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Carvalho ND Arentshorst M Kooistra R Stam H Sagt CM van den Hondel CA Ram AF 《Applied microbiology and biotechnology》2011,89(2):357-373
Endoplasmic reticulum associated degradation (ERAD) is a conserved mechanism to remove misfolded proteins from the ER by targeting
them to the proteasome for degradation. To assess the role of ERAD in filamentous fungi, we have examined the consequences
of disrupting putative ERAD components in the filamentous fungus Aspergillus niger. Deletion of derA, doaA, hrdC, mifA, or mnsA in A. niger yields viable strains, and with the exception of doaA, no significant growth phenotype is observed when compared to the parental strain. The gene deletion mutants were also made
in A. niger strains containing single- or multicopies of a glucoamylase–glucuronidase (GlaGus) gene fusion. The induction of the unfolded
protein response (UPR) target genes (bipA and pdiA) was dependent on the copy number of the heterologous gene and the ERAD gene deleted. The highest induction of UPR target
genes was observed in ERAD mutants containing multiple copies of the GlaGus gene. Western blot analysis revealed that deletion
of the derA gene in the multicopy GlaGus overexpressing strain resulted in a 6-fold increase in the intracellular amount of GlaGus protein
detected. Our results suggest that impairing some components of the ERAD pathway in combination with high expression levels
of the heterologous protein results in higher intracellular protein levels, indicating a delay in protein degradation. 相似文献
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Jørgensen TR Park J Arentshorst M van Welzen AM Lamers G Vankuyk PA Damveld RA van den Hondel CA Nielsen KF Frisvad JC Ram AF 《Fungal genetics and biology : FG & B》2011,48(5):544-553
A characteristic hallmark of Aspergillus niger is the formation of black conidiospores. We have identified four loci involved in spore pigmentation of A. niger by using a combined genomic and classical complementation approach. First, we characterized a newly isolated color mutant, colA, which lacked pigmentation resulting in white or colorless conidia. Pigmentation of the colA mutant was restored by a gene (An12g03950) which encodes a putative 4'phosphopantetheinyl transferase protein (PptA). 4'Phosphopantetheinyl transferase activity is required for the activation of Polyketide Synthases (PKSs) and/or Non-Ribosomal Peptide Synthases (NRPSs). The loci whose mutation resulted in fawn, olive, and brown color phenotypes were identified by complementation. The fawn phenotype was complemented by a PKS protein (FwnA, An09g05730), the ovlA mutant by An14g05350 (OlvA) and the brnA mutant by An14g05370 (BrnA), the respective homologs of alb1/pksP, ayg1 and abr1 in A. fumigatus. Targeted disruption of the pptA, fwnA, olvA and brnA genes confirmed the complementation results. Disruption of the pptA gene abolished synthesis of all polyketides and non-ribosomal peptides, while the naphtho-γ-pyrone subclass of polyketides were specifically dependent on fwnA, and funalenone on fwnA, olvA and brnA. Thus, secondary metabolite profiling of the color mutants revealed a close relationship between polyketide synthesis and conidial pigmentation in A. niger. 相似文献
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Kwon MJ Arentshorst M Roos ED van den Hondel CA Meyer V Ram AF 《Molecular microbiology》2011,79(5):1151-1167
Rho GTPases are signalling molecules regulating morphology and multiple cellular functions including metabolism and vesicular trafficking. To understand the connection between polarized growth and secretion in the industrial model organism Aspergillus niger, we investigated the function of all Rho family members in this organism. We identified six Rho GTPases in its genome and used loss-of-function studies to dissect their functions. While RhoA is crucial for polarity establishment and viability, RhoB and RhoD ensure cell wall integrity and septum formation respectively. RhoC seems to be dispensable for A. niger. RacA governs polarity maintenance via controlling actin but not microtubule dynamics, which is consistent with its localization at the hyphal apex. Both deletion and dominant activation of RacA (Rac(G18V)) provoke an actin localization defect and thereby loss of polarized tip extension. Simultaneous deletion of RacA and CftA (Cdc42) is lethal; however, conditional overexpression of RacA in this strain can substitute for CftA, indicating that both proteins concertedly control actin dynamics. We finally identified NoxR as a RacA-specific effector, which however, is not important for apical dominance as reported for A. nidulans but for asexual development. Overall, the data show that individual Rho GTPases contribute differently to growth and morphogenesis within filamentous fungi. 相似文献
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Damveld RA Arentshorst M VanKuyk PA Klis FM van den Hondel CA Ram AF 《Fungal genetics and biology : FG & B》2005,42(10):873-885
Glycosylphosphatidylinositol (GPI)-anchored proteins in fungi are found at the cell surface, either as plasma membrane proteins (GPI-PMPs) or attached by a remnant of the GPI-anchor to the cell wall (GPI-CWPs). GPI-CWPs can be extracted from the cell wall by treatment with hydrofluoric acid (HF), which cleaves the phosphodiester bond that is present in the remnant of the GPI-anchor. The filamentous fungus Aspergillus niger contains at least seven HF-extractable cell wall mannoproteins. One gene encoding an HF-extractable cell wall mannoprotein, cwpA, was cloned and further characterised. The protein sequence of CwpA indicated the presence of two hydrophobic signal sequences both at the N-terminus and C-terminus of the protein, for entering the ER and the addition of a GPI-anchor, respectively. A CwpA-specific antiserum was raised and in combination with fractionation experiments, we show that this protein was abundantly present as an HF-extractable protein in the cell wall of A. niger. 相似文献
