Nitric oxide (NO) is a toxic reactive nitrogen species that induces microbial adaption mechanisms. Screening a genomic DNA library identified a new gene, ntpA, that conferred growth tolerance upon Aspergillus nidulans against exogenous NO. The gene encoded a cysteine-rich 23-amino-acid peptide that reacted with NO and S-nitrosoglutathione to generate an S-nitrosated peptide. Disrupting ntpA increased amounts of cellular S-nitrosothiol and NO susceptibility. Thioredoxin and its reductase denitrosated the S-nitrosated peptide, decreased cellular S-nitrosothiol and conferred tolerance against NO, indicating peptide-mediated catalytic NO removal. The peptide binds copper(I) in vitro but is dispensable for metal tolerance in vivo. NO but not metal ions induced production of the peptide and ntpA transcripts. We discovered that the thionein family of peptides has NO-related functions and propose that the new peptide be named NO-inducible nitrosothionein (iNT). The ubiquitous distribution of iNT-like polypeptides constitutes a potent NO-detoxifying mechanism that is conserved among various organisms.

Microorganisms have developed mechanisms to combat reactive nitrogen species (RNS); however, only a few of the fungal genes involved have been characterized. Here we screened RNS-resistant Aspergillus nidulans strains from fungal transformants obtained by introducing a genomic DNA library constructed in a multicopy vector. We found that the AN0121.3 gene (hemC) encodes a protein similar to the heme biosynthesis enzyme porphobilinogen deaminase (PBG-D) and facilitates RNS-tolerant fungal growth. The overproduction of PBG-D in A. nidulans promoted RNS tolerance, whereas PBG-D repression caused growth that was hypersensitive to RNS. PBG-D levels were comparable to those of cellular protoheme synthesis as well as flavohemoglobin (FHb; encoded by fhbA and fhbB) and nitrite reductase (NiR; encoded by niiA) activities. Both FHb and NiR are hemoproteins that consume nitric oxide and nitrite, respectively, and we found that they are required for maximal growth in the presence of RNS. The transcription of hemC was upregulated by RNS. These results demonstrated that PBG-D is a novel NO-tolerant protein that modulates the reduction of environmental NO and nitrite levels by FHb and NiR. N itric oxide (NO) is the best-characterized of the reactive nitrogen species (RNS), which react to and damage DNA, lipids, and enzymes, as do reactive oxygen species (31, 39). Both enzymatic and chemical reactions can generate NO under physiological conditions. Mammalian NO synthase oxidizes arginine to produce NO, which transduces signals for neuronal communication, inflammation, and smooth-muscle relaxation (24). Macrophages produce NO by inducible NO synthase to fight infective microorganisms and parasites (5). Microorganisms generate NO as an intermediate of denitrification (10). Bacterial nitrification and denitrification processes involve nitrite (NO 2 Ϫ ) as an intermediate, and improper aeration often results in the accumulation of NO 2 Ϫ , which is concentration-dependently dismutated to NO, especially in an acidic environment (43). Thus, NO is ubiquitous in the microbial milieu, and exposure to NO 2 Ϫ and NO should induce microbial mechanisms to survive RNS toxicity.Microorganisms have developed constitutive and inducible strategies for combating RNS. Flavohemoglobin (FHb) is the best known enzyme that is involved in the RNS response, and it is widespread in bacteria, yeast, and filamentous fungi (29). It comprises protoheme-containing hemoglobin-like and flavincontaining ferredoxin-NAD(P)H reductase-like domains that are located in the amino-and carboxy-terminal regions, respectively (16). Flavohemoglobin functions as a nitric oxide dioxygenase (NOD), converts oxygen and NO to less-toxic nitrate, and facilitates NO tolerance (9). Besides FHb, bacteria and fungi produce cytochrome b-and cytochrome P450-type NO reductases (Nor), which reduce NO to nitrous oxide and consequently minimize NO toxicity (34, 42). Another bacterial flavorubredoxin-type Nor is NorVW, which contains a di-iron catalytic center and reduces and detoxifies NO (...