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Article

Protein Kinase A Negatively Regulates the Acetic Acid Stress Response in S. cerevisiae

by
Natasha M. Bourgeois
1,2,
Joshua J. Black
1,3,
Manika Bhondeley
1,4 and
Zhengchang Liu
1,*
1
Department of Biological Sciences, University of New Orleans, New Orleans, LA 70148, USA
2
Center for Global Infectious Disease Research, Seattle Children’s Research Institute, Seattle, WA 98109, USA
3
Department of Molecular Biology and Genetics, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA
4
Kudo Biotechnology, 117 Kendrick Street, Needham, MA 02494, USA
*
Author to whom correspondence should be addressed.
Microorganisms 2024, 12(7), 1452; https://doi.org/10.3390/microorganisms12071452
Submission received: 30 June 2024 / Revised: 11 July 2024 / Accepted: 15 July 2024 / Published: 17 July 2024
(This article belongs to the Special Issue Yeast Fermentation 2.0)
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Abstract

:
Bioethanol fermentation from lignocellulosic hydrolysates is negatively affected by the presence of acetic acid. The budding yeast S. cerevisiae adapts to acetic acid stress partly by activating the transcription factor, Haa1. Haa1 induces the expression of many genes, which are responsible for increased fitness in the presence of acetic acid. Here, we show that protein kinase A (PKA) is a negative regulator of Haa1-dependent gene expression under both basal and acetic acid stress conditions. Deletions of RAS2, encoding a positive regulator of PKA, and PDE2, encoding a negative regulator of PKA, lead to an increased and decreased expression of Haa1-regulated genes, respectively. Importantly, the deletion of HAA1 largely reverses the effects of ras2∆. Additionally, the expression of a dominant, hyperactive RAS2A18V19 mutant allele also reduces the expression of Haa1-regulated genes. We found that both pde2Δ and RAS2A18V19 reduce cell fitness in response to acetic acid stress, while ras2Δ increases cellular adaptation. There are three PKA catalytic subunits in yeast, encoded by TPK1, TPK2, and TPK3. We show that single mutations in TPK1 and TPK3 lead to the increased expression of Haa1-regulated genes, while tpk2Δ reduces their expression. Among tpk double mutations, tpk1Δ tpk3Δ greatly increases the expression of Haa1-regulated genes. We found that acetic acid stress in a tpk1Δ tpk3Δ double mutant induces a flocculation phenotype, which is reversed by haa1Δ. Our findings reveal PKA to be a negative regulator of the acetic acid stress response and may help engineer yeast strains with increased efficiency of bioethanol fermentation.

1. Introduction

Weak organic acids are commonly used to preserve foods by inhibiting the growth of microorganisms. The budding yeast, Saccharomyces cerevisiae, has multiple regulators that counteract the inhibitory effect of weak acids on growth, including Msn2/4, Rim101, Haa1, War1, and Pdr1/3, among others (reviewed in [1]). Haa1 is specifically involved in cellular adaptation to short chain, less lipophilic weak acids, such as acetic acid and propionic acid, while War1 is involved in adapting cells to more lipophilic weak acids such as sorbic acid and benzoic acid [2,3]. Understanding the mechanisms of yeast adaptation to weak organic acids can help inform strategies to improve food preservation when microbial growth is to be limited and engineer yeast strains to increase the efficiency of industrial biofermentations where robust growth is desired.
Acetic acid is a common inhibitor in industrial fermentation processes, and the adaptive response in the presence of sublethal concentrations of acetic acid in S. cerevisiae has been actively researched in recent years [1,3,4,5,6,7,8,9]. In the presence of lethal concentrations of acetic acid, yeast cell death and apoptosis ensue [10,11,12]. S. cerevisiae has evolved a complex network of physiological and cellular responses to mitigate the effect of acetic acid. A key component of this adaptative response is the transcription factor Haa1, which activates the expression of many genes that contribute to detoxification and adaptation to acetic acid [4,6,13]. One of the primary responses is the upregulation of genes encoding an activator of the plasma membrane H+-ATPase, Hrk1, and membrane transporters Tpo2 and Tpo3, which help to export protons and possibly acetate to restore intracellular pH balance [3,14,15]. Other Haa1 targets, such as Spi1 and Ygp1, may contribute to the remodeling of the cell envelope and decrease the diffusion of acetic acid across the plasma membrane [3,16], while Yro2 localizes to the plasma membrane and is required for adaptation to acetic acid, but its mechanism is unclear [3,17].
Haa1 shuttles between the cytoplasm and nucleus [18] and is mostly localized in the cytoplasm of unstressed cells. Weak organic acid treatment induces its nuclear translocation and transcriptional activation of its target genes. Haa1 is a multiply phosphorylated protein [18,19]. Haa1 phosphorylation is partially mediated by Hrr25, one of the four isoforms of casein kinase protein in S. cerevisiae [19]. In hrr25 mutant cells, Haa1 is hypophosphorylated, which correlates with its increased nuclear localization and higher levels of expression of its target genes. Nucleocytoplasmic shuttling is mediated by specific importins and exportins (reviewed in [20]) and Msn5 is required for the nuclear export of Haa1 [18].
Protein kinase A is an important regulator of cell growth, metabolism, and stress responses (reviewed in [21]). In the presence of high-quality nutrients, PKA increases the expression of ribosome biogenesis and positively regulates cell growth and proliferation [22,23,24,25]. When nutrients are limited, reduced PKA activity allows cells to adapt to various stress conditions. PKA has been implicated in yeast cell death and apoptosis in the presence of lethal concentrations of acetic acid [10,26,27]. There is a report suggesting that PKA is involved in Haa1-mediated acetic acid stress response, but its role has not been directly examined [28,29]. The protein kinase, Yak1, has been proposed to work downstream or in a parallel pathway of PKA [28,30,31], and Malcher et al. [28] reported that Haa1 and Yak1 regulate the expression of a few common target genes. Here, we analyzed the effect of mutations that alter PKA activity in S. cerevisiae on the expression of Haa1-regulated genes and cellular fitness in response to acetic acid treatment. We uncovered a negative regulatory role of PKA in the acetic acid stress response in yeast.

