Generation and phenotypic characterization of Aspergillus nidulans methylisocitrate lyase deletion mutants: methylisocitrate inhibits growth and conidiation.
Propionate is a very abundant carbon source in soil, and many microorganisms are able to use this as the sole carbon source. Nevertheless, propionate not only serves as a carbon source for filamentous fungi but also acts as a preservative when added to glucose containing media. To solve this contradiction between carbon source and preservative effect, propionate metabolism of Aspergillus nidulans was studied and revealed the methylcitrate cycle as the responsible pathway. Methylisocitrate lyase is one of the key enzymes of that cycle. It catalyzes the cleavage of methylisocitrate into succinate and pyruvate and completes the alpha-oxidation of propionate. Previously, methylisocitrate lyase was shown to be highly specific for the substrate (2R,3S)-2-methylisocitrate. Here, the identification of the genomic sequence of the corresponding gene and the generation of deletion mutants is reported. Deletion mutants did not grow on propionate as sole carbon and energy source and were severely inhibited during growth on alternative carbon sources, when propionate was present. The strongest inhibitory effect was observed, when glycerol was the main carbon source, followed by glucose and acetate. In addition, asexual conidiation was strongly impaired in the presence of propionate. These effects might be caused by competitive inhibition of the NADP-dependent isocitrate dehydrogenase, because the K(i) of (2R,3S)-2-methylisocitrate, the product of the methylcitrate cycle, on NADP-dependent isocitrate dehydrogenase was determined as 1.55 microM. Other isomers had no effect on enzymatic activity. Therefore, methylisocitrate was identified as a potential toxic compound for cellular metabolism.
Brock
Matthias
M
Institute for Microbiology, University Hannover, Herrenhäuser Strasse 2, 30419 Hannover, Germany. Matthias.brock@hki-Jena.de
D000595:Amino Acid Sequence; D001233:Aspergillus nidulans / Q000145:classification* / Q000201:enzymology / Q000235:genetics / Q000254:growth & development*; D002244:Carbon / Q000378:metabolism; D019755:Carbon-Carbon Lyases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D003470:Culture Media; D017353:Gene Deletion; D015966:Gene Expression Regulation, Fungal; D007521:Isocitrate Dehydrogenase / Q000378:metabolism; D007523:Isocitrates / Q000378:metabolism; D008969:Molecular Sequence Data; D009249:NADP / Q000378:metabolism; D010641:Phenotype; D011422:Propionates / Q000378:metabolism
D003470:Culture Media; D007523:Isocitrates; D011422:Propionates; D009249:NADP; D002244:Carbon; D007521:Isocitrate Dehydrogenase; D019755:Carbon-Carbon Lyases; C417277:methylisocitrate lyase