Quantitative analysis of phosphate accumulation in PHO regulatory system-mutant strains of Saccharomyces cerevisiae

Abstract

PHO-mutant strains of Saccharomyces cerevisiae, NOF-1 and NBD82-1, which constitutively express PHO81 and PHO4, respectively, have been reported to accumulate phosphate in high-phosphate conditions. However, detailed analysis, including a quantitative evaluation of the accumulated phosphate, has not been performed for these mutants. In this study, NOF-1 and NBD82-1 mutant and double mutant strains were cultured in a high-phosphate medium to quantitatively analyze the amount, accumulation form, and physiological use of the accumulated phosphate in the cells. In control strains (BY4741 and NBW7), the percentage of phosphorus in total dry weight of cell was approximately 2%TDW; for the NBD82-1 mutant and double mutant strains, it was approximately 6%TDW; and for strain NOF-1, it was 8.5%TDW. When cells of the mutant strains were stained with 4',6-diamidino-2-phenylindole (DAPI), they showed a fluorescence peak at 540nm, suggesting that phosphate accumulated as polyphosphoric acid (polyP). Quantitative evaluation revealed that for strain NOF-1, the percentage of phosphorus exiting as polyP in total dry weight of cell was approximately 5.0%TDW, equivalent to 60% of the total phosphorus in the cells. We also demonstrated that the mutant strains could grow well in phosphate-free medium, suggesting that phosphate accumulated in the cells was used as a phosphorus source. This is the first report concerning the quantitative analysis of phosphate accumulation and utilization of PHO regulatory system-mutant strains of Saccharomyces cerevisiae.