Engineering Inorganic Pyrophosphate Metabolism as a Strategy to Generate a Fluoride-Resistant Saccharomyces cerevisiae Strain

Academic Editors: Juan M. Gonzalez and Alba Cuecas Received: 23 December 2024 Revised: 13 January 2025 Accepted: 16 January 2025 Published: 21 January 2025 Citation: Perez-Castiñeira, J.R.; Ávila-Oliva, F.J.; Serrano, A. Engineering Inorganic Pyrophosphate Metabolism as a Strategy to Generate a Fluoride-Resistant Saccharomyces cerevisiae Strain. Microorganisms 2025, 13, 226. https://doi.org/10.3390/ microorganisms13020226 Copyright: © 2025 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/ licenses/by/4.0/). Article Engineering Inorganic Pyrophosphate Metabolism as a Strategy to Generate a Fluoride-Resistant Saccharomyces cerevisiae Strain José R. Perez-Castiñeira 1,* , Francisco J. Ávila-Oliva 2and Aurelio Serrano 1 1Instituto de Bioquímica Vegetal y Fotosíntesis, Universidad de Sevilla-CSIC, Av. Américo Vespucio 49, 41092 Sevilla, Spain; [email protected] 2Aplive, 41703 Dos Hermanas, Spain; [email protected] *Correspondence: jr[email protected] Abstract: Fluorine accounts for 0.3 g/kg of the Earth’s crust, being widely distributed in the environment as fluoride. The toxic effects of this anion in humans and other organisms have been known for a long time. Fluoride has been reported to alter several cellular processes although the mechanisms involved are largely unknown. Inorganic pyrophosphatases (PPases) are ubiquitous enzymes that hydrolyze inorganic pyrophosphate (PPi), a metabolite generated from ATP. In Saccharomyces cerevisiae, the enzyme responsible for PPi hydrolysis in the cytosol (IPP1) is strongly inhibited by fluoride in vitro . The essentiality of IPP1 for growth has been previously demonstrated using YPC3, a yeast mutant with conditional expression of the corresponding gene. Here, YPC3 was used to generate cells that tolerate high concentrations of fluoride by (a) the overexpression of IPP1 or its human ortholog, or (b) the substitution of IPP1 by the fluoride-insensitive PPase from Streptococcus mutans. The results obtained suggest that maintaining appropriate levels of PPase activity in the cytosol is essential for the adaptation of S. cerevisiae to high fluoride concentrations. The increase in fluoride tolerance allows YPC3 cells transformed with suitable plasmids to be selected on rich non-selective medium supplemented with this anion. Keywords: fluoride resistance; inorganic pyrophosphatase; overexpression; Saccharomyces cerevisiae;Streptococcus mutans 1. Introduction Fluoride, the smallest of the halide anions, has the highest electronegativity of all the elements of the Periodic Table. It is very abundant in the Earth’s crust (an average of 0.32 g/kg), being found in soils, water and the air. Fluoride concentrations range from 25 µ M to 100 mM in water resources, and it may be the most abundant anion in certain groundwaters [ 1 , 2 ]. Consumption of water with a fluoride content close to 0.2 mM can produce dental fluorosis in children, while skeletal fluorosis, a more severe disease, may result from regularly drinking water with higher levels of fluoride (0.2 to 0.8 mM). Skeletal fluorosis causes back pain and rigidity as well as neurological disorders, being endemic in at least 25 countries including India, Mexico, Kenya and Tanzania. The number of people suffering from this disease is estimated to be in the range of tens of millions [ 3 ]. Fluoride also exerts effects on other organisms such as bacteria and fungi. The antimicrobial activity of this anion has been studied in recent decades, mainly on bacteria causing dental caries, and it has been reported to be toxic to yeast and other fungi, some of them pathogenic [4]. Due to its ubiquity in the environment and its toxic effects, many organisms have developed strategies of fluoride resistance, although little is known about these strategies Microorganisms 2025,13, 226 https://doi.org/10.3390/microorganisms13020226 Microorganisms 2025,13, 226 2 of 18 and the biochemical pathways involved. Fluoride-responsive riboswitches that regulate the expression of genes in response to this anion have been identified in bacteria and archaea, fluoride exporters being the most common proteins found in operons with these riboswitches [ 5 , 6 ]. Two different fluoride export proteins have been identified in prokaryotes: (a) CLC F s, fluoride/proton antiporters, which harness the proton gradient to expel fluoride from the cytoplasm, and (b) Fluc proteins (fluoride channels), passive channels that drive fluoride extrusion down its electrochemical gradient across the plasma membrane. Among the eukaryotes, fungi (including