Weak organic acid stress inhibits aromatic amino acid uptake by yeast, causing a strong influence of amino acid auxotrophies on the phenotypes of membrane transporter mutants Bettina E. Bauer 1, *, Danielle Rossington 2, *, Mehdi Mollapour 3, *, Yasmine Mamnun 1 , Karl Kuchler 1 and Peter W. Piper 3 1 Department of Molecular Genetics, University and BioCenter of Vienna, Austria; 2 Unilever Research Colworth, Sharnbrook, Bedford, UK; 3 Department of Biochemistry and Molecular Biology, University College London, UK The ability of yeasts to grow in the presence of weak organic acid preservatives is an important cause of food spoilage. Many of the determinants of acetate resistance in Sac- charomyces cerevisiae differ from the determinants of resistance to the more lipophilic sorbate and benzoate. Interestingly, we show in this study that hypersensitivity to both acetate and sorbate results when the cells have auxo- trophic requirements for aromatic amino acids. In trypto- phan biosynthetic pathway mutants, this weak acid hypersensitivity is suppressed by supplementing the medium with high levels of tryptophan or, in the case of sorbate sensitivity, by overexpressing the Tat2p high affinity tryp- tophan permease. Weak acid stress therefore inhibits uptake of aromatic amino acids from the medium. This allows auxotrophic requirements for these amino acids to strongly influence the resistance phenotypes of mutant strains. This property must be taken into consideration when using these phenotypes to attribute functional assignments to genes. We show that the acetate sensitivity phenotype previously ascribed to yeast mutants lacking the Pdr12p and Azr1p plasma membrane transporters is an artefact arising from the use of trp1 mutant strains. These transporters do not confer resistance to high acetate levels and, in prototrophs, their presence is actually detrimental for this resistance. Keywords: Saccharomyces cerevisiae; weak organic acid food preservatives; plasma membrane transporters; Pdr12p; Azr1p. The resistance of yeasts to the small number of weak organic acids allowed in food preservation allows these organisms to cause large-scale spoilage of preserved foods and beverages [1]. Saccharomyces cerevisiae is able to grow in the presence of sorbate due to the War1p transcription factor-dependent induction of a single ATP-binding cassette (ABC) transporter, Pdr12p [2,3]. So strong is this Pdr12p induction in sorbate-stressed cells, that levels of this transporter in the plasma membrane approach those of the most abundant plasma membrane protein, plasma membrane H + -ATPase [1,2]. Pdr12p appears to be acting as an efflux pump for weak organic carboxylate anions. It has been directly shown that it lowers the intracellular levels of benzoate and fluorescein by catalysing an active efflux of these compounds from the cell [2,4]. The inhibitory effects of different organic acids on cells of the Dpdr12 mutant indicate that Pdr12p confers resistance to sorbate, benzoate and aliphatic short chain (C 3)8 ) carboxylic acids of reasonable water solubility [5,6]. A similar spectum of acids is also capable of mediating the War1p-dependent induction of Pdr12p [3,6]. These are acids that cannot generally be degraded by S. cerevisiae, instead exerting appreciably cytotoxic or cytostatic effects mainly through their ability to disrupt membrane structures [1]. Pdr12p does not confer resistance to either acetate (this study) or highly lipophilic, long chain acids [5]. Acetate is far less inhibitory to yeasts than the more lipophilic sorbate (trans,trans-hexanedienoate), even though these are two carboxylate compounds of identical pKa (4.76) [2,7–9]. This is thought to be because the latter compound has a much higher capacity to dissolve in membranes and so disorder membrane structure [1]. Nev- ertheless S. cerevisiae is often inhibited by the presence of high acetate levels in wine