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Chapter 23 : Nitrogen Metabolism in Filamentous Fungi
Category: Microbial Genetics and Molecular Biology; Fungi and Fungal Pathogenesis
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Among the filamentous fungi, the genetic basis of nitrogen metabolism has been most intensively studied in the model ascomycetes Aspergillus nidulans and Neurospora crassa by utilizing the excellent classical and molecular genetic systems provided by these species. Much of one's current knowledge is based on classical genetic analysis of mutants affected in specific aspects of the enzymology or the regulation of nitrogen metabolism. There are also instances where significant differences across species provide fascinating insights into the evolutionary divergence of nitrogen metabolism within the filamentous fungi. In this chapter, the molecular genetics of the ammonium assimilatory pathways is considered as the starting point for the biosynthesis of complex nitrogenous macromolecules. The switch from anabolism to catabolism requires the relief of nitrogen metabolite repression, a global control system that modulates the expression of large sets of nitrogen-catabolic enzymes. Recent studies suggest some diversity in the complex molecular mechanisms underlying this regulation among different fungal groups. Details of several nitrogen-catabolic systems are reviewed to illustrate the metabolic and regulatory strategies employed by fungi in the acquisition of nitrogen metabolites. The catabolism of certain amino acids, such as proline and arginine, provides a good source of nitrogen metabolites and supports strong growth in A. nidulans, whereas other amino acids, such as histidine and leucine, are very poor sources of nitrogen for the wild-type organism.
Key Concept Ranking
- L-Amino Acid Oxidase
Ammonium assimilation pathway in fungi. Ammonium can be assimilated by NADPGDH to form glutamate or by the GOGAT cycle through the action of GS and GOGAT to form glutamine and glutamate for the biosynthesis of nitrogenous molecules. The interconversion of ammonium, glutamate, and glutamine catalyzed by NADP-GDH, GS, GOGAT, and NAD-GDH is central to nitrogen metabolism.
Components of nitrogen regulation in A. nidulans. The expression of genes subject to nitrogen metabolite repression is regulated by changes in the levels and transcriptional activity of AreA in response to the nitrogen status of the cells (see the text). The levels of areA mRNA are influenced by autoregulation of areA transcription (1) and differential stability of the areA mRNA (2) such that the transcript is degraded more rapidly under nitrogen-sufficient conditions than when nitrogen is limiting or absent. The levels of AreA available to activate transcription within the nucleus are determined by a balance between nuclear import and export (3). Under conditions of nitrogen starvation, AreA accumulates in the nucleus due to a block in CrmA-dependent nuclear export. Once inside the nucleus, the transcriptional activity of AreA is influenced by interaction with the NmrA corepressor (4). The extent to which AreA activity is inhibited by NmrA under nitrogen-sufficient conditions is determined indirectly by the bZIP transcription factor MeaB (5). Under nitrogen-sufficient conditions, MeaB activates nmrA expression, leading to increased levels of NmrA and greater inhibition of AreA activity. Thus, the activity of AreA is determined by the relative levels of AreA and NmrA (6), with active AreA predominating under nitrogen-limiting or nitrogen starvation conditions.
Genomic arrangement of nitrate-assimilatory genes in sequenced fungal genomes. (A) In the nitrate-assimilatory pathway, nitrate is transported into the cell by nitrate permeases (encoded by nrtA and nrtB in A. nidulans). Endogenous nitrate is catabolized by nitrate reductase (encoded by niaD in A. nidulans) to nitrite, which in turn is converted by nitrite reductase (encoded by niiA in A. nidulans) to ammonium for synthesis of nitrogen-containing biomolecules. (B) Evolution of the nitrate-assimilatory gene cluster in filamentous fungi. The phylogenetic relationships of the various fungi are shown on the left ( Fitzpatrick et al., 2006 ). The niaD, niiA, and nrtA orthologues were identified by blastp and tblastn searches ( Altschul et al., 1997 ) against the respective genome databases, using the A. nidulans NiaD, NiiA, and NrtA sequences. The relative orientation of the open reading frames of niaD (black rectangles), niiA (grey rectangles), and nrtA (white rectangles) orthologues in various fungi is indicated by arrows. Thick lines connecting the rectangles indicate that the genes are linked. Nil means that sequence with high similarity could not be found in the respective genome sequences by blastp and tblastn searches. “Aspergillus spp.” includes A. nidulans, A. fumigatus, A. flavus, A. clavatus, A. terreus, A. oryzae, A. niger, and Neosartorya fischeri. “Fusarium spp.” includes F. graminearum, F. oxysporum, and F. verticillioides.
