Yeast response and tolerance to benzoic acid involves the Gcn4- and Stp1-regulated multidrug/multixenobiotic resistance transporter Tpo1

Abstract

The action of benzoic acid in the food and beverage industries is compromised by the ability of spoilage yeasts to cope with this food preservative. Benzoic acid occurs naturally in many plants and is an intermediate compound in the biosynthesis of many secondary metabolites. The understanding of the mechanisms underlying the response and resistance to benzoic acid stress in the eukaryotic model yeast is thus crucial to design more suitable strategies to deal with this toxic lipophilic weak acid. In this study, the Saccharomyces cerevisiae multidrug transporter Tpo1 was demonstrated to confer resistance to benzoic acid. TPO1 transcript levels were shown to be up-regulated in yeast cells suddenly exposed to this stress agent. This up-regulation is under the control of the Gcn4 and Stp1 transcription factors, involved in the response to amino acid availability, but not under the regulation of the multidrug resistance transcription factors Pdr1 and Pdr3 that have binding sites in TPO1 promoter region. Benzoic acid stress was further shown to affect the intracellular pool of amino acids and polyamines. The observed decrease in the concentration of these nitrogenous compounds, registered upon benzoic acid stress exposure, was not found to be dependent on Tpo1, although the limitation of yeast cells on nitrogenous compounds was found to activate Tpo1 expression. Altogether, the results described in this study suggest that Tpo1 is one of the key players standing in the crossroad between benzoic acid stress response and tolerance and the control of the intracellular concentration of nitrogenous compounds. Also, results can be useful to guide the design of more efficient preservation strategies and the biotechnological synthesis of benzoic acid or benzoic acid-derived compounds.

Keywords: Adaptation and tolerance to benzoic acid; Multidrug/multixenobiotic resistance transporters; Polyamines; Saccharomyces cerevisiae; TPO1/Tpo1; Weak acid food preservatives.

Fig. 1

TPO1 expression is required for a more rapid yeast adaptation to benzoic acid stress but not to low pH when a strong acid is used as the acidulant. a Growth curves of S. cerevisiae BY4741 (filled square, open square) or of the derived deletion mutant tpo1Δ (filled circle, open circle) in MM4 growth medium (at pH 4.0) either (open symbols) or not (closed symbols) supplemented with benzoic acid (0.9 mM) or b in this same basal medium acidified at pH 2, 3.5 or 5 using HCl as the acidulant (lower panel). c Growth curves of S. cerevisiae BY4741 and tpo1Δ harbouring an empty vector (open square and open circle, respectively) or the same pYEP351 vector with the TPO1 gene cloned (open triangle and open diamond, respectively) supplemented (open symbols) or not (closed symbols) with 0.5 mM benzoic acid. The growth curves shown are representative of, at least, three independent experiments that gave rise to the same growth patterns

Fig. 2

TPO1 overexpression leads to an enhanced tolerance to benzoic acid induced stress. Growth curves of S. cerevisiae BY4741.GPD (filled square, open square) and BY4741.GPD_TPO1 (filled triangle, open triangle) in MM4 growth medium (pH 4.0) either or not supplemented with benzoic acid (0.7–1.1 mM). The growth curves shown are representative of, at least, three independent experiments that gave rise to the same results

Fig. 3

Subcultivation of benzoic acid-adapted cells shows yeast cell adaptation independently of Tpo1 expression. Growth curves of wild-type (open square, grey square) and derived deletion mutant tpo1Δ (open circle, grey circle) in the presence of 0.9 mM benzoic acid (open symbols) in MM4 (pH 4.0). Grey symbols represent the subcultivation in fresh medium MM4 supplemented with 0.9 mM benzoic acid of cells harvested in the time-point marked with the dashed line. Cells cultures were followed by measuring OD_600nm (a) and colony forming units per millilitre (b). The results are representative of, at least, three independent experiments, and error bars represent standard deviation