fermentations [10], partly through the glyoxylate cycle enzymes needed for the assimilation of acetate and propionate in aerobic cultures being glucose- repressed ([11] and references therein). Our initial studies on Dpdr12 and wild-type cells indicated the former mutant to be more sensitive to high acetate levels [2]. A mutant lacking another plasma membrane transporter, Azr1p, was also shown to be acetate sensitive [12]. We show here that these effects on acetate resistance are only seen with auxotrophic mutants that need to assimilate aromatic amino acids from the culture medium. This appears to have led to incorrect assignments of function to the yeast Pdr12p and Azr1p transporters in studies that have used trp mutant backgrounds. Our data indicate that certain auxotrophic markers present in widely used yeast strains Correspondence to P. W. Piper, Department of Biochemistry and Molecular Biology, University College London, London WC1E 6BT. Fax: + 44 207 6797193, Tel.: + 44 207 6792212, E-mail: piper@bsm.bioc.ucl.ac.uk Abbreviation: ABC, ATP-binding cassette. Note: *The first three authors contributed equally to this work. (Received 30 April 2003, revised 30 May 2003, accepted 3 June 2003) Eur. J. Biochem. 270, 3189–3195 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03701.x may have led to other incorrect assignments of gene function, in studies where these assignments are based on drug susceptibility or resistance phenotypes. Materials and methods Yeast strains The yeast strains used in this study are listed in Table 1. The AZR1 gene was deleted using the kanMX4 cassette system [13]. Construction of a TAT2 overexpression plasmid The TAT2 gene was amplified from genomic DNA by PCR, using primers that introduced SalIsitesat)12 relative to the ATG and at 162 bp downstream of the stop codon. This PCR fragment was then Sal1-digested and cloned into the SalI site located between the ADH1 promoter and termi- nator of expression vector pAD4M [14]. The correct DNA sequence of the TAT2 insert of the resulting vector was confirmed by sequencing. Yeast growth Yeast was cultured aerobically at 30 °C in liquid YP medium [1% (w/v) Difco Yeast Extract, 2% Bacto peptone, 20 mgÆL )1 adenine], containing as carbon source either 2% glucose (YPD); or 2% galactose (YPGalactose). Media were titrated to pH 4.5 or pH 6.8 prior to autoclaving. Bioscreen culture of strains on pH 4.5 YPD (30 °C) in the presence of weak acid stress and the plating of strains on pH 4.5 YPD plates containing stress agents were both as described previously [2,4,5]. For Bioscreen culture cells were diluted in fresh YPD, pH 4.5, and inoculated into the wells of a Bioscreen microtitre plate (100 well honeycomb; Life Sciences International) to give an inoculum size of 5.0 · 10 3 cellsÆml )1 as described previously [5,15]. The stated concentrations of acetic or sorbic acid were then added to the wells. Growth at 30 °C with continuous shaking was then monitored by change in 600 nm optical density using a Labsystems Bioscreen automated turbidometric analyser (Life Sciences International, Basingstoke, UK). Analysis of Pdr12p levels Pdr12p levels were analysed by immunoblotting, as des- cribed earlier [2,3,6]. Results and discussion Loss of Pdr12 or Azr1p only decreases acetate resistance in strains with auxotrophic requirements for aromatic amino acids Our earlier studies of the growth of Dpdr12 and wild type cells indicated that the former mutant is more sensitive to acetate at pH 4.5 [2]. Another study reported that Azr1p, a plasma membrane transporter of the major facilitator superfamily, also confers acetate resistance [12]. We there- fore constructed a double Dpdr12 Dazr1 mutant (Materials and methods; Table 1), initially with the intention of determining which of these proteins, Pdr12p or Azr1p, might be more important for acetate resistance. We then investigated the resistances of this double deletant