Participation of CWI, HOG and Calcineurin pathways in the tolerance of Saccharomyces cerevisiae to low pH by inorganic acid

Authors

  • R.M. de Lucena,

    1. Interdepartmental Research Group in Metabolic Engineering, Federal University of Pernambuco, Recife, Brazil
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  • C. Elsztein,

    1. Interdepartmental Research Group in Metabolic Engineering, Federal University of Pernambuco, Recife, Brazil
    2. Department of Genetics, Federal University of Pernambuco, Recife, Brazil
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  • D.A. Simões,

    1. Interdepartmental Research Group in Metabolic Engineering, Federal University of Pernambuco, Recife, Brazil
    2. Department of Biochemistry, Federal University of Pernambuco, Recife, Brazil
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  • M.A. de Morais Jr

    Corresponding author
    1. Department of Genetics, Federal University of Pernambuco, Recife, Brazil
    • Interdepartmental Research Group in Metabolic Engineering, Federal University of Pernambuco, Recife, Brazil
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Correspondence

Marcos A. Morais Jr, Departamento de Genética, Universidade Federal de Pernambuco, Av. Moraes Rego, 1235, Cidade Universitária, 50670-901 Recife, PE, Brasil. E-mail: marcos.morais@pq.cnpq.br

Abstract

Aims

The present work aimed at identifying the metabolic response to acid stress and the mechanisms that lead to cell tolerance and adaptation.

Methods and Results

Two strategies were used: screening deletion mutants for cell growth at neutral and acid pH compared to wild type and measurement by qPCR of the expression of yeast genes involved in different pathways.

Conclusions

The results complement our previous findings and showed that the Cell Wall Integrity pathway is the main mechanism for cell tolerance to acid pH, and this damage triggers the protein kinase C (PKC) pathway mainly via the Wsc1p membrane sensor. In addition, cell wall injury might mimic the effects of high osmotic shock and activates the High Osmolarity Glycerol pathway, which amplifies the signal in the upper part of PKC pathway and leads to the activation of Ca2+ channels by SLT2 overexpression and this Ca2+ influx further activates calcineurin. Together, these mechanisms induce the expression of genes involved in cell cycle regulation and cell wall regeneration.

Significance and Impact of the Study

These interactions are responsible for long-term adaptation of yeast cells to the acidic environment, and the results could drive future work on the genetic modification of yeast strains for high tolerance to the stresses of the bioethanol fermentation process.

Introduction

As bioethanol is a commoditized high-volume low-value product, reduction in production costs continues to be a major goal. In Brazil, as in other countries, the fermentation process involves the recycling of the yeast population to reduce time and production costs. Thus, an important task is to maintain the yeast population at high viability and high fermentation capacity. As an open process, the fermentation wort is often contaminated with wild yeasts and bacteria (Skinner and Leathers 2004; Silva-Filho et al. 2005a; Liberal et al. 2007; Schell et al. 2007; Basílio et al. 2008; Basso et al. 2008; Schaber et al. 2010). Contamination by yeasts is rarely treated, although some biocides have been proposed (Elsztein et al. 2008). On the other hand, to keep the bacterial population under control the production plants either use large amounts of antibiotic or treat the yeast biomass in between fermentations with sulphuric acid at a pH in the range of 2·5–2 (Wheals et al. 1999; Silva Filho et al. 2005b; Basso et al. 2008). This treatment can impose a metabolic burden that could lead a loss of yeast cell viability (Carmelo et al. 1998; Melo et al. 2010), especially when used in synergy with other stress conditions such as higher temperature and high ethanol concentrations (Silva Filho et al. 2005b; Melo et al. 2010).

Many different metabolic responses are observed when Saccharomyces cerevisiae cells are exposed to weak organic acids such as acetic, lactic, succinic, sorbic or citric (Causton et al. 2001; Kapteyn et al. 2001; Lawrence et al. 2004; Schüller et al. 2004; Kawahata et al. 2006), which are used as food preservatives, or when they are exposed to strong inorganic acids such as hydrochloric or sulphuric acids (Claret et al. 2005; Kawahata et al. 2006; Chen et al. 2009; Melo et al. 2010). Recently, evidence has been reported shows that yeast cells respond promptly to a reduction in pH by inducing genes of the General Stress Response (GSR) pathway (Melo et al. 2010), the primary response to any environmental changes. In addition, it has been shown that yeast cells that fail to activate the protein kinase A (PKA) regulatory signalling cascade effectively are more tolerant to low pH sulphuric acid (Melo et al. 2010).