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A novel screening method for cell wall mutants in Aspergillus niger identifies UDP-galactopyranose mutase as an important protein in fungal cell wall biosynthesis 总被引:1,自引:0,他引:1 下载免费PDF全文
Damveld RA Franken A Arentshorst M Punt PJ Klis FM van den Hondel CA Ram AF 《Genetics》2008,178(2):873-881
To identify cell wall biosynthetic genes in filamentous fungi and thus potential targets for the discovery of new antifungals, we developed a novel screening method for cell wall mutants. It is based on our earlier observation that the Aspergillus niger agsA gene, which encodes a putative alpha-glucan synthase, is strongly induced in response to cell wall stress. By placing the agsA promoter region in front of a selectable marker, the acetamidase (amdS) gene of A. nidulans, we reasoned that cell wall mutants with a constitutively active cell wall stress response pathway could be identified by selecting mutants for growth on acetamide as the sole nitrogen source. For the genetic screen, a strain was constructed that contained two reporter genes controlled by the same promoter: the metabolic reporter gene PagsA-amdS and PagsA-H2B-GFP, which encodes a GFP-tagged nuclear protein. The primary screen yielded 161 mutants that were subjected to various cell wall-related secondary screens. Four calcofluor white-hypersensitive, osmotic-remediable thermosensitive mutants were selected for complementation analysis. Three mutants were complemented by the same gene, which encoded a protein with high sequence identity with eukaryotic UDP-galactopyranose mutases (UgmA). Our results indicate that galactofuranose formation is important for fungal cell wall biosynthesis and represents an attractive target for the development of antifungals. 相似文献
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Reijngoud Jos Arentshorst Mark Ruijmbeek Claudine Reid Ian Alazi Ebru Demirci Punt Peter J. Tsang Adrian Ram Arthur F. J. 《Biotechnology letters》2021,43(7):1323-1336
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Vera Meyer Mark Arentshorst Cees A M J J van den Hondel Arthur F J Ram 《Fungal genetics and biology : FG & B》2008,45(2):152-164
Hyphal tip growth is a key feature of filamentous fungi, however, the molecular mechanism(s) that regulate cell polarity are poorly understood. On the other hand, much more is known about polarised growth in the yeast Saccharomyces cerevisiae. Here, the proteins Spa2p, Bni1p, Bud6p and Pea2p form a protein complex named the polarisome known to be important for the assurance of polar growth. We searched the genome of Aspergillus niger and identified homologues for Spa2p, Bni1p, Bud6p but not for Pea2p. We characterised the function of the Spa2p homologue SpaA by determining its cellular localisation and by constructing deletion and overexpressing mutant strains. SpaA was found to be localised exclusively at the hyphal tip, suggesting that SpaA can be used as marker for polarisation. Deletion and overexpression of spaA resulted in reduced growth rate, increased hyphal diameter and polarity defects, indicating that one of the functions of SpaA is to ensure polarity maintenance. In addition, we could show that SpaA is able to complement the defective haploid invasive growth phenotype of a S. cerevisiae SPA2 null mutant. We suggest that the function of SpaA is to ensure maximal polar growth rate in A. niger. 相似文献
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Expanding the ku70 toolbox for filamentous fungi: establishment of complementation vectors and recipient strains for advanced gene analyses 总被引:1,自引:0,他引:1
Neuza D. S. P. Carvalho Mark Arentshorst Min Jin Kwon Vera Meyer Arthur F. J. Ram 《Applied microbiology and biotechnology》2010,87(4):1463-1473
Mutants with a defective non-homologous-end-joining (NHEJ) pathway have boosted functional genomics in filamentous fungi as
they are very efficient recipient strains for gene-targeting approaches, achieving homologous recombination frequencies up
to 100%. For example, deletion of the ku70 homologous gene kusA in Aspergillus niger resulted in a recipient strain in which deletions of essential or non-essential genes can efficiently be obtained. To verify
that the mutant phenotype observed is the result of a gene deletion, a complementation approach has to be performed. Here,
an intact copy of the gene is transformed back to the mutant, where it should integrate ectopically into the genome. However,
ectopic complementation is difficult in NHEJ-deficient strains, and the gene will preferably integrate via homologous recombination
at its endogenous locus. To circumvent that problem, we have constructed autonomously replicating vectors useful for many
filamentous fungi which contain either the pyrG allele or a hygromycin resistance gene as selectable markers. Under selective conditions, the plasmids are maintained, allowing
complementation analyses; once the selective pressure is removed, the plasmid becomes lost and the mutant phenotype prevails.
Another disadvantage of NHEJ-defective strains is their increased sensitivity towards DNA damaging conditions such as radiation.
Thus, mutant analyses in these genetic backgrounds are limited and can even be obscured by pleiotropic effects. The use of
sexual crossings for the restoration of the NHEJ pathway is, however, impossible in imperfect filamentous fungi such as A. niger. We have therefore established a transiently disrupted kusA strain as recipient strain for gene-targeting approaches. 相似文献