yeast), plants, and some ocean-dwelling animals possess fluoride exporters that belong to a third protein family known as FEX, structurally related to the Flucs [ 6 , 7 ]. Besides extrusion systems, alternative factors have been reported to influence fluoride tolerance [6,8]. Fluoride is known to be an inhibitor of certain major intracellular proteins, and its effects are believed to be exerted by charge interaction with cations such as Mg 2+ and/or Ca 2+ . In the case of enolase, structural data suggest that the inhibition is due to the formation of a magnesium-fluoride–phosphate complex [ 9 ]. Fluoride also has the capacity to form complexes with aluminum and beryllium cations that may act as phosphate emulators, thus inhibiting enzymes involved in the transfer of phosphoryl groups [ 10 ]. This large class of enzymes include the so-called inorganic pyrophosphatases (PPases, EC 3.6.1.1), which hydrolyze inorganic pyrophosphate (PPi) [ 11 ]. PPi is a by-product of many anabolic reactions, and its efficient removal allows their shift toward biosynthesis according to the law of mass action [ 12 ]; as a consequence, accumulation of PPi can collapse anabolism in cellular compartments like cytosol or mitochondria. Two main classes of structurally different PPases have been identified to date: (a) soluble PPases (sPPases), ubiquitous proteins that hydrolyze PPi, releasing the chemical energy of the phosphoanhydride bond as heat, and (b) ion (H + and/or Na + )-translocating inorganic pyrophosphatases (mPPases), integral membrane proteins that couple PPi hydrolysis to proton and/or sodium pumping across biological membranes [ 13 , 14 ]. Among the sPPases, two major non-homologous families, known as I and II, have been characterized [ 15 – 17 ] (Figures S1 and S2). Family I sPPases occur in all types of organisms, both prokaryotic and eukaryotic [ 18 ]. In this family of sPPases, PPi binding to the active site is a complex process that depends on divalent cation cofactors, Mg 2+ being the most efficient. The evolutionarily unrelated family II sPPases, which belong to the DHH-DHHA2 phosphoesterase proteins, are activated by other heavy-metal cations such as Mn 2+ or Co 2+ (although Mg 2+ acts as a co-factor). Family II sPPases include those from Streptococcus mutans (a bacterium that contributes to tooth decay in the human oral cavity), Bacillus subtilis and other Grampositive bacteria (Firmicutes), as well as some other lineages of bacteria (Chloroflexi, Spirochaetes, Thermotogae) and archaea (Euryarchaeota). Family I sPPases are strongly inhibited by 0.5 mM fluoride in vitro , whereas much higher concentrations of this anion are needed to inhibit family II sPPases and mPPases [19–21]. Saccharomyces cerevisiae has two genes encoding sPPases, both belonging to family I: IPP1 and IPP2 (also known as PPA2) (Figure S1). IPP1 is considered an essential nucleocytoplasmic protein while IPP2 is reportedly located in the mitochondrial lumen, where it plays a role in respiratory metabolism [ 22 – 24 ]. Previous work from our group in budding yeast showed that cells devoid of IPP1 undergo different fates depending on their energy metabolism; thus, fermenting cultures show massive cell death by autophagy, while respiring cultures do not die but undergo growth arrest in the S-phase of the cell cycle [ 25 ]. In human cells, there are two family I sPPase paralogs—PPA1 and PPA2—according to the UNIPROT database. These proteins are located at the cytoplasm and mitochondria, respectively, a situation similar to that of S. cerevisiae. Microorganisms 2025,13, 226 3 of 18 The essentiality of IPP1 for yeast growth, along with previous reports showing the high sensitivity of family I sPPases to fluoride in vitro , prompted us to check whether this interaction could have implications in vivo . The yeast mutant strain YPC3, previously generated in our laboratory [ 26 ], was used to accomplish this task. YPC3 cells have their IPP1 gene (SGD systematic name, YBR011C) under the control of the yeast galactokinase gene (GAL1) promoter; consequently, they only express their nucleocytosolic sPPase, when grown on galactose, being unable to grow on other carbon sources. YPC3 cells regain the capacity to grow on glucose (fermentative conditions) or glycerol (respiratory conditions) by transformation with plasmids bearing PPase genes under the control of constitutive promoters [ 24 – 28 ]. In practical terms, YPC3 allows the substitution of IPP1 by different PPi-hydrolyzing enzymes, hence the comparison between different types of PPases within the same cellular context. Here, we show that the resistance of S. cerevisiae cells to fluoride can be increased by overexpressing IPP1 or its human family I ortholog, PPA1. Furthermore, yeast cells in which IPP1 is functionally substituted by relatively low levels of the fluoride-resistant family II sPPase from S. mutans may also result in cells with higher tolerance to this anion. These results suggest a major role of IPP1 in the adaptation of S. cerevisiae to high concentrations of fluoride and shed light on the cytotoxic effects exerted by this anion at the molecular level. Possible implications for other eukaryotic and prokaryotic cells are discussed. 