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Adaptation of Saccharomyces cerevisiae to toxic manganese concentration triggers changes in inorganic polyphosphates

Nadezhda Andreeva, Lubov Ryazanova, Vladimir Dmitriev, Tatiana Kulakovskaya, Igor Kulaev
DOI: http://dx.doi.org/10.1111/1567-1364.12049 463-470 First published online: 1 August 2013

Abstract

The ability of Saccharomyces cerevisiae to adapt to toxic Mn2+ concentration (4 mM) after an unusually long lag phase has been demonstrated for the first time. The mutants lacking exopolyphosphatase PPX1 did not change the adaptation time, whereas the mutants lacking exopolyphosphatase PPN1 reduced the lag period compared with the wild-type strains. The cell populations of WT and ΔPPN1 in the stationary phase at cultivation with Mn2+ contained a substantial number of enlarged cells with a giant vacuole. The adaptation correlated with the triggering of polyphosphate metabolism: the drastic increase in the rate and chain length of acid-soluble polyphosphate. The share of this fraction, which is believed to be localized in the cytoplasm, increased to 76%. Its average chain length increased to 200 phosphate residues compared with 15 at the cultivation in the absence of manganese. DAPI-stained inclusions in the cytoplasm were accumulated in the lag phase during the cultivation with Mn2+.

Keywords
  • polyphosphate
  • Saccharomyces cerevisiae
  • manganese
  • adaptation
  • exopolyphosphatase
  • DAPI

Introduction

Inorganic polyphosphates (PolyP), the linear polymers of orthophosphoric acid, form complexes with metal cations (Kulaev et al., 2004). These complexes participate in many cellular functions (Reusch, 1992; Negoda et al., 2009; Rao et al., 2009; Docampo et al., 2010; Pavlov et al., 2010). The involvement of PolyP in heavy metal resistance was observed in many living organisms. PolyP sequestered Ni2+ in Staphylococcus aureus (Gonzales & Jensen, 1998). The Cd2+ tolerance of Escherichia coli depended on PolyP metabolism: the mutant strain carrying multiple copies of exopolyphosphatase and polyphosphate kinase encoding genes showed the enhanced resistance to Cd2+ (Keasling et al., 2000). PolyP sequestered heavy metals, on the one hand, and the entry of metal cations into the cells stimulated the exopolyphosphatase activity that releases Pi from PolyP, on the other hand (Keasling et al., 2000). The MeHPO4 (metal phosphate) ions were then transported out of the cells (Keasling et al., 2000). The archaea Sulfolobus metallicus with the high PolyP level was resistant to copper sulfate, while Sulfolobus solfataricus with the lower PolyP level could not grow in the presence of high concentrations of copper salts (Remonsellez et al., 2006). The earthworm Lumbricus rubellus inhabiting soils polluted with high concentrations of Pb2+ and Zn2+ accumulated inorganic phosphates in its tissues (Andre et al., 2009). In Trichoderma harzianum, the presence of cadmium induced a reduction in PolyP content (De Lima Freitas et al., 2011). In protozoa and some algae, cation sequestration is one of the functions of acidocalcisome, an organelle that contains pyrophosphate and PolyP bound with Ca2+ and other cations (Docampo et al., 2010). The addition of copper ions to the diet of the larva of Anticarsia gemmatalis, a lepidopteran pest of soybean, increased the PolyP content in cell lysate and spherites (specific vesicles similar to acidocalcisomes) (Gomes et al., 2012). The relationship between the Mn2+ homeostasis in yeast cells and PolyP was observed in short-time (1 h) uptake experiments: the content of acid-insoluble PolyP increased during Mn2+ uptake by the yeast cells (Okorokov et al., 1983).

As the content of heavy metals in the environment is continuously increasing as a result of natural and industrial contamination, living organisms have evolved the mechanisms for tolerating the presence of these compounds by efflux, complexation, or reduction in metal ions (Spain, 2003). The study of living organisms resistant to heavy metals ion, including manganese, is of interest due to the growing presence of these contaminants in the environment. Manganese is an essential trace element in living cells, because it is a cofactor of many enzymes including oxidases, dehydrogenases, DNA and RNA polymerases, and sugar transferases (Crowley et al., 1999). At high concentrations, manganese is also a toxic ion. Inhibition of the growth of Saccharomyces cerevisiae was observed at a concentration of 0.5 mM Mn2+ or higher (Blackwell et al., 1998; Jensen et al., 2003). Yeast cells possess many transport proteins for Mn2+ uptake, sequestration, and excretion; they are located in the plasma membrane, Golgi, vacuolar, and mitochondrial membranes (Culotta et al., 2005; Reddi et al., 2009). The phosphate transporter of the yeast plasma membrane, PHO84 (Reddi et al., 2009), is responsible for Mn2+ uptake via manganese-phosphate complexes. Disruption of the PHO84 gene results in a manganese-resistant phenotype (Jensen et al., 2003). This resistance is associated with the inability of yeast cells to take up a large amount of Mn2+. It is of interest to reveal the possibility of physiological adaptation of wild-type yeast cells to toxic Mn2+ concentrations. Such adaptation may be a useful model for understanding the tolerance of microorganisms to heavy metal cations.

