The Metabolic Regulation of Amino Acid Synthesis Counteracts Reactive Nitrogen Stress via Aspergillus nidulans Cross-Pathway Control

Abstract

Nitric oxide (NO) is a natural reactive nitrogen species (RNS) that alters proteins, DNA, and lipids and damages biological activities. Although microorganisms respond to and detoxify NO, the regulation of the cellular metabolic mechanisms that cause cells to tolerate RNS toxicity is not completely understood. We found that the proline and arginine auxotrophic proA5 and argB2 mutants of the fungus Aspergillus nidulans require more arginine and proline for normal growth under RNS stress that starves cells by accumulating fewer amino acids. Fungal transcriptomes indicated that RNS stress upregulates the expression of the biosynthetic genes required for global amino acids, including proline and arginine. A mutant of the gene disruptant, cpcA, which encodes the transcriptional regulation of the cross-pathway control of general amino acid synthesis, did not induce these genes, and cells accumulated fewer amino acids under RNS stress. These results indicated a novel function of CpcA in the cellular response to RNS stress, which is mediated through amino acid starvation and induces the transcription of genes for general amino acid synthesis. Since CpcA also controls organic acid biosynthesis, impaired intermediates of such biosynthesis might starve cells of amino acids. These findings revealed the importance of the mechanism regulating amino acid homeostasis for fungal responses to and survival under RNS stress.

Keywords: amino acid; nitric oxide; reactive species; starvation; stress.

Figure 1

Aspergillus nidulans proC gene tolerates NO. (A) Insertion of pRG3-1.7 and its derivatives. (B) Conidia incubated on MMN agar without or with 30 mM NaNO₂ (pH 5.5) generated 1 × 10¹–10⁵ colonies from YMS9 (parental strain), harboring indicated plasmids at 37 °C for 48 h. MMN, minimal nitric oxide medium; NaNO₂, sodium nitrite; NO, nitric oxide.

Figure 2

Nitric oxide-induced amino acid starvation in A. nidulans cells. (A) Biosynthetic pathways of proline and arginine in A. nidulans. (B) Morphology of colonies after incubating conidia (1 × 10¹–10⁵) at 37 °C for 48 h on MMN agar without or with indicated amounts of NaNO₂ (pH 5.5). Upper panel: A45 (proA5), A89 (argB2), and A26 (control; pro⁺, arg⁺) strains. Proline and arginine (1 mM each) were added to support auxotrophic growth, and 15 mM NaNO₂ (pH 5.5) was also added. Bottom panel: A26 strain cultured on MMN with exogenous proline, arginine (10 mM each), and 15 mM NaNO₂ (pH 5.5). (C) Cellular amino acid contents in liquid A. nidulans YMS9 cultures. Data are shown as means ± SD of three biological replicates (* p < 0.05, vs. 0 mM NaNO₂; Student t-tests). (D) YMS9 cells were incubated for 18 h, followed by NaNO₂ for 3 h. Then, changes in wet cell mass were evaluated.

Figure 3

CpcA controls the gene expression of Pro and Arg-biosynthetic genes. (A) Conidia (1 × 10¹–10⁵) were incubated at 37 °C for 48 h on MMN agar without or with 15 mM NaNO₂ (pH 5.5), then colony morphology was assessed. (B) Relative ratios of parental strain (YMS9) and ΔcpcA transcripts to actA. Strains were incubated in MMN at 37 °C for 18 h and then without or with 10 mM NaNO₂ (pH 5.5) for 3 h. Data are shown as means ± SD of data from three biological replicates (* p < 0.05 vs. YMS9 0 mM NaNO₂; † p < 0.05 vs. ΔcpcA 1 mM NaNO₂; Student t-tests). (C) Predicted CpcA-binding sequences in gene promoters. Nucleotides were predicted as numbers with translation start residues designated as 1. (D) Cellular amino acid content in liquid cultures of the parental strain (YMS9) and ΔcpcA incubated, as shown in (C). Data are shown as means ± SD of three biological replicates. (* p < 0.05, vs. YMS9 0 mM NaNO₂; † p < 0.05, vs. ΔcpcA 1 mM NaNO₂; Student t-tests). ΔcpcA, gene disruptant of cpcA.

Figure 4

Transcriptomes of genes involved in amino acid synthesis. Parental (YMS9) and cpcA disruptant (ΔcpcA) strains were incubated in MMN medium at 37 °C for 18 h and then with (+) or without (−) 1 mM NaNO₂ (pH 5.5) for 3 h. Genes were grouped based on the synthesized amino acids. +, CpcA-binding consensus on gene promoters. Red, cpcA-dependent genes.

Figure 5

Intracellular organic acid synthesis is regulated via CpcA. (A) Gene expression of pyruvate and TCA cycle metabolism. The reanalysis of the transcriptome shown in Figure 4. (B) Organic acids in liquid cultures of A. nidulans strains (as shown in Figure 4). Data are means ± SD of data from three biological replicates (* p < 0.05, vs. YMS9 0 mM NaNO₂; ^† p < 0.05, vs. ΔcpcA 1 mM NaNO₂; Student t-tests). (C) Activity of PDH and ACN in fungal cell-free extracts. Data are shown as means ± SD of three biological replicates (* p < 0.01, vs. 0 mM NaNO₂; Student t-tests). ACN, aconitase; NaNO₂, sodium nitrite PDH, pyruvate dehydrogenase.

Figure 6

Metabolic regulation mechanism counteracts RNS for fungal growth. AA, amino acid; CpcA, C-phycocyanin alpha subunit; NO, nitric oxide; OA, organic acid; RNS, reactive nitrogen species; TCA, tricarboxylic acid.