Isolation and characterization of two ammonium permease genes, meaA and mepA, from Aspergillus nidulans
Brendon J Monahan1, James A Fraser, Michael J Hynes
1Department of Genetics, University of Melbourne, Parkville, Victoria, Australia 3010.
Abstract:
Ammonium and the analogue methylammonium are taken into the cell by active transport systems which constitute a family of transmembrane proteins that have been identified in fungi, bacteria, plants, and animals. Two genes from Aspergillus nidulans, mepA and meaA, which encode ammonium transporters with different affinities have been characterized. The MepA transporter exhibits the highest affinity for methylammonium (Km, 44.3 microM); in comparison, the Km for MeaA is 3.04 mM. By use of targeted gene replacement strategies, meaA and mepA deletion mutants were created. Deletion of both meaA and mepA resulted in the inability of the strain to grow on ammonium concentrations of less than 10 mM. The single meaA deletion mutant exhibited reduced growth at the same concentrations, whereas the mepA deletion mutant displayed wild-type growth. Interestingly, multiple copies of mepA were found to complement the methylammonium resistance phenotype conferred by the deletion of meaA. The expression profiles for mepA and meaA differed; the mepA transcript was detected only in nitrogen-starved cultures, whereas meaA was expressed under both ammonium-sufficient and nitrogen starvation conditions. Together, these results indicate that MeaA constitutes the major ammonium transport activity and is required for the optimal growth of A. nidulans on ammonium as the sole nitrogen source and that MepA probably functions in scavenging low concentrations of ammonium under nitrogen starvation conditions.
Insights
Two Aspergillus nidulans genes, mepA and meaA, encode ammonium transporters. MeaA is crucial for growth on ammonium, while MepA scavenges low ammonium levels during nitrogen starvation.
Area of Science:
- Molecular biology
- Microbiology
- Biochemistry
Background:
- Ammonium and methylammonium are vital nutrients transported into cells via transmembrane proteins.
- These ammonium transporters are conserved across fungi, bacteria, plants, and animals.
- Understanding these transporters is key to nutrient uptake mechanisms.
Purpose of the Study:
- To characterize the roles of two Aspergillus nidulans ammonium transporter genes, mepA and meaA.
- To investigate the distinct affinities and functions of the MepA and MeaA transporters.
- To determine the impact of these transporters on fungal growth under varying nitrogen conditions.
Main Methods:
- Gene characterization of mepA and meaA in Aspergillus nidulans.
- Creation of meaA and mepA deletion mutants using targeted gene replacement.
- Analysis of growth phenotypes under different ammonium concentrations.
- Assessment of gene expression profiles via transcript detection.
Main Results:
- MepA exhibits high affinity for methylammonium (Km, 44.3 µM), while MeaA has lower affinity (Km, 3.04 mM).
- Deletion of both mepA and meaA prevents growth below 10 mM ammonium.
- meaA deletion reduces growth, whereas mepA deletion shows wild-type growth; mepA overexpression complements meaA deletion.
- meaA is expressed under ammonium-sufficient and starvation conditions; mepA is expressed only during nitrogen starvation.
Conclusions:
- MeaA is the primary ammonium transporter essential for optimal growth in Aspergillus nidulans.
- MepA likely functions in ammonium scavenging at low concentrations during nitrogen starvation.
- Differential expression and affinities highlight specialized roles for ammonium transporters.
