Proteomic analysis of Aspergillus nidulans cultured under hypoxic conditions

Abstract

The fungus Aspergillus nidulans reduces nitrate to ammonium and simultaneously oxidizes ethanol to acetate to generate ATP under hypoxic conditions in a mechanism called ammonia fermentation (Takasaki, K. et al.. J. Biol. Chem. 2004, 279, 12414-12420). To elucidate the mechanism, the fungus was cultured under normoxic and hypoxic (ammonia fermenting) conditions, intracellular proteins were resolved by 2-DE, and 332 protein spots were identified using MALDI MS after tryptic digestion. Alcohol and aldehyde dehydrogenases that play key roles in oxidizing ethanol were produced at the basal level under hypoxic conditions but were obviously provoked by ethanol under normoxic conditions. Enzymes involved in gluconeogenesis, as well as the tricarboxylic and glyoxylate cycles, were downregulated. These results indicate that the mechanism of fungal energy conservation is altered under hypoxic conditions. The results also showed that proteins in the pentose phosphate pathway as well as the metabolism of both nucleotide and thiamine were upregulated under hypoxic conditions. Levels of xanthine and hypoxanthine, deamination products of guanine and adenine were increased in DNA from hypoxic cells, indicating an association between hypoxia and intracellular DNA base damage. This study is the first proteomic comparison of the hypoxic responses of A. nidulans.

The Glutathione System of Aspergillus nidulans Involves a Fungus-specific Glutathione S-Transferase

“…A peroxiredoxin homologous to yeast Ahp1p (AN8692.3, 40% identical) as well as elongation factor 1B (AN9304.3), of which the Aspergillus fumigatus ortholog (elfA) reportedly has peroxiredoxin activity (40), were more than 2-fold up-regulated in the DGR1 strain. We previously constructed A. nidulans proteome maps comprising 300 intracellular proteins (35 Table 2). The Prx1p homolog was 1.8-fold more abundant in DGR1 than in the wild type strain, whereas CatA and CpeA was less induced in DGR1 ( Table 2).…”

“…Proteins (200 g) were isoelectrically focused, loaded onto precast 11.0% homogeneous polyacrylamide (slab) gels (PAGE) (20 ϫ 20 cm), and stained with SYPRO Ruby (BioRad) as described (35). Spots were detected using a ChemiDoc XRS (Bio-Rad).…”

“…Proteome Analysis-Samples for two-dimensional gel electrophoresis were prepared from A. nidulans A26 and DGR1 as described (35). Proteins (200 g) were isoelectrically focused, loaded onto precast 11.0% homogeneous polyacrylamide (slab) gels (PAGE) (20 ϫ 20 cm), and stained with SYPRO Ruby (BioRad) as described (35).…”

“…Proteins were excised from the slab, digested with trypsin gold (Promega), and used for matrix-assisted laser desorption ionization time-of-flight mass spectrometry analysis as described (35). Proteins were identified by peptide mass finger analysis using the MASCOT (Matrix Science Ltd.) search engines of the entire NCBI protein data base, and a protein sequence library was constructed in-house with the most recent annotation of the A. nidulans genome (Version 3, Broad Institute, Cambridge, MA).…”

Glutathione Reductase/Glutathione Is Responsible for Cytotoxic Elemental Sulfur Tolerance via Polysulfide Shuttle in Fungi

“…The blots were stained with Coomassie Brilliant Blue R-250, and amino-terminal amino acid sequences were determined in excised protein bands using an automated protein sequencer (Model Precise 492, Perkin Elmer, Waltham, MA). Purified SR was digested with trypsin, used for matrix-assisted laser desorption ionization time of flight-mass spectrometry (MALDI-TOF-MS), and peptide mass fingerprints were analyzed using the MASCOT search engine (Matrix Science Ltd., London, UK) as described (27). The template comprised nucleotide fragments corresponding to the gene for SR (glrA) were amplified by PCR using total DNA of F. oxysporum JCM11502 prepared as described by Takasaki et al (19) and the primers were fogr1 and fogr2 (supplemental Table S1).…”