A homologous transformation system for the opportunistic fungal pathogen Aspergillus fumigatus was developed. It is based on the A. fumigatus pyrG gene, encoding orotidine 5'-monophosphate decarboxylase, which was cloned and sequenced. Transformation of both Aspergillus (Emericella) nidulans and A. fumigatus pyrG mutant strains by the use of protoplasts or electroporation established the functionality of the cloned gene. DNA sequencing of the A. fumigatus pyrG1 mutant allele revealed that it encodes a truncated, non-functional, PyrG protein. Transformation of an A. fumigatus pyrG1 mutant with a plasmid carrying the novel pyrG2 allele constructed by in vitro mutagenesis yielded prototrophic transformants following recombination between both mutation sites. Analysis of transformants carrying the entire plasmid showed that up to 45% of integration had occurred at the pyrG locus. This provides a tool to target defined genetic constructs at a specific locus in the A. fumigatus genome in order to study gene regulation and function.
Aspergillus fumigatus is an important pathogen of the immunocompromised host, causing pneumonia and invasive disseminated disease with high mortality. In order to determine the importance of lysine biosynthesis for growth and pathogenicity, the A. fumigatus lysF gene, encoding a homologue of the A. nidulans homoaconitase LysF, was cloned and characterized. Cosmid cosGTM encoding lysF complemented a lysF mutant of Aspergillus nidulans. A. fumigatus lysF was deleted, resulting in a lysine-auxotroph. This phenotype was complemented to the wild-type by supplementation of the medium with both L-lysine and alpha-aminoadipic acid, or transformation using cosmid cosGTM. To study the virulence of the lysF deletion mutant of A. fumigatus, a low-dose intranasal mouse infection model of invasive aspergillosis was optimized for immunosuppressed BALB/c mice, allowing the application of an infection dose as low as 5 x 10(3) conidia per mouse. In this murine model, the Delta lysF mutant was avirulent, suggesting that lysine biosynthesis, or at least a functional homoaconitase, is important for survival of A. fumigatus in vivo and a potential target for antifungal drugs.
In filamentous fungi, lysine is synthesized via the alpha-aminoadipate pathway. In order to gain insight into this fungus-specific pathway (to date, no genes for enzymes of this pathway in filamentous fungi have been cloned) the lysine auxotrophic mutant LysF88 of Aspergillus nidulans was studied. HPLC and 1H-NMR analyses revealed that LysF88 accumulated homocitric acid in the culture supernatant. In addition, both the LysF88 mutant strain and LysF deletion strain (LysFKO) described here showed hardly any homoaconitase activity, indicating that lysF encodes homoaconitase. The lysF gene was cloned by complementation of the LysF88 mutant and sequenced. It has a size of 2397 bp, including a single intron of 72 bp. The two exons encode an open reading frame (ORF) of 2325 bp. The calculated M(r) of the homoaconitase protein (775 amino acids) is 83,943. A major and a minor transcript begin at positions -28 and -32, respectively. The 3' end of the lysF cDNA showed a poly(A) tail commencing at position +2647 following a 250 bp untranslated region after the lysF stop codon. A putative polyadenylation signal sequence (TATAAA) is located 49 bp upstream of the polyadenylation site. Computer analysis revealed 55% amino acid sequence identity between the products of the putative homoaconitase ORF of A. nidulans and that of the recently sequenced homologous Saccharomyces cerevisiae. The similarity was particularly obvious in a region of cysteine residues, which are characteristic of an iron-sulfur cluster, implying that homoaconitase contains such a cluster. The homoaconitases of A. nidulans and S. cerevisiae share only 20% sequence identity with S. cerevisiae aconitase. The pH optimum for the activity of A. nidulans homoaconitase in 0.1 M potassium phosphate buffer is between pH 8.1 and pH 8.6. Homoaconitase exhibited an apparent K(m) of 1.1 mM toward homoisocitric acid. The specific activity of homoaconitase was reduced by up to six-fold in mycelia grown in the presence of L-lysine, suggesting that it is regulated by lysine.
Formate is the major source of C1 units in many species of the genus Clostridium. In this study we have cloned and characterized the genes encoding pyruvate formate-lyase and its activating enzyme of Clostridium pasteurianum. The genetic and transcriptional organizations of the genes and the high level of homology exhibited by the respective gene products to their Escherichia coli counterparts indicate strong evolutionary conservation of these enzymes.
In beta-lactam-antibiotic-producing fungi, such as Aspergillus (Emericella) nidulans, L-alpha-aminoadipic acid is the branching point of the lysine and penicillin biosynthesis pathways. To obtain a deeper insight into the regulation of lysine biosynthesis genes, the regulation of the A. nidulans lysF gene, which encodes homoaconitase, was studied. Band-shift assays indicated that the A. nidulans multimeric CCAAT-binding complex AnCF binds to two of four CCAAT motifs present in the lysF promoter region. AnCF consists at least of three different subunits, designated HapB, HapC, and HapE. In both a delta hapB and a delta hapC strain, the expression of a translational lysF-lacZ gene fusion integrated in single copy at the chromosomal argB gene locus was two to three-fold higher than in a wild-type strain. These data show that AnCF negatively regulates lysF expression. The results of Northern blot analysis and lysF-lacZ expression analysis did not indicate a lysine-dependent repression of lysF expression. Furthermore, mutational analysis of the lysF promoter region revealed that two GATA sites matching the GATA consensus sequence HGATAR positively affected lysF-lacZ expression. Results of Northern blot analysis also excluded that the global nitrogen regulator AreA is the responsible trans-acting GATA-binding factor.
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