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

Abstract

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, aro4 , 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.

Characterization of a salt-induced DhAHP, a gene coding for alkyl hydroperoxide reductase, from the extremely halophilic yeast Debaryomyces hansenii

“…It has been clearly shown that sodium ions are less toxic for D. hansenii as compared with other organisms; therefore, it is considered a 'sodium-includer' organism [8]. The reduced toxic effect by Na + and its accumulation at high levels under high salt is probably indicative of an adaptive strategy of D. hansenii for growth in hypersaline environments [9]. The organism must posses an array of advantageous characteristics that collectively confer its high halotolerance.…”

DhARO4 induction and tyrosine nitration in response to reactive radicals generated by salt stress in Debaryomyces hansenii

“…We have reported (Calderón‐Torres et al , ) the induction of Dh ARO4 expression in response to 2 m NaCl; therefore in this study we wanted to define whether the enzymatic activity of Dh Aro4p also resulted in an increment; such activity was measured in cell extracts of D. hansenii grown in YPD (control), 1 m , and 2 m NaCl. Taking into account that Sc Aro4p and Sc Aro3p enzymes have a tyrosine and phenylalanine feedback inhibition, respectively (Helmstaedt et al , ), we measured the specific activity of Dh Aro4p in the presence of 2 m m phenylalanine; in order to evaluate whether the Dh Aro4p feedback inhibition is present during salt stress, phenylalanine and tyrosine were added to the medium (Figure ).…”

“…Specific activity of Sc Aro4p and Dh Aro4p during saline stress, with phenylalanine or tyrosine in the growth medium. A volume of 180 µl of crude extracts was used for the determination of Aro4p activity as reported by Calderón‐Torres et al (). The cells were grown in YPD (control) or YPD with 1 m NaCl for S. cerevisiae extracts, and also with 2 m NaCl for D. hansenii extracts, with amino acids: 5 m m phenylalanine (■) and 5 m m tyrosine (□)…”

“…The cell suspension was mechanically disrupted with glass beads at 4 °C. Protein concentration was measured by the method of Lowry (Markwell et al , ), and the enzymatic activity of Aro4p was determined as described by Calderón‐Torres et al ().…”

“…We have previously reported that putative DhARO4 mRNA increased 2-2.6 times during hypersaline stress (2 M NaCl), and DhARO4 could complement the aro4Δ null mutant of S. cerevisiae, restoring the specific activity of the enzyme ScAro4p to wild-type levels (Calderón-Torres et al, 2006). ARO4 codes for 3-deoxy-D-arabinoheptulosonate-7-phosphate synthase (DAHP synthase), which is the first enzyme in the shikiimate pathway leading to the biosynthesis of aromatic amino acids (Braus, 1991;Schnappauf et al, 1998).…”