2. Materials and Methods

2.1. Strains, Growth Media, and Growth Conditions

Yeast strains were grown at 30 °C in a YPD medium (1% bacto yeast extract (Fisher Scientific, Waltham, MA, USA), 2% bacto peptone (Fisher Scientific), 2% glucose (Fisher Scientific)), a YNBcasD medium (0.67% yeast nitrogen base (Fisher Scientific), 1% casamino acids (Fisher Scientific), 2% dextrose), synthetic dextrose minimal media (SD) (0.67% yeast nitrogen base without amino acids, 2% glucose), and an MM4 medium (0.17% yeast nitrogen base without amino acids and ammonium sulfate (Fisher Scientific), 0.265% ammonium sulfate (Fisher Scientific), 2% glucose, adjusted to pH 4) with or without 60 mM of acetic acid (Fisher Scientific). The SD and MM4 media were supplemented with uracil, leucine, histidine, methionine, and lysine (Sigma-Aldrich, St. Louis, MO, USA) at standard concentrations to cover auxotrophic requirements when required [32]. Agar (USBiological, Salem, MA, USA) was added at a final concentration of 2% for the solid medium. The yeast strains used in this study are listed in Table 1. Yeast strains carrying a hyperactive RAS2A18V19 allele were obtained by transformation with StuI-linearized pRS303-RAS2A18V19 (pZL3326). Transformants were selected on the SD medium without histidine. Yeast strains with TPO2, YRO2, YGP1–lacZ reporter genes integrated in the nuclear genome were obtained by transformation with respective lacZ reporter gene plasmids linearized by digesting with a restriction enzyme at a unique site. Transformants were selected on the SD medium without leucine (integrated TPO2–lacZ reporter gene) or on a YPD medium supplemented with 300 mg/L geneticin (integrated YRO2–lacZ or YGP1–lacZ reporter gene). Other deletion mutant strains were constructed by transforming yeast with respective gene knockout cassettes, dissecting sporulated heterozygous diploid strains, and/or crossing two mutant strains to obtain diploids for sporulation and dissection. Gene deletion mutations were confirmed by PCR genotyping.
Table 1. Yeast strains used in this study.
Table 1. Yeast strains used in this study.
StrainGenotypeSourceApplication
BY4741 MATa his3 Δ1 leu2Δ0 met17Δ0 ura3Δ0Lab. stockFigure 1A–D, Figure 2, Figure 3, Figure 4, Figure 5 and Figure 6
ZLY4370BY4741 ras1Δ::kanMX4This studyFigure 1A–D
ZLY4376BY4741 ras2Δ::kanMX4-2DThis studyFigure 1A–D, Figure 2 and Figure 3
ZLY4373BY4741 pde1Δ::kanMX4This studyFigure 1A–D
ZLY4368BY4741 pde2Δ::kanMX4This studyFigure 1A–D and Figure 3
ZLY4162BY4741 YRO2–lacZ::kanMX4This studyFigure 1E,F
ZLY4153BY4741 YGP1–lacZ::kanMX4This studyFigure 1E,F
ZLY4238BY4741 TPO2–lacZ::LEU2This studyFigure 1E,F
ZLY4322BY4741 YRO2–lacZ::kanMX4 RAS2::RAS2Ala18Val19::HIS3This studyFigure 1E,F
ZLY4319BY4741 YGP1–lacZ::kanMX4 RAS2::RAS2Ala18Val19::HIS3This studyFigure 1E,F and Figure 3
ZLY4323BY4741 TPO2–lacZ::LEU2 RAS2::RAS2Ala18Val19::HIS3This studyFigure 1E,F
ZLY4043BY4741 haa1Δ::kanMX4SGDP *Figure 2, Figure 3, Figure 5 and Figure 6
ZLY4470BY4741 haa1::HIS3 ras2::kanMX4This studyFigure 2
ZLY4419 ** MATa his3 Δ1 leu2Δ0 met17Δ0 ura3Δ0This studyFigure 3C
ZLY4424 ** MATa his3 Δ1 leu2Δ0 met17Δ0 ura3Δ0This studyFigure 3C
ZLY4411BY4741 ras2Δ::kanMX4-1DThis studyFigure 3C
ZLY4445BY4741 tpk1::kanMX4This studyFigure 4
ZLY4438BY4741 tpk2::kanMX4This studyFigure 4
ZLY4441BY4741 tpk3::kanMX4This studyFigure 4
ZLY4516 MATa ura3 Δ leu2Δ his3Δ tpk1::kanMX4 tpk2::kanMX4This studyFigure 4 and Figure 5
ZLY4518 MATα ura3 Δ leu2Δ his3Δ met17Δ lys2Δ tpk1::kanMX4 tpk2::kanMX4This studyFigure 4
ZLY4520 MATa ura3 Δ leu2Δ his3Δ lys2Δ tpk1::kanMX4 tpk3::kanMX4This studyFigure 4, Figure 5 and Figure 6
ZLY4521 MATα ura3 Δ leu2Δ his3Δ met17Δ tpk1::kanMX4 tpk3::kanMX4This studyFigure 4
ZLY4522 MATα ura3 Δ leu2Δ his3Δ met17Δ tpk2::kanMX4 tpk3::kanMX4This studyFigure 4 and Figure 5
ZLY4524BY4741 tpk2::kanMX4 tpk3::kanMX4This studyFigure 4
ZDY166BY4741 haa1::HIS3This studyFigure 5F
ZLY5264 MATa ura3 Δ leu2Δ his3Δ lys2Δ tpk1::kanMX4 tpk3::kanMX4 haa1::LEU2This studyFigure 5 and Figure 6
*, Saccharomyces genome deletion project [33]; **, this strain was obtained from tetrad analysis and had the same genotype as BY4741.

2.2. Plasmid Constructs

The plasmids used in this study are listed in Table 2. To generate the YGP1–lacZ reporter gene, two primers, 5′-GTCAGAATTCTTGGTAGACATGGTGGTGC-3′ and 5′-GTCAGTCGACCAGATAAAACAACTTGGAACTTCAT-3′, were used to amplify the DNA sequence from position −1980 to 25 in the promoter region of YGP1 using BY4741 genomic DNA as the template. The resulting PCR product was digested with EcoRI and SalI, and fused in frame to the E. coli LacZ gene in the centromeric plasmid pWEJ [34] to form plasmid pZL3170. To generate the TPO3–lacZ reporter gene in plasmid pZL3161, two primers, 5′-GTCAGGATCCTTGTCGAAATCGTCCAA-3′ and 5′-GTCAAAGCTTTGGATTCCTGTCTGTTCATTTC-3′, were used to amplify the DNA sequence from position −1976 to 19 in the promoter region of TPO3, which was cloned into the BamHI and HindIII sites of pWEJ. Similarly, two primers, 5′-GTCAGGATCCAATGGATACTCTTCGTATCG-3′ and 5′-GTCAAAGCTTGGAGCTTAGCGTTAGACAACAT-3′, were used to amplify the DNA sequence from position −980 to 22 in the promoter region of SPI1 for the construction of the SPI1–lacZ reporter gene plasmid pZL3155. pRS303-RAS2A18V19 (pZL3326) was constructed by cloning an EcoRI and BamHI-digested fragment encoding the hyperactive RAS2A18V19 allele from YCp50–RAS2A18V19 [35] into the corresponding cloning sites of pRS303. To generate YIp356–kanMX4, an integrative lacZ reporter gene plasmid carrying the kanMX4 selection marker, a portion of the URA3 open reading frame in YIp356 [36] was removed by digesting with NcoI and StuI and replaced by a Klenow-fragment-filled kanMX4 cassette digested with EcoRI and BglII. Integrative YRO2 and YGP1–lacZ reporter plasmids (pZL3203 and pZL3201) were constructed by cloning their respective promoters into YIp356–kanMX4. The integrative TPO2–lacZ reporter plasmid pZL3307 was generated by cloning the TPO2 promoter sequence into YIp366 [36]. The restriction enzymes were purchased from New England Biolabs (Ipswich, MA, USA).
Table 2. Plasmids used in this study.
Table 2. Plasmids used in this study.
PlasmidDescriptionReferenceApplication
pZL3164pRS416-YRO2–lacZ, expressing YRO2–lacZ reporter gene with 1929-bp YRO2 promoter sequence fused to lacZ coding sequence.[19]Figure 1A–D, Figure 2, Figure 4 and Figure 5
pZL3170pRS416-YGP1–lacZ, expressing YGP1–lacZ reporter gene with 1980 bp YGP1 promoter sequence fused to lacZ coding sequence.This studyFigure 1A–D, Figure 2, Figure 4 and Figure 5
YCp50-RAS2A18V19A hyperactive RAS2 mutant allele, RAS2A18V19, cloned in centromeric plasmid with a URA3 selection marker.[35]
pZL3326pRS303-RAS2A18V19, encoding the dominant active RAS2A18V19 allele on an integrative plasmid carrying a HIS3 selection marker.This studyFigure 1E,F and Figure 3
YIp356-kanMX4An integrative plasmid carrying a lacZ reporter gene and the kanMX4 selection marker.This study
pZL3203YIp356-kanMX4-YRO2, encoding an YRO2–lacZ reporter gene for integration into the genome.This studyFigure 1E,F
pZL3201YIp356-kanMX4-YGP1, encoding an YGP1–lacZ reporter gene for integration into the genome.This studyFigure 1E,F
pZL3307YIp366-TPO2, encoding an TPO2–lacZ reporter gene for integration into the genome.This studyFigure 1E,F
pZL3158pRS416-TPO2–lacZ, expressing TPO2–lacZ reporter gene with 1926 bp TPO2 promoter sequence fused to the lacZ coding sequence.[19]Figure 2 and Figure 5
pZL3161pRS416-TPO3–lacZ, expressing TPO3–lacZ reporter gene with 1976 bp TPO3 promoter sequence fused to the lacZ coding sequence.This studyFigure 5
pZL3155pRS416-SPI1–lacZ, expressing SPI1–lacZ reporter gene with 980 bp SPI1 promoter sequence fused to the lacZ coding sequence.This studyFigure 5