Saccharomyces cerevisiae cells contain other transduction mechanisms that allow them to sense and respond to environmental changes (Saito and Tatebayashi 2004). Among them, we can highlight the Cell Wall Integrity (CWI) pathway that is responsible for maintenance and function of the yeast cell wall (Levin 2005). This mechanism is controlled by the regulatory cascade led by protein kinase C (PKC) (Chen and Thorner 2007). Activation of the CWI pathway occurs during cell growth to adjust the cell wall during volume growth and division, and in the response to injuries to the cell envelope (Levin 2005). This pathway is trigged by the activation of membrane-associated sensor proteins Wsc1/2/3p that transduce the signal through the activation of the GTP/GDP-exchanging factor Rom2p, which in turn activates Rho1p (Fuchs and Mylonakis 2009). This protein acts directly by activating Fks1p, which is responsible for the synthesis of β1,3-glucan during maintenance or repair of the cell wall, or by activating Pkc1p that triggers the mitogen-activating protein kinase (MAPK) cascade (Fuchs and Mylonakis 2009). This kinase cascade will ultimately phosphorylate and activate the transcription factors Rlm1p and SBF (Swi4/6p complex) leading to the induction of genes involved in the CWI pathway and cell cycle regulation (Jung et al. 2002). The CWI pathway also responds to pH down-shifting (Kapteyn et al. 2001; Claret et al. 2005) and can interact with the High Osmolarity Glycerol (HOG) pathway, which is controlled by Hog1p in response to osmotic shock (Saito and Tatebayashi 2004; Hohmann 2009; Rodríguez-Peña et al. 2010). The HOG pathway recognizes and transduces the signal that is physically promoted by loss of cell turgor and works to maintain cell volume and homoeostasis (Lucena et al. 2010). Another interaction between cell pathways has been reported by Kullas et al. (2007) who showed that the Ca2+-dependent calmodulin pathway, which uses calcium as internal mediator of stress response via activation of calcineurin (Aramburu et al. 2004), is also important for growth of Candida albicans in acidic environments.

In the present study, we evaluated the participation of the components of those different pathways in the response and tolerance of S. cerevisiae cells to sulphuric acid. The results showed the interplay among those mechanisms that work to repair the damages in the yeast cell wall caused by low pH sulphuric acid.

Material and methods

Yeast strains and cell growth and maintenance

The laboratory strain BY4741 (MATa his3Δ leu2Δ met15Δ ura3Δ) and its isogenic deletion mutants were from the EUROSCARF (European Saccharomyces Cerevisiae Archive for Functional Analysis) collection, University of Frankfurt. The biological functions of the deleted genes are described in Table 1. The cells were maintained in solid YPD medium, and frozen stocks were prepared in 30% glycerol and stored at −80°C.

