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

De Lucas, JR Dominguez, AI Higuero, Y Martinez, O Romero, B Mendoza, A Garcia-Bustos, JF Laborda, F

Jr. De Lucas et al., Development of a homologous transformation system for the opportunistic human pathogen Aspergillus fumigatus based on the sC gene encoding ATP sulfurylase, ARCH MICROB, 176(1-2), 2001, pp. 106-113

31

INGLESE

Article

Microbiology

ARCHIVES OF MICROBIOLOGY

0302-8933 → ACNP

176

1-2

2001

106 - 113

ISI

0302-8933(200107)176:1-2<106:DOAHTS>2.0.ZU;2-3

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 muta nt strains were readily isolated by strong selection for selenate resistanc e. The coding region plus upstream and downstream regulatory sequences of t he A. fumigatus sC gene were cloned by inverse PCR and then sequenced. Sequ encing 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 5 74 amino acids which is highly similar to ATP sulfurylases from the filamen tous fungal species Aspergillus nidulans, Aspergillus terreus and Penicilli um 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 ortholo gues. A 3.8-kb DNA fragment amplified by PCR and containing the sC genes pl us 5' and 3' flanking regions was cloned into pUC19 to give the vector pSCF UM. Transformation of two different sC mutant isolates with the plasmid pSC FUM established the functionality of this new homologous transformation sys tem. Molecular analysis of sC(+) transformants showed that up to 44% of tra nsformed clones contained one or more copies of the entire plasmid integrat ed at the sC locus. This result also demonstrates the utility of the sC mar ker for targeting specific genetic constructs to the A. fumigatus sC locus, facilitating studies of gene regulation and function.