Citric acid inhibits growth of S. cerevisiae.
Growth of S. cerevisiae BY4741 was inhibited in the presence of increasing concentrations of citric acid in both ME broth (pH 3.5) (Fig. ) and on ME agar (pH 3.5) (Fig. ). Growth rate was reduced from a value of 0.406 in the control culture to 0.221 (45.6% inhibition) in the presence of 400 mM citric acid (Fig. ). Exposure to 300 mM citric acid resulted in a 30.2% reduction in the growth rate.
FIG. 1. Growth of S. cerevisiae BY4741 is inhibited in the presence of citric acid. (a) Cultures of S. cerevisiae BY4741 were grown to late exponential phase in ME medium alone (pH 3.5) and in the presence of 200, 300, and 400 mM citric acid (CA). (b) Serial (more ...) Screening the yeast disruptome identified many genes that confer resistance to citric acid stress.
To identify key genes involved in mediating resistance to citric acid stress, we conducted a phenotypic screen of all the 4,847 nonessential gene deletions in S. cerevisiae BY4741 MATa. Yeast deletion strains were spotted onto plates in the presence or absence of 400 mM citric acid. Of the 4,847 nonessential gene deletions tested; 69 (1.4%) showed sensitivity to citric acid compared to the parent (Table ). No citric acid-resistant phenotypes were detected. Of the 69 citric acid-sensitive gene deletions, 10 were of particular interest because they encode known regulatory proteins that could be involved in signaling an adaptive response to citric acid stress. The 10 gene deletions of interest are RCS1 (iron-regulated transcriptional repressor), MSN4 (transcriptional activator in response to stress), HOG1 (MAPK), BUB1 (serine/threonine protein kinase), CKA1 (casein kinase II), PTK2 (protein kinase involved in polyamine uptake); BCK1 (MEKK), SSK1 (two-component signal transducer), TPD3 (serine/threonine protein phosphatase 2A), and RRD1 (strong similarity to human phospho-tyrosyl phosphatase activator).
Screening the yeast disruptome identified genes required for optimal growth of S. cerevisiae in the presence of 400 mM citric acid (pH 3.5)a
Using the Gene Ontology (GO) database (http://fatigo.bioinfo.cnio.es/
), we analyzed the functional categories of citric acid-sensitive gene deletions. Of the total of 69 genes, 56 had GO at level 3, biological process, which resulted in the following GO profile: 55.1% metabolism, 46.4% cell growth and/or maintenance, 17.4% homeostasis, 11.6% response to external stimulus, 10.1% response to stress, 4.3% cell communication, 2.9% reproduction, 1.45% morphogenesis, 1.45% growth, and 15.94% GO terms present at other levels.
Growth in the presence of citric acid induces changes in protein expression.
We measured changes in the S. cerevisiae proteome occurring in response to growth in the presence of 300 mM citric acid (pH 3.5) over the pH separation ranges of 4.5 to 5.5, 4.0 to 7.0, and 6.0 to 11.0. These proteins were subsequently identified by matrix-assisted laser desorption ionization-time of flight mass spectrometry of the peptides produced by in-gel digestion with trypsin, followed by database searching using the peptide masses derived from each trypsinized protein (see Table S1 in the supplemental material).
Over the pH range 4.5 to 5.5, we reproducibly detected the up-regulation of six new proteins and the down-regulation of one protein due to the presence of 300 mM citric acid, pH 3.5 (Fig. ). The up-regulated proteins were identified as putative adenosine kinase (Ado1) (protein 1), dl-glycerol-3-phosphate (Gpp2) (protein 2), imidazole glycerol phosphate synthase (His7) (protein 4), stress-induced protein (Sti1) (protein 5), carbamyl phosphate synthase (Ura2) (protein 6), and pyruvate decarboxylase isozyme 1 (Pdc1) (protein 7) (Fig. ). The down-regulated protein was identified as an hypothetical ORF (Ylr301w) (protein 3 in Fig. ).