2. Materials and Methods 2.1. Bacteria and Yeast Strains Escherichia coli DH5 α strain (supE44 lacU169 (Ø80 lacZ M15) hsdR17 recA1 endA1 girA46 thi-1 rel A1) [ 29 ] was used for cloning purposes. S. cerevisiae haploid strain W303-1A (MATa, ade2-1 can1-100 his3-11,15 leu2-3,112 trp1-1, ura3-1) [ 30 ] was used both as a wild type and as a parental strain to generate a YPC3 mutant (W303-1A ipp1 UAS -ipp1 TATA ::HIS3GAL1 UAS -GAL1 TATA -IPP1) by the single-step transplacement procedure, as previously described [26]. 2.2. Plasmids Construction and Yeast Transformation Plasmids used for yeast transformation, listed in Table 1, were, in most cases, derivatives of the high-copy pRS699b plasmid [ 26 , 31 ] or the low-copy pRS416 plasmid [ 32 ]. The coding sequence of the IPP1 gene was amplified by PCR along with a 400 bp promoter region upstream the start codon, as previously described [ 26 ]. The latter has been shown to contain all the cis-acting elements essential for efficient IPP1 transcription [ 33 ]. Artificial EcoRI and SpeI restriction sites were introduced at the 5 ′ and 3 ′ ends, respectively, in order to directionally insert the resulting DNA fragment into the pRS699b plasmid. This yielded plasmid pIPP1-699, in which IPP1 is inserted between its own promoter and the PMA1 gene terminator region. The coding sequence of the S. mutans family II sPPase (SPP2; gene locus DQM59_RS02590) was amplified from genomic DNA by PCR with artificial EcoRV and SpeI sites at its 5 ′ and 3 ′ -ends, respectively, and introduced in pIPP1-699 using an EcoRV natural restriction site located just upstream the start codon of IPP1 and the unique SpeI site, thus yielding plasmid pSPP2-699. The expression cassettes containing the 400 bp long IPP1 promoter region, the coding sequences of IPP1 or SPP2, and the PMA1 terminator region were cleaved out of plasmids pIPP1-699 and pSPP2-699 with restriction enzymes EcoRI and HindIII and ligated to plasmid pRS416, digested with the same enzymes. This yielded plasmids pIPP1-416 and pSPP2-416. A similar strategy was followed to obtain plasmid piGMVP-416 from a previously published plasmid encoding the ion-translocating membrane PPase (mPPase) of the methanogenic archaeon Methanosarcina mazei (MVP) [ 28 ] (Figure S3), which is functionally and structurally very different to the sPPases used in this Microorganisms 2025,13, 226 4 of 18 work. High-copy plasmid pPPA1-426, bearing the cDNA coding for the cytosolic sPPase PPA1 from Homo sapiens (PPA1; HGNC, locus NC_000010.11) inserted in the high-copy plasmid pRS426 [ 34 ] under the control of the yeast glyceraldehyde-3-phosphate dehydrogenase (GPD) promoter, was generously donated by Dr. Agustín Hernández (Betternostic S. L., Noáin, Navarra, Spain). Finally, plasmid pIPPGFP-699 had been previously obtained by our group [24]. S. cerevisiae mutant strain YPC3 was transformed with the plasmids described above by using the method of Schiestl and Gietz [ 35 ]. Cells were initially grown at 30 ◦ C in rich medium containing galactose [YPGal: 1% (w/v) yeast extract, 2% (w/v) peptone, 2% (w/v) galactose] and transformants were selected by growing cells on 2% (w/v) agar plates made in synthetic medium [0.17% yeast nitrogen base without amino acids and ammonium sulfate, 2% galactose, 50 mM MES-TRIS pH 6, and a mixture of nucleotides and amino acids, described elsewhere [36], devoid of histidine and uracil]. 2.3. Phenotype Complementation Tests For complementation studies, 2 mL of YPD [1% (w/v) yeast extract, 2% (w/v) peptone, 2% (w/v) glucose] was inoculated with transformed cells from the plates and cultured overnight at 30 ◦ C with agitation (200 r.p.m. in a Infors Orbitron orbital shaker) until the stationary phase was reached. An amount of 20 µ L of these cultures was used to inoculate 2 mL of YPD and grown again overnight under the same conditions. This treatment was necessary to decrease the pyrophosphatase activity associated with IPP1 in YPC3 cells. For drop tests, ten-fold serial dilutions of the final cultures were made in sterile water, and 2.5 µ L drops of each dilution were spotted onto 2% (w/v) agar plates made in YPD or YPGly (made as YPD with 3% (w/v) glycerol substituting for glucose). Culture media were buffered by the addition of 50 mM MES (adjusted to the specified pH values with TRIS) and supplemented with NaF at the indicated concentrations. Plates were typically grown at 30 ◦C for 3–4 days. 2.4. Growth Curves in Liquid Medium Cells were cultured as described above, except that liquid YPD medium adjusted to pH 5.0 with 50 mM MES-TRIS and supplemented with 75 mM NaF was used instead of agar plates. Samples were collected at indicated times and their optical density at 660 nm (OD660) was measured. 