Transcriptome analysis of nitrate assimilation in Aspergillus nidulans reveals connections to nitric oxide metabolism

Abstract

Nitrate is a dominant form of inorganic nitrogen (N) in soils and can be efficiently assimilated by bacteria, fungi and plants. We studied here the transcriptome of the short-term nitrate response using assimilating and non-assimilating strains of the model ascomycete Aspergillus nidulans. Among the 72 genes positively responding to nitrate, only 18 genes carry binding sites for the pathway-specific activator NirA. Forty-five genes were repressed by nitrate metabolism. Because nirA- strains are N-starved at nitrate induction conditions, we also compared the nitrate transcriptome with N-deprived conditions and found a partial overlap of differentially regulated genes between these conditions. Nitric oxide (NO)-metabolizing flavohaemoglobins were found to be co-regulated with nitrate assimilatory genes. Subsequent molecular characterization revealed that the strongly inducible FhbA is required for full activity of nitrate and nitrite reductase enzymes. The co-regulation of NO-detoxifying and nitrate/nitrite assimilating systems may represent a conserved mechanism, which serves to neutralize nitrosative stress imposed by an external NO source in saprophytic and pathogenic fungi. Our analysis using membrane-permeable NO donors suggests that signalling for NirA activation only indirectly depends on the nitrate transporters NrtA (CrnA) and NrtB (CrnB).

Identification of Hypoxia-Inducible Target Genes of Aspergillus fumigatus by Transcriptome Analysis Reveals Cellular Respiration as an Important Contributor to Hypoxic Survival

“…Its role in detoxifying harmful RNIs during hypoxia remains unclear. Its slight upregulation under these conditions may also simply be explained by a coregulation with FhpA as reported from A. nidulans (40).…”

“…To elucidate the role of the flavohemoprotein FhpA during hypoxia, we characterized its regulation in more detail. In aspergilli, the NO dioxygenase activity of flavohemoproteins suggests a role in the detoxification of nitric oxide (NO), which is formed during nitrate assimilation (39,40). In agreement, it was shown that fhpA is expressed in the presence of nitrate but repressed when glutamine is used as sole nitrogen source (K. Lapp, M. Vödisch, K. Kroll, M. Strassburger, O. Kniemeyer, T. Heinekamp, and A.…”

Riboflavin and pantothenic acid biosynthesis are crucial for iron homeostasis and virulence in the pathogenic mold Aspergillus fumigatus

“…FAD is a crucial coenzyme of flavohemoglobins, which provide protection against NO and related reactive nitrogen species [24]. We therefore tested the growth of Δ riboB during NO stress, induced by addition of 5 mM NaNO 2 at a low pH of 4.0 (acidic conditions stimulate generation of nitric oxide from nitrate [25]), and limiting 2.5 µM riboflavin for growth. Growth of the mutant was inhibited compared to the wt and complemented strains, indicating an important role for riboflavin in nitric oxide (NO) detoxification (Figure 3(a)).…”

Heme-Biosynthetic Porphobilinogen Deaminase Protects Aspergillus nidulans from Nitrosative Stress

“…Aspergillus nidulans harbors two predicted paralogs that encode cytosolic FHbA and mitochondrial FHbB (32). We constructed an A. nidulans strain in which both paralogs were deleted (⌬fhb) (see Fig.…”

“…Recent reports indicate that A. oryzae produces two FHb isozymes that function as NODs and that they are located in the cytosol and mitochondria (40,41). Aspergillus nidulans is a powerful model organism of the genus Aspergillus that produces FHb in order to survive under nitrosative stress (32). We previously reported that A. nidulans dissimilated nitrate to generate ammonium via NO 2 Ϫ under hypoxic conditions (36).…”