An Investigation of TDA1 Deficiency in Saccharomyces cerevisiae During Diauxic Growth

Abstract

Tda1p is a protein kinase in Saccharomyces cerevisiae. Here we investigate the function of TDA1 during the diauxic shift using transcriptomics. We compared the gene expression in the deletion mutant tda1∆ and the reference strain (BY4741) during both the aerobic fermentation phase (log phase), and the respiratory phase (post-diauxic shift phase, PDS) in three separate independent experiments. We found: Differential gene expression analysis showed that compared to the reference strain, the tda1∆ mutant exhibited an upregulation of the glucose repressed hexose transporter HXT6 during the log phase, and upregulation of mitochondrial proteins and genes related to mitochondrial translation during the PDS phase. Gene set enrichment analysis showed an enrichment in mitochondrial translation in the PDS phase for the deletion mutant tda1∆, but not for the reference strain. Transcription factor analysis showed that the enrichment of Mig1p repressed genes was not statistically significant in TDA1 deletion mutants for neither log-phase nor PDS-phase. This conflicted with the previously suggested model that argued for an interaction between Tda1p and Mig1p. Instead, transcription factor analysis showed an enrichment of genes regulated by the HAP-complex, which regulates mitochondrial translation, during the PDS-phase in the tda1∆ mutant. The combined evidence from this study indicates that Tda1p does not participate in Mig1p-mediated glucose repression. Instead, we propose that it is involved in the regulation of mitochondrial translation by repressing the expression of HAP complex subunits.

Keywords: TDA1; diauxic growth; differential gene expression; glucose repression; mitochondrial translation; transcriptomics.

Figure 1

Growth curves and extracellular media over time of the tda1∆ mutant and reference strain. The green lines represent OD600, an indirect value of biomass accumulation, over time. The red lines represent glucose concentration over time while the blue lines represent ethanol concentration over time. The solid lines are measurements of the reference strain while the dashed lines belong to the tda1∆ mutant. The vertical dotted line represents the time when sampling took place (log phase sample = 9 h post inoculation, PDS phase sample = 26 h post inoculation). The cultivations were performed with three biological replicates (n = 3). Each point in the graph is the result of a rolling median (k = 4) of the median value from each time point.

Figure 2

Intersection analysis between contrasts and experiments. UpSet plot of DEGs in the intersections between the four contrasts for each experiment. The horizontal bars represent the total number of DEGs in the contrast while the vertical represent the number of DEGs in the specific contrast intersection, as shown in the matrix beneath the vertical bars.

Figure 3

Geneset enrichment analysis. (A) Dot plot of genome set enrichment analysis (FGSEA) on the log‐phase vs PDS‐phase contrasts for the tda1∆ strain and the reference strain. The y‐axis are the different experiments, x‐axis are GO‐terms that are enriched in at least one contrast, the facet rows separates distinct up‐ or downregulation, the facet columns separates the strains, the dot size is the ratio of DEGs found in the contrast over the total number of genes in the GO term, and the dot colour is set by the adjusted p‐value. Gene sets with an adjusted p‐value > 0.01 were filtered out. (B) Heatmap based on median of ratios normalised transcript counts within the biological process GO‐term “mitochondrial translation”. The heatmap is separated into experiments (columns) and the molecular function ontologies within the biological process GO‐term “mitochondrial translation” (rows). Genes with multiple molecular functions are shown repeatedly. Hierarchical clustering of genes was performed on the first experiment using Complete‐linkage clustering with Euclidian distance. The genes of the second and third experiments were then ordered based on the hierarchical clustering of the first experiment to highlight differences in expression pattern.

Figure 4

Distribution of genes that were differentially expressed over the diauxic shift. The x‐axis separates the three experiments, the y‐axis is the normalised gene counts, and the colour of the box plots signifies the phase (yellow are log‐phase and blue are PDS‐phase). The centre lines represent the median; box limits represent upper and lower quartiles; whiskers extend to the data points that are furthest from the box limits while within 1.5x interquartile range; dots are data points outside 1.5x interquartile range beyond the box limits. The upper and bottom rows are the gene expressions in the reference strain (green) and tda1∆ (magenta), respectively. (A) Genes that were consistently differentially expressed in the reference strain but not in tda1∆. (B) Genes that were consistently differentially expressed in the tda1∆ strain but not in the reference.

Figure 5

Graphical summary of the key findings in this study. The oval shapes represent significantly differentiated genes; colour represent in which contrast the genes were differentially expressed (yellow = Strains, log‐phase; blue = strains, PDS‐phase, green = Phases, reference but not tda1∆, and magenta = Phases, tda1∆ but not reference). The arrows represent regulation (red upwards arrow = upregulated in tda1∆; blue downwards arrow = downregulated in tda1∆; grey arrows = regulation that occurred in the reference strain but not tda1∆ during the diauxic shift). The location of the arrow represents the phase in which the gene was differentially expressed (left = log‐phase; right = PDS‐phase).