Table 1. List of yeast genes that were deleted for cell growth experiments or tested for gene expression by RT-qPCR
Yeast geneBiological function of the encoded protein according to Saccharomyces Genome Database (SGD)
SLG1/WSC1 Sensor-transducer of the stress-activated PKC1-MPK1 kinase pathway; involved in maintenance of cell wall integrity
WSC2 Partially redundant sensor-transducer of the stress-activated PKC1-MPK1 signalling pathway involved in maintenance of cell wall integrity and recovery from heat shock
WSC3 Partially redundant sensor-transducer of the stress-activated PKC1-MPK1 signalling pathway involved in maintenance of cell wall integrity
MID2 O-Glycosylated plasma membrane protein that acts as a sensor for cell wall integrity signalling and activates the pathway
ROM2 GDP/GTP exchange factor (GEF) for Rho1p and Rho2p
BCK1 Mitogen-activated protein (MAP) kinase kinase kinase (MAPKKK) acting in the protein kinase C (PKC) signalling pathway
MKK1 Mitogen-activated kinase kinase (MAPKK) involved in PKC pathway that controls cell integrity
MKK2 MAPKK involved in PKC pathway that controls cell integrity, functionally redundant with Mkk1p
SLT2 Serine/threonine MAP kinase (MAPK) of the PKC pathway involved in regulating maintenance of cell wall integrity, progression through the cell cycle and nuclear mRNA retention in heat shock
RLM1 MADS-box transcription factor, component of the PKC-mediated MAP kinase pathway involved in the maintenance of cell integrity
SWI4 DNA binding component of the SBF complex (Swi4p-Swi6p), a transcriptional activator that in concert with MBF (Mbp1-Swi6p) regulates late G1-specific transcription of targets including cyclins and genes required for DNA synthesis and repair
SWI6 Transcription cofactor, forms complexes with Swi4p and Mbp1p to regulate gene transcription at the G1/S transition
RGD1 GTPase-activating protein (RhoGAP) for Rho3p and Rho4p, possibly involved in control of actin cytoskeleton organization
HSP150 O-Mannosylated heat shock protein that is secreted and covalently attached to the cell wall via beta-1,3-glucan and disulfide bridges
YLR194C Structural constituent of the cell wall attached to the plasma membrane by a GPI-anchor
FKS1 Catalytic subunit of 1,3-beta-D-glucan synthase, functionally redundant with alternate catalytic subunit Gsc2p; binds to regulatory subunit Rho1p
CHS1 Chitin synthase I, requires activation from zymogenic form in order to catalyse the transfer of N-acetylglucosamine (GlcNAc) to chitin
GAS1 Beta-1,3-glucanosyltransferase, required for cell wall assembly and also has a role in transcriptional silencing
KRE6 Type II integral membrane protein required for beta-1,6 glucan biosynthesis
PKH1 Serine/threonine protein kinase involved in sphingolipid-mediated signalling pathway that controls endocytosis
MNN9 Subunit of Golgi mannosyltransferase complex also containing Anp1p, Mnn10p, Mnn11p and Hoc1p that mediates elongation of the polysaccharide mannan backbone
MSN2 Transcriptional activator related to Msn4p; activated in stress conditions, which results in translocation from the cytoplasm to the nucleus; binds DNA at stress response elements of responsive genes, inducing gene expression
MSN4 Transcriptional activator related to Msn2p; activated in stress conditions, which results in translocation from the cytoplasm to the nucleus; binds DNA at stress response elements of responsive genes, inducing gene expression
HOG1 Mitogen-activated protein kinase involved in osmoregulation
MID1 N-Glycosylated integral membrane protein of the ER membrane and plasma membrane, functions as a stretch-activated Ca2+-permeable cation channel required for Ca2+ influx stimulated by pheromone
CCH1 Voltage-gated high-affinity calcium channel involved in calcium influx in response to some environmental stresses as well as exposure to mating pheromones
CRZ1 Transcription factor that activates transcription of genes involved in stress response
YAP1 Basic leucine zipper (bZIP) transcription factor required for oxidative stress tolerance
CIN5 Basic leucine zipper (bZIP) transcription factor of the yAP-1 family
PDE2 High-affinity cyclic AMP phosphodiesterase, component of the cAMP-dependent protein kinase signalling system
RAS1 GTPase involved in G-protein signalling in the adenylate cyclase activating pathway, plays a role in cell proliferation
RAS2 GTP-binding protein that regulates the nitrogen starvation response, sporulation and filamentous growth

Cell growth assay

For the pre-inoculum preparation, yeast cells were grown in liquid YPD medium (20 g l−1 glucose, 20 g l−1 peptone and 10 g l−1 yeast extract) at 30°C under orbital agitation (120 rpm) for 16 h. Cultivation media were adjusted with concentrated sulphuric acid to different pH and then sterilized in an autoclave. pH was adjusted to 7·0 (whenever necessary) with 3 mol l−1 KOH and sterilized. Before inoculation, medium samples were withdrawn to check the initial pH. Yeast cells from the pre-inoculum were used to inoculate fresh medium to initial optical density of 0·1 (OD600nm) and cultivated to 0·5. Then, the cells were centrifuged and transferred to in new sterile flasks (initial 0·1 OD600nm) containing YPD medium adjusted to the pH of interest.

Absolute growth was measured by the optical density (OD600nm) of the culture. Cell density was measured at zero (starting point 0·1 OD600nm), 24 and 48 h of cell growth. Relative growth was plotted as the percentage of cell growth of the deletion strain at certain pH relative to its parental strain by the formula (ODM7/ODP7) for growth at pH 7·0 and (ODM2·5/ODP2·5) for pH 2·5, where OD is the cell density reached by deletion mutants (M) and parental (P) strains grown at pH 7·0 or 2·5 for 24 and 48 h of cultivation. All experiments were performed in biological triplicate with two technical replicates, and the results were presented as the average (±SD).

Cell viability and growth recovery

To test the loss of viability by low pH, yeast cells pregrown in YPD at pH 7 as above were centrifuged and suspended in YPD adjusted to pH 2·5 or pH 7 and incubated for 30 min. After appropriated dilution in saline, 100 μl of cell suspension was plated onto YPD medium at pH 7 and the plates were incubated at 30°C for 2 days. The number of CFUs were recorded and used to calculate the remaining viable cells. Afterwards, cell suspensions in YPD pH 2·5 or pH 7 were incubated for 24 h at 30°C (treated cells), the cells recovered by centrifugation, suspended in YPD pH 7 and incubated at 30°C for 24 and 48 h. Optical density of the cultures and the number of viable cells by plating onto YPD were recorded. All experiments were performed in biological triplicate with technical duplicate and referred as average (±SD).

