Abstract Detail

Molecular mechanisms of adaptation to host and environment

Downes, Damien [1], Davis, Meryl [2], Wong, Koon Ho [3], Hynes, Michael [2], Todd, Richard [1].

Transcriptional regulation of NADP-GDH in Aspergillus nidulans: A key step in the nitrogen assimilation pathway.

Assimilation of nitrogen nutrients in fungi occurs via the GOGAT pathway. For many fungi NADP-dependent glutamate dehydrogenase (NADP-GDH), is a key enzyme in nitrogen assimilation via this pathway. In Aspergillus nidulans NADP-GDH is encoded by gdhA, and expression of gdhA is regulated in response to available nitrogen nutrients. Expression of gdhA is coordinately regulated by three transcription factors: the GATA factor AreA, which regulates nitrogen metabolic genes and two Zn(II)2Cys6 proteins TamA and LeuB. TamA is the co-activator of AreA whereas LeuB is the regulator of leucine biosynthesis, however TamA and LeuB also interact. We investigated the effects of leucine biosynthesis on gdhA regulation and found that leucine abundance and biosynthesis regulates the levels of NADP-GDH activity and gdhA expression. We showed using leucine biosynthesis mutants with perturbed levels of α-isopropylmalate (α-IPM), that this pathway intermediate affects gdhA regulation. Regulation by leucine abundance is dependent upon the presence of functional LeuB with an intact Zn(II)2Cys6 DNA binding domain. Using promoter mutations we identified two sites of action for LeuB within the gdhA promoter. These two LeuB sites lack sequence identity, with one site conforming to the predicted LeuB DNA binding site consensus motif whereas the second site is a novel regulatory sequence element. This second novel site of action is also required for TamA regulation of gdhA. Previously it was thought that the Zn(II)2Cys6 motif of TamA was dispensable for function. We now show that activation of gdhA by TamA occurs primarily by a mechanism requiring the TamA DNA binding motif. Using chromatin-immunoprecipitation we show that the TamA DNA binding motif is required for DNA binding of FLAG-epitope-tagged TamA to the gdhA promoter. We show that the levels of TamA binding at gdhA are regulated by nitrogen source. Therefore expression of the key nitrogen assimilation enzyme NADP-GDH is regulated at the transcriptional level by sensing of both the levels of leucine and quality of available nitrogen nutrients.

1 - Kansas State University, Department of Plant Pathology, 4024 Throckmorton, Manhattan, Kansas, 66502, USA
2 - University of Melbourne, Department of Genetics, Melbourne, VIC, 3000, Australia
3 - University of Macau, Faculty of Health Science, Macau

Keywords:
Nitrogen metabolism
Leucine biosynthesis
Zn(II)2Cys6
Zinc finger
Transcriptional regulation.

Presentation Type: Symposium or Colloquium Presentation
Session: SY5
Location: Room 103 AB/Kellogg Hotel and Conference Center
Date: Tuesday, June 10th, 2014
Time: 3:30 PM
Number: SY5006
Abstract ID:166
Candidate for Awards:None