2.3. Yeast Transformation and β-Galactosidase Activity Assays

For transformation, yeast cells were freshly grown in YPD liquid medium and transformed using a high-efficiency method [37]. The YNBcasD medium, the SD medium supplemented with appropriate amino acids and uracil, and the YPD medium supplemented with geneticin were used to select yeast transformants based on the URA3, LEU2, and kanMX4 selection markers, respectively. For the β-galactosidase activity assays, the yeast strains were grown in the MM4 medium with or without 60 mM acetic acid at 30 °C for at least 6 generations to reach an OD600 of 0.5–0.8 before collection. The cells were collected by centrifugation, and the β-galactosidase activity assays were conducted as described [32]. During the pilot stage of the reporter gene analysis, a minimum of two independently obtained mutant strains were utilized in the analysis of the effect of a specific mutation on the expression of lacZ reporter genes and usually found to yield similar results. Two to six independent cultures were grown, and assays were carried out in duplicate for each sample. The data are presented as the mean ± standard deviation. The means of the β-galactosidase activity assay results were compared using the t-test. The “*” in figures indicates a significant difference in the means of two groups of data (p < 0.05). The two ends of a horizontal line under an “*” in the data figures mark the two strains under comparison.

2.4. Generation of Growth Curves

The wild-type and isogenic mutant strains were grown on the YPD solid medium for 2 to 3 days at 30 °C. The cells from the plate were then grown in the liquid MM4 medium overnight to an OD600 of 1~1.6 to obtain pre-cultures, which were diluted into the MM4 medium with and without 60 mM acetic acid with a starting OD600 of ~0.05. The OD600 values of the cell cultures were determined over time until they reached the stationary phase.

2.5. Serial Dilution of Cells for Growth Analysis

The wild-type and isogenic mutant strains were freshly grown on the YPD solid medium at 30 °C for 2–3 days. The cells were picked from a plate into sterile water and diluted to the same starting OD600 of 0.1. Five-fold serial dilutions were made using sterile 96-well plates and 8-channel pipettes. As described in the figure legend, 5 μL aliquots were spotted on solid medium. The cells were grown for 2 to 4 days at 30 °C before pictures were taken for cell growth analysis.

2.6. Differential Interference Contrast Microscopy of Yeast Cultures

The cells were grown in the MM4 medium without or with 60 mM acetic acid for a minimum of six generations to reach OD600 ≥ 0.6. Before coverslips were applied, 15 μL cell cultures were pipetted onto the slide for 3 min. Differential interference contrast (DIC) images of live cells were immediately captured using a Nikon Eclipse E800 microscope equipped with a Nikon Plan Fluor 40× DIC M objective lens (Nikon Inc., Melville, NY, USA). Images were acquired with a Photometrics Coolsnap fx charge-coupled device (CCD) camera and Metamorph imaging software (version 7.7) (Molecular Devices, Sunnyvale, CA, USA) and processed using ImageJ (version 1.53k) (National Institutes of Health, Bethesda, MD, USA).

3. Results

3.1. ras2Δ and pde2Δ Increase and Decrease the Expression of YRO2 and YGP1–lacZ Reporter Gene, Respectively