RNA extraction and cDNA synthesis

Cell from BY4741 strain and mutants hog1Δ and slt2Δ pre-inoculum were transferred to fresh YPD pH 7 at an initial cell density of 0·1 (OD600nm) and cultivated to 0·5 as above. The cultures were split into two samples, and each one diluted 1 : 1 with fresh YPD medium adjusted to pH 7·0 (with KOH) or pH 2·5 (with sulphuric acid). After incubation for 1 h at 30°C with orbital agitation (120 rpm), the cells were recovered by centrifugation and immediately frozen in liquid nitrogen and stored at −80°C until use.

For total RNA extraction, yeast cells were suspended in 200 μl AE buffer (50 m mol l−1 sodium acetate, 10 mmol l−1 EDTA, pH 5·3) and 50 μl 10% SDS. After homogenization, cells were lysed by incubation at 65°C for 5 min. The lysates were centrifuged at 13 000 g for 5 min at 4°C, and the supernatants transferred to a new tube. The RNA was purified with the NucleoSpin® RNA II kit (Macherey-Nagel, Düren, Germany), following the instructions of the manufacturer. Purified RNA was stored at −20°C until use. Preparation of cDNA used the ImProm-II Reverse Transcription System Promega II kit (Promega, Madison, WI, USA), following the instructions of the manufacturer.

Gene expression analysis by RT-qPCR

Coding regions of target genes were recovered from the Yeast Genome Database – SGD (http://www.yeastgenome.org). Primer design was performed using the online Genscript Primer Design in advanced mode (www.genscript.com/cgi-bin/tools) using the following parameters: the sizes of the primers between 17 and 25 bases, Tm value of 59°C and size of amplicons between 70 and 110 bp. The primer pairs were analysed using the Netprimer tool to determine the formation of self-hybrids, duplex, hairpins and loops (www.premierbiosoft.com/netprimer/netprlaunch/netprlaunch.html) to select those with a ranking greater than 90%. The primer pairs were matched with the coding regions of target genes (www.blast.org) and to PCR in silico (http://genome.ucsc.edu/cgi-bin/hgPcr) using the genome of S. cerevisiae as template. The primer sequences were synthesized by IDT Technologies (Coralville, IA, USA).

PCR was performed using the ABI Prism 7300 (Applied Biosystems, Foster City, CA, USA) detection system and the SYBR Green PCR Master Mix (Applied Biosystems) kit. The amplification conditions used were initial step at 50°C for 2 min, 95°C for 10 min, 40 cycles of 95°C for 15 s and 60 min for 1 min. For the determination of contamination by genomic DNA, the PCRs were carried out with RNA samples for each condition without being converted to cDNA. The results produced no detectable amplification in any condition. The values of Cq threshold cycle were given automatically for the independent amplification. Raw Cq values for all samples were then plotted in Microsoft Excel 2007 worksheets to create a suitable input file for the geNorm applet according to the User's Guide (Vandesompele et al. 2002). The first step was to determine the expression stability for the candidate reference genes ARO10, THI3, PDC1, PDC5, PDC6, ADH3, EFB1 and LEU4 (Table S1 – supplementary data). According to geNorm, at least two of them should be used for data normalization (Fig. S1 – supplementary data) and PDC1 and LEU4 were chosen according the M value (Figs S1 and S2 – supplementary data). All parameters were followed as described by Elsztein et al. (2011).

Results

Determination of Minimal Inhibitory pH (MIP) value for yeast growth

As the first step, we identified the minimal inhibitory pH (MIP), the lowest pH value that allows yeast cell to grow in medium adjusted with sulphuric acid. For this test, we chose the parental BY4741 strain and its isogenic slt2Δ mutant. This mutant was chosen for its severe growth inhibition, compared to other mutants, when exposed to acid pH (Fig. 2 and other data not shown). The results showed that absolute growth of the slt2Δ mutant was similar to parental strain at neutral or moderate acid pH (Fig. 1). The mutant strain displayed the same parental cell growth up to pH 4·0, while its growth was significantly affected at pH 3 (Fig. 1). At pH 2·5, cell growth for parental strain was severely affected, whereas growth of mutant cells was abolished (Fig. 1). No growth of both strains was observed at lower pH. Therefore, we established pH 2·5 as the MIP value for the quantitative experiments of cell growth and gene expression as the stressing condition by sulphuric acid.

Figure 1.

Effect of pH on the growth of parental strain BY4741 and the mutant slt2Δ. The strains were tested in YPD medium pH 7·0 (adjusted with KOH if necessary) and adjusted with sulphuric acid as indicated. The experiment started with inoculum 0·1 OD600nm at 30°C with shaking. The OD was measured after 24 h of growth (white columns) and after 48 h of growth (black columns).