and the corresponding Dpdr12 and Dazr1 single gene deletes to acetate and sorbate. Growth on plates containing increasing levels of sorbate or acetate indicated that the loss of Azr1p did not result in increased sensitivity to either of these acids (Fig. 1; strains with no auxotrophic requirements for aromatic amino acids). It appeared though that acetate and sorbate sensi- tivities were being enhanced with the loss of both Azr1p and Pdr12p (Fig. 1, Dpdr12 Dazr1 mutant). Remarkably, the loss of Pdr12p alone in this background resulted in slightly increased resistance to acetate (Fig. 1). To investigate the effects of high levels of Pdr12p induction on acetate resistance we used a strain (Table 1, Table 1. S. cerevisiae strains used in this study. Strain Genotype Source FY1679–11c MATa ura3-52 his3-D200 leu2-D1 Euroscarf FY73 MATa ura3-52 his3-D200 Euroscarf FY1679–28c MATa ura3-52 his3-D200 leu2-D1 trp1-D63 Euroscarf YYM19 MATa ura3-52 his3-D200 leu2-D1 trp1-D63 Dpdr12::hisG [2] FY809 MATa ura3-52 his3-D200 leu2-D1 Dpdr12::hisG FY1679–11c x YYM19 11c-azr1D MATa ura3-52 his3-D200 leu2-D1 Dazr1::kanMX4 This study 11c-pdr12D azr1D MATa ura3-52 his3-D200 leu2-D1 Dpdr12::hisG, Dazr1::kanMX4 This study GAL1-PDR12 MATa ura3-52 his3-D200 leu2-D1 trp1-D63 PDR12::kanMX4-proGAL1 [6] BY4741 MATa ura3-0 his3-D1 leu2-D0 met15-D0 Euroscarf trp1D MATa ura3-0 his3-D1 leu2-D0 met15-D0 Dtrp1-kanMX4 Euroscarf trp2D MATa ura3-0 his3-D1 leu2-D0 met15-D0 Dtrp2-kanMX4 Euroscarf trp3D MATa ura3-0 his3-D1 leu2-D0 met15-D0 Dtrp3-kanMX4 Euroscarf trp4D MATa ura3-0 his3-D1 leu2-D0 met15-D0 Dtrp4-kanMX4 Euroscarf trp5D MATa ura3-0 his3-D1 leu2-D0 met15-D0 Dtrp5-kanMX4 Euroscarf YPH499 MATa ura3-52 lys2-801 am ade2-101 oc trp1-D63 his3-D200 leu2-D1 [20] YPH499 TRP + YPH499 trp1-D63::pRS304(TRP1) This study war1-42 YPH499 war1-42 This study 3190 B. E. Bauer et al.(Eur. J. Biochem. 270) Ó FEBS 2003 GAL1-PDR12) in which the PDR12 gene is not under the control of its normal promoter, but controlled instead by the GAL1 promoter. This was because the PDR12 gene is not normally induced by acetate [6], but during growth of this GAL1-PDR12 strain on galactose can be induced to levels similar to those seen in wild-type cells exposed to sorbic acid stress [6]. Growth of normal yeast on galactose relative to glucose slightly decreases acetate resistance (Fig. 2), yet the strong Pdr12p induction with the growth of the GAL1-PDR12 strain on galactose decreases acetate resistance still further (Fig. 2). This contrasts with the greatly increased sorbate resistance that results from same GAL1 promoter-directed induction of Pdr12p [6]. It provides yet further evidence that the induction of Pdr12p, though beneficial for resistance to C 3)8 aliphatic carboxylic acids and to sorbate [2,6], is actually somewhat detrimental for resistance to acetate. These results appeared to contradict our previous find- ings of a decreased acetate resistance with the loss of Pdr12p [2]. The strains used in these earlier studies were identical to those used to obtain the data in Fig. 1, except that they possessed the additional trp1-D63 mutation (Table 1). When we retested the trp1-D63 strains used in our earlier work [2], we found that they were generally less weak acid resistant than the corresponding TRP + strains in Fig. 1. Nevertheless, were still able to confirm our earlier findings of a decreased acetate resistance with the loss of Pdr12p in the trp1-D63 genetic background. We therefore investigated Fig. 1. Growth of strains on YPD (pH 4.5) in the presence of the indicated levels of sorbate or acetate. Wild-type (W+), Dpdr12 (FY809) and Dazr1 single mutants, and a Dpdr12 Dazr1 double mutant (all isogenic to FY1679–11c; Table 1) were tested. An undiluted overnight culture, and 1 : 10 and 1 : 100 serial dilutions, were spotted onto solid pH 4.5 YPD and the plates photographed after 3 days at 30 °C. Fig. 2. The effects of Pdr12p overexpression on growth in the presence of increased levels of acetate. The two trp1 strains analysed are isogenic but for the PDR12 gene being under native promoter control in the wild-type (FY1679–28c; W+) and under GAL1 promoter control in the strain GAL1- PDR12. An undiluted overnight culture, and 1 : 10 and 1 : 100 serial dilutions, were spotted onto solid pH 4.5 YPD or YPGalactose and the plates photographed after 3 days at 30 °C. Ó FEBS 2003 Auxotrophy effects on yeast transporter mutants (Eur. J. Biochem. 