Alternative CUG Codon Usage in the Halotolerant Yeast Debaryomyces hansenii: Gene Expression Profiles Provide New Insights into Ambiguous Translation.

The halotolerant yeast Debaryomyces hansenii belongs to the CTG-Ser1 clade of fungal species that use the CUG codon to translate as leucine or serine. The ambiguous decoding of the CUG codon is relevant for expanding protein diversity, but little is known about the role of leucine-serine ambiguity in cellular adaptations to extreme environments. Here, we examine sequences and structures of tRNACAG from the CTG-Ser1 clade yeasts, finding that D. hansenii conserves the elements to translate ambiguously. Then, we show that D. hansenii has tolerance to conditions of salinity, acidity, alkalinity, and oxidative stress associated with phenotypic and ultrastructural changes. In these conditions, we found differential expression in both the logarithmic and stationary growth phases of tRNASer, tRNALeu, tRNACAG, LeuRS, and SerRS genes that could be involved in the adaptive process of this yeast. Finally, we compare the proteomic isoelectric points and hydropathy profiles, detecting that the most important variations among the physicochemical characteristics of D. hansenii proteins are in their hydrophobic and hydrophilic interactions with the medium. We propose that the ambiguous translation, i.e., leucylation or serynation, on translation of the CUG-encoded residues, could be linked to adaptation processes in extreme environments.

Tolerance to Oxidative Stress in Budding Yeast by Heterologous Expression of Catalases A and T from Debaryomyces hansenii.

The function of catalases A and T from the budding yeast Saccharomyces cerevisiae (ScCta1 and ScCtt1) is to decompose hydrogen peroxide (H2O2) to mitigate oxidative stress. Catalase orthologs are widely found in yeast, suggesting that scavenging H2O2 is crucial to avoid the oxidative damage caused by reactive oxygen species (ROS). However, the function of catalase orthologs has not yet been experimentally characterized in vivo. Here, we heterologously expressed Debaryomyces hansenii DhCTA1 and DhCTT1 genes, encoding ScCta1 and ScCtt1 orthologs, respectively, in a S. cerevisiae acatalasemic strain (cta1Δ ctt1Δ). We performed a physiological analysis evaluating growth, catalase activity, and H2O2 tolerance of the strains grown with glucose or ethanol as carbon source, as well as under NaCl stress. We found that both genes complement the catalase function in S. cerevisiae. Particularly, the strain harboring DhCTT1 showed improved growth when ethanol was used as carbon source both in the absence or presence of salt stress. This phenotype is attributed to the high catalase activity of DhCtt1 detected at the exponential growth phase, which prevents intracellular ROS accumulation and confers oxidative stress resistance.

Contribution of the mitogen-activated protein kinase Hog1 to the halotolerance of the marine yeast Debaryomyces hansenii.

Halotolerant species are adapted to dealing continually with hyperosmotic environments, having evolved strategies that are uncommon in other organisms. The HOG pathway is the master system that regulates the cellular adaptation under these conditions; nevertheless, apart from the importance of Debaryomyces hansenii as an organism representative of the halotolerant class, its HOG1 pathway has been poorly studied, due to the difficulty of applying conventional recombinant DNA technology. Here we describe for the first time the phenotypic characterisation of a null HOG1 mutant of D. hansenii. Dhhog1Δ strain was found moderately resistant to 1 M NaCl and sensitive to higher concentrations. Under hyperosmotic shock, DhHog1 fully upregulated transcription of DhSTL1 and partially upregulated that of DhGPD1. High osmotic stress lead to long-term inner glycerol accumulation that was partially dependent on DhHog1. These observations indicated that the HOG pathway is required for survival under high external osmolarity but dispensable under low and mid-osmotic conditions. It was also found that DhHog1 can regulate response to alkali stress during hyperosmotic conditions and that it plays a role in oxidative and endoplasmic reticulum stress. Taken together, these results provide new insight into the contribution of this MAPK in halotolerance of this yeast.