Additional experiments were carried out by adding sorbitol to the medium, as a protectant of the yeast cell wall, because it recovered the growth slt2Δ mutant at pH 3 (Claret et al. 2005). The results in the present work showed that growth of slt2Δ mutant was unaffected at pH 2·5 but increased by 2·7-fold at pH 3 (Fig. S3 – supplementary data).

Identification of PKC/CWI genes important for tolerance to low pH

Mutant strains in which genes had been deleted from the PKC/CWI pathway were tested for cell growth at the MIP value and compared to growth at pH 7·0 and to the growth of the parental strain (Fig. 2). Among the sensors-encoding genes, slg1Δ/wsc1Δ mutant was more affected by acid pH after 24 h, followed by mid2Δ and wsc2Δ (Fig. 2a). However, these mutants tended to achieve the parental biomass after 48 h of cultivation, which indicated that there was adaptation to the acid environment (Fig. 2b). The strains with the wsc3Δ mutation showed higher growth than the parental strain (Fig. 2a,b). Similar results were observed for the mutant strain rom2Δ (Fig. 2a,b), indicating that inactivation of these two genes result in a de-repression of growth inhibition caused by low pH. For the MAPK-encoding genes, the results showed that bck1Δ was as sensitive to acid pH as slt2Δ, and no adaptation was observed after the longer cultivation time whatsoever. The strain mkk1Δ showed intermediary growth reduction by 50% of the parental strain at 24 h of cultivation, while the growth of mutant mkk2Δ was hardly affected at acid pH. Both mutants achieved parental growth after 48 h of cultivation (Fig. 2b). Among the transcription factors-encoding genes, the mutant rlm1Δ was severely affected by acid pH, followed by swi6Δ and swi4Δ (Fig. 2a). They all presented some sort of recovery of cell growth after longer cultivation period (Fig. 2b), although the rlm1Δ mutant exhibited only 25% of the final parental growth.

Figure 2.

Effect of low pH on the growth of mutant strains with deletion to genes related to the Cell Wall Integrity pathway. The strains were tested in YPD medium pH 7·0 (white columns) and YPD adjusted with sulphuric acid to pH 2·5 (black columns). The experiment started with inoculum 0·1 OD600nm at 30°C with agitation. The results were referred as percentage of cell growth of mutant strains related to BY4741 parental strain after 24 h (a) or 48 h (b) of cultivation, in which 100% refers to growth at pH 7·0.

Involvement of other regulatory pathways in the tolerance to acid pH

Yeast cells with deleted regulatory genes other than PKC/CWI were also tested (Fig. 3). The yeast mutants hog1Δ, mid1Δ, cch1Δ and rgd1Δ were severely affected by low pH, and no recovery of cell growth was observed if cells were left for longer cultivation times. Thus, no adaptation mechanism was observed for these mutants. Moreover, their phenotypes were similar to those observed for bck1Δ and slt2Δ mutants, which is a clear indication of the cross-participation of those pathways in the tolerance to acid pH. Sorbitol added to the medium was itself very toxic for hog1Δ mutant, and no growth was observed in any pH tested. Moreover, the cells produced pseudo-hyphae in the presence of that compound (data not shown), as it has been reported in the literature (Hohmann 2002). The mutant hsp150Δ displayed only 50% of the parental cell growth, while the yap1Δ and cin5Δ/yap4Δ mutants grew normally at pH 2·5 (Fig. 3). Similar to what observed for wsc3Δ and rom2Δ mutants (Fig. 2a,b), mutations in upstream genes of the PKA pathway (RAS1, RAS2 and PDE2) showed higher overall growth than the parental strain (Fig. 3), indicating that inactivation of these two genes also resulted in a sort of de-repression mechanism.

Figure 3.

Effect of low pH on the growth yeast with deletion genes to High Osmolarity Glycerol pathway, calcineurin and other regulatory pathways. The strains were tested in YPD medium pH 7·0 (white columns) and YPD adjusted with sulphuric acid to pH 2·5 (black columns). The experiment started with inoculum 0·1 OD600nm at 30°C with agitation. The results were recorded as the percentage of cell growth of mutant strains relative to BY4741 parental strain after 24 h (a) or 48 h (b) of cultivation, in which 100% refers to growth at pH 7·0.

Growth recovery after acid stress

The parental strain BY4741 and its mutants hog1Δ and slt2Δ were evaluated for viability and recovery of cell growth after shock with sulphuric acid. We have found that the effect of low pH leads to loss of cell viability in the tested strains, particularly in slt2Δ mutant that showed 95% mortality after 30 min of treatment (Table 2). That loss of viability leads to reduced cell growth after 24 h of cultivation on YPD pH 2·5 relative to cultivation on YPD pH 7 (Fig. 4). However, cells of BY4741 and hog1Δ strains recovered their growth upon transference to YPD pH 7 (Figs 2, 3 and 4). On the other hand, slt2Δ cells did not grow even after 48 h of cultivation.