270) 3191 whether trp1-D63, and other mutations leading to require- ments for aromatic amino acids, might be causing an unusually high sensitivity to weak organic acid stress. Auxotrophic requirements for aromatic amino acids dramatically increase sensitivity to weak organic acid stress, a sensitivity suppressed by amino acid supplementation Platings of an isogenic series of strains (haploids derived from a YYM19 X FY73 cross; Table 1) on both sorbate- and benzoate-containing agar confirmed that trp1-D63 enhances sensitivities to these two acids (Fig. 3). This was apparent even in those cells that lacked Pdr12p (Fig. 3), showing that trp1-D63 acts to increase weak acid sensitivity independently of the Pdr12p ABC transporter. Similar results (not shown) were obtained when these same strains were plated in the presence of high concentrations of acetate. Next, mutants were obtained from the Euroscarf collec- tion that lack specific enzymes of the aromatic amino acid biosynthetic pathway. These were then tested for their sensitivities to both acetate and sorbate. Growth of the wild-type (BY4741), together with its aro and trp mutant derivatives on acetate and sorbate plates at pH 4.5 revealed that the aro1D, aro2D, aro7D, trp1D, trp2D, trp3D, trp4D and trp5D mutations all (in a similar manner to trp1-D63; Fig. 3) hypersensitize cells to acetate and sorbate (not shown). Defects in several of the enzymes of aromatic amino acid biosynthesis therefore hypersensitize yeast to weak organic acid stress. We also investigated the growth of this same series of strains after they had been inoculated into pH 4.5 liquid medium, either with no weak acid present or with acetate or sorbate (Materials and methods). Figure 4A,B shows representative culture data for the TRP + strain BY4741 and its trp5D mutant derivative, either in the absence or the presence of acetate stress. The trp5D mutant is clearly much more sensitive than the wild-type to the inhibitory effects of acetate under these conditions (Fig. 4A,B). The trp1D, Fig. 3. Both the Dtrp1 and Dpdr12 mutations increase sensitivity to sorbate and benzoate. Isogenic haploid strains with the indicated abbreviated genotypes (all of mating type a, and also ura3) were spotted (undiluted; also as 1 : 10 and 1 : 100 serial dilutions) onto pH 4.5 YPD containing the indicated level of organic acid. The plates were photographed after 3d at 30 °C. Fig. 4. Bioscreen culture of the TRP + BY4741 wild-type (A,C) and its Dtrp5 mutant derivative (B,D), in the presence of no weak acid (j), 40m M acetate (n) and 80 m M acetate (m). Theculturesin(A)and(B) were grown in the absence of any medium tryptophan supplement, whereas the cultures in (C) and (D) were grown with a 50 m M tryp- tophan supplementation. 3192 B. E. Bauer et al.(Eur. J. Biochem. 270) Ó FEBS 2003 trp2D, trp3D and trp4D mutants were also compromised under these conditions (not shown), their growth being essentially similar to that of the trp5D mutant cells shown in Fig. 4. All of these mutants, unlike the BY4741 parent, must catalyse an uptake of tryptophan from the medium in order to grow. Suspecting that it might be this tryptophan uptake that is inhibited strongly by the weak acid stress, we investigated the effects of supplementing the medium with a high level of tryptophan (Fig. 4). While the trp5D mutant is extremely acetate-sensitive, this hypersensitivity