2.5. Preparations of Soluble Protein Extracts from Yeast Yeast colonies were collected from a plate and liquid-grown up to the stationary phase in selective medium; then, 2–4 mL of YPD was inoculated with a 1:100 volume of stationary culture. After overnight growth at 30 ◦ C with agitation as described above, cells were sedimented by centrifugation in a bench centrifuge (3000 × g, 5 min), washed thoroughly with water, resuspended in 0.2 mL of ice-cold buffer A (25 mM Tris-HCl, pH 8, 10% (w/v) glycerol, 2 mM DTT, 1 mM EDTA, 1 mM benzamidine, 2 mM ε -aminocaproic acid, 1 mM PMSF), and homogenized by vigorous shaking with glass beads. The homogenate was diluted up to 0.5 mL with buffer B (10 mM Tris-HCl, pH 8, 10% glycerol, 2 mM DTT, 1 mM EDTA) and centrifuged in a microfuge (20,000 × g, 20 min) at 4 ◦ C to remove beads, debris, and most of the membrane fraction. The resulting supernatant was used as the soluble protein extract. 2.6. Enzymatic Assay, Protein Determinations and Western Blotting Pyrophosphatase activity was assayed at 30 ◦ C in a final volume of 200 mL in a medium containing 50 mM MOPS-TRIS pH 7.2, 1 mM MgCl 2 and 0.5 mM Na 4 PPi in the presence and absence of 0.5 mM NaF. The amount of released orthophosphate was deter- Microorganisms 2025,13, 226 5 of 18 mined spectrophotometrically, as previously described [ 37 ]. In every assay, the amount of sample and/or the incubation time (usually 3–5 min) were adjusted so that the absorbance values fell within the linear part of a calibration curve made with a commercial orthophosphate standard solution. Absorbances were measured every 20 s, plotted versus time, and the activities were calculated from the slopes of the resulting straight lines. Protein concentration was estimated using a Coomassie-blue dye binding-based assay from Bio-Rad (München, Germany) according to the manufacturer’s instructions using ovalbumin as a standard. Immunodetection by Western blot was performed as described elsewhere [ 38 ] using a commercial rabbit polyclonal antibody against S. cerevisiae IPP1 (AP21326F_N, OriGene EU, Herford, Germany). 2.7. Determination of Internal PPi Levels in Yeast Cells YPC3 cells transformed with the plasmids shown in Table 1were grown in YPD buffered at pH 5, as described above. The initial OD 660 values of the cultures were individually adjusted so that after 8–9 h, all of them had a final OD 660 of around 0.5 (roughly 10 9 cells). Cultures were then split into two aliquots, and NaF (final concentration 75 mM) was added to one of them. Cells were grown for two more hours under the same conditions, after which they were collected by centrifugation, washed with ice-cold deionized water and broken as described in Section 2.5, except that a 4% (v/v) perchloric acid aqueous solution was used instead of buffer A. Beads, debris, and denatured proteins were removed by centrifugation (20 min, 20,000 × g, 4 ◦ C). PPi concentrations were determined in the supernatants as previously described [39]. Table 1. Plasmids used in this communication. Plasmid Description References pRS699b 1 S.cerevisiae/E. coli shuttle plasmid bearing the constitutive promoter and terminator of yeast gene PMA1 and selection marker URA3. It has a 2-micron (2 m) origin of replication, which yields a high number of plasmid copies (14–34) per cell. [31] pIPP1-699 1,2 Plasmid derived from pRS699b bearing the promoter and coding sequence of gene IPP1 from S. cerevisiae inserted upstream of the PMA1 terminator. [40] pIPP1-416 1 Plasmid derived from pRS416 bearing the promoter and coding sequence of gene IPP1 from S. cerevisiae. Its main difference from pIPP1-699 is that it has a yeast centromeric sequence (CEN) and an autonomously replicating sequence (ARS) that results in a low number of plasmid copies (2–5) per yeast cell. This study pSPP2-699 1Plasmid derived from pIPP1-699 bearing gene SPP2 from S. mutans.This study pSPP2-416 1Plasmid derived from pIPP1-416 bearing gene SPP2 from S. mutans.This study pPPA1-426 1 Plasmid derived from high-copy plasmid pRS426 that bears the cDNA coding for the cytosolic sPPase from Homo sapiens (PPA1). Donated by Dr. Hernández piGMVP-416 1,2 Plasmid derived from pIPP1-416 bearing the sequence that codes for the putative N-terminal signal peptide of yeast Suc2p followed by those of yEGFP and the Na+-translocating mPPase from M. mazei (MVP). This study and [28] pIPP1GFP-699 2 Plasmid derived from pIPP1-699 bearing the coding sequence of gene IPP1 from S. cerevisiae followed in-frame by that of yEGFP. [24] 1 Plasmid used for complementation and fluoride resistance studies. 