The aim of this study was to reveal the changes in PolyP content and cellular localization during the adaptation of S. cerevisiae to the toxic concentration of Mn2+.

Materials and methods

Strains and growth conditions

The wild-type strain of S. cerevisiae VKM Y-1173 was obtained from the All-Russian Collection of Microorganisms. The parent strain CRY (WT) and the strains with altered PolyP metabolism (Table 1) were kindly provided by N. Rao and A. Kornberg (Sethuraman et al., 2001).

View this table:
1

The mutant strains of Saccharomyces cerevisiae (Sethuraman et al., 2001) used in the study

StrainNameGenotypeAlterations in PolyP metabolism
CRYWTMATa ade2 his3 leu2 trp1 ura3Parent strain
CRXΔPPX1MATa ade2 his3 trp1 ura3 ppx1Δ::LEU2No activity of exopolyphosphatase PPX1
CRNΔPPN1MATa ade2 his3 ura3 ppn1Δ:: CgTPR1No activity of exopolyphosphatase PPN1
CNXΔPPX1ΔPPN1MATa ade2 his3 ura3 ppn1Δ:: CgTPR1 ppx1Δ::LEU2No activities of exopolyphosphatase PPX1 and PPN1

The yeast was cultivated in the YPD (2% glucose, 2% peptone, 1% yeast extract) phosphate-deficient medium to the stationary growth stage. The phosphate-deficient medium containing 0.02 mM of Pi was prepared according to Rubin (1973). The cells were harvested at 5000 g for 10 min, washed with sterile distilled water, and transferred into the YPD medium with 14 mM potassium phosphate. MnSO4 was added to a final concentration of 4 mM. In the control YPD medium, Mn2+ concentration was estimated to be 0.0036 mM.

Polyphosphate extraction and assay

For PolyP extraction and assay, the cells were harvested by centrifugation at 5000 g for 10 min and washed twice with distilled water. Five PolyP fractions were extracted according to Vagabov et al., 2000; : PolyP1 with 0.5 N HClO4; PolyP2 with NaClO4 (2 g) and 1 N HClO4 (0.5 mL) per 1 g of wet biomass; PolyP3 with a weak NaOH solution, pH 9-10; and PolyP4 with 0.05 N NaOH, pH 12. All fractions were extracted at 0 °C. PolyP5 was assayed by the appearance of Pi during the hydrolysis of residual biomass in 0.5 N HClO4 at 90 °C for 20 min. After each extraction procedure, the residual biomass was separated by centrifugation, and supernatants were analyzed. PolyP and Pi were assayed as described previously (Kulakovskaya et al., 1999; Vagabov et al., 2000). The total PolyP content was calculated as a sum of all fractions. For electrophoresis, PolyP5 fraction was extracted with cold water according to Vagabov et al., 2008.

PolyP was precipitated from the extracts with saturated Ba(NO3)2 solution, dissolved (Vagabov et al., 2000), and subjected to electrophoresis in 30% polyacrylamide gel with 7 M urea (Kumble & Kornberg, 1995). The gels were stained with 0.05% toluidine blue, a specific dye to PolyP, in a water solution containing 25% methanol and 1% glycerol, and then washed with distilled water (Kumble & Kornberg, 1995). Commercial PolyP with average chain lengths of 15, 25, and 75 phosphate residues from Sigma and the one with an average chain length of 208 phosphate residues from Monsanto were used as markers. The average chain lengths of PolyP were taken as the values indicated by Sigma and Monsanto.

The manganese content was assayed by atomic absorption spectroscopy after samples burning at 180 °C in 32% HClO4 (Lichko & Okorokov, 1976).

The average data of three independent experiments with the standard deviation are presented.

