W ability to grow on media containing phenethyl alcohol (PEA; 0.3% v/v),

Transcription

1 Copyright by the Genetics Society of America SENSITIVITY OF TRYPTOPHAN, TYROSINE AND PHENYLALANINE MUTANTS OF SACCHAROMYCES CEREVISIAE TO PHENETHYL ALCOHOL JAMES H. MEADE' AND THOMAS R. MANNEY Department of Physics, Kansas State University, Manhattan, Kansas Manuscript received August 3,1981 Revised copy accepted February 23,1983 ABSTRACT Phenethyl alcohol inhibits the growth of many microorganisms. It is believed that the growth inhibition is mediated by its effect on the cell membrane. Differences between sensitive and resistant strains are suggested to be due to alterations in membrane structure. We report that, in some strains, an unexpected relationship exists between auxotrophy for tryptophan, tyrosine and phenylalanine and sensitivity to phenethyl alcohol. HILE testing several of our strains of Saccharomyces cerevisiae for the W ability to grow on media containing phenethyl alcohol (PEA; 0.3% v/v), we observed that all of our trp5 mutants (tryptophan synthetase deficient) were sensitive to PEA. This observation was surprising since to our knowledge no previous studies of the effects of PEA had shown any correlation between the biosynthesis of tryptophan and resistance to PEA, PEA is a growth inhibitor of many microorganisms (LESTER 1965; YURA and WADA 1968; TAKAHASHI 1972; SILVA et al. 1976) and has been postulated to primarily affect the cytoplasmic membrane (SILVER and WENDT 1967). Strains resistant to PEA have been reported in Escherichia coli (YURA and WADA 1968; WADA and YURA 1974) and in S. cerevisiae (TAKAHASHI 1972). In E. coli (YURA and WADA 1968), a comparison of PEA-resistant and -sensitive strains showed a difference in permeability for potassium ions between the strains, and it was suggested that an alteration in membrane structure was responsible for the difference between resistant and sensitive strains. In this paper we describe the isolation and characterization of sensitive mutants from resistant strains and of resistant mutants from sensitive strains of S. cerevisiae. We have found that many genes, including those required for aromatic amino acid biosynthesis, are involved in the expression of the wildtype level of resistance to PEA. MATERIALS AND METHODS Strains: The four principal strains of S. cerevisiae used in this study are: X2180-1A (a gal2), X2180-1B (a go&), XP300-26C (a thr4 his6 lysl ga12) and XP300-29B (a trp5-18 ade2-1 his6 lysl gal2). Current address: Cetus Corporation, 600 Bancroft Way, Berkeley, California Genetics 1Q4: June, 1983.

