Relationships between rDNA, Nop1 and Sir complex in biotechnologically relevant distillery yeasts

Abstract

Distillery yeasts are poorly characterized physiological group among the Saccharomyces sensu stricto complex. As industrial yeasts are under constant environmental stress during fermentation processes and the nucleolus is a stress sensor, in the present study, nucleolus-related parameters were evaluated in 22 commercially available distillery yeast strains. Distillery yeasts were found to be a heterogeneous group with a variable content and length of rDNA and degree of nucleolus fragmentation. The levels of rDNA were negatively correlated with Nop1 (r = -0.59, p = 0.0038). Moreover, the protein levels of Sir transcriptional silencing complex and longevity regulators, namely Sir1, Sir2, Sir3 and Fob1, were studied and negative correlations between Sir2 and Nop1 (r = -0.45, p = 0.0332), and between Sir2 and Fob1 (r = -0.49, p = 0.0211) were revealed. In general, S. paradoxus group of distillery yeasts with higher rDNA pools and Sir2 level than S. bayanus group was found to be more tolerant to fermentation-associated stress stimuli, namely mild cold/heat stresses and KCl treatment. We postulate that rDNA state may be considered as a novel factor that may modulate a biotechnological process.

Keywords: Distillery yeasts; Nop1; Nucleolus; Sirtuins; rDNA.

Fig. 1

Analysis of selected nucleolus parameters in 22 distillery yeasts. a Southern blot analysis of the rDNA length. gDNA, genomic DNA after digestion with BamHI; lanes 1–22, 22 distillery strains. The length of rDNA calculated per amount of DNA is also shown (bottom). b Polymerase chain reaction (PCR)-based analysis of rDNA levels. Lane M, DNA molecular size marker; lanes 1–22, 22 distillery strains. Template amount-dependent PCR results are also shown (bottom). DNA from strain 10 (12.5, 25, 125 and 250 ng) was subjected to PCR-based analysis of rDNA content (lanes 12.5, 25, 125 and 250). c Fluorescence in situ hybridization (FISH)-based analysis of rDNA content. rDNA was visualized using whole chromosome painting probe (WCPP) specific to chromosome XII that contains rDNA locus in yeast (green, bottom). Fluorescence signals of chromosome XII were quantified using ImageJ software (top). The integrated fluorescence density is presented in relative fluorescence units (RFUs). The box-and-Tukey whisker plots are shown, n = 100. Typical micrographs are also presented (bottom). The cells were labeled with FITC to detect chromosome XII-specific signals (green). DNA was visualized using DAPI staining (blue). d Comparative analysis of rDNA pools between two distillery yeast groups, namely S. bayanus group (strains 1, 2, 3, 4, 5, 6 and 16) and S. paradoxus group (strains 7, 8, 10, 11, 12, 13, 14, 15, 17, 18, 20, 21 and 22) using Southern blotting (left), FISH (middle) and PCR (right). The bars indicate SD or SEM, n = 3, **p < 0.01, ***p < 0.001 compared to S. bayanus group (Student’s t test). e Analysis of chromosome I, III, XI and XII signals using fluorescence in situ hybridization (FISH) and whole chromosome painting probes (WCPPs). Chromosome-specific signals were scored in 100 nuclei and presented as a percentage; namely, more than two chromosome-specific signals are shown, n = 100. f Silver staining of nucleolar organizer region-based analysis of nucleolus fragmentation. Fragmented nucleoli were scored (%). The typical micrographs are also shown (right) (color figure online)

Fig. 2

a Western blot analysis of Nop1, Fob1, Sir1, Sir2 and Sir3 protein contents in 22 distillery yeasts (lanes from 1 to 22). Anti-Act1 antibody served as a loading control. b Comparative analysis of Nop1, Fob1 and Sir2 protein levels between two distillery yeast groups, namely S. bayanus group (strains 1, 2, 3, 4, 5, 6 and 16) and S. paradoxus group (strains 7, 8, 10, 11, 12, 13, 14, 15, 17, 18, 20, 21 and 22). The bars indicate SD, n = 3, *p < 0.05, **p < 0.01, ***p < 0.001 compared to S. bayanus group (Student’s t test). c Correlation analysis between the length of rDNA and Nop1 (r = −0.59, p = 0.0038), Sir2 and Nop1 (r = −0.45, p = 0.0332) and Sir2 and Fob1 (r = −0.49, p = 0.0211). The 95 % confidence interval is shown. Results represent the mean from three independent experiments. Correlation analysis of the data was performed using a linear correlation (Pearson r) test. Protein content is presented in arbitrary units (a.u.)

Fig. 3

Analysis of the utilization of non fermentable carbon sources (a) and sensitivity to fermentation-associated stress stimuli (b) in distillery yeasts (strains from 1–22 as denoted on a scheme) using spot assay. a Yeast cells at the logarithmic phase of growth were diluted (1 × 10⁵ cells/ml), and growth on solid YPG and YPE media was inspected after 48 h. b Yeast cells at the logarithmic phase of growth were diluted (1 × 10⁵ cells/ml), and growth on solid YPD medium in the presence of different stress stimuli was inspected after 48 h. In the case of hydrogen peroxide, cells were incubated with hydrogen peroxide for 40 min and then transferred to solid YPD medium. Representative photographs are shown. Strains 1, 2, 3, 4, 5, 6 and 16, S. bayanus; strains 7, 8, 10, 11, 12, 13, 14, 15, 17, 18, 20, 21 and 22, S. paradoxus; strain 9, S. cerevisiae; strain 19, S. kudriavzevii