To determine the potential role of PKA in the regulation of Haa1-dependent gene expression, we analyzed the expression of lacZ reporter genes driven by the promoters of two well-established target genes of Haa1, YRO2 and YGP1 [3,38], in mutants with altered PKA activities. PKA is activated by the second messenger cyclic AMP, which is produced by adenylate cyclase (reviewed in [21]). Adenylate cyclase is activated by two Ras proteins, Ras1 and Ras2 [39]. The downregulation of PKA is achieved via two cyclic AMP phosphodiesterases, Pde1 and Pde2, which convert cyclic AMP to AMP [40]. We generated pde1Δ, pde2Δ, ras1Δ, and ras2Δ mutants by dissecting sporulated heterozygous diploid mutant strains and transformed them with centromeric plasmids encoding a YRO2–lacZ or YGP1–lacZ reporter gene. The transformants were grown in MM4 medium without or with 60 mM acetic acid for a minimum of six generations to reach the logarithmic phase, and the cells were collected for β-galactosidase activity assays. In the cells grown in the absence of acetic acid supplementation, pde2Δ reduced the expression of YRO2–lacZ by 4.3-fold, while ras2Δ increased its expression by 2.8-fold (Figure 1A). In the cells grown in the presence of 60 mM acetic acid, the overall effect of pde2Δ and ras2Δ on the expression of YRO2–lacZ reporter gene was still present, albeit at reduced levels: pde2Δ reduced YRO2–lacZ expression by 27%, while ras2Δ increased its expression by 2.1-fold (Figure 1B). Under both growth conditions, pde1Δ and ras1Δ did not significantly affect the expression of the YRO2–lacZ reporter gene. Similar results were obtained for the YGP1–lacZ reporter gene (Figure 1C,D). In the MM4 medium without acetic acid supplementation, pde2Δ reduced YGP1–lacZ expression by 8-fold, while ras2Δ increased its expression by 4.2-fold. In the MM4 medium supplemented with 60 mM acetic acid, pde2Δ reduced YGP1–lacZ expression by 20%, and ras2Δ increased its expression by 2.2-fold. As was observed for the YRO2–lacZ reporter gene, pde1Δ and ras1Δ did not result in significant changes in the expression of the YGP1–lacZ reporter gene. Our findings are consistent with the notion that PKA negatively regulates the expression of Haa1-regulated genes.
Microorganisms 12 01452 g001
Figure 1. The opposite effects of pde2Δ and RAS2A18V19 versus ras2Δ on the expression of YRO2–lacZ, YGP1–lacZ, and/or TPO2–lacZ reporter genes. (A–D) pde2Δ and ras2Δ have opposite effects on the expression of YRO2–lacZ and YGP1–lacZ reporter genes. Wild type (BY4741) and isogenic pde1Δ (ZLY4373), pde2Δ (ZLY4368), ras1Δ (ZLY4370), and ras2Δ (ZLY4376) mutant strains carrying a centromeric plasmid encoding a YRO2–lacZ (pZL3164, panels (A,B)) or YGP1–lacZ reporter gene (pZL3170, panels (C,D)) were grown in the MM4 medium without acetic acid (panels (A,C)) and with 60 mM acetic acid (panels (B,D)) to mid-logarithmic phase. β-galactosidase activity assays were conducted as described in Materials and Methods. The data are presented as the mean ± standard deviation. The means of the results were compared by t-test. “*” indicates a significant difference (p < 0.05) in the means of two groups of data indicated by the beginning and end of a horizontal line throughout this manuscript. (E,F) A dominant, hyperactive allele of RAS2, RAS2A18V19, decreases the expression of YRO2–, YGP1–, and TPO2–lacZ reporter genes. The yeast strains carrying an integrated reporter gene as indicated (YRO2–lacZ, ZLY4162; YGP1–lacZ, ZLY4153; TPO2–lacZ, ZLY4238) were transformed with an integrative plasmid encoding RAS2A18V19. The resultant transformants and non-transformed control strains were grown in the MM4 medium without (panel (E)) and with (panel (F)) 60 mM acetic acid. β-galactosidase activity assays were conducted.
Figure 1. The opposite effects of pde2Δ and RAS2A18V19 versus ras2Δ on the expression of YRO2–lacZ, YGP1–lacZ, and/or TPO2–lacZ reporter genes. (A–D) pde2Δ and ras2Δ have opposite effects on the expression of YRO2–lacZ and YGP1–lacZ reporter genes. Wild type (BY4741) and isogenic pde1Δ (ZLY4373), pde2Δ (ZLY4368), ras1Δ (ZLY4370), and ras2Δ (ZLY4376) mutant strains carrying a centromeric plasmid encoding a YRO2–lacZ (pZL3164, panels (A,B)) or YGP1–lacZ reporter gene (pZL3170, panels (C,D)) were grown in the MM4 medium without acetic acid (panels (A,C)) and with 60 mM acetic acid (panels (B,D)) to mid-logarithmic phase. β-galactosidase activity assays were conducted as described in Materials and Methods. The data are presented as the mean ± standard deviation. The means of the results were compared by t-test. “*” indicates a significant difference (p < 0.05) in the means of two groups of data indicated by the beginning and end of a horizontal line throughout this manuscript. (E,F) A dominant, hyperactive allele of RAS2, RAS2A18V19, decreases the expression of YRO2–, YGP1–, and TPO2–lacZ reporter genes. The yeast strains carrying an integrated reporter gene as indicated (YRO2–lacZ, ZLY4162; YGP1–lacZ, ZLY4153; TPO2–lacZ, ZLY4238) were transformed with an integrative plasmid encoding RAS2A18V19. The resultant transformants and non-transformed control strains were grown in the MM4 medium without (panel (E)) and with (panel (F)) 60 mM acetic acid. β-galactosidase activity assays were conducted.
Microorganisms 12 01452 g001

3.2. A Hyperactive Allele of RAS2, RAS2A18V19, Reduces the Expression of YRO2, YGP1, and TPO2–lacZ Reporter Genes

Ras2 and Pde2 are a positive and a negative regulator of PKA, respectively. To further test the role of PKA in the regulation of the expression of Haa1-dependent genes, we examined the effect of a hyperactive allele of RAS2, RAS2A18V19 [41,42] expressed from the low-copy centromeric plasmid YCp50. The glycine 19 to valine mutation in RAS2 leads to constitutive activation of PKA signaling. The lacZ reporter genes described in Figure 1A–D are encoded on the centromeric plasmid pRS416, which has the same URA3 selection marker as YCp50. Thus, we generated YRO2–lacZ and YGP1–lacZ reporters on integrative plasmids using the kanMX4 selection marker and integrated them into the nuclear genome. We added another Haa1-regulated gene, TPO2, and constructed an integrative TPO2–lacZ reporter gene carrying the LEU2 selection marker. The yeast strains carrying these integrated reporter genes showed a more than 12-fold increase in β-galactosidase activities in response to the treatment with 60 mM acetic acid (compare Figure 1E and Figure 1F), indicating that these three integrated lacZ reporter genes were valid readouts of the acetic acid stress response. Our initial analysis showed that the expression of RAS2A18V19 on YCp50 significantly reduced the expression of all three lacZ reporter genes in cells grown in the absence of acetic acid treatment. However, we noticed it took 2–3 times longer for the cells carrying the plasmid YCp50–RAS2A18V19, in comparison with an empty vector, to undergo six cell divisions in the MM4 medium supplemented with 60 mM acetic acid to reach the same OD600 reading of ~0.6. The growth defects of yeast strains grown in the presence of the 60 mM acetic acid due to the expression of RAS2A18V19 will be described later in Figure 3. We were concerned that the growth defect might affect the yeast cell’s ability to maintain the YCp50–RAS2A18V19 plasmid, which could complicate the interpretation of reporter gene activity data. Therefore, we generated an integrative plasmid encoding RAS2A18V19 and integrated it into the RAS2 genomic locus. The β-galactosidase activity assay showed that RAS2A18V19 significantly reduced the expression of YRO2–, YGP1–, and TPO2–lacZ reporter genes in cells grown in MM4 medium without acetic acid treatment (Figure 1E). In cells grown in the MM4 medium supplemented with 60 mM acetic acid, RAS2A18V19 marginally reduced the expression of these three reporter genes. Together, these data suggest that the activation of PKA due to pde2Δ or RAS2A18V19 reduces the expression of Haa1-regulated genes. Conversely, reduced PKA activity due to a ras2Δ mutation increases the expression of Haa1-regulated genes.