2 236 J. H. MEADE AND T. R. MANNEY Media: The following standard culture media were used in these experiments. YEPAD: Difco yeast extract, 10 g/liter, Difco Bacto-Peptone, 20 g/liter; dextrose, 20 g/liter; adenine, 80 mg/liter; agar, 20 g/liter. MV (minimal + vitamins): Difco yeast nitrogen base without amino acids and ammonium sulfate, 1.45 g/liter; ammonium sulfate, dextrose, 20 g/liter; agar, 20 &liter (after WICKERHAM 1946). For some experiments MV was supplemented as follows: adenine (ade), 30 mg/liter; L- histidine (his), 20 mg/liter; L-lysine (lys), 40 mg/liter; L-phenylalanine (phe), 50 mg/liter; L-threonine (thr), 150 mg/liter; L-tryptophan (trp), 30 mg/liter; or L-tyrosine (tyr) 20 mg/liter. MB (minimal + biotin): dextrose agar, 20 &liter; ammonium sulfate, 5 g/liter; potassium phosphate monobasic, 1 g/liter; magnesium sulfate, 0.5 g/liter; sodium chloride, 0.1 g/liter; calcium chloride, 0.1 g/liter; biotin, 20 mg/liter, boric acid, 500 pg/liter; copper sulfate, 40 &liter; manganese sulfate, 400 pg/liter; molybdic acid, sodium salt, 200 pg/liter; and zinc sulfate, 400 pg/liter. Unless stated otherwise, phenethyl alcohol (Eastman Kodak, Rochester, New York) was added to the media at a concentration of 0.3% (v/v). Indole was added to the media at a concentration of 10 mg/liter. Genetic methods: All mutants were isolated by plating appropriately diluted samples of yeast cells on agar medium, irradiating with ultraviolet radiation from a 15-watt GE germicical lamp for 17 s at a distance of 26 cm and, after 2 days of growth, replica plating to an appropriate test medium. Complementation was scored by a test employing replica plating. Cultures were mixed on YEPAD, incubated overnight to permit mating, and then replica plated to the appropriate test medium. RESULTS Association of sensitivity to PEA with a requirement for tryptophan: An examination of the ability of four of our strains to grow in the presence of various concentrations of PEA (Table 1) yielded results similar to those reported by TAKAHASHI (1972). Although none of the strains could grow on a medium with 0.4% PEA, XP300-29B was more sensitive than the rest in that it could not grow at 0.3% PEA. We examined the segregation of sensitivity (no growth at 0.3% PEA) and resistance (growth at 0.3% PEA) in spores from tetrads previously isolated from crosses of XP300-29B with XP300-26C or with strains related to XP300-26C and observed a 22 segregation pattern indicative of a single nuclear gene. In all of the tetrads, sensitivity to PEA segregated with the trp5-18 allele (PD = 173, NPD = 0, TT = 0). We then tested 47 independent trp5 mutants which had previously been isolated from X2180-1A, X2180-1B or strains isogenic to these two strains for their sensitivity to PEA. All of the trp5 mutants, in contrast to their parent strains, were sensitive to PEA. Therefore, sensitivity to PEA can be correlated with mutations at the TRP5 locus. Examination of five TABLE 1 Effect of PEA on growth of several yeast strains Growth in presence of various concentrations of PEA Strain 0% 0.2% 0.3% 0.4% - X2180-1A X2180-1B - XP300-26C XP300-29B

3 PHENETHYL ALCOHOL SENSITIVITY 237 other mutants that had been isolated from X2180-1A or X2180-1B, representing the other four TRP loci, showed that they were also sensitive to PEA. Complementation tests showed that both sensitivity to PEA and auxotrophy for tryptophan are expressed as recessive traits. The TRP5 gene contains the genetic information for an enzyme that catalyzes the last three reactions in the production of tryptophan. Strains containing mutations in any of the genes coding for earlier steps in the tryptophan biosynthetic pathway (TRPI-TRP4) and those trp5 mutants that can still convert indole plus L-serine to L-tryptophan are able to grow on minimal medium if it is supplemented with indole. We, therefore, examined the trp mutants to see whether the sensitive strains could be made resistant to PEA by the addition of indole to the medium. We found that, if a mutant could utilize indole to make tryptophan, it could grow in the presence of 0.3% PEA when indole was also present in the medium. The presence of tryptophan in the media in concentrations up to 90 mg/liter did not alter the sensitivity of trp mutants to PEA even when proline replaced ammonium sulfate as the sole nitrogen source; under these conditions, the general amino acid permease is not repressed (GRENSON, Hou and CRABEEL 1970). To determine whether other genes besides those required for tryptophan biosynthesis are needed for expression of resistance to PEA, we isolated from the PEA-resistant strain XP300-26C two classes of mutant strains. First, since tryptophan biosynthesis shares several common steps with phenylalanine and tyrosine biosynthesis, we wanted to determine whether only those steps necessary for the production of tryptophan were essential for resistance to PEA or whether the genes required for tyrosine or phenylalanine were also involved. Second, to determine whether other genes besides those required for aromatic amino acid biosynthesis might be involved, we isolated mutants based on their sensitivity to PEA. Isolation of aro mutants: Twenty-five mutants unable to make one or more of the aromatic amino acids (aro mutants) were isolated. Their specific requirements were determined by replica plating to MV supplemented with various combinations of amino acids. The ability of these mutants to grow on media containing PEA or PEA plus indole was also determined. These results show that a mutation in any of the genes for aromatic amino acid biosynthesis can make a strain sensitive to PEA. Further, the ability to make a sensitive strain resistant by the addition of indole to the medium was confined solely to certain mutants that require only tryptophan. Complementation analysis of these aro mutants with strains obtained from the yeast Genetics Stock Center (Berkeley, California) showed that our mutants contained mutations in AROI, AR02, AR07, TRP3 or TRP5. Isolation of mutants sensitive to PEA: We isolated 19 mutants that were unable to grow on medium with 0.3% PEA and determined their growth requirements (Table 2). In addition to aro mutants, we found that 13 of the mutants had no new auxotrophic requirement since they grew on MV + lys + his + thr. Since these 13 mutants could have mutations in one or more of the aro genes affecting only the gene product s response to PEA while leaving its