FIG. 2. Identification of changes in protein expression induced in S. cerevisiae BY4741 due to growth in the presence of 300 mM citric acid, pH 3.5. Total soluble proteins were separated by IEF over the pH range 4.5 to 5.5. Proteins were prepared from control (more ...)
Similarly, over the pH ranges 4 to 7 and 6 to 11, we detected the up-regulation of eight (Fig. ) and seven proteins (Fig. ), respectively. The up-regulated proteins on the pH 4 to 7 gels were identified as heat shock protein 70 isoform (Ssa1) (protein 8), vacuolar ATPase V1 domain subunit E (Vma4) (protein 9), 40S ribosomal protein S0-A (Rps0ap) (protein 10), dl-glycerol-3-phosphatase (Gpp1) (protein 11), inorganic pyrophosphatase (Ipp1) (protein 12), enolase 2 (Eno2) (protein 13), heat shock protein 70 isoform (Ssb2) (protein 14), and heat shock protein 26 (Hsp26) (protein 15) (Fig. ).
FIG. 3. Identification of changes in protein expression induced in S. cerevisiae BY4741 due to growth in the presence of 300 mM citric acid, pH 3.5. Total soluble proteins were separated by IEF over the pH range 4 to 7. Proteins were prepared from control cells (more ...)
FIG. 4. Identification of changes in protein expression induced in S. cerevisiae BY4741 due to growth in the presence of 300 mM citric acid, pH 3.5. Total soluble proteins were separated by IEF over the pH range 6 to 11. Proteins were prepared from control cells (more ...)
The up-regulated proteins identified on the pH 6 to 11 gels were mitochondrial F1F0-ATPase alpha subunit (Atp1) (protein 16), IMP dehydrogenase homolog (Imd3) (protein 17), aceto-hydroxyacid reductoisomerase (Ilv5) (protein 18), mitochondrial malate dehydrogenase (Mdh1) (protein 19), Hsp40 family chaperone (Sis1) (protein 20), 3-deoxy-d-arabino-heptulosonate-7-phosphate synthase (Aro3) (protein 21), and isocitrate dehydrogenase 1 alpha-4-beta-4 subunit (Idh1) (protein 22) (Fig. ).
A number of the up-regulated proteins on the gels were identified as polypeptide fragments (e.g., Pdc1, Ssa1, Eno2, and Ssb2). However, like all the other proteins listed, expression of the proteins represented by these fragments was altered in duplicate experiments. Comparison of the experimental versus predicted molecular weights and pIs of the identified proteins is shown in Table S1 in the supplemental material. The predicted and experimental molecular weights were mostly in agreement, apart from the polypeptide fragments discussed previously. However, the experimental and predicted pIs varied, even in intact proteins, which could be explained by the presence of different posttranslational modifications.
As before, we analyzed the GO database functional categories of the citric acid-induced changes in protein expression. Of the total of 24 proteins with up-regulated expression, 22 had GO at level 3, biological process, which resulted in the following GO profile: 83.3% metabolism, 20.8% cell growth and/or maintenance, 8.3% response to stress, 4.17% response to external stimulus, and 4.17% homeostasis.
Comparison of the above proteins with the genome-wide, citric acid-sensitive, gene deletion screen in Table revealed no correlation between proteins with altered expression due to growth in the presence of citric acid and the citric acid-sensitive gene deletions.
Growth in the presence of citric acid induces changes in gene expression.
The gene expression profile of S. cerevisiae grown in the presence of 300 mM citric acid (pH 3.5) for 20 min was compared to that of cells grown in ME medium (pH 3.5) alone. Genes which showed a greater than 1.5-fold induction due to the presence of citric acid are shown in Table S2 in the supplemental material. The data are listed according to putative functional categories, as described in the MIPS database, and are expressed as the means and standard deviations from three independent experiments.
