeast, in which SIN components operate downstream of actin ring formation and are necessary for ring contraction and later events of septation. We conclude that A. nidulans has components of a SIN,MEN pathway, one of which, SEPH, is required for early events during cytokinesis. [source] SREA is involved in regulation of siderophore biosynthesis, utilization and uptake in Aspergillus nidulansMOLECULAR MICROBIOLOGY, Issue 5 2001Harald Oberegger Under conditions of low iron availability, most fungi excrete siderophores in order to mobilize extracellular iron. We show that lack of the GATA-type transcription factor SREA in Aspergillus nidulans not only leads to derepression of siderophore biosynthesis but also to deregulation of siderophore-bound iron uptake and ornithine esterase expression. Furthermore, SREA deficiency causes increased accumulation of ferricrocin, the siderophore responsible for intracellular iron storage. In sreA deletion strains, extracellular siderophore production is derepressed but still regulated negatively by iron availability, indicating the presence of an additional iron-regulatory mechanism. In contrast, iron affects ferricrocin accumulation in a positive way, suggesting a protective role for this siderophore in detoxification of intracellular iron excess. The harmfulness of deregulated iron uptake in this mutant is demonstrated by increased expression of genes encoding the antioxidative enzymes catalase CATB and the superoxide dismutases SODA and SODB. It is noteworthy that iron starvation was found to repress catB expression in wild-type (wt) and SREA-deficient strains, consistent with catB being subject to SREA-independent iron regulation. Differential display led to the identification of putative SREA target genes amcA and mirA. The deduced MIRA amino acid sequence displays significant similarity to recently characterized siderophore permeases of Saccharomyces cerevisiae. amcA encodes a putative mitochondrial carrier for the siderophore precursor ornithine, indicating cross-regulation of siderophore and ornithine metabolism. [source] Comparison of the sequences of the Aspergillus nidulans hxB and Drosophila melanogaster ma-l genes with nifS from Azotobacter vinelandii suggests a mechanism for the insertion of the terminal sulphur atom in the molybdopterin cofactorMOLECULAR MICROBIOLOGY, Issue 1 2000Laïla Amrani The molybdopterin cofactor (MoCF) is required for the activity of a variety of oxidoreductases. The xanthine oxidase class of molybdoenzymes requires the MoCF to have a terminal, cyanolysable sulphur ligand. In the sulphite oxidase/nitrate reductase class, an oxygen is present in the same position. Mutations in both the ma-l gene of Drosophila melanogaster and the hxB gene of Aspergillus nidulans result in loss of activities of all molybdoenzymes that necessitate a cyanolysable sulphur in the active centre. The ma-l and hxB genes encode highly similar proteins containing domains common to pyridoxal phosphate-dependent cysteine transulphurases, including the cofactor binding site and a conserved cysteine, which is the putative sulphur donor. Key similarities were found with NifS, the enzyme involved in the generation of the iron,sulphur centres in nitrogenase. These similarities suggest an analogous mechanism for the generation of the terminal molybdenum-bound sulphur ligand. We have identified putative homologues of these genes in a variety of organisms, including humans. The human homologue is located in chromosome 18.q12. [source] Identification of medically important Aspergillus species by single strand conformational polymorphism (SSCP) of the PCR-amplified intergenic spacer region Identifizierung humanmedizinisch relevanter Aspergillus-Arten durch Analyse der Einzelstrang-Konformations-Polymorphismen der amplifizierten Intergenic-Spacer-RegionMYCOSES, Issue 11-12 2000P.-M. Rath Aspergillus; Identifizierung; ITS-Region; PCR; SSCP Summary., The amplified 5.8S RNA coding DNA with the neighbouring internal transcribed spacers ITS I and ITS II (ITS I,5.8S rDNA , ITS II) of 27 culture collection strains of Aspergillus fumigatus, Aspergillus flavus, Aspergillus nidulans, Aspergillus niger, and Aspergillus terreus were investigated by single strand conformational polymorphism (SSCP) analysis. All strains showed a polymerase gel electrophoresis (PCR) product of 0.6 kb. Separation of DNA single strands of the PCR product in an acrylamide-bisacrylamide gel containing formamide SSCP resulted in individual patterns for each of the species. A minor variability within the species A. fumigatus and A. flavus did not affect the correct species identification. The results were confirmed when investigating 55 wild