Rapid and sensitive plate method for detection of Aspergillus fumigatus.
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The routine identification of Aspergillus fumigatus in clinical samples involves, apart from direct examination, the isolation of the organism on a plate followed by its microscopic characterization. This approach lacks sensitivity, specificity, and speed. A new procedure has been developed combining microcolony formation on a nylon membrane filter at 45 degrees C with the detection of a specific 4-methylumbelliferyl-alpha-L-arabinopyranoside cleaving enzyme activity in digitonin permeabilized cells. The test takes approximately 14 h and has an efficiency of 98.2% and false-positive and -negative rates of 0 and 3.1%, respectively. When applied to 188 clinical samples taken from patients with proven or nonproven presence of Aspergillus species, a good agreement with the conventional plate-microscopy method was obtained. (+info)
The DNA-binding domain of the gene regulatory protein AreA extends beyond the minimal zinc-finger region conserved between GATA proteins.
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The AreA protein of Aspergillus nidulans regulates the activity of over 100 genes involved in the utilisation of nitrogen, and has a limited region of homology with the vertebrate family of GATA proteins around a zinc finger (Zf) motif. A 66 amino acid (a.a.) residue fragment (Zf(66)) corresponding to the zinc finger, a 91 a.a fragment (Zf(91)) containing an additional 25 a.a. at the C-terminus, and a much larger 728 a.a. sequence (3'EX) corresponding to the 3'exon have been over-expressed as fusion proteins in E. coli and purified. The DNA-protein complexes formed by these proteins have been examined by gel retardation analysis. The 91 a.a. protein forms a discrete shifted species with a GATA-containing DNA fragment with high affinity (K(d)=0.15 nM), whereas the 66 a.a. protein has very low ( approximately microM) affinity for the same sequence. The results show that the region of AreA required for high affinity DNA binding extends beyond the zinc finger motif that is homologous to GATA-1, requiring in addition a region within the 25 a.a. sequence C-terminal to the zinc finger. Using hydroxyl radical and ethylation interference footprinting, the minimal Zinc finger protein (Zf(66)) shows no appreciable interference effects whereas Zf(91) shows much stronger interference effects, identical to those of the larger protein. These effects extend over sequences up to two nucleotides either side of the GATA site, and indicate contacts additional to those observed in the three-dimensional structure of the complex of the minimal zinc-finger protein with DNA. We suggest that these additional contacts are responsible for the enhanced DNA binding affinity of the extended zinc-finger protein Zf(91). (+info)
The Aspergillus nidulans cysA gene encodes a novel type of serine O-acetyltransferase which is homologous to homoserine O-acetyltransferases.
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The Aspergillus nidulans cysA gene was cloned by functional complementation of the cysA1 mutation that impairs the synthesis of O:-acetylserine. The molecular nature of cysA1 and cysA103 alleles was characterized; a nucleotide substitution and a frame shift were found in the former and a deletion mutation in the latter. The CYSA protein is 525 amino acids long and is encoded by an uninterrupted open reading frame. Expression of the cysA gene appears not to be regulated by sulfur, carbon and nitrogen sources. Protein sequence analysis reveals extensive similarity to homoserine O:-acetyltransferases, particularly the bacterial ones, and no homology with known serine O:-acetyltransferases. The authors propose that the CYSA protein is analogous to serine O:-acetyltransferases, i.e. it catalyses the same reaction but has an independent evolutionary origin. (+info)
Enzymatic lesions in methionine mutants of Aspergillus nidulans: role and regulation of an alternative pathway for cysteine and methionine synthesis.
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In Aspergillus nidulans the pathway involving cystathionine formation is the main one for homocysteine synthesis. Mutants lacking cystathionine gamma-synthase or beta-cystathionase are auxotrophs suppressible by: (i) mutations in the main pathway of cysteine synthesis (cysA1, cysB1, and cysC1), (ii) mutations causing stimulation of cysteine catabolism (su101), and (iii) mutations in a presumed regulatory gene (suAmeth). A relative shortage of cysteine in the first group of suppressors causes a derepression of homocysteine synthase, the enzyme involved in the alternative pathway of homocysteine synthesis. A similar derepression is observed in the suAmeth strain. Homocysteine synthesized by this pathway serves as precursor for cysteine and methionine synthesis. A mutant with altered homocysteine synthase is a prototroph, indicating that this enzyme is not essential for the fungus. (+info)
Amide utilization in Aspergillus nidulans: evidence for a third amidase enzyme.