2 Plasmid used for transformation method based on selection in YPD. Microorganisms 2025,13, 226 6 of 18 2.8. Spontaneous Generation of Fluoride-Resistant Cells W303-1A cells were grown up to the exponential phase (OD 660 between 0.6 and 0.9) in liquid unbuffered YPD medium devoid of NaF; then, ten-fold serial dilutions of these cultures were carried out in sterile water and 100 µ L of each dilution was extended onto 2% (w/v) agar plates made in YPD buffered at pH 5 and supplemented with 60 to 70 mM NaF. Plates were incubated at 30 ◦C for at least one week. 2.9. Transformation of YPC3 Cells and Selection of Transformants in YPD Transformation was performed by the method of Schiestl and Gietz [ 35 ] with some modifications: 2 mL of YPGal was inoculated with YPC3 cells from an agar plate prepared in the same medium and cultured overnight at 30 ◦ C with agitation, as in Section 2.3. The following day, 15 mL of YPGal was inoculated with the overnight stationary culture so that the initial OD 660 was around 0.3. Cells were grown for 4 h under the same conditions, collected by centrifugation, resuspended in 15 mL of YPD, and grown for 2 more hours until the OD660 was between 1 and 1.3. The last step was necessary to allow YPC3 cells to adapt from galactose to glucose metabolism before the transformation/selection procedure. Glucose-adapted YPC3 cells were collected by centrifugation (3000 × g, 1 min in a bench centrifuge), washed with 15 mL of sterile water, resuspended in 1 mL of sterile water, transferred to a microtube, and centrifuged again in a microfuge (13,000 × g, 30 s). The final washed pellet was resuspended in 1 mL of sterile water, and 100 mL aliquots were transformed with plasmids pRS699b (control), pIPP1-699, pIPP1GFP-699 and piGMVP416 by the lithium acetate/polyethylene glycol method [ 35 ]. After heat-shock lasting 20–25 min at 42 ◦ C, transformed cells were sedimented in a microfuge and resuspended in 1 mL of sterile water, and 20 µ L aliquots were extended onto YPD agar plates buffered with 50 mM MES-TRIS, pH 5, with the optional addition of NaF (up to 40 mM final concentration). 2.10. Fluorescence Microscopy Individual colonies of YPC3 transformed as described in the previous paragraph were grown in liquid YPD for 4 h and directly visualized with a fully automated Leica DM6000B microscope (Leica Microsystems) with FITC green fluorescence filters (excitation filter 480/40 nm, dichromatic mirror 505 nm, suppression filter 527/30 nm), a 40 × objective, and equipped with a cooled CCD (charge-coupled device) camera (ORCAAG, Hamamatsu Photonics). 3. Results YPC3 cells were transformed with plasmids bearing gene coding for different evolutionarily unrelated PPases under the control of constitutive promoters (Table 1). Transformants recovered the capacity to grow under fermentative conditions (2% (w/v) glucose) with the only exception being cells transformed with plasmid pRS699b. Moreover, YPC3 cells transformed with plasmids pIPP1-699, pSPP2-699, pSPP2-416 and piGMVP-416 could also grow in the presence of up to 75 mM NaF, unlike the parental strain W303-1A. Interestingly, cells transformed with the plasmids bearing the gene coding for the fluoride-insensitive family II sPPase from S. mutans showed the highest tolerance to this anion. Plasmids pIPP1-416 and pPPA1-426 were less effective at conferring resistance to this salt (Figure 1). Identical results were obtained with KF, demonstrating that the toxicity of NaF was a specific effect of fluoride. Microorganisms 2025,13, 226 7 of 18 Microorganisms 2025, 13, x FOR PEER REVIEW 7 of 19 Moreover, YPC3 cells transformed with plasmids pIPP1-699, pSPP2-699, pSPP2-416 and piGMVP-416 could also grow in the presence of up to 75 mM NaF, unlike the parental strain W303-1A. Interestingly, cells transformed with the plasmids bearing the gene coding for the fluoride-insensitive family II sPPase from S. mutans showed the highest tolerance to this anion. Plasmids pIPP1-416 and pPPA1-426 were less effective at conferring resistance to this salt (Figure 1). Identical results were obtained with KF, demonstrating that the toxicity of NaF was a specific effect of fluoride. Figure 