4 238 J. H. MEADE AND T. R. MANNEY TABLE 2 PEA-sensitive mutants isolated from XP300-26C" New requirement trp tyr tyr and phe trp, tyr, and phe none No. of mutants 3* Nineteen mutants were identified among 2.35 X lo4 surviving cells that formed colonies (55.3% survival). One of these mutants will grow on a medium containing PEA and indole. biosynthetic function intact, we crossed each of these 13 mutants with strains containing mutations in TRPI, TRP2, TRP3, TRP4, TRP5, AROZ, AR02, AR07 or TYRI. All of the resulting diploids were tested for their growth response to PEA. Whereas all of the haploid strains were sensitive, all of the diploids were resistant to PEA. Therefore, we conclude that these 13 mutants represent additional genes distinct from those required for aromatic amino acid biosynthesis. These complementation results also indicate that in all of these strains sensitivity to PEA is recessive to resistance. Chorismic acid, in addition to being the central metabolite in the pathway of aromatic amino acid biosynthesis, is the branch point for biosynthesis of p- aminobenzoic acid, vitamin K and ubiquinone. To determine whether any of the 13 mutants that grew on MV + his + lys + thr required one or more of the vitamins normally supplied in MV, we examined the ability of these mutants to grow on MB + his + lys + thr, a minimal medium supplemented with biotin. Twelve of the 13 mutants grew on MB. Preliminary studies with the one strain that did not grow on MB indicate that it probably requires two or more of the vitamins normally supplied by MV (calcium pantothenate, folic acid, inositol, niacin, riboflavin, p-aminobenzoic acid, pyridoxine hydrochloride and thiamine hydrochloride). Whether the sensitivity to PEA and the new multiple vitamin requirements are due to mutation of a single gene or represent two independent mutations has not been determined. To determine the number of additional genes represented by these 13 mutants, we crossed them with strains of a mating type, sporulated the diploids and isolated a strains containing the mutation of PEA sensitivity. Nine of the mutations were successfully crossed into a strains, but we were not able to isolate a strains for four mutants due to problems with poor sporulation and poor spore viability. Complementation tests were made between the nine a PEA-sensitive strains and the 13 mutants. Every diploid combination, except those in which the diploid was formed between an a strain and its corresponding a mutant, was able to grow on YEPAD + PEA. Therefore, there are at least ten complementation groups represented by these 13 mutants. TAKAHASHI (1972) has identified one gene conferring resistance to PEA (PEAZ); this allele has since been lost (T. TAKAHASHI, personal communication). Since PEA1 has been mapped on chromosome 111 distal to THR4 and none of our mutants map in this