Of the 6,144 ORFs, 68 (1.1%) showed greater than a 1.5-fold induction (see Table S2 in the supplemental material) and 12 of these genes showed an increase greater than 2.5-fold: glyceraldehyde-3-phosphate dehydrogenase 2 (TDH2), alpha-trehalose-phosphate synthase (TPS1), glycerol-3-phosphate dehydrogenase (GPD1), cell surface glycoprotein (SED1), stress-inducible aldehyde dehydrogenase (ALD3), three cell wall-associated proteins (SPI1, CWP1, and CCW14), and four ORFs of unknown function (YDL204w, YGL037c, YGR086c, and YFR001w).
Of the 68 up-regulated genes, GO database analysis, at level 3 of biological process, revealed 43 genes with the following profile: 44.1% metabolism, 26.5% cell growth and/or maintenance, 13.2% response to stress, 8.8% response to external stimuli, 8.8% cell communication, 4.41% reproduction, 2.94% morphogenesis, 2.94% homeostasis, 2.94% growth, 1.47% death, 1.47% cell death, 1.47% ageing, 1.47% conjugation, and 36.8% with GO terms at other levels.
Furthermore, 54 (0.9%) of the 6,144 ORFs showed a decrease in expression greater than 1.5-fold (see Table S3 in the supplemental material). Of these ORFs, 33 showed repression more than 2.5-fold and were nearly all structural components of the ribosome, apart from pyruvate decarboxylase 1 (PDC1), a putative ATP-dependent RNA helicase (ECM16), and a gene mediating glucose repression (GSF2). GO analysis of these genes, performed exactly as described above, revealed 63.0% metabolism, 11.1% cell growth and/or maintenance, 1.85% homeostasis, 1.85% response to external stimulus, and 11.1% GO terms present at other levels. The majority of genes that were repressed mediate protein synthesis and were largely structural constituents of the ribosome.
Comparison of these genes induced or repressed in response to citric acid with the genome-wide, citric acid-sensitive, gene deletion screen in Table revealed 10 genes whose expression was altered upon exposure to citric acid and whose corresponding gene deletion strain also exhibited sensitivity to citric acid: TPS1 (α, α-trehalose-phosphate synthase), FBP1 (fructose-1,6-bisphosphate), IMP1 (protease, mitochondrial), VMA2 (H+-ATPase V1 domain 60-kDa subunit, vacuolar), BMH2 (suppressor of clathrin deficiency), CCW14 (secretory stress response protein 1), CYC8 (general repressor of transcription), YAR1 (ankyrin repeat-containing protein), and YDL204W and YOR161c both encoding proteins of unknown function. These 10 genes represent 8.2% of the total of 122 genes that displayed altered expression in response to citric acid.
Detailed phenotypic analysis of deletion mutants reveals a role for the MAPK HOG pathway in resistance to citric acid.
Results from the genome-wide screen (sensitivity of HOG1, RRD1, SSK1, and MSN4 deletion strains), protein expression analysis (up-regulation of Gpp1p and Gpp2p), and transcript analysis (induction of GPD1, GPP2, TPS1, and CTT1) clearly suggested a role for the HOG pathway in mediating resistance to citric acid stress.
To further investigate the possibility that citric acid resistance may be regulated via the HOG pathway, we investigated the growth sensitivity or resistance of the nonessential gene deletions within the entire HOG pathway, including general stress genes, and genes known to be regulated by HOG1 that are involved in glycerol biosynthesis. This experiment was performed in more detail than the initial genome deletion screen by assaying sensitivity to citric acid using shaking flask cultures and calculating change in growth rate. This was necessary because we have detected discrepancies in the results from sensitivity assays performed on agar compared to liquid culture (for example, Δpbs2, Δptp2 and Δptp3). In some cases, strains show sensitivity in liquid culture but not on agar (see below). Growth of the parent strain and the following HOG pathway gene deletion strains was monitored in ME broth (pH 3.5) with and without 300 mM citric acid: regulatory sensor (Δsho1), upstream control system (Δssk1), upstream kinases (Δste20 and Δste50), MAP kinase kinase kinase (Δste11 and Δssk2), MAP kinase kinase (Δpbs2), MAP kinase (Δhog1), phosphatases (Δptp2 and Δptp3), transcriptional regulators (Δmsn1, Δmsn2, Δmsn4, Δhot, Δsmp1, Δsko1, Δgen4, and Δskn7), and the target genes (Δctt1, Δhsp12, Δgpd1, Δgpd2, Δgpp1, and Δgpp2).