1. Drop tests of transformed YPC3 cells and parental strain W303-1A grown at different pH values and concentrations of NaF under fermentative conditions. YPC3 cells were transformed with plasmids shown in Table 1 and grown as described in the main text. Serial dilutions of the cultures were prepared in sterile water, spotted onto YPD agar plates containing 50 mM MES (adjusted to the specified pH values with TRIS) and supplemented with the indicated concentrations of NaF. Growth was recorded after 3 days, except for plates containing 75 mM NaF, which were grown for 4 days before the photograph was taken. Cell viability declined with NaF concentrations above 75 mM until the complete halting of growth was observed at 100 mM NaF. The results obtained with YPD agar plates were confirmed by growing cells in liquid culture media (Figure 2). For the sake of clarity, only the growth of W303-1A cells and strain YPC3 transformed with plasmid pIPP1-699 in liquid YPD, pH 5.0, with 75 mM NaF is shown, but similar results were obtained with YPC3 cells transformed with plasmids pSPP2-699 and pSPP2-416. Figure 1. Drop tests of transformed YPC3 cells and parental strain W303-1A grown at different pH values and concentrations of NaF under fermentative conditions. YPC3 cells were transformed with plasmids shown in Table 1and grown as described in the main text. Serial dilutions of the cultures were prepared in sterile water, spotted onto YPD agar plates containing 50 mM MES (adjusted to the specified pH values with TRIS) and supplemented with the indicated concentrations of NaF. Growth was recorded after 3 days, except for plates containing 75 mM NaF, which were grown for 4 days before the photograph was taken. Cell viability declined with NaF concentrations above 75 mM until the complete halting of growth was observed at 100 mM NaF. The results obtained with YPD agar plates were confirmed by growing cells in liquid culture media (Figure 2). For the sake of clarity, only the growth of W303-1A cells and strain YPC3 transformed with plasmid pIPP1-699 in liquid YPD, pH 5.0, with 75 mM NaF is shown, but similar results were obtained with YPC3 cells transformed with plasmids pSPP2-699 and pSPP2-416. Microorganisms 2025, 13, x FOR PEER REVIEW 8 of 19 Figure 2. Growth of W303-1A cells and YPC3 cells transformed with plasmid pIPP1-699 in liquid YPD adjusted to pH 5.0 in the absence and presence of 75 mM NaF. (-■-) W303-1A cells grown without NaF, (-●-) YPC3 cells transformed with plasmid pIPP1-699 without NaF, (--♦--) W303-1A grown with 75 mM NaF, and (- -▲- -) YPC3 cells transformed with plasmid pIPP1-699 grown with 75 mM NaF. The OD 660 values are the averages of three independent experiments. YPC3 cells transformed with plasmids bearing different PPases also increased fluoride resistance in agar plates with medium containing glycerol, considered a ‘non-fermentable’ carbon source in yeast [41] (Figure 3). Figure 3. Drop tests of transformed yeast mutant YPC3 cells and parental strain W303-1A grown at different pH values and concentrations of NaF under respiratory conditions. Experiments were performed as in Figure 1, except that 3% (w/v) glycerol substituted for glucose in the plates. Crude extracts from YPC3 cells transformed with plasmid pIPP1-699 showed a 10fold increase in soluble PPase-specific activity as compared to those obtained from the parental strain W303-1A, the increase being only around 50% higher in the case of cells transformed with centromeric plasmid pIPP1-416. YPC3 cells transformed with high-copy Figure 2. Growth of W303-1A cells and YPC3 cells transformed with plasmid pIPP1-699 in liquid YPD adjusted to pH 5.0 in the absence and presence of 75 mM NaF. (- ■ -) W303-1A cells grown without NaF, (- • -) YPC3 cells transformed with plasmid pIPP1-699 without NaF, (-- ♦ --) W303-1A grown with 75 mM NaF, and (- - ▲ - -) YPC3 cells transformed with plasmid pIPP1-699 grown with 75 mM NaF. The OD660 values are the averages of three independent experiments. Microorganisms 2025,13, 226 8 of 18 YPC3 cells transformed with plasmids bearing different PPases also increased fluoride resistance in agar plates with medium containing glycerol, considered a ‘non-fermentable’ carbon source in yeast [41] (Figure 3). Microorganisms 2025, 13, x FOR PEER REVIEW 8 of 19 Figure 2. Growth of W303-1A cells and YPC3 cells transformed with plasmid pIPP1-699 in liquid YPD adjusted to pH 5.0 in the absence and presence of 75 mM NaF. (-■-) W303-1A cells grown without NaF, (-●-) YPC3 cells transformed with plasmid pIPP1-699 without NaF, (--♦--) W303-1A grown with 75 mM NaF, and (- -▲- -) YPC3 cells transformed with plasmid pIPP1-699 grown with 75 