Role of the Regulatory Gene areA of Aspergillus oryzae in Nitrogen Metabolism

Abstract

ABSTRACT The areA gene of Aspergillus oryzae was cloned by cross-hybridization with the Aspergillus nidulans areA gene and was found to encode an 866-amino-acid protein that is very similar to other fungal nitrogen regulatory proteins. The A. oryzae areA gene can complement A. nidulans areA loss-of-function mutations. Functional analyses indicated that the N-terminal region of the A. oryzae AreA protein was dispensable for function and revealed a probable acidic activation domain in the protein. C-terminal truncation of the protein resulted in derepression of several nitrogen-controlled activities in A. nidulans , while deletions extending into the conserved GATA type zinc finger region abolished the activator function. The A. oryzae areA gene was inactivated by replacement with the A. oryzae pyrG gene. Strains containing the resulting areA deletion grew as well as the wild-type strain on glutamine but were unable to grow vigorously on other nitrogen sources, including ammonium. While A. oryzae exhibited reduced growth on 10 mM ammonium, the results of growth tests indicated that areA mutants of both A. oryzae and A. nidulans were affected in utilization of low concentrations of ammonium. The levels of the major nitrogen assimilatory enzymes, NADP-linked glutamate dehydrogenase (EC 1.4.1.4 ) and glutamine synthetase (EC 6.3.1.2 ), were determined. In both A. oryzae and A. nidulans areA mutants, the NADP-glutamate dehydrogenase levels were reduced, whereas the glutamine synthetase levels were not affected. These results suggest that the AreA protein may play an important role in the regulation of nitrogen assimilation in addition to its previously established regulatory role in nitrogen catabolism.