Fluorescence microscopy

Staining with fluorochrome 4′,6′-diamino-2-phenylindole 2HCl (DAPI) is one of the effective methods of PolyP detection (Serafim et al., 2002; Kulakova et al., 2011). DAPI is used for DNA detection at 0.1 to 1 μg mL−1 and for PolyP detection at 3–50 μg mL−1 (Serafim et al., 2002). The emission maximum of DAPI and DAPI-DNA complexes is 456 nm. The emission maximum of DAPI-PolyP complexes is 525 nm. The fluorescence of pure DAPI-DNA complexes was blue and white, while that of pure DAPI-PolyP complexes was yellow. Living cells may have mixed fluorescence spectra. DAPI fluoresces green when PolyP is colocalized with DNA or when DAPI and DAPI-PolyP have a mixed spectrum.

For examination under fluorescent and phase-contrast microscopes (AXIO Imager A1, ZEISS, Germany), the cells were incubated in PBS, pH 7.4, with 10 μg mL−1 DAPI (Sigma) at 30° for 15 min, centrifuged, and resuspended in PBS. Filter set 49 (ZEISS) was used with the excitation maximum at 359 nm and the emission maximum at 460 nm.

Results

Saccharomyces cerevisiae adapts to manganese after an unusually long lag phase

We have observed for the first time the ability of wild-type strain S. cerevisiae VKM Y-1173 (see Fig. S1, Supporting Information) and some mutant strains (Fig. 1a) to grow in the presence of toxic Mn2+ concentration (4 mM). This growth was characterized by an unusually long lag phase. All mutants under study had a 2-h lag phase in the medium without Mn2+. The lag phases of the strains WT (Fig. 1a) and ΔPPX1 (data not shown) lasted for about 96 h in the presence of Mn2+. The lag phases of the strains ΔPPN1 (Fig. 1a) and ΔPPX1ΔPPN1 (data not shown) lasted for about 65 h in the presence of Mn2+. If re-inoculated into a fresh medium with Mn2+, the adapted cells showed only a slight growth delay (Fig. 1a). The strain ΔPPX1 lacks exopolyphosphatase PPX1 (Wurst et al., 1995) hydrolyzing mainly short-chained PolyP, while the strain ΔPPN1 lacks exopolyphosphatase PPN1 (Sethuraman et al., 2001) hydrolyzing mainly long-chained PolyP (Andreeva et al., 2006). The strain ΔPPX1ΔPPN1 is a double mutant in both enzymes (Sethuraman et al., 2001). Thus, the absence of exopolyphosphatase PPX1 did not change the adaptation time, whereas the absence of exopolyphosphatase PPN1 did reduce the lag period.

1

(a) Growth curves of the strains of Saccharomyces cerevisiae in YPD medium: ○ –WT strain, without Mn2+; ● – ΔPPN1 strain, without Mn2+; ◻ – WT strain, with 4 mM Mn2+; ◼ – ΔPPN1 strain, with 4 mM Mn2+; ♢ – WT strain, the cells adapted to Mn2+ were placed into the fresh medium with 4 mM Mn2+. (b) Manganese concentration in the medium during cultivation of strains of S. cerevisiae:△ –WT strain; ▲ – ΔPPN1 strain.

Manganese uptake

The absorption of Mn2+ by the cells of the strains WT and ΔPPN1 was estimated by the decrease in its concentration in the cultivation medium (Fig. 1b). Both strains absorbed Mn2+ from the medium. Absorption begins as early as in the lag phase. The difference in the kinetics of decrease in Mn2+ concentration in the medium corresponded to the difference between the growth curves of strains under study. The Mn2+ content in the cells of both strains was ~1.4 mmol g−1 dry biomass in 24 h of cultivation. It was ~0.56 mmol g−1 dry biomass for both strains in 120 h of cultivation. It should be noted that the yield of biomass in 120 h was 2.3 and 1.8 g dry biomass L−1 for WT and ΔPPN1, respectively. So, there were no significant differences in manganese content in the biomass of the two strains.

Changes in PolyP during adaptation to manganese

As the ΔPPN1 mutant lacking exopolyphosphatase PPN1 adapted to manganese before WT, we compared manganese-induced changes in PolyP in these two strains. It is known that PolyP of separate fractions in S. cerevisiae differs in the average chain length (Vagabov et al., 2008). For example, the average chain length of acid-soluble PolyP1 is usually ~15 phosphate residues, while that of acid-insoluble PolyP5 is more than 200 phosphate residues (Vagabov et al., 2008). The amounts of PolyP fractions are shown in Fig. 2. In the stationary phase in the presence of Mn2+, the content of PolyP increased in both strains compared with the cultivation without Mn2+ (Fig. 2a and b). This enhancement was due to the increase in acid-soluble PolyP1: The contribution of PolyP1 to total PolyP was unusually high (~76% in both strains). The PolyP1 content in the WT cells was enhanced during the cultivation with Mn2+ already in the lag phase. This increase was not observed in the ΔPPN1 strain.

