Abstract
Inorganic polyphosphate (polyP) is crucial for adaptive reactions and stress response in microorganisms. A convenient model to study the role of polyP in yeast is the Saccharomyces cerevisiae strain CRN/PPN1 that overexpresses polyphosphatase Ppn1 with stably decreased polyphosphate level. In this study, we combined the whole-transcriptome sequencing, fluorescence microscopy, and polyP quantification to characterize the CRN/PPN1 response to manganese and oxidative stresses. CRN/PPN1 exhibits enhanced resistance to manganese and peroxide due to its pre-adaptive state observed in normal conditions. The pre-adaptive state is characterized by up-regulated genes involved in response to an external stimulus, plasma membrane organization, and oxidation/reduction. The transcriptome-wide data allowed the identification of particular genes crucial for overcoming the manganese excess. The key gene responsible for manganese resistance is PHO84 encoding a low-affinity manganese transporter: Strong PHO84 down-regulation in CRN/PPN1 increases manganese resistance by reduced manganese uptake. On the contrary, PHM7, the top up-regulated gene in CRN/PPN1, is also strongly up-regulated in the manganese-adapted parent strain. Phm7 is an unannotated protein, but manganese adaptation is significantly impaired in Δphm7, thus suggesting its essential function in manganese or phosphate transport.
Highlights
Inorganic polyphosphate is a linear anionic polymer containing from several to hundreds of orthophosphate residues linked by energy-rich phosphoanhydride bonds
The polyphosphatase activity in the cell-free extracts was higher in the respective overproducing strains (Table S1)
The polyP level in control strain (CRN)/PPN1 cells was lower as compared to CRN and CRN/PPX1 (Figure 1B)
Summary
Inorganic polyphosphate (polyP) is a linear anionic polymer containing from several to hundreds of orthophosphate residues linked by energy-rich phosphoanhydride bonds. PolyP and its associated enzymes are involved in stress response and virulence, in addition to the function of phosphate and energy reserve [2,18,19,20,21]. The commonly used models are the ppk or ppk2-null strains lacking polyphosphate kinases and having a significantly decreased polyP level [2,31,32] They provide the possibility of direct studying of the effect of a decreased polyP level on bacterial cells, including stress response. The V-ATPase itself plays a key role in the response to oxidative and heavy metal stress [37,38,39], its dysfunction hides possible polyP functions in stress response This is why we had to design strains with enhanced expression of polyP hydrolyzing enzymes. The pre-adaptive state was characterized by up-regulation of the oxidative stress response genes, which provided enhanced resistance to oxidative stress
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