ABB  Vol.2 No.4 , August 2011
The early stage of polyphosphate accumulation in saccharomyces cerevisiae: comparative study by extraction and DAPI staining
ABSTRACT
Inorganic polyphosphate (PolyP), a bioactive polymer with multiple functions, plays a key role in biomineralizaion and phosphorus homeostasis in yeasts. After phosphate starvation, the cells of Saccharomyces cerevisiae restored their pool of PolyP during the first 30 min of incubation in the media containing phosphate and carbon sources. The cells of parent strain accumulated PolyP both in glucose and ethanol-containing media. In the medium with glucose, the strain with inactivated PPX1 and PPN1 genes (encoding two major yeast polyphosphatases) accumulated 2-fold more PolyP than the parent strain. The PolyP in the mutant cells had a greater average chain length compared to the parent strain. The strain with inactivated exopolyphosphatase genes РРХ1 and PPN1 was incapable of PolyP synthesis in the medium with ethanol. The in vivo staining of cells with DAPI show that in the cells of parent strain PolyP appeared first in cytoplasm and mitochondria under cultivation in glucose-containing medium and in cytoplasm and vacuoles in the medium with ethanol. In the ΔPPX1ΔPPN1double mutant PolyP accumulated in cytoplasm and vacuoles under cultivation in glucose-contained medium.

Cite this paper
nullRyazanova, L. , Andreeva, N. , Kulakovskaya, T. , Valiakhmetov, A. , Yashin, V. , Vagabov, V. and Kulaev, I. (2011) The early stage of polyphosphate accumulation in saccharomyces cerevisiae: comparative study by extraction and DAPI staining. Advances in Bioscience and Biotechnology, 2, 293-297. doi: 10.4236/abb.2011.24042.
References
[1]   Kulaev, I.S., Vagabov, V.M., Kulakovskaya, T.V. (2004) The Biochemistry of Inorganic Polyphosphates. John Wiley & Sons Ltd., Chichester.

[2]   Rao, N.N., Gomez-Garcia, M.R. and Kornberg, A. (2009) Inorganic polyphosphate: essential for growth and survival. Ann. Rev. Biochem., 78, 605–647.

[3]   Docampo, R., Ulrich, P. and Moreno, S.N. (2010) Evolution of acidocalcisomes and their role in polyphosphate storage and osmoregulation in eukaryotic microbes. Philos. Trans. R. Soc. Lond. B Biol. Sci., 365, 775-784.

[4]   Chaves, F.P., Lunsdorf, H.L. and Jerez, C.A.(2004) Growth of polychlorinated-biphenyl-degrading bacteria in the presence of biphenyl and chlorobiphenyls generates oxydative stress and massive accumulation of inorganic polyphosphate. Appl. Envinron. Microbiol. 70, 3064-3072.

[5]   Campos, E; Fa?anha, A; Moraes, J. et al. (2007) A mitochondrial exopolyphosphatase activity modulated by phosphate demand in Rhipicephalus (Boophilus) microplus embryo. Insect Biochem. Mol. Biol., 37, 1103-1107.

[6]   Gebremariam, S.Y., Beutel, M.W., Christian, D. and Hess, T.F. (2011) Research advances and challenges in the microbiology of enhanced biological phosphorus removal-a critical review. Water Environ Res., 83, 195-219.

[7]   Omelon, S.J. and Grynpas, M.D. (2008) Relationships between polyphosphate chemistry, biochemistry and apatite biomineralization. Chem. Rev., 108, 4694-4715.

[8]   Vagabov, V.M., Trilisenko, L.V. and Kulaev I.S. (2000) Dependence of inorganic polyphosphate chain length on the orthophosphate content in the culture medium of the yeast Saccharomyces cerevisiae. Biochemistry (Moscow), 65, 349-355.

[9]   Vagabov, V.M., Trilisenko, L.V., Kulakovskaya, T.V. et al.. (2008) Еffect of carbon source on polyphosphate accumulation in Saccharomyces cerevisiae. FEMS Yeast Res., 8, 877-882.