Fig. 4

TPO1 is up-regulated under benzoic acid stress in a Gcn4- and Stp1-dependent manner a Growth curve of S. cerevisiae BY4741 (filled square, open square) and of the deletion mutants BY4741_gcn4Δ (filled triangle, open triangle) and BY4741_stp1Δ (filled circle, open circle) in MM4 growth medium (at pH 4.0) (closed symbols) or in this same basal medium supplemented with 0.9 mM benzoic acid (open symbols). Quantification of TPO1 mRNA levels during the growth curve of the three strains in the presence or absence of benzoic acid was based on quantitative real-time RT-PCR. For each strain, the transcript levels of the TPO1 gene shown are relative to the transcript levels registered in exponential-phase cells (at an OD_600nm of 0.4) cultivated in unsupplemented MM4 growth medium (at pH 4.0). In all samples, TPO1 mRNA levels were normalized using ACT1 transcript levels. b Effect of the Gcn4 response elements (GRE) and Stp1 response elements (SRE) located in the TPO1 promoter in the benzoic acid-induced up-regulation of TPO1, measured, through RT-PCR, as the mRNA level of the LacZ gene, expressed under the control of the TPO1 promoter. Wild-type cells harbouring the pYEP354w_TPO1::lacZ plasmid (which contains natural TPO1 promoter) or the derived mutant constructs having the GRE and SRE motifs individually inactivated were cultivated in MM4 growth medium (at pH 4) (black bars) or in this same growth medium supplemented with 0.9 mM benzoic acid (white bars) and harvested after 3 h of growth

Fig. 5

Effect of benzoic acid stress and TPO1 expression in the internal pool of polyamines and amino acids. a Spermidine, spermine and putrescine intracellular levels after 1 h of incubation in the absence (black bars) or presence (white bars) of benzoic acid (0.9 mM). b Intracellular amino acid pools of BY4741 (black bars) and BY4741_tpo1Δ (white bars) in the presence or absence of 0.9 mM benzoic acid. The amino acids added to the MM4 growth medium to suppress the auxotrophies of the BY4741 strain are underlined. The arrows indicate amino acids whose concentration was below the detection limit in the acid-challenged cells. Wt wild-type

Fig. 6

TPO1 is up-regulated under ammonium or leucine limitation. a Expression of the TPO1 gene during the first 3 h of growth of the prototrophic strain 2344c in minimal growth medium supplemented with a limiting (lim; open triangle and grey bars) (0.0265 g L⁻¹) or a saturating (sat; open square and black bars) (2.65 g L⁻¹) concentration of ammonium sulphate as the sole nitrogen source. Growth curve of the 2344c strain in the two conditions of ammonium availability is also shown. b Expression of TPO1 during growth of S. cerevisiae BY4741 (filled square, open square) or of gcn4Δ (filled circle, open circle) in MM4 growth medium. Cell samples were harvested during exponential growth (sample 1) and when cells approached the stationary phase at an OD_600nm of 1.0 (sample 2). At this point, the cultures were split in two, and fresh leucine was added to one of the cultures (closed symbols) while the other remained in the leucine-exhausted growth medium (open symbols, dashed curve). After 1 h of incubation in the leucine-exhausted (sample 3) or in the leucine-replete growth medium (sample 4), cells were harvested and TPO1 transcription levels were assessed. The results presented are means of at least three independent experiments, and error bars represent standard deviation

Fig. 7

Effect of Gcn4 and Stp1 in the regulation of genes involved in synthesis, uptake and excretion of polyamines. Documented regulatory associations between the genes involved in synthesis, uptake and excretion of polyamines and Gcn4 and Stp1 are shown according with the information available in the YEASTRACT database (dashed lines), plus the data described herein on the effect of Stp1 and Gcn4 over TPO1 expression (full lines). Genes up-regulated by benzoic acid stress are highlighted in grey boxes while down-regulated genes are shown in white boxes (Abbott et al. 2007)