It is clear that important gene deletions within the HOG MAPK pathway induce sensitivity to citric acid (Fig. ). In fact, upon exposure to 300 mM citric acid, deletion of the protein kinases SSK1, PBS2, and HOG1 resulted in approximately 20% additional reduction in growth rate relative to the growth rate of the parent (Fig. ). Furthermore, deletion of the transcription factor MSN4 gene showed approximately 25% additional inhibition of growth rate and deletion of the protein phosphatases PTC2, PTP2, and PTP3 also showed enhanced sensitivity upon exposure to citric acid. Interestingly, deletion of GPP1 resulted in approximately 20% increase in growth rate, or resistance, to citric acid relative to the parent.
FIG. 5. Growth inhibition of S. cerevisiae BY4741 MATa (BY4741/a) and single deletion mutants of genes that constitute the HOG pathway or are known to regulate or be regulated by the HOG pathway. (a) Cultures were grown to late exponential phase in ME medium (more ...)
To confirm that deletion of Δhog1 caused sensitivity to citric acid, we performed a complementation experiment by introducing HOG1 into the Δhog1 deletion mutant on a single-copy plasmid (pRS313::HOG1) under the control of its own promoter. Upon transformation of this plasmid into the Δhog1 strain, sensitivity to citric acid was abolished (Fig. ). Furthermore, we overexpressed HOG1 by cloning the ORF into the multicopy vector pRS413 and transforming BY4741. Subsequently, transformants were found not to display any significant phenotype upon exposure to citric acid (data not shown).
Exposure to citric acid results in the phosphorylation and activation of Hog1p.
Ultimately, stimulation of the HOG pathway results in the activation of the MAP kinase Hog1p via the dual phosphorylation of threonine-174 and tyrosine-176 (33
). To determine whether exposure to citric acid resulted in the activation of Hog1p, we used an antibody that specifically detects the dually phosphorylated form of Hog1p, and thus, the active kinase (38
). Phosphorylation of Hog1p was determined by Western blot analysis in the parent strain and the Δhog1
, and Δpbs2
deletions after a 10-min exposure to 300 mM citric acid. For a positive control, we also determined the degree of phosphorylation of Hog1p after exposure to 0.4 M NaCl, a concentration previously shown to result in Hog1p phosphorylation (17
). Protein loading was checked using an antibody against Hog1p, and as expected, no Hog1p was detected in the Δhog1
strain, but Hog1p was present at similar levels in the parent strain and both the Δssk1
strains (Fig. ).
FIG. 6. Western blot demonstrating the dual phosphorylation of Hog1p upon exposure to citric acid. Soluble protein extracts were prepared from S. cerevisiae BY4741 MATa (BY4741/a) and Δhog1, Δssk1, and Δpbs2 mutants with or without a 10-min (more ...)
Hog1p is phosphorylated, and thus activated, upon exposure to either 300 mM citric acid or 0.4 M NaCl (Fig. ). Upon exposure to citric acid or NaCl, we observed no phosphorylation of Hog1p in either the Δssk1 or Δpbs2 strain. These results confirm that both Ssk1p and Pbs2p are important upstream components of the HOG pathway that are required for optimal activation of Hog1p in response to both citric acid and NaCl stress. This also explains why the Δssk1 and Δpbs2 strains are almost as sensitive to citric acid stress as the Δhog1 strain.
Activation of the HOG pathway by citric acid is not due to osmotic stress.
