Genetics - Theses
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ItemRegulation of amidase genes in Aspergillus nidulans : mechanisms of nitrogen metabolite repression(University of Melbourne, 2002)The filamentous fungus Aspergillus nidulans can utilise a wide variety of amides as the sole nitrogen source. The expression of the required catabolic enzymes is tightly regulated in response to nitrogen availability. The global mechanism responsible for this response is termed nitrogen metabolite repression, and is mediated by the GATA zinc finger activator protein AreA. During nitrogen limitation, AreA activates the expression of genes required for the utilisation of alternative nitrogen sources. If a more easily assimilated source such as ammonium is present changes in areA mRNA stability and interaction with the negatively acting NmrA protein prevent AreA activation of catabolic gene expression. This thesis describes the isolation of three genes involved in amide utilisation in A. nidulans and an analysis of their regulation. The fmdS gene encodes a formamidase unlike the characterised acetamidase of A. nidulans, and is required for the hydrolysis of formamide. The gmdA and bzuA genes are required for the utilisation of benzamide as a nitrogen source, and encode general amidase and benzoate para-hydroxylase, respectively. Like the acetamidase, the general amidase belongs to the amidase signature family of enzymes. In addition to benzamide, the general amidase also mediates the hydrolysis of a variety of long chain amides. BzuA is required to prevent benzoate toxicity following the hydrolysis of benzamide by GmdA. The transcription of both fmdS and gmdA is highly regulated by AreA-mediated nitrogen metabolite repression, with a low level of expression during growth on preferred nitrogen sources such as ammonium and increased levels during growth on an alternative source such as alanine. In addition, this activation was shown to be enhanced during nitrogen starvation, defining an additional level of AreA function. The transcription of these genes is affected by carbon availability, with transcription halted upon carbon starvation irrespective of nitrogen availability. Detailed deletion analysis of the fmdS promoter was performed, and the only changes in fmdS transcription revealed were though deletion of putative AreA recognition sequences. This suggested the mechanism behind the carbon starvation response is probably inactivation of AreA. Expression of fmdS was also found to be affected by transcriptional interference from an upstream gene (usgS), whose transcript overlaps the fmdS coding region. Beyond its affect on reducing fmdS transcription when in cis, gene inactivation revealed no indication of the role of the usgS gene. The nitrogen starvation and carbon starvation responses of nitrogen metabolite repression are independent of the two mechanisms already characterised in this global regulatory phenomenon - NmrA interaction and areA transcript stability. In an attempt to identify factors which may play a role in the starvation mechanisms, a possible A. nidulans homologue of the S. cerevisiae URE2 gene was cloned. In yeast, Ure2 is a negatively acting factor that interacts with and represses the AreA homologue Gln3. Several ESTs with similarity to URE2 were identified, allowing the gstA gene to be cloned. Gene inactivation showed gstA encodes a functional glutathione S-transferase involved in resistance to xenobiotics and heavy metals. The evidence presented here strongly suggests the A. nidulans genome does not contain a true URE2 homologue that is involved in nitrogen metabolite repression. A gene replacement strategy was developed to allow epitope tagging of AreA, yielding a variety of strains with which this regulatory factor could be studied at the protein level. Western blot analysis has indicated that AreA is differentially phosphorylated under different conditions of nitrogen and carbon availability. The starvation responses in AreA regulated structural gene expression correspond to the post-translational modifications observed suggesting that it is functionally significant.