is almost totally suppressed by a high level of tryptophan (Fig. 4B,D). Such tryptophan supplementation could restore the growth of the acetate-stressed trp5D mutant cells to that of control, acetate-stressed wild-type (BY4741) cells lacking such supplementation (Fig. 4A,D). Liquid culture of strain BY4741 and its trpD mutant derivatives in the absence or presence of sorbate showed that trp mutants are also hypersensitized to sorbate stress (consistent with the effects of the trp1-D63 mutation on growth on pH 4.5 YPD agar containing sorbate; Fig. 3). Figure 5 shows sample data for the TRP + BY4741 strain and its trp5D mutant derivative grown in the presence of 0.9 m M or 1.8 m M sorbate. Again, sorbate hypersensitivity was substantially suppressed with the addition of a high level of tryptophan to the growth medium. The latter supplementation almost restored the growth of the trp5D mutant to that of sorbate-stressed wild-type cells lacking such supplementation (Fig. 5). Control experiments showed that these high tryptophan levels have no effect on Pdr12p induction by sorbate (Fig. 6A). Furthermore, because exogenous tryptophan suppresses both the acetate (Fig. 4) and sorbate (Fig. 5B) sensitivities of trp mutant cells (only the latter acid, not the former, being a Pdr12p inducer [6]) it is clear that this suppression by exogenous tryptophan bears no relationship to the weak acid inducibility of Pdr12p. Overexpression of Tat2p increases sorbate resistance, but only in trp mutant backgrounds The above data reveals that a requirement for uptake of aromatic amino acids from the culture medium leads to unusually high sensitivity to weak organic acid stress. An increased capacity for cells to catalyse uptake of aromatic amino acids might therefore suppress this sensitivity. To obtain evidence of whether this is the case, we studied the effects of overexpressing the high affinity tryptophan perm- ease, Tat2p [16]. The TAT2gene was placed under the control of the strong, constitutive ADH1 promoter in the multicopy vector pAD4M, thereby yielding the plasmid pTAT2 (Materials and methods). This construct and the empty vector pAD4M were then introduced into both trp1-D63 and TRP1 versions of strain YPH499 (Table 1). The increased sorbate sensitivity due to trp1-D63 was substantially sup- pressed by the pTAT2 overexpression vector, a plasmid which could almost restore the growth of the sorbate-stressed trp1-D63 cells to the level of growth displayed by an isogenic TRP + prototroph (Fig. 7A). Furthermore, Pdr12p levels were not affected by TAT2 overexpression (Fig. 6B), indicating that this rescue of acid-stressed cells by pTAT2 acts independently of the Pdr12p transporter. Fig. 5. Bioscreen culture of BY4741 wild-type (h, j) and Dtrp5 mutant cells (m, n)atpH4.5.Cells were grown in the presence of either 0.9 m M (A) or 1.8 m M (B) sorbic acid, either in the absence (h, n)or the presence (j, m) of tryptophan supplementation. Fig. 6. Measurements of Pdr12p levels. (A) Both basal and sorbate- induced levels of Pdr12p are not altered by a tryptophan supplemen- tation (+). (B) Pdr12p levels do not undergo significant change in Tat2p-overexpressing cells; also Tat2p overexpression in the war1-42 mutant does not restore Pdr12p induction. Ó FEBS 2003 Auxotrophy effects on yeast transporter mutants (Eur. J. Biochem. 