Figure 4.

Growth recovery of yeast cells of BY4741 (grey columns), hog1Δ (hatched columns) and slt2Δ (dotted columns) strains by upshifting medium pH. Cells grown in YPD pH 7·0 were transferred to YPD pH 2·5 and cultivated for 24 h. Afterwards they were washed and suspended in YPD pH 7·0 and cultivated for 24 h and 48 h. The results of cell density (OD600nm) were relative to growth in YPD pH 7·0 during all stages of the experiment.

Table 2. Lethal effect of sulphuric acid at pH 2·5 in yeast cells
StrainCFU (105 cell ml−1)% mortality
0 min30 min
BY4741 9·5 ± 1·66·6 ± 0·830·8
hog1Δ 7·8 ± 0·72·9 ± 0·862·4
slt2Δ10·1 ± 0·40·5 ± 0·0695·0

Gene expression in response to low pH with sulphuric acid

Expression of genes of CWI pathway was measured by comparing the incubation of BY4741 strain cells at pH 2·5 relative to pH 7·0. All genes involved in membrane and cell wall integrity and maintenance were induced by acid stress (YLR194c > FKS1 > CHS1 > HSP150 > KRE6 > MNN9 = PKH1 > GAS1) (Fig. 5). The 10 times overexpression of MSN2 and MSN4 genes indicated that GSR is involved in the sulphuric acid response. In addition, the four times overexpression of the CRZ1 gene indicated that the Ca2+-calmodulin pathway is also responsive to this type of stress. The overexpression of RLM1 and SLT2 genes confirmed the importance of the downstream part of the PKC pathway involved in the cell response to acid stress (Fig. 5) in addition to their importance for cell tolerance (Fig. 2). On the other hand, the results showed that the CIN5 gene was very responsive to acid stress (Fig. 5), although it was not essential for cell tolerance (Fig. 3). The high gene expression together with the growth defect phenotype of the corresponding mutants place these RLM1 and SLT2 genes in a key position of cell response and tolerance to acid pH.

Figure 5.

Relative gene expression after the yeast strain BY4741 was stressed with sulphuric acid. The graph shows the induction of genes related to the Cell Wall Integrity pathway, to the overall response to stress and to the calcineurin pathway. The strain BY4741 grown in YPD medium pH 7·0–0·5 OD600nm at 30°C with agitation and afterwards was transferred to new flasks containing YPD pH 7·0 and 2·5, where it remained growing for 1 h at 30°C with agitation. Incubation at pH 7·0 was used as reference condition. All normalization procedure was detailed in Material and Methods following Vandesompele et al. (2002) and Elsztein et al. (2011).

Relative gene expression analysis was performed in cells with deletion in HOG1 or SLT2 gene exposed to pH 2·5 with sulphuric acid. Overall, these mutants showed decreased expression of all genes tested for different signalling mechanisms. It is noteworthy that the expression of MSN4 and CRZ1 genes were abolished both in hog1Δ (Fig. 6) and slt2Δ (Fig. 7) mutants. Moreover, it was confirmed that the integrity of HOG1 and SLT2 genes was essential for the expression of CIN5/YAP4 (Fig. 6) and RLM1 (Fig. 7) genes, respectively. And lastly, and most importantly, the results clearly showed that the expression of SLT2 gene was totally dependent on the presence of HOG1 gene (Fig. 6), which corroborates the PKC-HOG cross-talk hypothesis raised above.

Figure 6.

Relative gene expression after the yeast strain hog1Δ was stressed with sulphuric acid. The graph shows the induction of genes related to the Cell Wall Integrity pathway, to the overall response to stress and to the calcineurin pathway. The strain BY4741 grown in YPD medium pH 7·0–0·5 OD600nm at 30°C with agitation and afterwards was transferred to new flasks containing YPD pH 7 and 2·5, where it remained growing for 1 h at 30°C with agitation. Incubation at pH 7·0 was used as reference condition. All normalization procedure was detailed in Material and Methods following Vandesompele et al. (2002) and Elsztein et al. (2011).

Figure 7.

Relative gene expression after the yeast strain slt2Δ was stressed with sulphuric acid. The graph shows the induction of genes related to the Cell Wall Integrity pathway, to the overall response to stress and to the calcineurin pathway. The strain BY4741 grown in YPD medium pH 7·0–0·5 OD600nm at 30°C with agitation and afterwards was transferred to new flasks containing YPD pH 7 and 2·5, where it remained growing for 1 h at 30°C with agitation. Incubation at pH 7·0 was used as reference condition. All normalization procedure was detailed in Material and Methods following Vandesompele et al. (2002) and Elsztein et al. (2011).

