A. niger strains used in this study are listed in Table 1. Strains were cultivated in minimal medium (MM; (Bennett and Lasure 1991 )) containing 55 mM glucose, 7 mM KCl, 11 mM KH2PO4, 70 mM NaNO3, 2 mM MgSO4, 76 nM ZnSO4, 178 nM H3BO3, 25 nM MnCl2, 18 nM FeSO4, 7.1 nM CoCl2, 6.4 nM CuSO4, 6.2 nM Na2MoO4, 174 nM EDTA; or in complete medium containing, in addition to MM, 0.1% (w/v) casamino acids and 0.5% (w/v) yeast extract. When required, 10 mM uridine and/or 100 µg/ml of hygromycin was added. When using the amdS as selection marker, strains were grown in MM without NaNO3 and supplemented with 10 mM acetamide and 15 mM cesium chloride.

Aspergillus niger strains used in this study

NameGenotypeReference
N402cspA1, amdS−Bos et al. (1988 (link))
AB4.1pyrG−, amdS−van Hartingsveldt et al. (1987 (link))
MA70.15aΔkusA, pyrG−, amdS+Meyer et al. (2007 (link))
MA78.6aΔkusA, amdS+This study
NC4.1aΔkusA, pyrG−, amdS−This study
NC5.1aΔkusA, pyrG+, amdS−This study
NC6.2ΔkusA, pyrG+, amdS+, ΔhacAThis study
NC7.1ΔkusA, pyrG+, amdS+, ΔireA/ireAThis study
NC8.1ΔkusA, pyrG+, amdS+, ΔhacA, pAMA-hacAThis study
NC9.1ΔkusA, pyrG+, amdS+, ΔireA, pAMA-ireAThis study
MA169.4akusA::DR-amdS-DR, pyrG−This study
MA171.1kusA::DR-amdS-DR, pyrG+, ΔracAThis study
MA172.1kusA+, pyrG+, ΔracAThis study
MK15.AΔkus, pyrG+, ΔsrgAThis study
MK18.AkusA::DR-amdS-DR, pyrG+, ΔsrgAThis study

aStrains have been deposited at the Fungal Genetics Stock Center (www.fgsc.net)

To obtain pyrG− strains, 2 × 107 spores were inoculated on MM agar plates supplemented with 0.75 mg/ml 5′-fluoroorotic acid (FOA), 10 mM uridine and 10 mM proline as nitrogen source. Plates were incubated for 1–2 weeks at 30°C. FOA-resistant mutants were isolated, purified and tested for uridine auxotrophy on MM with and without uridine (mutants should not grow on medium lacking uridine). To obtain amdS− strains, 2 × 107 spores were inoculated on MM agar plates supplemented with 0.2% 5′-fluoroacetamide (FAA) and 10 mM urea as nitrogen source. After 1–2 weeks incubation at 30°C, FAA-resistant mutants were isolated, purified and tested for growth on acetamide medium (mutants should not grow on medium containing acetamide as sole nitrogen source).
All basic molecular techniques were performed according to standard procedures (Sambrook and Russel 2001 ). Transformation of A. niger, genomic DNA extraction, screening procedures, diagnostic PCR and Southern analysis were conducted as recently described in detail (Meyer et al. 2010 ).