The HOG pathway is involved in the adaptation of cells to a high-osmolarity environment and is required for the growth of S. cerevisiae
in media supplemented with high concentrations of NaCl (0.9 M) or sorbitol (1.5 M) (13
). Therefore, we considered the possibility that because relatively high concentrations of citric acid were required to inhibit yeast growth, the observed inhibitory effect of citric acid could be a consequence of osmotic stress. If true, this would also explain our observation that the HOG pathway plays a role in mediating resistance to citric acid.
To investigate this possibility, we measured the osmolarity of 150 and 300 mM citric acid in ME medium (pH 3.5) using a vapor pressure osmometer. The osmolarity, calculated from two independent readings, of ME medium (pH 3.5) was determined to be 83 mmol kg−1 (Table ). The addition of 150 and 300 mM citric acid increased the osmolarity to 263 and 460 mmol kg−1, respectively. To compare this osmolarity with concentrations of compounds commonly used to study osmotic stress in S. cerevisiae, we generated a standard curve of increasing concentrations of sorbitol, KCl, and NaCl in ME medium (pH 3.5) versus measured osmolarity (data not shown). From this standard curve, the osmolarity of 150 mM citric acid was found to be equivalent to that of 230 mM sorbitol, 100 mM KCl, and 125 mM NaCl, and the osmolarity of 300 mM citric acid was equivalent to that of 460 mM sorbitol, 210 mM KCl, and 250 mM NaCl (Table ).
Effects of equivalent osmolarities of citric acid, sorbitol, KCl, and NaCl on glycerol production and growth rate of S. cerevisiae BY4741 MATa and Δhog1 deletion mutant
Next, we measured the effects of 150 and 300 mM citric acid (pH 3.5) and equivalent osmolarities of sorbitol, KCl, and NaCl on the growth rate of the parent strain and the Δhog1 deletion strain, which is unable to grow in high-osmolarity media. Table shows the percentage growth inhibition calculated by comparing the growth rate in ME medium (pH 3.5) and the growth rate during exposure to equivalent osmolarities of citric acid, sorbitol, NaCl, and KCl.
The growth rate of the parent strain was inhibited by 10 and 34% in the presence of 150 and 300 mM citric acid, respectively, but no growth inhibition was observed upon exposure to equivalent osmolarities of sorbitol, NaCl, or KCl. Thus, we can conclude that the growth inhibitory effect of citric acid at these concentrations is not due to osmotic stress. Importantly, the Δhog1 strain was very sensitive to citric acid, and growth was inhibited by 33 and 59% by 150 and 300 mM citric acid, respectively. Exposure of Δhog1 to an osmolarity of sorbitol, NaCl, or KCl equivalent to that of 150 mM citric acid resulted in no growth inhibition. There was minor inhibition of Δhog1 upon exposure to an osmolarity of sorbitol, NaCl, or KCl equivalent to that of 300 mM citric acid but only between 9 and 14% compared to 59% with 300 mM citric acid (Table ). Thus, these results clearly show that the principal inhibitory effect of citric acid is not due to osmotic stress.
To test this conclusion further, we determined whether any citric acid-sensitive gene deletions we identified in our genome-wide screen were known to be sensitive to osmotic stress according to the MIPS mutant phenotype catalogue entry osmotic sensitivity. Of the 43 genes known to display osmotic sensitivity, only genes that encode proteins that are components of the HOG pathway also showed sensitivity to citric acid. All the other osmotically sensitive genes, apart from Δvps33, were not sensitive to citric acid, including Δsat4, Δrvs161, Δgpd1, Δppz1, and Δrts1 (Table ).
