Strain-specific nuclear genetic background differentially affects mitochondria-related phenotypes in Saccharomyces cerevisiae

Abstract

In the course of our studies on mitochondrial defects, we have observed important phenotypic variations in Saccharomyces cerevisiae strains suggesting that a better characterization of the genetic variability will be essential to define the relationship between the mitochondrial efficiency and the presence of different nuclear backgrounds. In this manuscript, we have extended the study of such relations by comparing phenotypic assays related to mitochondrial functions of three wild-type laboratory strains. In addition to the phenotypic variability among the wild-type strains, important differences have been observed among strains bearing identical mitochondrial tRNA mutations that could be related only to the different nuclear background of the cells. Results showed that strains exhibited an intrinsic variability in the severity of the effects of the mitochondrial mutations and that specific strains might be used preferentially to evaluate the phenotypic effect of mitochondrial mutations on carbon metabolism, stress responses, and mitochondrial DNA stability. In particular, while W303-1B and MCC123 strains should be used to study the effect of severe mitochondrial tRNA mutations, D273-10B/A1 strain is rather suitable for studying the effects of milder mutations.

Keywords: Diseases; nuclear background; oxidative stress; respiration; yeast model.

Figure 1

Growth capability of WT strains. Serial dilutions of cultures from three WT strains were spotted on YP plates containing 2% glucose or 3% glycerol as carbon sources and incubated at 28°C. Pictures were acquired after for 2–3 days of growth for plates containing glucose, and after 5 days of growth for plates containing glycerol as carbon source.

Figure 2

Cultivation in bioreactor of WT cells and mt tRNA-mutant cells. Comparison of growth curves and of O₂ consumption rates of WT strains (panels A and B) and of WValC25T, MValC25T, and DValC25T mutants (panels C and D) performed in YP 0.25% glucose-containing medium. In panels B and D, the times of respiration arrest are indicated by arrows flagged with time values. The same flagged symbols are reported in panels A and C at the corresponding time points.

Figure 3

Oxygen consumption curves of resting WT strains. Comparison of Oxygen consumption rates in reaction chamber of WT cells in resting condition. Cells were previously grown in YP medium containing 2% (A) or 0.25% glucose (B).

Figure 4

Influence of three different nuclear backgrounds on the glycerol growth phenotype of mt tRNA mutants. Serial dilutions of LeuT20C, LeuA29G and ValC25T yeast mutant strains, having MCC123, W303-1B and D273-10B/A1 as nuclear background, grown overnight in YP liquid medium containing 2% glucose, were spotted on a YP plate containing 2% glucose or 3% glycerol and incubated at 28°C for 7 days.

Figure 5

Influence of three different nuclear backgrounds associated to the same WT mt DNA on the glycerol growth phenotype. Serial dilutions of the three strains MCC123, W303-1B and D273-10B/A1, endowed with WT mitochondria of the strain FF1210-6C, were grown overnight in YP liquid medium containing 2% glucose, spotted on a YP plate containing 2% glucose or 3% glycerol and incubated at 28°C for 5 days.

Figure 6

Hydrogen peroxide sensitivity of WT, rho° and mt tRNA mutants in MCC123, W303-1B, and D273-10B/A1 nuclear backgrounds. Comparison of the halo sizes of H₂O₂ growth inhibition (disks saturated with 50 mM H₂O₂) obtained by plating 2 × 10⁹ cells from exponential cultures. The values of halo sizes, expressed in cm as halo radius after subtraction of disk radius, are means (with standard deviations) obtained from three independent experiments. *(p < 0.05) and **(p < 0.01) indicate the statistical significance of differences calculated by comparing the strains linked by the bars.

Figure 7

EGTA sensitivity of WT, rho° and mt tRNA mutants in MCC123, W303-1B, and D273-10B/A1 nuclear backgrounds. Exponential cultures grown overnight in YP liquid medium containing 2% glucose, were spotted on plates containing EGTA as indicated. Plates are incubated at 28°C for 4 days.

Figure 8

Chronological life span and petite formation. Panel A: comparison of chronological viability of WT strains grown in YP liquid medium containing 2% glucose, reported as percentages of colony forming units. Stationary phases were taken as 100%. Panel B: comparison of petite colonies formation during chronological survival as in panel A. For each analysis (A, B) the same aliquots of cultures were diluted and plated on solid medium. Large and petite colonies forming units were counted after 3 days. Panel C: comparison of chronological viability of rho° strains performed as in panel A. Data are means of at least three independent experiments, with reported standard deviations bars.