RESUMO
The movement of ammonium across biological membranes is mediated in both prokaryotes and eukaryotes by ammonium transport proteins (AMT/MEP) that constitute a family of related sequences. We have previously identified two ammonium permeases in Aspergillus nidulans, encoded by the meaA and mepA genes. Here we show that meaA is expressed in the presence of ammonium, consistent with the function of MeaA as the main ammonium transporter required for optimal growth on ammonium as a nitrogen source. In contrast, mepA, which encodes a high-affinity ammonium permease, is expressed only under nitrogen-limiting or starvation conditions. We have identified two additional AMT/MEP-like genes in A. nidulans, namely, mepB, which encodes a second high-affinity ammonium transporter expressed only in response to complete nitrogen starvation, and mepC, which is expressed at low levels under all nitrogen conditions. The MepC gene product is more divergent than the other A. nidulans AMT/MEP proteins and is not thought to significantly contribute to ammonium uptake under normal conditions. Remarkably, the expression of each AMT/MEP gene under all nitrogen conditions is regulated by the global nitrogen regulatory GATA factor AreA. Therefore, AreA is also active under nitrogen-sufficient conditions, along with its established role as a transcriptional activator in response to nitrogen limitation.
Assuntos
Aspergillus nidulans/genética , Proteínas Fúngicas/metabolismo , Fatores de Transcrição GATA/metabolismo , Regulação Fúngica da Expressão Gênica , Genes Fúngicos/genética , Proteínas de Membrana Transportadoras/genética , Fatores de Transcrição/metabolismo , Sequência de Aminoácidos , Aspergillus nidulans/citologia , Aspergillus nidulans/efeitos dos fármacos , Proteínas de Ligação a DNA/metabolismo , Deleção de Genes , Perfilação da Expressão Gênica , Proteínas de Membrana Transportadoras/química , Proteínas de Membrana Transportadoras/metabolismo , Dados de Sequência Molecular , Compostos de Amônio Quaternário/metabolismo , Compostos de Amônio Quaternário/farmacologia , Homologia de Sequência de AminoácidosRESUMO
The filamentous fungus Aspergillus nidulans can use a wide range of compounds as nitrogen sources. The synthesis of the various catabolic enzymes needed to breakdown these nitrogen sources is regulated by the areA gene, which encodes a GATA transcription factor required to activate gene expression under nitrogen-limiting conditions. The areA102 mutation results in pleiotropic effects on nitrogen source utilization, including better growth on certain amino acids as nitrogen sources. Mutations in the sarA gene were previously isolated as suppressors of the strong growth of an areA102 strain on l-histidine as a sole nitrogen source. We cloned the sarA gene by complementation of a sarA mutant and showed that it encodes an l-amino acid oxidase enzyme with broad substrate specificity. Elevated expression of this enzyme activity in an areA102 background accounts for the strong growth of these strains on amino acids that are substrates for this enzyme. Loss of function sarA mutations, which abolish the l-amino acid oxidase activity, reverse the areA102 phenotype. Growth tests with areA102 and sarA mutants show that this enzyme is the primary route of catabolism for some amino acids, while other amino acids are metabolized through alternative pathways that yield either ammonium or glutamate for growth.