3.3. Increased Expression of YRO2–, YGP1–, and TPO2–lacZ Reporter Genes Due to ras2Δ Requires Haa1

Our data from Figure 1 suggest that PKA negatively regulates the expression of YRO2, YGP1, and TPO2, which are known target genes of Haa1. These target genes may require transcription factors other than Haa1. Thus, PKA may negatively regulate Haa1 or other transcription factors. To differentiate these two possibilities, we determined whether the increased expression of YRO2–, YGP1–, and TPO2–lacZ reporter genes due to ras2Δ required Haa1. Consistent with published findings, haa1Δ reduced the expression of these three reporter genes under both basal and acetic acid-inducing conditions (Figure 2). The increased expression of YRO2–lacZ and TPO2–lacZ due to ras2Δ was largely reversed by haa1Δ under both basal and acetic acid-inducing conditions. In contrast, the ras2Δ-induced expression of the YGP1–lacZ reporter gene was only partially reversed by haa1Δ. YGP1–lacZ has higher basal activity (compare Figure 2C with Figure 2A,E), suggesting that other transcription factors also contribute to YGP1 expression. The (partial) reversal of ras2Δ-induced expression of YRO2, TPO2, and YGP1 by haa1Δ suggest that ras2Δ haa1Δ mutant cells may be sensitive to acetic acid treatment. Figure 2G shows that this is indeed the case. ras2Δ led to slight growth defects on the solid MM4 medium. Surprisingly, ras2Δ mutant cells did not exhibit increased resistance to acetic acid despite having an increased expression of YRO2, YGP1, and TPO2. This paradox will be explained in the section below. Together, our data suggest that ras2Δ-induced expression of YRO2, YGP1, and TPO2 is (partially) dependent on Haa1.
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Figure 2. Haa1 is required or partially required for increased expression of YRO2–lacZ, YGP1–lacZ, and TPO2–lacZ reporter genes due to ras2Δ. Wild type (BY4741) and isogenic haa1Δ (ZLY4043), ras2Δ (ZLY4376), and ras2Δ haa1Δ (ZLY4470) mutant strains carrying a centromeric plasmid encoding a YRO2–lacZ (pZL3164, panels (A,B)), YGP1–lacZ (pZL3170, panels (C,D)), or TPO2–lacZ (pZL3158, panels (E,F)) reporter gene were grown in the MM4 medium without (panels (A,C,E)) and with 60 mM acetic acid (panels (B,D,F)). β-galactosidase activity assays were conducted. The data are presented as the mean ± standard deviation. (G) ras2Δ haa1Δ double mutant cells are sensitive to acetic acid stress. Wild-type and isogenic mutant strains described for panels (A–F) were serially diluted and spotted on MM4 medium without and with 60 mM acetic acid. “*” indicates a significant difference (p < 0.05) in the means of two groups of data indicated by the beginning and end of a horizontal line.
Figure 2. Haa1 is required or partially required for increased expression of YRO2–lacZ, YGP1–lacZ, and TPO2–lacZ reporter genes due to ras2Δ. Wild type (BY4741) and isogenic haa1Δ (ZLY4043), ras2Δ (ZLY4376), and ras2Δ haa1Δ (ZLY4470) mutant strains carrying a centromeric plasmid encoding a YRO2–lacZ (pZL3164, panels (A,B)), YGP1–lacZ (pZL3170, panels (C,D)), or TPO2–lacZ (pZL3158, panels (E,F)) reporter gene were grown in the MM4 medium without (panels (A,C,E)) and with 60 mM acetic acid (panels (B,D,F)). β-galactosidase activity assays were conducted. The data are presented as the mean ± standard deviation. (G) ras2Δ haa1Δ double mutant cells are sensitive to acetic acid stress. Wild-type and isogenic mutant strains described for panels (A–F) were serially diluted and spotted on MM4 medium without and with 60 mM acetic acid. “*” indicates a significant difference (p < 0.05) in the means of two groups of data indicated by the beginning and end of a horizontal line.
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3.4. pde2Δ and RAS2A18V19 Increase Acetic Acid Sensitivity While ras2Δ Improves Cellular Fitness in Response to Acetic Acid Stress

Haa1 is required for cellular adaptation to acetic acid stress. Our data thus far suggest that PKA negatively regulates Haa1. Altered Haa1 activity in mutants that affect PKA signaling may result in changes in cellular fitness in the presence of acetic acid. To test this possibility, we generated and compared the growth curves of wild-type, haa1Δ, RAS2A18V19, pde2Δ, and ras2Δ mutant strains grown in the MM4 medium without and with 60 mM acetic acid. In the absence of acetic acid treatment, haa1Δ, pde2Δ, and RAS2A18V19 mutant cells exhibited similar growth to the wild-type strain (Figure 3A). In contrast, in response to acetic acid treatment, pde2Δ and RAS2A18V19 mutant cells had similar growth defects to haa1Δ mutant cells (Figure 3B). In the absence of acetic acid treatment, ras2Δ led to slight growth defects (Figure 3A), which is consistent with the growth defects of ras2Δ mutants on the MM4 solid medium lacking acetic acid (Figure 2G). In the presence of acetic acid treatment, ras2Δ abolished the lag phase in the initial stage of acetic acid treatment, and ras2Δ mutant cells outperformed wild type (Figure 3B). We further compared the growth curves of four wild-type strain cultures (three independently obtained wild-type strains) and three ras2Δ mutant strain cultures (two independently obtained ras2 mutant strains) in the MM4 medium supplemented with 60 mM acetic acid and found that the phenotype was reproducible (Figure 3C).
Sensitivity to acetic acid treatment due to haa1Δ is one of the most prominent phenotypes in the acetic acid stress response pathway in S. cerevisiae. Reduced cellular fitness in pde2Δ and RAS2A18V19 mutant cells in response to acetic acid treatment is consistent with our model that PKA is a negative regulator of Haa1. Improved cellular fitness of ras2Δ mutant cells in response to acetic acid stress further lends support to this model. When yeast cells encounter weak organic acids at inhibitory concentrations, cells arrest growth initially and may resume exponential growth after an extended lag phase (reviewed in [1]). When the adapted cells are transferred to a fresh medium with the same level of weak acids, no growth delay is detected. ras2Δ cells in Figure 3 resemble wild-type cells that have adapted to acetic acid stress. This is consistent with our notion that PKA is a negative regulator of acetic acid stress in yeast.
If ras2Δ abolishes the lag phase as seen in Figure 3B,C, should not the ras2Δ mutant cells show a relative growth advantage over the wild-type on the MM4 solid medium supplemented with 60 mM acetic acid in Figure 2G? This could be reconciled by the difference in yeast cell growth on the solid medium versus the liquid medium. In the serial dilution analysis of cell growth presented in Figure 2G, cells were picked from the plate after 2–3 days on the YPD medium and transferred into water for determining OD600. Almost all cells from the YPD plate were unbudded at this stage. It is possible that both the wild-type and ras2Δ mutant cells have low PKA activity at this stage of cell growth. Their transfer to water for serial dilutions may further reduce the difference, if any, in PKA activity. This would lead to the observed result in Figure 2G: the ras2Δ cells do not lead to a growth advantage over the wild type on the MM4 medium supplemented with acetic acid. In Figure 3, the precultures were grown in the MM4 liquid medium overnight to the mid–late logarithmic phase before they were diluted into the MM4 medium without and with acetic acid to initiate the experiment for the generation of growth curves. It is expected that the PKA pathway activities are different between the wild-type and ras2Δ mutant cells in the logarithmic phase of cell growth in the dextrose medium.
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Figure 3. Mutations in PDE2 and HAA1, and expression of the hyperactive RAS2A18V19 allele reduce cellular fitness in response to acetic acid stress, while ras2Δ increases it. (A,B) The growth curves of wild-type and isogenic mutant strains as indicated were generated from cells grown in MM4 medium without acetic acid (panel (A)) and with 60 mM acetic acid (panel (B)). The growth curves are representative of data from two independent cultures. WT, BY4741; haa1Δ, ZLY4043; RAS2A18V19, ZLY4319; pde2Δ, ZLY4368; ras2Δ, ZLY4376. (C) The growth curves of four cultures of wild type and three cultures of haa1Δ mutant strains in MM4 medium supplemented with 60 mM acetic acid. WT #1 and #2, BY4741; WT #3, ZLY4419; WT #4, ZLY4424; ras2 #1 and #2, ZLY4376; ras2 #3, ZLY4411.
Figure 3. Mutations in PDE2 and HAA1, and expression of the hyperactive RAS2A18V19 allele reduce cellular fitness in response to acetic acid stress, while ras2Δ increases it. (A,B) The growth curves of wild-type and isogenic mutant strains as indicated were generated from cells grown in MM4 medium without acetic acid (panel (A)) and with 60 mM acetic acid (panel (B)). The growth curves are representative of data from two independent cultures. WT, BY4741; haa1Δ, ZLY4043; RAS2A18V19, ZLY4319; pde2Δ, ZLY4368; ras2Δ, ZLY4376. (C) The growth curves of four cultures of wild type and three cultures of haa1Δ mutant strains in MM4 medium supplemented with 60 mM acetic acid. WT #1 and #2, BY4741; WT #3, ZLY4419; WT #4, ZLY4424; ras2 #1 and #2, ZLY4376; ras2 #3, ZLY4411.
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3.5. Mutations in TPK1 (TPK3) and TPK2 Have Opposite Effects on YRO2 and YGP1 Expression