5 PHENETHYL ALCOHOL SENSITIVITY 239 region, we will designate the wild-type resistant alleles in our strains PEA2 to PEA11. Isolation of PEA-resistant mutants from a sensitive strain: To determine whether a sensitive strain could be made resistant by additional mutations, other than back mutations, we isolated 23 PEA-resistant strains from XP300-29B. Among the 23 strains, we found that ten still required tryptophan. The change from PEA sensitive to PEA resistant in these ten mutants has resulted from a recessive mutation in one gene. Complementation tests were made between these ten mutants and an (Y trp5 strain. In all cases the diploids were sensitive to PEA, indicating that the mutation for resistance carried in these ten mutants is expressed as a recessive trait. Three of the ten resistant strains were crossed with XP300-26C and sporulated, and asci were dissected. The new mutations conferring resistance to PEA segregated as a single gene and showed no linkage to the trp5-18 marker. Two a strains containing the PEA-resistant allele and the trp5-18 allele were used for complementation tests with the ten mutants. All of the diploids were resistant to PEA. We tested these PEAresistant mutants to determine whether they might be resistant to a higher concentration of PEA than the other resistant strains but found that they also grew on media with 0.3% but not 0.4% PEA. To determine whether this mutant allele suppresses PEA sensitivity only for this trp5 mutation or whether its suppression is more general in action, we crossed a strain containing the sensitivity suppressor with six PEA-sensitive strains. The diploids were sporulated, asci were dissected, and the spores were tested. The suppressor was found to suppress PEA sensitivity of a different trp5 allele and also a trp3 mutation. It did not suppress a tyrl mutation nor three of the PEA-sensitive mutations that had no new auxotrophic requirement (pea2, pea3 and pea4). DISCUSSION All of the yeast strains used in our study are, in a strict sense, sensitive to PEA, since none of them can grow on media containing 0.4% PEA. However, if we examine the growth of our strains on media containing 0.3% PEA, we can operationally divide them into two groups, resistant and sensitive. The 13 PEAsensitive mutants we isolated that had no new auxotrophic requirement are not surprising. These mutants may have altered cell membranes or membrane proteins and be similar to some mutants isolated in E. coli. What was surprising was finding that a relationship existed between auxotrophy for tyrosine, phenylalanine and tryptophan and sensitivity to PEA. This sensitivity was not reversed by the presence of these amino acids in the media, but the sensitivity of certain trp mutants could be reversed by the addition of indole to the medium. Also, a recessive suppressor mutation was isolated that made the trp mutants resistant to PEA while having no effect on other mutants. At this point we are unable to speculate on the mechanism by which aro mutants are made sensitive to the effect of PEA. This research was supported by grant GM awarded by the Public Health Service.

6 240 J. H. MEADE AND T. R. MANNEY LITERATURE CITED GRENSON, M., C. Hou and M. CRABEEL, 1970 Multiplicity of the amino acid permeases in Saccharomyces cerevisiae. IV. Evidence for a general amino acid permease. J. Bacteriol LESTER, G., 1965 Inhibition of growth, synthesis, and permeability in Neurospora crassa by phenethyl alcohol. J. Bacterol. 90: SILVA, M. T., J. C. F. SOUSA, M. A. E. MACEDO, J. POLONIA and A. M. PARENTE, 1976 Effects of phenethyl alcohol on Bacillus and Streptococcus. J. Bacteriol SILVER, S. and L. WENDT, 1967 Mechanism of action of phenethyl alcohol: breakdown of the cellular permeability barrier. J. Bacteriol. 93: TAKAHASHI, T., 1972 Phenethyl alcohol resistance in Saccharomyces cerevisiae. Bull. Brew. Sci. 18: WADA, C. and T. YURA, 1974 Phenethyl alcohol resistance in Escherichia coli A temperaturesensitive mutation (dnap) affecting DNA replication. Genetics WICKERHAM, L. H., 1946 A critical evaluation of the nitrogen assimilation tests commonly used in the classification of yeasts. J. Bacteriol YURA, T. and C. WADA, 1968 Phenethyl alcohol resistance in Escherichia coli I. Resistance of strain C600 and its relation to azide resistance. Genetics Corresponding editor: E. JONES