Metabolic and Developmental Effects Resulting from Deletion of thecitAGene Encoding Citrate Synthase in Aspergillus nidulans

Abstract

Citrate synthase is a central activity in carbon metabolism. It is required for the tricarboxylic acid (TCA) cycle, respiration, and the glyoxylate cycle. In Saccharomyces cerevisiae and Arabidopsis thaliana, there are mitochondrial and peroxisomal isoforms encoded by separate genes, while in Aspergillus nidulans, a single gene, citA, encodes a protein with predicted mitochondrial and peroxisomal targeting sequences (PTS). Deletion of citA results in poor growth on glucose but not on derepressing carbon sources, including those requiring the glyoxylate cycle. Growth on glucose is restored by a mutation in the creA carbon catabolite repressor gene. Methylcitrate synthase, required for propionyl-coenzyme A (CoA) metabolism, has previously been shown to have citrate synthase activity. We have been unable to construct the mcsA⌬ citA⌬ double mutant, and the expression of mcsA is subject to CreA-mediated carbon repression. Therefore, McsA can substitute for the loss of CitA activity. Deletion of citA does not affect conidiation or sexual development but results in delayed conidial germination as well as a complete loss of ascospores in fruiting bodies, which can be attributed to loss of meiosis. These defects are suppressed by the creA204 mutation, indicating that McsA activity can substitute for the loss of CitA. A mutation of the putative PTS1-encoding sequence in citA had no effect on carbon source utilization or development but did result in slower colony extension arising from single conidia or ascospores. CitA-green fluorescent protein (GFP) studies showed mitochondrial localization in conidia, ascospores, and hyphae. Peroxisomal localization was not detected. However, a very low and variable detection of punctate GFP fluorescence was sometimes observed in conidia germinated for 5 h when the mitochondrial targeting sequence was deleted.There has been increased interest in primary carbon metabolism in fungi in recent years. There are two main reasons for this. As fungal pathogens establish infection they must adapt their utilization of carbon sources to the substrates present in the new environment of the host cells (reviewed in reference 6). With many of the fungal genomes available, the number of genes encoding enzymes and transporters potentially involved in central metabolism has become apparent and is greater than might have been anticipated (for example, see reference 16). Deciphering this complexity requires not only genome-wide studies but also detailed studies of individual genes encoding these proteins in order to determine their regulation and the cellular localization of the proteins, as well as their roles in metabolism and development. Here we report molecular genetic analysis of the citA gene encoding citrate synthase (EC 4.1.3.7), a central enzyme of carbon metabolism, in the filamentous ascomycete Aspergillus nidulans.Citrate synthase is required for the formation of citrate from acetyl-coenzyme A (CoA) and oxaloacetate in the tricarboxylic acid (TCA) cycle and is therefore necessary for respirat...

Role of Carnitine Acetyltransferases in Acetyl Coenzyme A Metabolism in Aspergillus nidulans

“…AcuJ-GFP was detected using mouse anti-GFP (at 1/4,000) and anti-mouse IgG-horseradish peroxidase ([HRP] at 1/4,000) antibodies (Promega Corp.) as primary and secondary antibodies, respectively. Anti-␣-tubulin was used to detect ␣-tubulin as a loading control as previously described (39). Signals were detected using Fujifilm image reader LAS-3000 (Berthold Australia Pty Ltd).…”

“…Strains were incubated for 45 min to 1 h with 25 to 50 nM MitoTracker Red CMXRos (Invitrogen) before being fixed as previously described (61). Confocal microscopy was performed as described previously (39).…”

“…The phenotype was equivalent to that observed for the acuJ221 mutant (3,57). It is important to note that, unlike some other mutants affected in acetyl-CoA and peroxisome metabolism (26,29,39), acuJ⌬ strains have no obvious developmental phenotypes.…”

“…In A. nidulans CAT activity is crucial for growth on sources of acetyl-CoA. Citrate synthase, encoded by citA, is mitochondrial, and no major function for a minor peroxisomal form has been found (39). Mutations in facC result in loss of growth on acetate but not on fatty acids (3,57), and facC encodes a CAT with no predicted MTS or PTS, strongly indicating that cytoplasmic activity is not essential for fatty acid utilization (57).…”

Differential expression of citA gene encoding the mitochondrial citrate synthase of Aspergillus nidulans in response to developmental status and carbon sources

“…We have reported the purification and characterization of the citrate synthase from A. nidulans (Maeng et al, 1993), and also have analyzed the nucleotide sequence of the citrate synthase gene, named citA, cloned from the chromosomespecific genomic library of this fungus (Park et al, 1997). It has also been reported that deletion of citA results in poor growth on glucose but not on derepressing carbon sources, and that methyl citrate synthase (McsA) can substitute for the loss of CitA activity (Murray and Hynes, 2010). In the present study, we isolated the cDNA of citA and analyzed its nucleotide sequence to confirm the primary structure of the citrate synthase.…”