[10]   Allan, R.A. and Miller, J.J. (1980) Influence of S-adenosylmethionine on DAPI-induced fluorescence of polyphosphate in the yeast vacuole. Can. J. Microbiol., 26, 912-920.

[11]   Tijssen, J.P.F., Beekes, H.W. and Van Steveninck, J. (1982) Localization of polyphosphate in Saccharomyces fragilis, as revealed by 4’6’-diamidino-2-phenylindole fluorescence. Biochem.Biophys. Acta, 721, 394-398.

[12]   Serafim, L.S., Lemos, O.C., Levantesi, C. et al. (2002) Methods for detection and visualization of intracellular polymers stored by polyphosphate-accumulating microorganisms. J. Microbiol. Meth., 51, 1-18.

[13]   Puchkov, E.O. (2006) The viability assessment of ethanol-producing yeast by computer-aided fluorescence microscopy. Mikrobiologiia, 75, 193-200.

[14]   Puchkov, E.O. (2010) Brownian motion of polyphosphate complexes in yeast vacuoles:characterization by fluorescence microscopy with image analysis. Yeast, 27, 309–315.

[15]   Wurst, H., Shiba, T. and Kornberg, A. (1995) The gene for a major exopolyphosphatase of Saccharomyces cerevisiae. J. Bacteriol., 177, 898-906.

[16]   Sethuraman, A., Rao, N.N. and Kornberg, A. (2001) The endopolyphosphatase gene: essential in Saccharomyces cerevisiae. Proc. Natl. Acad. Sci. USA, 98, 8542– 8547.

[17]   Lichko, L.P., Andreeva, N.A., Kulakovskaya, T.V. et al.. (2003) Exopolyphosphatases of the yeast Saccharomyces cerevisiae. FEMS Yeast Res., 3, 233-238.

[18]   Andreeva, N.A., Kulakovskaya, T.V. and Kulaev, I.S. (2006) High molecular mass exopolyphosphatase from the cytosol of the yeast Saccharomyces cerevisiae is encoded by the PPN1 gene. Biochemistry (Moscow), 71, 975-977.

[19]   Rubin, G.M. (1973) The nucleotide sequence of Saccharomyces cerevisiae 5.8 S ribosomal ribonucleic acid. J. Biol. Chem., 11, 3860–3875.

[20]   Kulakovskaya, T.V., Andreeva, N.A., Karpov, A.V. et al. (1999) Hydrolysis of tripolyphosphate by purified exopolyphosphatase of Saccharomyces cerevisiae cytosol: kinetic model. Biochemistry (Moscow), 64, 990-993.

[21]   Pestov, N.A., Kulakovskaya, T.V. and Kulaev I.S. (2004) Inorganic polyphosphate in mitochondria of Saccharomyces cerevisiae at phosphate limitation and phosphate excess. FEMS Yeast Res., 4, 643-648.

[22]   Andreeva, N.A., Kulakovskaya, T.V., Kulakovskaya, E.V. et al. (2008) Polyphosphates and exopolyphosphatases in cytosol and mitochondria of Saccharomyces cerevisiae during growth on glucose or ethanol under phosphate surplus. Biochemistry (Moscow), 73, 65-69.

[23]   Pestov, N.A., Kulakovskaya, T.V. and Kulaev I.S. (2005) Effects of inactivation of the PPN1 gene on exopolyphosphatases, inorganic polyphosphates and function of mitochondria in the yeast Saccharomyces cerevisiae. FEMS Yeast Res., 5, 823-828.

[24]   Trilisenko, L.V., Vagabov, V.M. and Kulaev, I.S. (2002) The content and chain length of polyphosphate from vacuoles of Saccharomyces cerevisiae VKM Y-1173. Biochemistry (Moscow), 67, 592-596.

[25]   Lichko, L., Kulakovskaya, T., Pestov, N. and Kulaev, I. (2006) Inorganic polyphosphates and exopolyphosphatases in cell compartments of the yeast Saccharomyces cerevisiae under inactivation of PPX1 and PPN1 genes. Biosci Rep., 26, 45-54.

 
 
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