Isolation and characterization of two ammonium permease genes, meaA and mepA, from Aspergillus nidulans

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.

FIG. 1.

mepA and meaA have different gene structures but similar predicted protein secondary structures. (A and B) Exon-intron structures, restriction maps, and gene replacement strategies for meaA (A) and mepA (B) are shown. Black boxes represent exons, and the direction of transcription is indicated by an arrow. The positions in meaA and mepA of the argB and riboB selectable markers, respectively, used for targeted gene replacement are indicated. The portion of the benA gene located on the mepA clone is also shown. (C) meaA and mepA are both predicted to encode an 11-transmembrane-domain protein. The positions of putative transmembrane (TM) helices (shaded boxes numbered above with Roman numerals) and topology were assigned by using the prediction programs HMMTOP (43) and TMHMM (39). These programs gave the same numbers of transmembrane domains and very similar positions, although the helix-to-tail transition coordinates did vary slightly. The coordinates given here for each transmembrane helix are from the HMMTOP output. Arrows represent the direction of each transmembrane helix. The putative extracellular (E) and intracellular (I) locations of the N-terminal and C-terminal tails, respectively, are indicated.

FIG. 2.

Relatedness of MepA and MeaA to known MEP/AMT and rhesus protein sequences. The tree was constructed by the neighbor-joining method (35) with ClustalX (42). Data were corrected for multiple substitutions, and gaps in the alignment were ignored for the tree building. The name of the gene or open reading frame (ORF), the organism, and the GenBank accession number are indicated. N. crassa sequences were identified by database screening, and the contig in which the nucleotide sequence was present is indicated (assembly version 1, Neurospora Sequencing Project, Whitehead Institute/MIT Center for Genome Research, http://www-genome.wi.mit.edu).

FIG. 3.

Phenotypic analysis of meaA and mepA single- and double-deletion mutants. (A) Growth on a range of ammonium concentrations. At each ammonium concentration, tests were performed at pH 4.5 and pH 6.5. (B) Complementation analysis of the meaA mepA double-deletion mutant. Transformants were tested for both growth on ammonium and sensitivity to methylammonium (MACl). (C) Multiple-copy analysis of the meaA and mepA genes. An increase in the sensitivity of the transformants to methylammonium (MACl), depending on the copy number of the gene, is shown. The increase in copy number is represented by the grey triangle.

FIG. 4.

[¹⁴C]methylammonium uptake of meaA and mepA single- and double-deletion mutants. (A) [¹⁴C]methylammonium transport activity of the wild type (▵), meaA deletion mutant (□), mepA deletion mutant (•), and meaA mepA double-deletion mutant (○) grown in 20 mM ammonium compared to that of the wild type subjected to a 4-h nitrogen starvation period (▴). The final methylammonium concentration was 1 mM. (B and C) Methylammonium transport activity of nitrogen-starved samples at final methylammonium concentrations of 0.2 mM (B) and 1 mM (C). The symbols for the strains are the same as those for panel A.

FIG. 4.

[¹⁴C]methylammonium uptake of meaA and mepA single- and double-deletion mutants. (A) [¹⁴C]methylammonium transport activity of the wild type (▵), meaA deletion mutant (□), mepA deletion mutant (•), and meaA mepA double-deletion mutant (○) grown in 20 mM ammonium compared to that of the wild type subjected to a 4-h nitrogen starvation period (▴). The final methylammonium concentration was 1 mM. (B and C) Methylammonium transport activity of nitrogen-starved samples at final methylammonium concentrations of 0.2 mM (B) and 1 mM (C). The symbols for the strains are the same as those for panel A.

FIG. 5.

Velocity-substrate concentration curves for MepA and MeaA. [¹⁴C]methylammonium uptake rates measured at the substrate concentrations (conc.) indicated (see Materials and Methods) are shown for the meaA and mepA deletion mutants.

FIG. 6.

Northern blot analysis of meaA and mepA expression. RNA was isolated from wild-type (MH1), mepA single-deletion (MH9861), meaA single-deletion (MH9965), and mepA meaA double-deletion (MH9829) strains. Mycelium was grown in ANM with 20 mM ammonium at 37°C for 16 h (+) and then transferred to nitrogen-free medium for 4 h (−). Northern blots were hybridized with probes specific for mepA, meaA, or A. nidulans histone H3 as a loading control (13).