Molecular cloning and functional characterization of the pathway-specific regulatory gene nirA, which controls nitrate assimilation in Aspergillus nidulans

Abstract

We have cloned an 11-kbp segment of the genomic DNA of Aspergillus nidulans which complements mutations in nirA, the pathway-specific regulatory gene of the nitrate assimilation pathway. Gene disruption in the corresponding region of the nuclear DNA leads to a phenotype and a gene complementation pattern indistinguishable from that observed in known noninducible nirA mutants. Transformation studies with subclones of the 11-kbp genomic segment showed that a nonreverting null mutation nirA87, maps to a 1.5-kbp stretch within that segment. These data confirm that the cloned segment contains the nirA gene. The gene is completely encompassed in the 11-kbp genomic segment, as a plasmid carrying the corresponding insert gives rise to multicopy transformants exhibiting better growth than wild type on nitrate or nitrite as the sole nitrogen source. Southern and genetic analyses of transformants obtained with various plasmid subclones established a gene size of at most 5.9 kbp. Northern (RNA) hybridization experiments revealed a 4-kb nirA transcript which is barely visible in the wild type but clearly seen in a transformant carrying about 10 gene copies. In both strains, nirA mRNA is synthesized constitutively. Upstream of nirA, a neighboring transcript about 2.8 kbp in length which is transcribed from the opposite strand with respect to nirA was localized. The transcript levels of niaD and niiA, encoding the nitrate and nitrite reductase core proteins, respectively, were investigated in nirA mutants and a nirA multicopy transformant. The results show that the nirA product regulates the transcript steady-state level of these structural genes and that it is a limiting factor for their expression.