PKA has three catalytic subunits, Tpk1, Tpk2, and Tpk3 [43]. Although they are redundant for cell viability, these yeast Tpk isoforms are known to have nonredundant, pathway-specific roles (reviewed in [44]). To determine which catalytic subunit is involved in the regulation of acetic acid stress responsive genes, we analyzed the expression of YRO2– and YGP1–lacZ reporter genes in strains carrying a single deletion of TPK1, TPK2, or TPK3. In cells grown in the absence of acetic acid treatment, tpk1Δ and tpk3Δ single deletion strains led to an increased expression of both YRO2– and YGP1–lacZ reporter genes, while tpk2Δ reduced their expression (Figure 4A,C). Between tpk1Δ and tpk3Δ, tpk3Δ had a stronger effect than tpk1Δ, suggesting that Tpk3 is a stronger inhibitor of YRO2 and YGP1 expression than Tpk1. In the cells grown in the presence of 60 mM acetic acid, the inducing effects due to tpk1Δ and tpk3Δ single mutations were largely maintained, and the effect of tpk2Δ on the expression of YRO2– and YGP1–lacZ reporter genes was abolished or reversed (Figure 4B,D).
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Figure 4. Mutations in TPK1 (TPK3) and TPK2 have opposite effects on the expression of YRO2 and YGP1. (A–D) The effect of tpk1Δ, tpk2Δ, and tpk3Δ single mutations on the expression of YRO2–lacZ and YGP1–lacZ reporter genes. Wild-type (BY4741) and isogenic tpk1Δ (ZLY4445), tpk2Δ (ZLY4438), and tpk3Δ (ZLY4441) mutant strains carrying a centromeric plasmid encoding a YRO2–lacZ (pZL3164, panels (A,B)) or YGP1–lacZ (pZL3170, panels (C,D)) reporter gene were grown in MM4 medium without acetic acid (panels (A,C)) and with 60 mM acetic acid (panels (B,D)) to mid-logarithmic phase. β-galactosidase activity assays were conducted. The data are presented as the mean ± standard deviation. (E–H) The effect of tpk1/2Δ, tpk1/3Δ, and tpk2/3Δ double mutations on the expression of YRO2–lacZ and YGP1–lacZ reporter genes. Wild type (BY4741) and isogenic tpk1/2Δ (ZLY4516 and ZLY4518), tpk1/3Δ (ZLY4520 and ZLY4521), and tpk2/3Δ (ZLY4522 and ZLY4524) were analyzed for the expression of YRO2–lacZ and YGP1–lacZ reporter genes as described for panels (A–D). The data are presented as the mean ± standard deviation. “*” indicates a significant difference (p < 0.05) in the means of two groups of data indicated by the beginning and end of a horizontal line.
Figure 4. Mutations in TPK1 (TPK3) and TPK2 have opposite effects on the expression of YRO2 and YGP1. (A–D) The effect of tpk1Δ, tpk2Δ, and tpk3Δ single mutations on the expression of YRO2–lacZ and YGP1–lacZ reporter genes. Wild-type (BY4741) and isogenic tpk1Δ (ZLY4445), tpk2Δ (ZLY4438), and tpk3Δ (ZLY4441) mutant strains carrying a centromeric plasmid encoding a YRO2–lacZ (pZL3164, panels (A,B)) or YGP1–lacZ (pZL3170, panels (C,D)) reporter gene were grown in MM4 medium without acetic acid (panels (A,C)) and with 60 mM acetic acid (panels (B,D)) to mid-logarithmic phase. β-galactosidase activity assays were conducted. The data are presented as the mean ± standard deviation. (E–H) The effect of tpk1/2Δ, tpk1/3Δ, and tpk2/3Δ double mutations on the expression of YRO2–lacZ and YGP1–lacZ reporter genes. Wild type (BY4741) and isogenic tpk1/2Δ (ZLY4516 and ZLY4518), tpk1/3Δ (ZLY4520 and ZLY4521), and tpk2/3Δ (ZLY4522 and ZLY4524) were analyzed for the expression of YRO2–lacZ and YGP1–lacZ reporter genes as described for panels (A–D). The data are presented as the mean ± standard deviation. “*” indicates a significant difference (p < 0.05) in the means of two groups of data indicated by the beginning and end of a horizontal line.
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We next examined the effect of tpk1Δ tpk2Δ, tpk1Δ tpk3Δ, and tpk2Δ tpk3Δ double deletions on the expression of YRO2 and YGP1 using β-galactosidase activity assays. Consistent with a mild induction in the expression of YRO2– and YGP1–lacZ reporter genes in the tpk1Δ and tpk3Δ single deletion strains, a tpk1Δ tpk3Δ double deletion further increased their expression under both basal and acetic acid stress conditions (compare Figure 4E,F to Figure 4A,B and Figure 4G,H to Figure 4C,D). Additionally, tpk2Δ reversed the increased expression of YRO2– and YGP1–lacZ reporter genes in tpk3Δ mutant cells to the wild-type levels under both growth conditions. tpk2Δ also reversed the increased expression of YRO2– and YGP1–lacZ reporters due to tpk1Δ below wild-type levels in the absence of acetic acid stress. In cells grown in the presence of acetic acid, tpk2Δ reversed their expression in tpk1Δ mutant cells to the wild-type levels. Taken together, our data indicate that Tpk1 and Tpk3 play a redundant, negative regulatory role in the expression of YRO2 and YGP1, while Tpk2 antagonizes the effect of Tpk1 and Tpk3.
The opposing roles of the three catalytic subunits of PKA in a cellular process have been reported previously [28,45,46]. Tpk2 activates filamentous growth while Tpk1 and Tpk3 inhibit it. This is achieved through the interaction of the three catalytic subunits with different downstream effectors (reviewed in [47]). The mechanism behind the opposite effects of Tpk1 (Tpk3) and Tpk2 on the expression YRO2 and YGP1 is currently unknown.