strains from patients and the environment. It is concluded that the analysis of the amplified ITS I,5.8S rDNA , ITS II region by SSCP allows the differentiation of the medically most relevant aspergilli. As the method does not require morphologically fully developed fungal colonies, it yields species diagnosis faster than the conventional macroscopic and microscopic identification. Zusammenfassung., Die amplifizierte 5,8S RNA kodierende DNA mit den benachbarten Internal Transcribed Spacern ITS I und ITS II (ITS I,5,8S rDNA , ITS II) von 27 Referenzstämmen der Spezies Aspergillus fumigatus, A. flavus, A. nidulans, A. niger und A. terreus wurde durch Analyse der Einzelstrang-Konformations-Polymorphismen (SSCP) untersucht. Alle Stämme zeigten ein PCR-Produkt mit einer Größe von 0,6 kb. Die SSCP-Muster nach Auftrennung der DNA-Einzelstränge dieses Produktes in einem Acrylamid-Bisacrylamid Gel mit Formamid waren für jede der untersuchten Spezies charakteristisch. Eine geringfügige Variabilität der Muster bei den Spezies A. fumigatus und A. flavus schränkte die Interpretation nicht ein. Die Ergebnisse wurden bei der Analyse von 55 Isolaten von Patienten und aus der Umwelt bestätigt. Die SSCP-Analyse der amplifizierten ITS I,5,8S rDNA , ITS II Region erlaubt somit eine Differenzierung der humanmedizinisch wichtigsten Aspergillus -Spezies vor der Ausbildung charakteristischer makro- und mikromorphologischer Strukturen. [source] Evolutionary history of the ancient cutinase family in five filamentous Ascomycetes reveals differential gene duplications and losses and in Magnaporthe grisea shows evidence of sub- and neo-functionalizationNEW PHYTOLOGIST, Issue 3 2008Pari Skamnioti Summary ,,The cuticle is the first barrier for fungi that parasitize plants systematically or opportunistically. Here, the evolutionary history is reported of the multimembered cutinase families of the plant pathogenic Ascomycetes Magnaporthe grisea, Fusarium graminearum and Botrytis cinerea and the saprotrophic Ascomycetes Aspergillus nidulans and Neurospora crassa. ,,Molecular taxonomy of all fungal cutinases demonstrates a clear division into two ancient subfamilies. No evidence was found for lateral gene transfer from prokaryotes. The cutinases in the five Ascomycetes show significant copy number variation, they form six clades and their extreme sequence diversity is highlighted by the lack of consensus intron. The average ratio of gene duplication to loss is 2 : 3, with the exception of M. grisea and N. crassa, which exhibit extreme family expansion and contraction, respectively. ,,Detailed transcript profiling in vivo, categorizes the M. grisea cutinases into four regulatory patterns. Symmetric or asymmetric expression profiles of phylogenetically related cutinase genes suggest subfunctionalization and neofunctionalization, respectively. ,,The cutinase family-size per fungal species is discussed in relation to genome characteristics and lifestyle. The ancestry of the cutinase gene family, together with the expression divergence of its individual members provides a first insight into the drivers for niche differentiation in fungi. [source] Microscopy reveals disease control through novel effects on fungal development: a case study with an early-generation benzophenone fungicide,PEST MANAGEMENT SCIENCE (FORMERLY: PESTICIDE SCIENCE), Issue 5 2006Mark R Schmitt Abstract The benzophenones are a new class of agricultural fungicides that demonstrate protectant, curative and eradicative/antisporulant activity against powdery mildews. The chemistry is represented in the marketplace by the fungicide metrafenone, recently introduced by BASF and discussed in the following paper. The benzophenones show no evidence of acting by previously identified biochemical mechanisms, nor do they show cross-resistance with existing fungicides. The value of microscopy in elucidating fungicide mode of action is demonstrated through identification of the effects of an early benzophenone, eBZO, on mildew development. eBZO caused profound alterations in the morphology of powdery mildews of both monocotyledons and dicotyledons, affecting multiple stages of fungal development, including spore germination, appressorial formation, penetration, surface hyphal morphology and sporogenesis. Identification of analogous effects of eBZO on sporulation in the model organism Aspergillus nidulans (Eidam) Winter provides a unique opportunity to elucidate important morphogenetic regulatory sites in the economically important obligate pathogens, the powdery mildews. Benzophenones provide a further example of the benefits of whole-organism testing in the search for novel fungicide modes of action. Copyright © 2006 