Finally, activation of the HOG pathway results in an increase in intracellular glycerol concentration that allows the cells to grow in a high-osmolarity environment. This response is mediated by an increase in the expression of GPD1
). Therefore, we also measured the concentrations of intracellular glycerol in cells exposed to 150 and 300 mM citric acid and compared them to those of cells grown in equivalent osmolarities of sorbitol, KCl, and NaCl. Table shows that after a 3-h exposure to 150 and 300 mM citric acid, the intracellular concentration of glycerol does increase from 2.9 to 4.3 and 6.8 mmol g−1
protein, respectively. This correlates with our observation that exposure to citric acid results in increased expression of GPD1
. These values were very similar to the intracellular glycerol concentrations measured when S. cerevisiae
was exposed to equivalent osmolarities of sorbitol, KCl, and NaCl (Table ). Therefore, despite our observation that citric acid activates the HOG pathway via a mechanism distinct from osmotic stress, one of the major consequences of HOG pathway activation, namely, production of intracellular glycerol, does occur during citric acid stress.
Protein expression is up- and down-regulated via the HOG pathway on exposure to citric acid.
We have presented evidence that the HOG pathway mediates adaptation to citric acid. To further investigate how Hog1p is regulating the cellular resistance mechanism to citric acid, we studied changes in protein expression occurring in response to citric acid in the Δhog1 deletion strain in the presence and absence of 300 mM citric acid, pH 3.5. Changes in the Δhog1 proteome were then compared with the changes in the proteome already observed for the parent strain in the presence and absence of citric acid.
Over the pH range 4 to 7, we reproducibly detected the up-regulation of seven new proteins due to growth in the presence of citric acid; expression of the seven new proteins was dependent on deletion of HOG1 (Fig. ). These proteins were identified by peptide mass fingerprinting (see Table S4 in the supplemental material). The seven new proteins were brain modulosignalin homologue (Bmh1p) (protein 23), beta subunit of pyruvate dehydrogenase (Pdb1p) (protein 24), dihydroorotate dehydrogenase (Ura1p) (protein 25), fructose bisphosphate aldose (Fba1p) (protein 26), hypothetical protein (Ydr533cp) (protein 27), 6-phosphogluonate dehydrogenase (Gnd1p) (protein 28), and arginase (Car1p) (protein 29) (Fig. ). We can conclude that HOG1 negatively regulates the expression of these proteins during exposure to citric acid stress.
FIG. 7. Identification of citric acid-induced proteins whose expression was dependent on deletion of the HOG1 gene. Protein expression in wild-type (WT) S. cerevisiae BY4741 and Δhog1 mutant in the presence or absence of 300 mM citric acid (CA), pH 3.5, (more ...)
We also observed that two of the proteins previously identified to be up-regulated upon exposure to citric acid were no longer up-regulated in the Δhog1 deletion strain (Fig. ). These proteins were identified as heat shock protein 70 isoform (Ssa1p) (protein 8) (Fig. ) and enolase 2 (Eno2p) (protein 13) (Fig. ) (see Table S1 in the supplemental material). Thus, citric acid-induced expression of these proteins must be under the control of Hog1p.
FIG. 8. Identification of two citric acid-induced proteins whose expression is dependent on fully functional HOG1. Protein expression in wild-type (WT) S. cerevisiae BY4741 and Δhog1 mutant in the presence or absence of 300 mM citric acid (pH 3.5) is (more ...)
Finally, we detected two proteins whose expression was unaffected by exposure to citric acid alone but whose expression was repressed in the Δhog1 deletion strain only in the presence of citric acid. These proteins were identified as 40S ribosomal protein S0-B (Rps0bp) (protein A) (Fig. ) and GAL4 enhancer protein (Egd2p) (protein B) (Fig. ). Thus, a functional Hog1p is required to maintain the expression of these two proteins during exposure to citric acid.
None of these Hog1p-regulated proteins correlated with HOG1
-regulated genes detected after treatment with 0.7 M NaCl (31
). Growth sensitivity assays of the corresponding deletion strains of the above genes did not reveal any sensitivity to citric acid stress, indicating that despite the fact that these proteins are regulated by Hog1p during citric acid stress, they are not themselves essential for optimal adaptation.