2

The content of PolyP of separate fractions in the cells of WT strain (a) and ΔPPN1 strain (b) of Saccharomyces cerevisiae at cultivation without manganese for 24 h (stationary stage) and with 4 mM MnSO4 for 24 h (lag stage) and 72 and 120 h (stationary stage). 1– PolyP1, 2 – PolyP2, 3 – PolyP3, 4 – PolyP4, 5 – PolyP5.

It is notable that the content of the highest molecular weight PolyP5 fraction is considerably enhanced in the cells of both strains during the cultivation with Mn2+ in the lag phase (Fig. 2a and b). The cells of the WT strain at this stage also show the enhanced content of PolyP3 fraction.

The average PolyP chain lengths in different fractions were assessed by electrophoresis in polyacrylamide gel. The average chain length of PolyP1 was ~15 phosphate residues during the cultivation of both strains without Mn2+ (Fig. 3). There was a drastic increase in the chain length of PolyP1 in the presence of Mn2+: The average chain length of PolyP1 was estimated as ~45-65 phosphate residues in the lag phase and ~200 phosphate residues in the stationary phase in both strains (Fig. 3).

3

Polyacrylamide gel electrophoresis of PolyP1 fraction from the cells of WT strain and ΔPPN1 strain: 1 – cultivation without Mn2+, stationary phase; 2 – cultivation with 4 mM Mn2+, lag phase; 3 – cultivation with 4 mM Mn2+, stationary stage. Commercial PolyP with average chain lengths of 15, 25, and 75 phosphate residues from Sigma and the one with an average chain length of 208 phosphate residues from Monsanto was used as markers.

The average chain lengths of PolyP2, PolyP3, and PolyP4 fractions were ~ 65, 75, and 75-200 phosphate residues, respectively; the average chain length of PolyP5 fraction was more than 200 phosphate residues in the stationary phase of the parent strain and the ΔPPN1 strain without Mn2+ (data not shown). These values are similar to those obtained earlier for the yeast S. cerevisiae (Vagabov et al., 2008; Breus et al., 2012). In the presence of Mn2+, the average chain lengths of PolyP2, PolyP3, and PolyP4 increased but insignificantly, while that of PolyP5 remained the same in both strains (data not shown).

Excess Mn2+ similarly alters the PolyP1 content and chain length in the stationary phase in the WT and ΔPPN1 strains. When the adapted cells were placed into a fresh medium with Mn2+, the PolyP content, the proportion, and the average chain length of PolyP1 remained at the same high level (data not shown).

Changes in cell morphology and DAPI staining with excess Mn2+

Light microscopy revealed the changes in cell morphology in the presence of excess Mn2+. The damaged and deformed cells were predominant in the population of WT strains after 24 h of cultivation with Mn2+ (Fig. 4c). The cell population of the ΔPPN1 mutant was more heterogeneous after 24-h cultivation in the presence of Mn2+: Large round cells and budding cells appeared together with the damaged cells (Fig. 5c). This observation is consistent with the earlier adaptation of the ΔPPN1 mutant to Mn2+. The cell populations of both strains in the stationary phase during the cultivation with Mn2+ contained a significant number of cells twice as large (Figs 4e and 5e) as the cells grown in the medium without Mn2+ (Figs 4a and 5a). The enlarged cells had a giant vacuole. It was probably due to Mn2+ accumulation in these organelles. It is known that the vacuoles of S. cerevisiae accumulate manganese (Lichko et al., 1980; Culotta et al., 2005). The cells re-inoculated in the fresh medium with Mn2+ retained this morphological feature (see Fig. S2, Supporting Information).

4

Micrographs of the cells of WT strain of Saccharomyces cerevisiae. (a, c, e) phase-contrast microscopy; (b, d, f) fluorescence microscopy of the DAPI-stained cells. (a, b) cultivation without Mn2+, 24 h; (c, d) cultivation in the presence of Mn2+; 24 h; (e, f) cultivation in the presence of Mn2+, 120 h. The bar line is 5 μm. 1 – mineralized cell with high PolyP content, 2 – nucleus, 3 – vacuole, 4 – cell wall.