Society of Chemical Industry [source] Impact of fungal drug transporters on fungicide sensitivity, multidrug resistance and virulence,PEST MANAGEMENT SCIENCE (FORMERLY: PESTICIDE SCIENCE), Issue 3 2006Maarten A de Waard Abstract Drug transporters are membrane proteins that provide protection for organisms against natural toxic products and fungicides. In plant pathogens, drug transporters function in baseline sensitivity to fungicides, multidrug resistance (MDR) and virulence on host plants. This paper describes drug transporters of the filamentous fungi Aspergillus nidulans (Eidam) Winter, Botrytis cinerea Pers and Mycosphaerella graminicola (Fückel) Schroter that function in fungicide sensitivity and resistance. The fungi possess ATP-binding cassette (ABC) drug transporters that mediate MDR to fungicides in laboratory mutants. Similar mutants are not pronounced in field resistance to most classes of fungicide but may play a role in resistance to azoles. MDR may also explain historical cases of resistance to aromatic hydrocarbon fungicides and dodine. In clinical situations, MDR development in Candida albicans (Robin) Berkhout mediated by ABC transporters in patients suffering from candidiasis is common after prolonged treatment with azoles. Factors that can explain this striking difference between agricultural and clinical situations are discussed. Attention is also paid to the risk of MDR development in plant pathogens in the future. Finally, the paper describes the impact of fungal drug transporters on drug discovery. Copyright © 2006 Society of Chemical Industry [source] Genotoxicity of Achillea millefolium essential oil in diploid cells of Aspergillus nidulansPHYTOTHERAPY RESEARCH, Issue 2 2009Juliane Rocha de Sant'Anna Abstract The essential oil of Achillea millefolium is commonly used in folk medicine for the treatment of several diseases and has been demonstrated previously to exert an in vitro antimicrobial activity against human pathogens. Current study investigates the genotoxic activity of A. millefolium oil. The oil's major constituents are: chamazulene (42.15%), sabinene (19.72%), terpin-4-ol (5.22%), , -caryophyllene (4.44%) and eucalyptol (3.10%), comprising 74.63% of the total. The oil's genotoxic evaluation was performed at concentrations of 0.13 µL/mL, 0.19 µL/mL and 0.25 µL/mL with a heterozygous diploid strain of Aspergillus nidulans, named A757//UT448, with green conidia. A statistically significant increasing number of yellow and white mitotic recombinants, per colony, of the diploid strain was reported after oil treatment with 0.19 µL/mL and 0.25 µL/mL concentrations. The genotoxicity of the oil was associated with the induction of mitotic non-disjunction or crossing-over by oil. Copyright © 2008 John Wiley & Sons, Ltd. [source] Proteomic analysis of Aspergillus nidulans cultured under hypoxic conditionsPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 1 2009Motoyuki Shimizu Abstract The fungus Aspergillus nidulans reduces nitrate to ammonium and simultaneously oxidizes ethanol to acetate to generate ATP under hypoxic conditions in a mechanism called ammonia fermentation (Takasaki, K. et al.. J. Biol. Chem. 2004, 279, 12414,12420). To elucidate the mechanism, the fungus was cultured under normoxic and hypoxic (ammonia fermenting) conditions, intracellular proteins were resolved by 2-DE, and 332 protein spots were identified using MALDI MS after tryptic digestion. Alcohol and aldehyde dehydrogenases that play key roles in oxidizing ethanol were produced at the basal level under hypoxic conditions but were obviously provoked by ethanol under normoxic conditions. Enzymes involved in gluconeogenesis, as well as the tricarboxylic and glyoxylate cycles, were downregulated. These results indicate that the mechanism of fungal energy conservation is altered under hypoxic conditions. The results also showed that proteins in the pentose phosphate pathway as well as the metabolism of both nucleotide and thiamine were upregulated under hypoxic conditions. Levels of xanthine and hypoxanthine, deamination products of guanine and adenine were increased in DNA from hypoxic cells, indicating an association between hypoxia and intracellular DNA base damage. This study is the first proteomic comparison of the hypoxic responses of A. nidulans. [source] Induction of Cell Wall Thickening by the Antifungal Compound Dihydromaltophilin Disrupts Fungal Growth and is Mediated by Sphingolipid BiosynthesisTHE JOURNAL OF EUKARYOTIC MICROBIOLOGY, Issue 2 2009SHAOJIE LI ABSTRACT. Dihydromaltophilin (heat-stable antifungal factor [HSAF]) is an antifungal metabolite produced in Lysobacter enzymogenes biocontrol strain C3. This compound induces