Discussion

Using two different experimental strategies, cell growth and gene expression, we have shown that the genes involved in the cell wall integrity mechanism and the PKC-MAPK pathways are essential for yeast cells to grow in acidic environments, such as those imposed by the industrial yeast recycling process for bioethanol production.

The key component in the response to cell wall injuries is MAP kinase Slt2p, the last component of the regulatory PKC phosphorylation cascade that phosphorylates and activates the transcription factors Rlm1p and Swi4/6p (SBF) complex. It has been shown that Slt2p is activated in response to heat shock, hyperosmotic, oxidative and alkaline stresses (Davenport et al. 1995; Hahn and Thiele 2002; Vilella et al. 2005; Serrano et al. 2006). Moreover, this gene is essential for cell tolerance to hydrochloric acid (Claret et al. 2005). In the present work, these findings have been corroborated by the severe growth defect observed for the slt2Δ mutant (Figs. 1, 2 and 4) and by the overexpression of SLT2 gene upon downshifting the pH to 2·5 with sulphuric acid (Fig. 5). The pH value of 3·0, equivalent to 1 mmol l−1 of dissociated H in the medium, was the threshold for slt2Δ mutant growth in hydrochloric acid (Claret et al. 2005) and sulphuric acid (Fig. 1). A further decrease to pH 2·5, the equivalent of raising the concentration of dissociated H in the medium to 3·16 mmol l−1, reduced the growth of the parental BY4741 cells and impaired the growth of the slt2Δ mutant. We recorded a growth rate of 0·4 h for S. cerevisiae JP1 industrial strain in YPD medium adjusted to pH 2·5 with sulphuric acid, which decreased to 0·13 h when using synthetic YNB medium (Melo et al. 2010). At pH 2, no growth was observed for BY4741 cell in YPD (Fig. 1) or for JP1 cells in YNB, whereas the industrial strain grew at 0·34 h in YPD-pH 2 (Melo et al. 2010). In addition to the sensitivity of the slt2Δ mutant to low pH, deletion of the BCK1 gene also impaired cell growth at pH 2·5 with both sulphuric (Fig. 2) and hydrochloric acids (Claret et al. 2005). This latter gene encodes the first MAP kinase in the PKC cascade (Chen and Thorner 2007). On the other hand, deletion of one of the intermediary kinases encoding genes MKK1 or MKK2 had only a mild effect on cell growth, which indicates the complementary function of their products. Additionally, deletion of RGD1 gene also inhibited cell growth at pH 2·5 (Fig. 3) and increased cell mortality in hydrochloric acid (Claret et al. 2005). Rgd1p activates the GTPase activity of the small G-protein Rho1p and as a consequence is involved in the activation of the PKC pathway. Therefore, together those results indicate that PKC-MAP kinase cascade is the core mechanism for cell tolerance of low pH.

But how do the yeast cells sense this environmental stress? In S. cerevisiae, five membrane-located sensor proteins named Wsc1p, Wsc2p, Wsc3p, Mid2p and Mtl1p are responsible for transducing to Rho1p the condition of the cell wall (Verna et al. 1997). The lost of Wsc1p causes cell sensitivity to several stress conditions such as heat, oxidative, ethanolic and alkaline stresses and the presence of antifungal drugs (Zu et al. 2001; Serrano et al. 2006). Mid2p is also an important sensor of cell wall damage, although it only responds to a narrow range of injuries (de Nobel et al. 2000). Despite its parental cell viability phenotype, the mutant mid2Δ was deficient in the activation of CWI mechanism (Claret et al. 2005). It was concluded that Mid2p works on the transduction of acid pH stress, while Wsc1p is more involved in the activation of the GSR mechanism (Claret et al. 2005). In the present study, the mutant cells wsc1Δ and mid2Δ, and to a lesser extent wsc2Δ, showed lower growth at pH 2·5 after 24 h (Fig. 2a). As growth continued, they reached parental cell density (Fig. 2b) probably because of the activity of the remaining functional sensors in a sort of hierarchical order. Thus, the yeast cells adapt to that acid environment by triggering the coordinated action of the different pathways.

