Figure 5.
The production of aromatic alcohols is controlled by cell density. (A) The accumulation of aromatic alcohols in the medium increases with increasing cell density. Saccharomyces haploid cells were inoculated at 105 cells/mL in minimum medium (YNB) containing 5 mM L-Pro as nitrogen source. At indicated time points the cell density (◆) was measured by OD600 and the concentration of aromatic alcohols (●, phenylethanol; ▴, tyrosol; ◼, tryptophol) was determined by HPLC. The diploid cells have very similar growth and alcohol production curves to haploid cells. (B) The rate of aromatic alcohol production is controlled by cell density. Wild-type (WT) haploid cells were incubated in SLAD containing 50 μM specific aromatic amino acids either at low density (5 × 105 cells/mL) or high density (5 × 107 cells/mL) for 2 h. One milliliter of CM from the low-density culture and 10 μL of CM from the high-density culture were lyophilized and loaded on the TLC silica gel. Wild-type diploid cells show similar results. (C) High cell density up-regulates the level of ARO9 and ARO10 transcripts, and the induction requires ARO80. Wild-type (WT) and aro80 cells were incubated in liquid SLAD medium either at low (105 cells/mL, white bars) or high (5 × 107 cells/mL, black bars) cell densities for 60 min. Cells were collected by filtration and centrifugation. Total RNA was purified and subjected to qRT–PCR analysis. The transcript level in wild-type cells at low density was used as the baseline for comparison. The results shown here were obtained from haploid strains. The diploid strains show the same pattern. (D) Tryptophol, but not tyrosol and phenylethanol, induces the expression of ARO9 and ARO10 genes, and the autostimulation depends on ARO80. Wild-type (WT) and aro80 cells were cultured at OD600 0.2 in SD with or without the specified aromatic alcohol (500 μM) for 4 h. RNA was isolated and analyzed by qRT–PCR. The transcript level of the wild-type strain in SD was the baseline for calculating fold changes. Tryptophol has the same effects on the expression of ARO9 and ARO10 in both haploid and diploid strains. The results shown here were obtained from haploid strains. (White bars) SD alone; (light-gray bars) tyrosol; (dark-gray bars) phenylethanol; (black bars) tryptophol. (E) Tryptophol (TrpOH) stimulates the growth of a reporter strain PARO9-URA3 on uracil-minus (SD) plates, and the stimulation requires ARO80. Five microliters of 10-fold titrated Saccharomyces cells (L8199 for wild-type and L8220 for aro80 mutant) was spotted on specified plates for 2 d at 30°C. (F) Tryptophol up-regulates the transcript levels of ARO9 and ARO10 in cells at low density (only ARO9 is shown). Wild-type (WT) or aro80 cells were incubated in SLAD with (black bars) or without (white bars) 500 μM tryptophol at low (105 cells/mL) or high (5 × 107 cells/mL) cell densities for 60 min. RNA was isolated and analyzed by qRT–PCR. The transcript level of ARO9 in wild-type cells at low density without tryptophol was the baseline for comparison. Both haploid and diploid strains show the same pattern of gene expression. Only the results from haploid strains are shown. (G) The Saccharomyces aro80 mutant is defective in the biosynthesis of aromatic alcohols. Saccharomyces wild-type (WT) and aro80 cells were incubated in YNB + 2% glucose with either 37 mM (lanes 1,3) or 50 μM ammonium sulfate (lanes 2,4). CM was analyzed by TLC. The aro80/aro80 mutant shows the same defect in the production of aromatic alcohols as the aro80 haploid mutant. The results from haploid strains are shown.