Growth of A. nidulans and N. crassa on various amino acids as nitrogen source. (A) A. nidulans wild-type, areAΔ, and areA102 strains were grown at 37°C for 2 days on ANM solid media ( Cove, 1966 ) containing 1% glucose and ammonium (NH4), glutamine (Gln), glutamate (Glu), alanine (Ala), nitrate (NO3), proline (Pro), γ-amino butyric acid (GABA), arginine (Arg), uric acid (UA), or histidine (His) at a final concentration of 10 mM as the sole nitrogen source. (B) Wild-type A. nidulans was grown on solid ANM minimal media ( Cove, 1966 ) containing 1% glucose and the indicated amino acid at a concentration of 10 mM. The relative levels of growth after 2 days at 37°C are ranked from strongest (++++) to weakest (–/+). Wild-type N. crassa was grown on liquid Vogel’s medium lacking NH4 and NO3 with 2% sucrose and the indicated amino acid at a concentration of 10 mM. The relative strength of growth is determined by dry mass weight after 3 days of growth at 30°C ( Facklam and Marzluf, 1978 ). The N. crassa data are grouped and expressed with symbols as follows: ++++, >30 mg; +++, <30 mg; ++, <15 mg; +, 5 mg; –/+, <2 mg. ND, not determined.
Evolution of L-amino acid oxidase in filamentous fungi. The phylogenetic relationships of the various filamentous ascomycetes are indicated on the left ( Fitzpatrick et al., 2006 ; Broad Institute website [http://www.broad.mit.edu/annotation/genome/aspergillus_group/]). L-Amino acid oxidase and related sequences in various fungi were obtained by blastp and tblastn searches ( Altschul et al., 1997 ) of the respective fungal genome database (Broad Institute [http://www.broad.mit.edu/annotation/fgi/]), using the A. nidulans sequences (L-amino acid oxidase, SarA [accession number, AAT84085], and L-amino acid oxidase-like sequence [accession number, EAA64973]). A minus sign (–) represents the absence of any orthologous sequence. An asterisk (*) indicates that the identified sequence was almost equally similar to both L-amino oxidase and L-amino oxidase-like sequences but has a slightly higher percentage of identity and similarity to the indicated sequence. Accession numbers or gene locus (GL) numbers of L-amino acid oxidase sequences are as follows: A. flavus (GL, AFL2G_08801.2); A. terreus (EAU31805); A. niger (CAK45753); A. clavatus (EAW12261); S. nodorum (EAT79476); M. grisea (EDK01261); N. crassa (CAD21325); F. graminearum (FGSG_13802.3); F. oxysporum (GL, FOXG_15820.2 and FOXG_15290.2); and F. verticillioides (GL, FVEG_13289.3 and FVEG_12615.3). Accession numbers or GL numbers of L-amino acid oxidase-like sequences are as follows: A. flavus (GL, AFL2G_11781.2); A. oryzae (BAC55901); A. terreus (EAU36894); N. fischeri (EAW19737); A. fumigatus (EAL86792); S. nodorum (EAT82237); F. oxysporum (GL, FOXG_05815.2); and F. verticillioides (FVEG_03694.3).
Conservation of nitrogen regulators in filamentous ascomycetes a