5

Micrographs of the cells of ΔPPN1 strain of Saccharomyces cerevisiae. (a, c, e) phase-contrast microscopy; (b, d, f) fluorescence microscopy of the DAPI-stained cells. (a, b) cultivation without Mn2+, 24 h; (c, d) cultivation in the presence of Mn2+; 24 h; (e, f) cultivation in the presence of Mn2+, 72 h. The bar line is 5 μm. 1 – mineralized cell with high PolyP content, 2 – nucleus, 3 – vacuole, 4 – cell wall, 5 – PolyP containing inclusion, 6 – round low-fluorescent cell

Intense yellow-orange fluorescence of DAPI-stained cells was observed after 24 h of cultivation in the presence of Mn2+ (Figs 4d and 5d). This fluorescence is typical for DAPI-PolyP complexes (Serafim et al., 2002; Kulakova et al., 2011). Some cells were wholly fluorescent, while fluorescent granules could be seen in the cytoplasm of other cells. The fluorescence of the cell envelope was observed as well. It confirms the multiple localization of PolyP in yeast cells (Kulaev et al., 2004). However, weakly fluorescent cells were already observed in the population of the ΔPPN1 mutant (Fig. 5d).

The cells of both strains of S. cerevisiae were weakly stained by DAPI in the stationary phase independent of the presence or absence of Mn2+ (Figs 4 and 5b and f). It is noteworthy that the cell of S. cerevisiae does not always show intense fluorescence with DAPI even at high concentrations of PolyP (Ryazanova et al., 2011). The absence of DAPI-fluorescence is not always indicative of the low PolyP level in the yeast, and therefore, chemical extraction of this polymer is still necessary for its quantification. We suppose that intensive fluorescence with DAPI in the lag phase of growth with Mn2+ correlates with the accumulation of alkali-soluble PolyP3 and acid-insoluble PolyP5. Similar coincidence was observed earlier in S. cerevisiae during PolyP accumulation in a nitrogen-limited medium (Breus et al., 2012).

Discussion

In this study, we have demonstrated for the first time the ability of S. cerevisiae to adapt to toxic Mn2+ after an unusually long lag phase. The adaptation correlates with the triggering of PolyP metabolism: the drastic increase in the rate and chain length of acid-soluble PolyP1. This fraction is supposed to be localized in the cytoplasm (Kulaev et al., 2004).

We believe that excess manganese affects PolyP accumulation in several ways. First, Mn2+ may stimulate Pi uptake via the transport protein, PHO84, taking up manganese-phosphate complexes (Jensen et al., 2003). Second, Mn2+ stimulates the PolyP-synthase activity of Vtc4, the protein of the vacuolar membrane of S. cerevisiae (Hothorn et al., 2009), whereas Mn2+ inhibits the activity of some yeast exopolyphosphatases (Andreeva et al., 1993). The accumulated PolyP may contribute to Mn2+ detoxification by forming complexes with these metal cations.

There is still no obvious answer to the question why the mutation in the PPN1 gene results in acceleration of adaptation to Mn2+. As PolyP biosynthesis is an energy-consuming process, it is probable that the ΔPPN1 mutant with decreased PolyP hydrolysis saves energy and can use it to start growth earlier. This is another pleiotropic effect of the PPN1 gene in addition to its previously known effects. The ΔPPN1 mutants are unable to grow on ethanol and lactate (Pestov et al., 2005) and have the lower expression of exopolyphosphatase PPX1 (Lichko et al., 2004). The cause of the lower level of PolyP in the ΔPPN1 mutant cells in the lag phase in the presence of Mn2+ is still unknown. Indirect effects of the PPN1 gene products on PolyP synthesis are not improbable.

The functional significance of exopolyphosphatase PPN1 for yeasts cannot be considered as completely understood. The mutants in this gene do not show any growth variations during cultivation on the media with phosphate limitation or excess (Lichko et al., 2008). The products of this gene are processed by proteinases (Sethuraman et al., 2001) and have multiple localizations in the cell, including vacuolar and mitochondrial membranes (Lichko et al., 2006), and cytoplasm (Andreeva et al., 2006); the protein sequence contains a transmembrane domain (http://www.uniprot.org/uniprot/Q04119). Further experiments on transformation of the mutants by the PPN1 gene may clear up the question about the functions of this presumably polyfunctional protein.

Supporting Information

Fig. S1. Growth curves of Saccharomyces cerevisiae wild strain VKM Y-1173.

Fig. S2. Micrographs of the cells of parent strain CRY of Saccharomyces cerevisiae.

Acknowledgements

The authors were supported by the Russian Foundation for Basic Research (grant 11-04-01009).

Footnotes

  • Editor: Monique Bolotin-Fukuhara

References

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