Microorganisms combat the ubiquitous toxic 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, and identified two novel RNS-resistant genes (hemC,mthA) and studied their RNS resistant mechanisms. hemC encodes a protein similar to the heme biosynthesis enzyme porphobilinogen deaminase (PBG-D). PBG-D is known to be critical for synthesizing heme, but there is no report about PBG-D involved in tolerance of nitrosative stress. We found that overproduction of PBG-D tolerated A. nidulans growth against RNS whereas repressed PBG-D resulted in hypersensitive growth to RNS. Level of PBG-D was comparable to those of cellular heme synthesis as well as flavohemoglobin (FHb) and nitrite reductase (NiR) 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 RNS-tolerant protein that modulates the reduction of environmental NO and nitrite levels by FHb and NiR. The mthAgene encodes a small peptide containing cysteine-rich motif, which is characteristic of metallothionein. Metallothionein is a well known heavy metal tolerating peptide although few report showed its function in RNS-defense mechanism. We found that transcription of mthA in A. nidulans was induced by RNS. Disrupting mthA(ΔmthA) increased growth sensitivity to RNS, and overexpressing mthA (HmthA) increased RNS tolerance. Western blot analysis using anti-S-nitrosocysteine antibody showed that the amounts of S-nitrosated proteins in the RNS-treated A. nidulans strains were in the order of ΔmthA > WT > HmthA. Spectroscopic and the Biotin-switch methods detected S-nitroso-MthA upon exposing MthA to NO, indicating that MthA bound to and detoxified NO in the fungus.
Full conference title:
- ASM 112th (2012)