FIG. 1.
Regulation of expression of amdS-lacZ in response to different nitrogen sources. (A) AreA was HA tagged and the areA promoter replaced with the gpdA promoter in a two-step strategy (panel 1). 5′ areA (−18 to +811) was gene replaced with riboB by transformation of a riboflavin auxotroph with linearized pJAF5224 and selection for Ribo+ prototrophs (panel 2). areA was reconstructed with modifications by transformation of the areA::riboB(5′) strain from panel 1 with linearized pJAF5200 and selection on 10 mM nitrate. pJAF5200 contains gpdA(p)areAHA truncated at +1475 and confers AreA function only by homologous integration at the areA locus. (B) Strains of the indicated genotypes were grown for 16 h in 1% glucose medium with the nitrogen sources 10 mM ammonium tartrate (NH4), 10 mM l-glutamine (GLN), and 10 mM l-alanine (ALA) or subjected to nitrogen starvation (−N) by transferring mycelium pregrown in 1% glucose-10 mM ammonium tartrate for 16 h to 1% glucose medium lacking a nitrogen source for 4 h. Mycelium was harvested, extracted, and assayed for β-galactosidase. Specific activities with standard errors for at least three experiments are shown. (C) Wild-type and areA::riboB strains were grown for 16 h in 1% glucose-10 mM ammonium tartrate medium and then transferred to glucose medium lacking a nitrogen source, harvested at the indicated times, and assayed for β-galactosidase. (D) The indicated strains were grown for 16 h in glucose-ammonium medium (0 min), transferred to glucose medium containing various nitrogen sources (10 mM) for the indicated times, and then harvested and assayed for β-galactosidase. GLN, l-glutamine; NH4, ammonium tartrate; GLU, l-glutamate; ALA, l-alanine; PRO, l-proline; UREA, urea; ASN, l-asparagine; UA, uric acid; −N, no added nitrogen source.