270) 3193 To corroborate the independence of the Tat2p effect from Pdr12p function, we took advantage of a loss-of-function mutant in War1p, the transcription factor responsible for PDR12 induction upon acid stress challenge [3]. This mutant, war1-42, fails to induce PDR12 expression when challenged with sorbate and is therefore sorbate-hypersen- sitive. We transformed this war1-42 mutant and its YPH499 parent (both trp1-D63 strains) with pTAT2 and the empty pAD4M vector. Transformants were then tested for their ability to grow in the presence of sorbate. While pAD4M could not sustain the growth of the war1-42 mutant on 0.75 m M sorbate, pTAT2 could enable these cells to tolerate sorbate at up to 1 m M (Fig. 7B). Hence, Tat2p overexpres- sion can partially rescue the sorbate sensitivity of the war1- 42 mutant, a mutant lacking Pdr12p induction, in a trp1 genetic background. Loss of Pdr12p or Azr1p increases acetate resistance in prototrophic backgrounds This investigation into the influences of aromatic amino acid auxotrophy on weak acid resistance was initiated in response to the finding that losses of Pdr12p or of Azr1p appeared to be exerting opposite effects on acetate resistance in trp1 and TRP + genetic backgrounds. In trp1 mutants the losses of these transporters increase sensitivity to acetate [2,12], whereas in TRP + cells the same losses are associated with either neutral effects or a decreased sensitivity to acetate (Fig. 1). Notably, neither Pdr12p nor Azr1p is actually induced by acetate stress [6] (M. Mollapour, unpublished results), although a GAL1 promoter-directed induction of Pdr12p clearly results in a reduced resistance to acetate (Fig. 2). Bioscreen culture (Fig. 8) confirmed that the loss of Azr1p alone does not increase the sorbate sensitivity of TRP + cells, though this loss slightly increased the sorbate sensitivity of the Dpdr12 mutant (Fig. 8B). Unexpectedly, the Dpdr12 and Dazr1 single gene deletes grew considerably better than the wild-type at pH 4.5 in the presence of 120 m M acetate (Fig. 8). After an extended lag phase, the Dpdr12 Dazr1 double mutant also grew considerably better than the wild-type (Fig. 8C). Loss of either Pdr12p or Azr1p appears therefore to be beneficial for growth in the presence of high concentrations of acetate. The reasons for this improved growth are not yet clear, but one possibility is that these mutants are not displaying the apoptotic events normally seen in yeast cells treated with high levels of acetate [17]. Such improved growth after an extended lag phase was not indicated by the initial plating (Fig. 1), underlining the importance of progressing from initial plating assays (Figs 1–3) to detailed studies of growth kinetics for any full characterization of the effects of a mutation on growth in the presence of a stress agent (Fig. 8). Importantly, this and other studies [18] strike a note of caution, as they highlight the strong influences that certain mutations in the genetic background can exert on stress resistances. It is unlikely that Pdr12p or Azr1p confers acetate resistance as suggested by earlier work [2,12]; indeed their loss may actually elevate this resistance (Fig. 8). Fig. 7. Effects of Tat2p overexpression on sorbate resistance. (A) Overexpression of Tat2p increases sorbate resistance, but only in a trp mutant background. (B) The same overexpression suppresses the sorbate sensitivity of a mutant (war1-42) that is defective in Pdr12p induction. Undiluted overnight cultures of the indicated transformants were serially diluted, spotted onto pH 4.5 YPD, and the plates incu- bated 3 days at 30 °C. Fig. 8. Bioscreen culture of TRP + wild-type (j), Dpdr12 (r) and Dazr1 (m) single mutants and a Dpdr12 Dazr1 double mutant (s)atpH4.5. Experiments were carried out in the presence of no stress agent (A), 0.9 m M sorbate (B), 80 m M acetate (C) or 120 m M acetate (D). 3194 B. E. Bauer et al.(Eur. J. Biochem. 270) Ó FEBS 2003 The bacterium Escherichia coli cannot synthesize aromatic amino acids when it suffers severe oxidative stress. This auxotrophy, the result of oxidation of the 1,2-dihydroxy- ethyl thiamine pyrophosphate intermediate of transketolase, is suppressed when cultures are supplemented with inter- mediates (e.g. shikimate) that allow aromatic amino acid synthesis to occur independently of the transketolase reac- tion [19]. Weak acid stress in yeast is acting in a fundament- ally different way. 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