After being transduced by the MAP kinase pathway, the cellular response to low pH reaches the target transcription factors that induce the expression of CWI genes. In this study, we have shown that yeast cells lacking Rlm1p, Swi4p and Swi6p show different growth defect phenotypes, which suggests different levels of participation of these proteins in the activation of CWI genes. Rlm1p is one the main targets of Slt2p and directly regulates the expression of CWI genes (Jung et al. 2002). As expected, CWI genes involved in cell wall biogenesis (FKS1, GAS1, KRE6 and CHS1) (Levin 2005) and structure (HSP150 and YLR194c) (Kapteyn et al. 1999; García et al. 2004) were induced by sulphuric acid treatment (Fig. 5) and, their inductions were diminished in stl2Δ mutant (Fig. 7). This induction was followed by the huge induction of SLT2 gene itself (Fig. 5). It has been reported that transcription of the SLT2 gene is dependent on Hog1p and Rlm1p upon hyperosmotic shock (Hahn and Thiele 2002), which was corroborated herein (Fig. 6). Thus, under acidic stress, an amplification cascade could be created at downstream part of the PKC pathway: Slt2p activates Rlm1p which in turn, along with Hog1p, back increases the expression of SLT2 gene. It creates a positive feedback of gene expression and protein activation that ensures the expression of complete set of CWI genes. This coordinated work between CWI and HOG pathways in response to environmental stress has already been reported (Hahn and Thiele 2002; Bermejo et al. 2008; Rodríguez-Peña et al. 2010; Elsztein et al. 2011), and the in vivo biochemical interaction of Slt2p and Hog1p in a complex with Cdc37p was shown (Hawle et al. 2007).

The HOG pathway responds to osmotic stress (Saito and Tatebayashi 2004; Hohmann 2009; Rodríguez-Peña et al. 2010), and its leading protein Hog1p is also responsible for the activation of the protein complex Msn2/4p, which in turns induces the expression of GSR genes and the RGD1 gene (Schüller et al. 1994). Furthermore, Rgd1p activates Pkc1p under acid stress (Claret et al. 2005). Therefore, Hog1p would indirectly amplify the signal also at the upstream part of PKC pathway under acid stress, in addition to its above indicated interference in the lower part of that pathway. It was also shown Hog1p also takes part of the expression of RLM1 under acid stress (Fig. 6). Interestingly, Claret et al. (2005) did not observe the growth defect of hog1Δ and rlm1Δ mutants in hydrochloric acid that we observed in sulphuric acid. The reason could be the pH difference of pH 3·0 (Claret et al. 2005) and 2·5 (this work), meaning an increase of three times in the H concentration. It could make a relevant difference if HOG pathway acts as amplifier of the response to acid stress. The damage caused by sulphuric acid to cell wall could also expose the cell membrane, as happens during protoplast formation, resulting in decreasing of cell turgor pressure that physically mimic a hyperosmotic shock transduced through the HOG pathway, as has been proposed recently (Lucena et al. 2010). Thus, the stress signal may be transduced by PKC and HOG parallel pathways.

Additionally, the induction of MSN2 and MSN4 genes observed in the present work (Fig. 5) can be related to that observed induction of GSR genes upon cultivation at low pH (Melo et al. 2010). It was also shown that CIN5 gene was 10-times induced at acid pH (Fig. 5). This gene, an allele of YAP4, encodes for a transcription factor involved in the resistance to antifungal drugs and tolerance of high osmolarity (Rodrigues-Pousada et al. 2010). Under high osmolarity stress, CIN5/YAP4 is regulated by Msn2p via two STRE motifs in its promoter, whereas under oxidative stress, it is co-regulated by Msn2p and Yap1p through STRE and YRE motifs, respectively (Nevitt et al. 2004a,b). We propose that under acid stress the induction CIN5 gene is regulated by Msn2p.

Growth inhibition assays provided evidence on the participation of Ca2+ calmodulin-dependent calcineurin pathway in the tolerance to acid pH. Mutations in MID1 and CCH1 genes, which encode the cell membrane Ca2+ influx channel (Cyert 2003), impaired cell growth at pH 2·5 (Fig. 3). Moreover, CRZ1 gene that is induced by calcineurin in response to Ca2+ influx (Cyert 2003) was also induced by acid stress (Fig. 5). There are three ways in which the CWI pathway and the Ca2+-mediated response can interact: activation of Cch1p/Mid1p channel by Slt2p (Bonilla and Cunningham 2003); activation of Crz1p by Rho1p-Skn7p (Tsuchiya et al. 1998); and through cooperation between Slt2p and Crz1p in the expression of FKS2 in response to injuries in the yeast cell wall (Zhao et al. 1998). Together, Slt2p, Hog1p and Crz1p ensure the post-translation activation of all transcription factors needed for complete expression of genes involved in cell wall regeneration, cell cycle progression (the SBF complex) and GSR for yeast cell to survive at low pH.

Acknowledgements

This work was performed in the context of the Bioethanol Research Network of the State of Pernambuco and was supported with grants by the Brazilian agencies FACEPE (PRONEM programme and PhD scholarship programme) and CNPq (Biofuel research programme).

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