mM NaF. The OD 660 values are the averages of three independent experiments. YPC3 cells transformed with plasmids bearing different PPases also increased fluoride resistance in agar plates with medium containing glycerol, considered a ‘non-fermentable’ carbon source in yeast [41] (Figure 3). Figure 3. Drop tests of transformed yeast mutant YPC3 cells and parental strain W303-1A grown at different pH values and concentrations of NaF under respiratory conditions. Experiments were performed as in Figure 1, except that 3% (w/v) glycerol substituted for glucose in the plates. Crude extracts from YPC3 cells transformed with plasmid pIPP1-699 showed a 10fold increase in soluble PPase-specific activity as compared to those obtained from the parental strain W303-1A, the increase being only around 50% higher in the case of cells transformed with centromeric plasmid pIPP1-416. YPC3 cells transformed with high-copy Figure 3. Drop tests of transformed yeast mutant YPC3 cells and parental strain W303-1A grown at different pH values and concentrations of NaF under respiratory conditions. Experiments were performed as in Figure 1, except that 3% (w/v) glycerol substituted for glucose in the plates. Crude extracts from YPC3 cells transformed with plasmid pIPP1-699 showed a 10-fold increase in soluble PPase-specific activity as compared to those obtained from the parental strain W303-1A, the increase being only around 50% higher in the case of cells transformed with centromeric plasmid pIPP1-416. YPC3 cells transformed with high-copy plasmid pPPA1-426, bearing the gene coding for the human cytosolic sPPase, also showed a significant increase in soluble PPase activity (around three-fold) with respect to W303-1A cells. In all these cases, the respective activities were strongly inhibited by the presence of 0.5 mM NaF in the assay (Figure 4). Lower levels of specific PPase activity, between 5and 10-fold less than those observed with multi-copy plasmids bearing family I sPPases, were detected in extracts obtained from cells transformed with pSPP2-699 and pSPP2-416, which encode a family II sPPase. In these cases, the soluble PPase activity was insensitive to fluoride (Figure 4). Cells transformed with piGMVP-416, encoding the Na + -translocating mPPase from M. mazei (MVP), only showed membrane-associated fluoride-insensitive PPase activity that increased by sixto seven-fold in the presence of 100 mM KCl, as previously described [ 28 ]. Immunodetection performed in cell extracts with a polyclonal antibody against S. cerevisiae IPP1 showed that, in YPC3 cells transformed with plasmids pIPP1-699, pIPP1-416 and pPPA1-426, increases in PPase activity correlated with the levels of a polypeptide of 32 kDa, the expected size for eukaryotic family I sPPase polypeptides [ 18 ]. No band was Microorganisms 2025,13, 226 9 of 18 detected in cells transformed with control plasmid pRS699b or with pSPP2-699, pSPP2-416, and piGMVP-416 (Figure 5). Microorganisms 2025, 13, x FOR PEER REVIEW 9 of 19 plasmid pPPA1-426, bearing the gene coding for the human cytosolic sPPase, also showed a significant increase in soluble PPase activity (around three-fold) with respect to W3031A cells. In all these cases, the respective activities were strongly inhibited by the presence of 0.5 mM NaF in the assay (Figure 4). Lower levels of specific PPase activity, between 5and 10-fold less than those observed with multi-copy plasmids bearing family I sPPases, were detected in extracts obtained from cells transformed with pSPP2-699 and pSPP2-416, which encode a family II sPPase. In these cases, the soluble PPase activity was insensitive to fluoride (Figure 4). Figure 4. Levels of hydrolytic sPPase activity in protein extracts observed from transformed yeast mutant YPC3 cells and parental strain W303-1A. Activity assays were performed in the absence (black columns) and presence (white columns) of 0.5 mM NaF, as described in the Section 2. Values of specific activities are averages ± SE corresponding to at least 5 independent experiments. Student’s unpaired t tests were performed using the T test calculator on the webpage https://www.graphpad.com/quickcalcs/ttest1/?format=SEM (accessed on 10 January 2025). Legend: (1) statistically significant difference (p = 0.0133), (2) very statistically significant difference (p = 0.0042), (3) very statistically significant difference (p = 0.0024), (4) extremely statistically significant difference (p = 0.0008), (5) very statistically significant difference (p = 0.0045), (6) very statistically significant difference (p = 0.0070), and (7) statistically significant difference (p = 0.0211). Cells transformed with piGMVP-416, encoding the Na + -translocating mPPase from M. mazei (MVP), only showed membrane-associated fluoride-insensitive PPase activity that increased by sixto seven-fold in the presence of 100 mM KCl, as previously described [28]. Immunodetection performed in cell extracts with a polyclonal antibody against S. cerevisiae IPP1 showed that, in YPC3 cells transformed with plasmids pIPP1-699, pIPP1416 and pPPA1-426, increases in PPase activity correlated with the levels of a polypeptide of 32 kDa, the expected size for eukaryotic family I sPPase polypeptides [18]. No band was detected in cells transformed with control plasmid pRS699b or with pSPP2-699, pSPP2-416, and piGMVP-416 (Figure 5). Figure 4. Levels of hydrolytic sPPase activity in protein extracts observed from transformed yeast mutant YPC3 cells and parental strain W303-1A. Activity assays were performed in the absence (black columns) and presence (white columns) of 0.5 mM NaF, as described in the Section 2. Values of specific activities are averages ± SE corresponding to at least 5 independent experiments. Student’s unpaired ttests were performed using the Ttest calculator on the webpage https://www.graphpad.com/ quickcalcs/ttest1/?format=SEM (accessed on 10 January 2025). Legend: (1) statistically significant difference (p= 0.0133), (2) very statistically significant difference (p= 0.0042), (3) very statistically significant difference (p= 0.0024), (4) extremely statistically significant difference (p= 0.0008), (5) very statistically significant difference (p= 0.0045), (6) very statistically significant difference (p= 0.0070), and (7) statistically significant difference (p= 0.0211). Microorganisms 2025, 13, x FOR PEER REVIEW 10 of 19 Figure 5. Immunodetection of family I sPPases in protein extracts obtained transformed YPC3 cells and parental strain W303-1A (upper panel). A polyclonal antibody against S. cerevisiae IPP1 was used. In total, 50 µg of total protein was loaded per lane. The lower panel shows the Ponceau S staining of the nitrocellulose filter after transferring the proteins from the SDS-PAGE gel . Internal levels of PPi significantly increased in soluble extracts of W303-1A cells and YPC3 cells transformed with plasmid pIPP1-416 after growing for only 2 h in the presence of NaF. The overexpression of family I PPases or expression of the S. mutans family II PPase SPPA2 prevented this scenario (Figure 6). Figure 6. Internal levels of PPi measured in transformed YPC3 cells and parental strain W303-1A after 2 h of growing in the presence of 75 mM NaF (black columns). White columns show PPi levels of the respective control cells grown in standard YPD (see the Section 2 for further details of the procedure). Values are averages ± SE corresponding to 4 independent experiments. Student’s unpaired t tests were performed using the T test calculator at on webpage https://www.graphpad.com/quickcalcs/ttest1/?format=SEM (accessed on 10 January 2025) Legend: (*) statistically significant difference (p = 0.0457) and (**) very statistically significant difference (p = 0.0025). Fluoride-resistant yeast cells spontaneously appeared after the growth of W303-1A cells for 6–7 days in YPD agar plates buffered at pH 5 and supplemented with 70 mM NaF. These experiments were carried out as described in the Section 2 with six independent W303-1A clones. Around 0.003% of cells were estimated to spontaneously develop Figure 5. Immunodetection of family I sPPases in protein extracts obtained transformed YPC3 cells and parental strain W303-1A (upper panel). A polyclonal antibody against S. cerevisiae IPP1 was used. In total, 50 µ g of total protein was loaded per lane. The lower panel shows the Ponceau S staining of the nitrocellulose filter after transferring the proteins from the SDS-PAGE gel. Microorganisms 2025,13, 226 16 of 18 Abbreviations The following abbreviations are used in this manuscript: IPP1 nucleocytosolic inorganic pyrophosphatase from S. cerevisiae PPi inorganic pyrophosphate PPase inorganic pyrophosphatase m-PPase membrane-bound ion-translocating inorganic pyrophosphatase MVP Na+-translocating m-PPase from the archaea Methanosarcina mazei OD660 optical density at 660 nm PPA1 cytosolic inorganic pyrophosphatase from Homo sapiens sPPase soluble inorganic pyrophosphatases SPP2 inorganic pyrophosphatase from S. mutans yEGFP yeast-enhanced green fluorescent protein References 1. Barbier, O.; Arreola-Mendoza, L.; Del Razo, L.M. Molecular Mechanisms of Fluoride Toxicity. Chem. Biol. Interact. 2010,188, 319–333. [CrossRef] [PubMed] 2. Smith, F.A.; Hodge, H.C.; Dinman, B.D. Airborne Fluorides and Man: Part I. Crit. Rev. Environ. Sci. 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