cell wall thickening in Aspergillus nidulans. Here we show that the cell wall thickening is a general response to HSAF in diverse fungal species. In the A. nidulans model, the thickened cell wall negatively affects hyphal growth. Growth of HSAF-pre-treated hyphae failed to resume at hyphal tips with thick cell wall and the actin cable could not re-polarize at the thickened region of the cell wall, even after the treated hyphae were transferred to drug-free medium. Moreover, HSAF-induced cell wall thickening is mediated by sphingolipid synthesis: HSAF failed to induce cell wall thickening in the absence of ceramide synthase BarA and the sphingolipid synthesis inhibitor myriocin was able to suppress HSAF-induced cell wall thickening. The thickened cell wall could be digested by chitinase suggesting that chitin contributes to the HSAF-induced thickening. Furthermore, HSAF treatment activated the transcription of two chitin synthase encoding genes chsB and chsC. [source] The structure and domain organization of Escherichia coli isocitrate lyaseACTA CRYSTALLOGRAPHICA SECTION D, Issue 9 2001K. L. Britton Enzymes of the glyoxylate-bypass pathway are potential targets for the control of many human diseases caused by such pathogens as Mycobacteria and Leishmania. Isocitrate lyase catalyses the first committed step in this pathway and the structure of this tetrameric enzyme from Escherichia coli has been determined at 2.1,Å resolution. E. coli isocitrate lyase, like the enzyme from other prokaryotes, is located in the cytoplasm, whereas in plants, protozoa, algae and fungi this enzyme is found localized in glyoxysomes. Comparison of the structure of the prokaryotic isocitrate lyase with that from the eukaryote Aspergillus nidulans reveals a different domain structure following the deletion of approximately 100 residues from the larger eukaryotic enzyme. Despite this, the active sites of the prokaryotic and eukaryotic enzymes are very closely related, including the apparent disorder of two equivalent segments of the protein that are known to be involved in a conformational change as part of the enzyme's catalytic cycle. [source] Asperfuranone from Aspergillus nidulans Inhibits Proliferation of Human Non-Small Cell Lung Cancer A549 Cells via Blocking Cell Cycle Progression and Inducing ApoptosisBASIC AND CLINICAL PHARMACOLOGY & TOXICOLOGY, Issue 1 2010Clay C. C. Wang To identity the anti-cancer mechanism of asperfuranone, we assayed its effect on apoptosis, cell cycle distribution, and levels of p53, p21 Waf1/Cip1, Fas/APO-1 receptor and Fas ligand. Enzyme-linked immunosorbent assay showed that the G0/G1 phase arrest might be due to p53-dependent induction of p21 Waf1/Cip1. An enhancement in Fas/APO-1 and its two form ligands, membrane-bound Fas ligand (mFasL) and soluble Fas ligand (sFasL), might be responsible for the apoptotic effect induced by asperfuranone. Our study reports here for the first time that the induction of p53 and the activity of Fas/Fas ligand apoptotic system may participate in the anti-proliferative activity of asperfuranone in A549 cells. [source] Nonribosomal Peptide Synthesis in Schizosaccharomyces pombe and the Architectures of Ferrichrome-Type Siderophore Synthetases in FungiCHEMBIOCHEM, Issue 4 2006Torsten Schwecke Dr. Abstract A nonribosomal peptide synthetase (NRPS) in Schizosaccharomyces pombe, which possesses an unusual structure incorporating three adenylation domains, six thiolation domains and six condensation domains, has been shown to produce the cyclohexapeptide siderophore ferrichrome. One of the adenylation domains is truncated and contains a distorted key motif. Substrate-binding specificities of the remaining two domains were assigned by molecular modelling to glycine and to N -acetyl- N -hydroxy- L -ornithine. Hexapeptide siderophore synthetase genes of Magnaporthe grisea and Fusarium graminearum were both identified and analyzed with respect to substrate-binding sites, and the predicted product ferricrocin was identified in each. A comparative analysis of these synthetase systems, including those of the basidiomycete Ustilago maydis, the homobasidiomycete Omphalotus olearius and the ascomycetes Aspergillus nidulans, Aspergillus fumigatus, Fusarium graminearum, Cochliobolus heterostrophus, Neurospora crassa and Aureobasidium pullulans, revealed divergent domain compositions with respect to their number and positioning, although all produce similar products by iterative processes. A phylogenetic analysis of both NRPSs and associated L - N5 -ornithine monooxygenases revealed that ferrichrome-type siderophore biosynthesis has coevolved in fungi with varying in trans interactions of NRPS domains. [source] |