Putative 3-nitrotyrosine detoxifying genes identified in the yeast Debaryomyces hansenii: in silico search of regulatory sequences responsive to salt and nitrogen stress

Background: During salt stress, the yeast Debaryomyces hansenii synthesizes tyrosine as a strategy to avoid the oxidation of proteins. Tyrosine reacts with nitrogen radicals to form 3-nitrotyrosine. 3-nitrotyrosine prevents the effects of associated oxidative stress and thus contributes to the high halotolerace of the yeast. However, the mechanism of how D. hansenii counteracts the presence of this toxic compound is unclear. In this work, we evaluated D. hansenii's capacity to assimilate 3-nitrotyrosine as a unique nitrogen source and measured its denitrase activity under salt stress. To identify putative genes related to the assimilation of 3-nitrotyrosine, we performed an in silico search in the promoter regions of D. hansenii genome. Results: We identified 15 genes whose promoters had binding site sequences for transcriptional factors of sodium, nitrogen, and oxidative stress with oxidoreductase and monooxygenase GO annotations. Two of these genes, DEHA2E24178g and DEHA2C00286g, coding for putative denitrases and having GATA sequences, were evaluated by RT-PCR and showed high expression under salt and nitrogen stress. Conclusions: D. hansenii can grow in the presence of 3-nitrotyrosine as the only nitrogen source and has a high specific denitrase activity to degrade 3-nitrotyrosine in 1 and 2 M NaCl stress conditions. The results suggest that given the lack of information on transcriptional factors in D. hansenii, the genes identified in our in silico analysis may help explain 3-nitrotyrosine assimilation mechanisms.

Cell wall involvement in the glycerol response to high osmolarity in the halotolerant yeast Debaryomyces hansenii.

Osmotic stress was studied through the induction of the gene coding for glycerol 3-phosphate dehydrogenase (DhGPD1) in the halotolerant yeast Debaryomyces hansenii. This yeast responded to modifications in turgor pressure by stimulating the transcription of DhGPD1 when exposed to solutes that cause turgor stress (NaCl or sorbitol), but did not respond to water stress mediated by ethanol. In contrast to what has been documented to occur in Saccharomyces cerevisiae, D. hansenii protoplasts did not show induction in the transcription of DhGPD1 showing a limitation in their response to solute stress. The results presented indicate that the presence of the cell wall is of significance for the induction of DhGPD1 and hence for osmotic regulation in halotolerant D. hansenii. It appears that the main osmosensor that links high osmolarity with glycerol accumulation may be of a different nature in this yeast.

DhARO4, an amino acid biosynthetic gene, is stimulated by high salinity in Debaryomyces hansenii.

The highly halotolerant yeast Debaryomyces hansenii when grown in the presence of 2M NaCl, increased the expression of ARO4 which is involved in the biosynthesis of aromatic amino acids. The function of the isolated gene was verified by complementation of a Saccharomyces cerevisiae null mutant, aro4Delta, restoring the specific activity of the enzyme (a 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase) to wild-type levels. DhARO4 transcript expression under high salinity was stimulated at the beginning of the exponential growth phase. As the DhARO4 promoter region presents putative GCRE and CRE sequences, its expression was evaluated under conditions of NaCl stress and amino acid starvation, showing similar expression levels for either condition. The combined effect of both stressors resulted in a further increase in transcript levels over the singly added stressors, indicating independent stimulatory events. Our results support the hypothesis that high salinity and amino acid availability are physiologically interconnected.

Heterologous expression of glycerol 3-phosphate dehydrogenase gene [DhGPD1] from the osmotolerant yeast Debaryomyces hansenii in Saccharomyces cerevisiae.

The role for the gene encoding glycerol 3-phosphate dehydrogenase (DhGPD1) from the osmotolerant yeast Debaryomyces hansenii, in glycerol production and halotolerance, was studied through its heterologous expression in a Saccharomyces cerevisiae strain deficient in glycerol synthesis (gpd1Delta). The expression of the DhGPD1 gene in the gpd1Delta background restored glycerol production and halotolerance to wild type levels, corroborating its role in the salt-induced production of glycerol. Although the gene was functional in S. cerevisiae, its heterologous expression was not efficient, suggesting that the regulatory mechanism may not be shared by these two yeasts.

Isolation of a GPD gene from Debaryomyces hansenii encoding a glycerol 3-phosphate dehydrogenase (NAD+).

A gene homologous to GPD1, coding for glycerol-3-phosphate dehydrogenase (sn-glycerol 3-phosphate: NAD(+) oxidoreductase, EC 1.1.1.8), has been isolated from the halophilic yeast Debaryomyces hansenii by complementation of a Saccharomyces cerevisiae gpd1 Delta mutant. DNA sequencing of the complementing genomic clone indicated the existence of an open reading frame encoding a protein with 369 amino acids. Comparative analysis of the deduced amino acid sequence showed high similarity to homologous genes described for other eukaryotic GPD enzymes. The sequence has been submitted to the GenBank database under Accession No. AY333427.