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A mutation in a gene designated gmdA has been found to lead to loss of ability of Aspergillus nidulans to use benzamide, phenylacetamide and several other amides as sole nitrogen sources for growth. The gmdA1 lesion results in low levels of an enzyme, called the general amidase, which has acitivity for a wide range of amide substrates. This enzyme is reressed by certain nitrogen-containing metabolites, including ammonium, but is probably not regulated by induction or by carbon catabolite repression. Evidence is presented for the general amidase being distinct from the previously characterized acetamidase and formamidase enzymes. The data also indicate that there is a fourth amidase capable of the hydrolysis of valeramide and hexanamide. (+info)
xylP promoter-based expression system and its use for antisense downregulation of the Penicillium chrysogenum nitrogen regulator NRE.
(70/1128)
A highly inducible fungal promoter derived from the Penicillium chrysogenum endoxylanase (xylP) gene is described. Northern analysis and the use of a beta-glucuronidase (uidA) reporter gene strategy showed that xylP expression is transcriptionally regulated. Xylan and xylose are efficient inducers, whereas glucose strongly represses the promoter activity. Comparison of the same expression construct as a single copy at the niaD locus in P. chrysogenum and at the argB locus in Aspergillus nidulans demonstrated that the xylP promoter is regulated similarly in these two species but that the level of expression is about 80 times higher in the Aspergillus species. The xylP promoter was found to be 65-fold more efficient than the isopenicillin-N-synthetase (pcbC) promoter in Penicillium and 23-fold more efficient than the nitrate reductase (niaD) promoter in Aspergillus under induced conditions. Furthermore, the xylP promoter was used for controllable antisense RNA synthesis of the nre-encoded putative major nitrogen regulator of P. chrysogenum. This approach led to inducible downregulation of the steady-state mRNA level of nre and consequently to transcriptional repression of the genes responsible for nitrate assimilation. In addition, transcription of nreB, which encodes a negative-acting nitrogen regulatory GATA factor of Penicillium, was found to be subject to regulation by NRE. Our data are the first direct evidence that nre indeed encodes an activator in the nitrogen regulatory circuit in Penicillium and indicate that cross regulation of the controlling factors occurs. (+info)
A rapid method for efficient gene replacement in the filamentous fungus Aspergillus nidulans.
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The construction of mutant fungal strains is often limited by the poor efficiency of homologous recombination in these organisms. Higher recombination efficiencies can be obtained by increasing the length of homologous DNA flanking the transformation marker, although this is a tedious process when standard molecular biology techniques are used for the construction of gene replacement cassettes. Here, we present a two-step technology which takes advantage of an Escherichia coli strain expressing the phage lambda Red(gam, bet, exo) functions and involves (i) the construction in this strain of a recombinant cosmid by in vivo recombination between a cosmid carrying a genomic region of interest and a PCR-generated transformation marker flanked by 50 bp regions of homology with the target DNA and (ii) genetic exchange in the fungus itself between the chromosomal locus and the circular or linearized recombinant cosmid. This strategy enables the rapid establishment of mutant strains carrying gene knock-outs with efficiencies >50%. It should also be appropriate for the construction of fungal strains with gene fusions or promoter replacements. (+info)
Cytochrome abnormalities and cyanide-resistant respiration in extranuclear mutants of Aspergillus nidulans.
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The cytochrome spectra of two extranuclear mutants of Aspergillus nidulans and the double-mutant recombinant formed from them have been examined both at room temperature and at the temperature of liquid N2 and compared with those of the wild-type strain. The oligomycin-resistant, slow growing mutant contained an increased amount of cytochrome c without any loss of cytochromes b and a,a3. The cold-sensitive mutant, apparently normal when grown at 37 C, showed an increased amount of cytochrome c and a partial loss of cytochromes b and a,a3 when grown at 20 C. A combination of these effects was observed in the double-mutant recombinant. Cyanide-resistant respiration was present in both mutant strains and in the recombinant at much higher levels than in the wild-type strain. In the oligomycin-resistant mutant, this was usually present together with cyanide-sensitive respiration, whereas in the cold-sensitive mutant and recombinant grown at 20 C cyanide-resistant approached 100%. Inhibitor and growth yield studies indicated that the cyanide-resistant pathway was not used by the cold-sensitive mutant during growth at 20 C. (+info)