3.6. haa1Δ Largely Abolishes the Increased Expression of YRO2, TPO2, TPO3, and SPI1 in tpk1Δ tpk3Δ Double Mutant Cells

A tpk1Δ tpk3Δ double mutation leads to the highest levels of expression of YRO2– and YGP1–lacZ reporter genes among tpk single and tpk double mutant cells grown in the absence of acetic acid treatment (Figure 4). To determine whether this is due to activation of Haa1, we introduced haa1Δ into tpk1Δ tpk3Δ double mutants and analyzed the expression of these reporter genes. We also included three more lacZ reporter genes under the control of the promoters of TPO2, TPO3, and SPI1 in the analysis. Figure 5 shows that haa1Δ largely abolished the increased expression of YRO2, TPO2, TPO3, and SPI1–lacZ reporter genes due to tpk1Δ tpk3Δ in cells grown in the absence of acetic acid treatment. haa1Δ had little effect in reducing the expression of the YGP1–lacZ reporter gene in the tpk1Δ tpk3Δ double mutant. haa1Δ also had the weakest effect in reducing YGP1–lacZ expression compared to YRO2– and TPO2–lacZ reporter genes in ras2Δ mutant cells (Figure 2), suggesting that the increased expression of YGP1–lacZ due to ras2Δ and tpk1Δ tpk3Δ is dependent on additional transcription factor(s) and/or regulatory mechanisms. The reversal of the tpk1Δ tpk3Δ-induced expression of these reporter genes by haa1Δ suggests that tpk1Δ tpk3Δ haa1Δ triple mutant cells are sensitive to acetic acid stress, which is confirmed by the growth data in Figure 5F. Together, our data suggest that Haa1 is activated in tpk1Δ tpk3Δ double mutant cells, resulting in the increased expression of Haa1-dependent genes.
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Figure 5. Haa1 is largely required for increased expression of YRO2–lacZ, TPO2–lacZ, TPO3–lacZ, and SPI1–lacZ reporter genes in tpk1/3Δ mutant cells grown in MM4 medium. Wild type (BY4741) and isogenic haa1Δ (ZLY4043), tpk1/3Δ (ZLY4520), and tpk1/3Δ haa1Δ (ZLY5264) mutant strains carrying a centromeric plasmid encoding a YRO2–lacZ (panel (A)), YGP1–lacZ (panel (B)), TPO2–lacZ (panel (C)), TPO3–lacZ (panel (D)), or SPI1–lacZ (panel (E)) reporter gene were grown in MM4 medium without acetic acid to mid-logarithmic phase. β-galactosidase activity assays were conducted. The data are presented as the mean ± standard deviation. (F) haa1Δ single and tpk1Δ tpk3Δ haa1Δ triple mutant cells are equally sensitive to acetic acid stress. Wild type (BY4741) and isogenic tpk1/2Δ (ZLY4516), tpk2/3Δ (ZLY4522), tpk1/3Δ (ZLY4520), haa1Δ (ZDY166), and tpk1/3Δ haa1Δ (ZLY5264) mutant strains were serially diluted and spotted on MM4 medium without and with 60 mM acetic acid. “*” indicates a significant difference (p < 0.05) in the means of two groups of data indicated by the beginning and end of a horizontal line.
Figure 5. Haa1 is largely required for increased expression of YRO2–lacZ, TPO2–lacZ, TPO3–lacZ, and SPI1–lacZ reporter genes in tpk1/3Δ mutant cells grown in MM4 medium. Wild type (BY4741) and isogenic haa1Δ (ZLY4043), tpk1/3Δ (ZLY4520), and tpk1/3Δ haa1Δ (ZLY5264) mutant strains carrying a centromeric plasmid encoding a YRO2–lacZ (panel (A)), YGP1–lacZ (panel (B)), TPO2–lacZ (panel (C)), TPO3–lacZ (panel (D)), or SPI1–lacZ (panel (E)) reporter gene were grown in MM4 medium without acetic acid to mid-logarithmic phase. β-galactosidase activity assays were conducted. The data are presented as the mean ± standard deviation. (F) haa1Δ single and tpk1Δ tpk3Δ haa1Δ triple mutant cells are equally sensitive to acetic acid stress. Wild type (BY4741) and isogenic tpk1/2Δ (ZLY4516), tpk2/3Δ (ZLY4522), tpk1/3Δ (ZLY4520), haa1Δ (ZDY166), and tpk1/3Δ haa1Δ (ZLY5264) mutant strains were serially diluted and spotted on MM4 medium without and with 60 mM acetic acid. “*” indicates a significant difference (p < 0.05) in the means of two groups of data indicated by the beginning and end of a horizontal line.
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3.7. tpk1Δ tpk3Δ Double Mutants Yield Flocculent Cultures in Response to Acetic Acid Stress

When growing cultures for β-galactosidase activity assays, we noticed that the tpk1Δ tpk3Δ mutant cells are flocculent in the MM4 medium supplemented with 60 mM acetic acid (Figure 6A). In the MM4 medium without the supplementation of acetic acid, the tpk1Δ tpk3Δ mutant cells were not flocculent. The tpk single mutants, tpk1Δ tpk2Δ, and tpk2Δ tpk3Δ double mutant strains did not yield flocculent cultures in the MM4 medium supplemented with acetic acid either. Surprisingly, despite similar phenotypes exhibited by the ras2Δ and tpk1Δ tpk3Δ mutant cells, ras2Δ did not result in flocculation in the MM4 medium supplemented with 60 mM acetic acid. Culture flocculence is noticeable when large aggregates of cells are formed. To determine whether the tpk1Δ tpk3Δ double mutant cells in the absence of acetic acid treatment form small clusters, we examined cell cultures under microscope. The microscopic analysis showed that the tpk1Δ tpk3Δ mutant cells grown in the absence of 60 mM acetic acid had similar clustering (or lack of) to the wild-type, while they formed large clusters in response to acetic acid stress (Figure 6B,C). Since the tpk1Δ tpk3Δ mutant cells formed clusters only in the presence of acetic acid, we investigated whether Haa1 was required for the flocculation phenotype. We found that the tpk1Δ tpk3Δ haa1Δ triple mutant cells did not yield flocculent cultures in the MM4 medium supplemented with 60 mM acetic acid (Figure 6). The flocculation phenotype observed in Figure 6 may be related to the expression of FLO11. Mutations in TPK1/3 and TPK2 are known to have opposite effects on FLO11 expression [45,46]. It is possible that the increased expression of FLO11 in tpk1Δ tpk3Δ double mutant cells somehow leads to flocculation in response to acetic acid stress. Together, our data suggest that Haa1 activation in tpk1Δ tpk3Δ mutant cells grown in the presence of acetic acid contributes to culture flocculence. Flocculation can be beneficial in industrial fermentation processes by facilitating the removal of yeast cells (reviewed in [48]). This phenotype may be useful for engineering acetic acid-resistant strains for bioethanol production and other industrial fermentation processes.
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Figure 6. A tpk1Δ tpk3Δ double mutation leads to a Haa1-dependent flocculation phenotype in response to acetic acid stress. (A) Pictures of culture tubes containing wild type (BY4741) and isogenic haa1Δ (ZLY4043), tpk1/3Δ (ZLY4520), and tpk1/3Δ haa1Δ (ZLY5264) mutant strains grown in the MM4 medium without and with 60 mM acetic acid in the mid–late log phase. (B) Differential interference contrast (DIC) images of yeast cells from cultures, as described in panel (A). (C) A differential interference contrast image of a large aggregate of tpk1/3Δ cells (flattened between the cover slip and slide) grown in the MM4 medium supplemented with 60 mM acetic acid.
Figure 6. A tpk1Δ tpk3Δ double mutation leads to a Haa1-dependent flocculation phenotype in response to acetic acid stress. (A) Pictures of culture tubes containing wild type (BY4741) and isogenic haa1Δ (ZLY4043), tpk1/3Δ (ZLY4520), and tpk1/3Δ haa1Δ (ZLY5264) mutant strains grown in the MM4 medium without and with 60 mM acetic acid in the mid–late log phase. (B) Differential interference contrast (DIC) images of yeast cells from cultures, as described in panel (A). (C) A differential interference contrast image of a large aggregate of tpk1/3Δ cells (flattened between the cover slip and slide) grown in the MM4 medium supplemented with 60 mM acetic acid.
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4. Discussion

