## 3 Results
### Transcriptome Profiling Reveals the Pre-Adapted State of the CRN/PPN1 Strain  
To explore the mechanism of CRN/PPN1 resistance to manganese, we performed transcriptome profiling (RNA-Seq) for the CRN and CRN/PPN1 strains in the control and in manganese excess media.  
To investigate global gene expression changes in CRN and CRN/PPN1 strains, we used a principal component analysis (Figure 2A) of the quantitative gene expression data (Table S3). The first and the second principal components (PCs) account for 70% of variance and clearly segregate the samples: PC1 reflects the expression changes arising from the adaptation to manganese, while PC2 reflects the changes resulting from Ppn1 overexpression. Interestingly, CRN/PPN1 samples were shifted towards the manganese adapted state even in the control medium. This observation is also supported by a comparison of gene sets up- and down-regulated in CRN/PPN1 and upon manganese adaptation (Figure 2B), which are strikingly similar, with only a few genes uniquely differentially expressed in CRN/PPN1.  
To investigate this phenomenon, we assessed differential gene expression (Table S4) for the samples grown in manganese versus control medium, and used the gene ontology enrichment analysis.  
Figure 1: Growth characteristics and polyphosphate content in the cells of CRN, CRN/PPN1, and CRN/PPX1 strains of _S. cerevisiae_ cultivated in control YPD medium with 14 mM KH\({}_{2}\)PO\({}_{4}\) and in the same medium supplemented with 5 mM MnSO\({}_{4}\). (**A**) growth curves; control medium—lines without dots; manganese excess medium—lines with dots; CRN is shown in gray, CRN/PPN1 in green, and CRN/PPX1 in blue; (**B**) growth velocities, logarithmic growth stage; (**C**) manganese content in the cells grown in the presence of 5 mM MnSO\({}_{4}\), logarithmic growth stage: CRN (40 h growth), CRN/PPN1 (16 h growth), and CRN/PPX1 (40 h growth); (**D**) the percentage of dead cells in the logarithmic growth stage in the presence of 5 mM MnSO\({}_{4}\); (**E**) total polyphosphate content in control and manganese grown cells, stationary growth stage. Three replicates were performed for each experiment; mean \(\pm\) s.d. is shown on panels (**B**,**D**,**E**). Two-sample t-test was used to assess the statistical significance: *** _p_-value < 0.001, **_p_-value < 0.01, * _p_-value < 0.05, n.s.—not significant.  
The adaptation of the parent CRN strain to manganese follows the same pattern as we have observed earlier for the CRY strain [47]. The up-regulated genes were enriched with multiple GO terms reflecting stress response, cell wall, and membrane organization (multiple GO terms, \(p<10^{-7}\)). The down-regulated genes were enriched with all types of protein biosynthesis and ribosome-related GO terms, which clearly agrees with growth suppression under manganese excess (Figure 1A,C).  
The CRN/PPN1 adaptation follows the same pattern as that of the CRN strain, with a few additions of GO-terms related to phosphate metabolic processes (GO:0006796 and GO:0006793, \(p<10^{-4}\)), which is associated with changes in the polyP content (Figure 1B).  
Additionally, we estimated differential gene expression in the control medium for CRN/PPN1 versus CRN samples. This analysis confirmed the pre-adapted state of the CRN/PPN1 strain, with up-regulation of the genes responsible for cellular response to external stimulus (GO:0071496 and GO:0009991, \(p<10^{-5}\)), plasma membrane components (GO:0005886, \(p<10^{-4}\)), and, importantly, oxidation-reduction process (GO:0055114, \(p<10^{-5}\)). Of note, in manganese-adapted CRN/PPN1 samples, slight enrichment of the genes responsible for oxidoreductase activity (GO:0016491, \(p\sim\)0.01) was also detected.  
Thus, Ppn1 overexpression leads to general adaptive processes and, specifically, oxidation-reduction pathways. This particular feature facilitates CRN/PPN1 adaptation to manganese excess.