Development of a homologous transformation system for the opportunistic human pathogen Aspergillus fumigatus based on the sC gene encoding ATP sulfurylase

Abstract

The development of a homologous transformation system for the opportunistic human pathogenic fungus Aspergillus fumigatus is described. The system is based on the sC gene encoding ATP sulfurylase. Several A. fumigatus sC mutant strains were readily isolated by strong selection for selenate resistance. The coding region plus upstream and downstream regulatory sequences of the A. fumigatus sC gene were cloned by inverse PCR and then sequenced. Sequencing of the sC cDNA revealed the presence of five introns located within the first half of the gene. The A. fumigatus sC gene encodes a protein of 574 amino acids which is highly similar to ATP sulfurylases from the filamentous fungal species Aspergillus nidulans, Aspergillus terreus and Penicillium chrysogenum. By contrast, ATP sulfurylases from the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe lack the C-terminal adenosine-5'-phosphosulfate kinase-like domain present in the filamentous fungal orthologues. A 3.8-kb DNA fragment amplified by PCR and containing the sC gene plus 5' and 3' flanking regions was cloned into pUC19 to give the vector pSCFUM. Transformation of two different sC mutant isolates with the plasmid pSCFUM established the functionality of this new homologous transformation system. Molecular analysis of sC+ transformants showed that up to 44% of transformed clones contained one or more copies of the entire plasmid integrated at the sC locus. This result also demonstrates the utility of the sC marker for targeting specific genetic constructs to the A. fumigatus sC locus, facilitating studies of gene regulation and function.

Study of the essentiality of the Aspergillus fumigatus triA gene, encoding RNA triphosphatase, using the heterokaryon rescue technique and the conditional gene expression driven by the alcA and niiA promoters

“…Also, in comparison with the CEA22 strain, a sevenfold higher protoplast yield (about 1.4 Â 10 7 protoplasts/g wet weight of mycelium) was obtained when the A. fumigatus sC29 strain was treated for 2 h with b-D-glucanase (8 mg/ml) (InterSpex) and driselase (5 mg/ml) (InterSpex). All these advantages highlight the suitability of this transformation system, based on the A. fumigatus sC29 recipient strain (De Lucas et al, 2001) and the A. nidulans sC marker, for future gene manipulations in A. fumigatus.…”

“…For it, we firstly constructed the vector pALSCRTF, a plasmid that contains the A. nidulans sC gene as the fungal selective marker and the A. nidulans alcA P fused to a non-functional 3 0 -truncated version of the triA ORF lacking the last 495 codons. Using this vector, we transformed the A. fumigatus sC29 mutant (De Lucas et al, 2001) to generate by homologous recombination strains carrying the A. nidulans alcA P -triA fusion gene (Fig. 2).…”

“…The A. fumigatus sC29 mutant (De Lucas et al, 2001), unable to use sulfate as the sole sulfur source, was utilized in transformation experiments using the A. nidulans sC gene as the fungal selective marker. The A. fumigatus KU80DpyrG (DakuB KU80 ; pyrG À ) mutant strain (da Silva Ferreira et al, 2006), was used in transformations that employed the Neurospora crassa pyr-4 gene as the fungal selective marker.…”

“…L-methionine (10 mM) was added to the medium when the A. fumigatus sC29 strain was cultivated. Transformation of A. fumigatus was done according to established procedures (De Lucas et al, 2001).…”

Marker recycling system using the <i>sC</i> gene in the white koji mold, <i>Aspergillus luchuensis</i> mut. <i>kawachii</i>

“…The sC gene encodes an ATP sulfurylase enzyme involved in sulfate metabolism (Arst, 1968). The applicability of the sC gene as a counter-selectable marker has been shown in a number of Aspergillus species, including A. nidulans, A. niger, A. fumigatus, A. oryzae, and A. aculeatus (Adachi et al, 2009;Arst, 1968;Buxton et al, 1989;De Lucas et al, 2001;Yamada et al, 1997). ATP sulfurylase is involved in the incorporation of sulfur during synthesis of sulfur-containing amino acids.…”