Haa1 plays an important role in mediating cellular adaptation to acetic acid stress by activating the expression of many acetic acid-responsive genes. In this report, we show that PKA is a negative regulator of the acetic acid stress response. Our conclusion is based on the observation that PKA-activating mutations, namely, pde2Δ and RAS2A18V19, reduce the expression of Haa1-regulated genes. Conversely, mutations that reduce the activity of PKA, ras2Δ, and tpk1Δ tpk3Δ increase the expression of Haa1-regulated genes. The reduced expression of Haa1-regulated genes in pde2Δ and RAS2A18V19 mutants correlates with increased sensitivity to acetic acid. In contrast, the increased expression of Haa1-regulated genes in ras2Δ mutants improves cellular fitness in response to acetic acid stress. PKA is an important regulator of cell growth, development, and stress response pathways. Our report cements a new role for PKA as an important player in the acetic acid stress response.
How do PKA and Haa1 work together in mediating the expression of Haa1-responsive genes? One possibility is that Haa1 acts downstream of PKA in a linear pathway. In this scenario, PKA negatively regulates the activity of Haa1. Alternatively, PKA and Haa1 may work in parallel pathways in an antagonistic manner. We failed to detect an interaction between Haa1 and Tpk1, Tpk2, or Tpk3 via yeast two-hybrid and/or coimmunoprecipitation analyses, as well as significant changes in the phosphorylation state of Haa1 in mutants that alter the PKA activity. Although it can be challenging to observe an interaction between a protein kinase and its target due to the inherent disengagement after the target is phosphorylated, the failure to detect an interaction between Haa1 and Tpk1/2/3 increases the possibility that PKA may regulate Haa1 indirectly or function in a parallel pathway of Haa1. Other regulatory factors, for example, Msn2/4 and Yak1, have been associated with the expression of acetic acid-responsive genes [1,16,28]. Msn2/4 and Yak1 have been proposed to function downstream or in a parallel pathway of PKA [21,28,49,50]. Future work is needed to elucidate the mechanism by which PKA regulates the expression of Haa1-dependent genes.
PKA regulates the expression of acetic acid-responsive genes, so it is conceivable that PKA activity may be regulated by acetic acid. If so, how does PKA sense acetic acid? PKA is under the control of many positive and negative regulators. There can be many possibilities. One facile explanation is that reduced cytoplasmic pH in response to acetic acid treatment regulates the activity of PKA. Cytoplasmic pH has been proposed to be a second messenger that regulates the PKA pathway [51]. In their study, Dechant et al. proposed that high cytoplasmic pH activates PKA while low pH inactivates it. Incubation of yeast cells with acetic acid in the growth medium at a pH below the pKa value of acetic acid can lead to a reduction in the cellular pH (reviewed in [1]). We propose that low cytoplasmic pH due to acetic acid stress leads to reduced PKA activity and consequently increased expression of Haa1-regulated genes. However, this notion seems to contradict the observation of PKA activation by low pH [52]. In their study, Colombo et al. found that the addition of the protonophore 2,4-dinitrophenol to yeast cells at low extracellular pH caused an activation of Ras2 and an increase in cAMP levels. Future work is needed to reconcile these discrepancies.
The involvement of PKA in the acetic acid stress response in yeast opens new possibilities for designing acetic acid-resistant strains for biofermentation applications. Research exploring genetic and biochemical strategies to improve yeast robustness against acetic acid stress can ultimately enhance the efficiency and productivity of fermentation-based industries.

Author Contributions

J.J.B. had the original idea to analyze the role of PKA on the expression of Haa1-regulated genes, conducted pilot experiments on the expression of lacZ reporter genes in mutants with altered PKA activity, and generated the data in Figure 1. N.M.B. and M.B. conducted the analysis of gene expression presented in Figure 2, Figure 4 and Figure 5. Z.L. generated the data for Figure 3 and Figure 6, coordinated the study, and wrote the paper. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the NIH grant 1R15GM094772-01A1.

Data Availability Statement

The original contributions presented in the study are included in the article, further inquiries can be directed to the corresponding author.

Acknowledgments

We thank Jeanne Hirsch and Kelly Tatchell for sharing the YCp50-RAS2A18V19 plasmid, Sylvester Tumusiime and Zhejun Dong for technical support.

Conflicts of Interest

Author Manika Bhondeley was employed by the company Kudo Biotechnology. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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Bourgeois, N.M.; Black, J.J.; Bhondeley, M.; Liu, Z. Protein Kinase A Negatively Regulates the Acetic Acid Stress Response in S. cerevisiae. Microorganisms 2024, 12, 1452. https://doi.org/10.3390/microorganisms12071452

AMA Style

Bourgeois NM, Black JJ, Bhondeley M, Liu Z. Protein Kinase A Negatively Regulates the Acetic Acid Stress Response in S. cerevisiae. Microorganisms. 2024; 12(7):1452. https://doi.org/10.3390/microorganisms12071452

Chicago/Turabian Style

Bourgeois, Natasha M., Joshua J. Black, Manika Bhondeley, and Zhengchang Liu. 2024. "Protein Kinase A Negatively Regulates the Acetic Acid Stress Response in S. cerevisiae" Microorganisms 12, no. 7: 1452. https://doi.org/10.3390/microorganisms12071452

APA Style

Bourgeois, N. M., Black, J. J., Bhondeley, M., & Liu, Z. (2024). Protein Kinase A Negatively Regulates the Acetic Acid Stress Response in S. cerevisiae. Microorganisms, 12(7), 1452. https://doi.org/10.3390/microorganisms12071452

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