JOURNAL OF BACTERIOLOGY, June 1987, p. 2440-2448 Vol. 169, No. 60021-9193/87/062440-09$02.00/0Copyright C 1987, American Society for Microbiology

Cloning and Characterization of Saccharomyces cerevisiae GenesThat Confer L-Methionine Sulfoximine and Tabtoxin Resistance

ELZBIETA T. MAREKt AND ROBERT C. DICKSON*

Department of Biochemistry, University of Kentucky Medical Center, Lexington, Kentucky 40536-0084

Received 10 October 1986/Accepted 9 March 1987

Pseudomonas tabaci produces a toxin, tabtoxin, that causes wildfire disease in tobacco. The primary targetof tabtoxin is presumed to be glutamine synthetase. Some effects of tabtoxin in tobacco can be mimicked bymethionine sulfoximine (MSO), a compound that is known to inactivate glutamine synthetase. To understandhow organisms can be made resistant to tabtoxin and MSO, we used Saccharomyces cerevisiae. We demonstratethat yeast strains carrying the glutamine synthetase gene, GLN1, on a multicopy plasmid overproducedglutamine synthetase and showed increased drug resistance. These and other data indicate that glutaminesynthetase is the primary target of tabtoxin and MSO in S. cerevisiae. We also isolated three S. cerevisiae DNAinserts of 2.1, 2.3, and 2.8 kilobases that conferred tabtoxin and MSO resistance when the inserts were presenton a multicopy plasmid. These plasmids conferred resistance to MSO by blocking intracellular transport of thedrug. Transport appeared to occur by one or more methionine permeases. Resistance to tabtoxin could alsooccur by blockage of intracellular transport, but the drug was transported by some permease other than amethionine permease. These drug resistance plasmids did not block transport of citrulline, indicating that theydid not affect the general amino acid permease.

Wildfire is an infectious leafspot disease of tobacco. Thechlorotic leaf symptom that characterizes the disease iscaused by tabtoxin (32), a phytotoxic dipeptide produced byPseudomonas tabaci. Tabtoxin is a novel ,-lactam-containing amino acid, 2-amino-4-(3-hydroxy-2-oxoazacyclo-butan-3-yl)butanoic acid, linked by a peptide bond to theamino group of either serine or threonine (32). Tabtoxininhibits glutamine synthetase activity in tobacco in vivo,causing accumulation of ammonium (8, 30, 34). Inhibition ofglutamine synthetase has at least two effects, either one ofwhich could cause cell death. First, the enzyme is probablythe only major route for glutamine synthesis in plants, andsecond, the glutamine synthetase-glutamate synthase path-way is the only efficient way to detoxify the ammoniareleased by nitrate reduction, amino acid degradation, orphotorespiration in most plants (17).

Inhibition of glutamine synthetase by tabtoxin can beprevented by glutamine (30), which suggests that glutaminesynthetase is the only cellular target in tobacco. Methioninesulfoximine (MSO), known to be a potent and highly specificinhibitor of glutamine synthetase (22), causes chlorotic le-sions in tobacco leaves, and these effects are also preventedby glutamine (30). Because it appears to mimic the effects oftabtoxin and because it is commercially available, whereastabtoxin is not, MSO has been used as a tool for studyingwildfire disease in tobacco. Carlson (3) isolated MSO-resistant tobacco cells from callus cultures and showed thatplants derived from them were resistant to the chloroticaffects of both MSO and P. tabaci. In some lines theMSO-resistant trait behaved as a single dominant locus. Themolecular mechanism underlying the resistance trait remainsunknown. Nicotiana longiflora is a species of tobacco that isnaturally resistant to wildfire. It has been possible to transferresistance to burley tobacco, but not to flue-cured commer-cial cultivars of tobacco, by standard breeding techniques

* Corresponding author.t Present address: Department of Plant Pathology, University of

Kentucky, Lexington, KY 40536.

(20). The wildfire resistance trait behaves as a single domi-nant locus. The molecular mechanism underlying this trait isnot known, nor is it known whether this natural resistancetrait is related to the MSO resistance trait described byCarlson.As part of a long-term study to understand how tabtoxin

and MSO kill tobacco cells and how the N. longifloraresistance gene functions, we undertook studies of tabtoxinand MSO resistance in Saccharomyces cerevisiae. Therationale for our experiments is based on the observations ofRine et al. (27) that S. cerevisiae can be made resistant tometabolic inhibitors by increasing the concentration of theenzyme or protein that is the target of the inhibitor. In-creased protein is obtained by molecularly cloning the genethat codes for the protein on a multicopy S. cerevisiaerecombinant DNA vector. This in effect is an example ofcellular resistance due to gene amplification. We reasonedthat S. cerevisiae could be made resistant to tabtoxin andMSO by putting its glutamine synthetase gene, GLNJ (24),on a multicopy vector. Our results confirmed this prediction.We also believed that there might be other yeast genes thatwould confer resistance and that these would be easy toselect from a yeast gene bank. This approach enabled us toselect yeast genes that confer MSO and tabtoxin resistanceon yeast when present on a multicopy vector. These genesfunction by preventing uptake of MSO.

MATERIALS AND METHODS

Bacterial and yeast strains. Escherichia coli DG75 (F-hsdSl leu-6 ara-14 galK2 xyl-5 mtl-i rpsL20 thi-1 supE44AlacZ39 X-) and HB101 (F- hsdS20 recA13 ara-14 proA2lacY1 galK2 rpsL20 xyl-5 mtl-l supE44 X-) were used ashosts for transformation and propagation of plasmids. S.cerevisiae strains used were DC5 (a leu2-3, leu2-112, his-3canl-11), DBY745 (a ura3-52, leu2-3, leu2-112, adel-100),745-glnl (a ura3-52, leu2-3, leu2-112, adel-100, glnl-754[24]), SJ21 (a gal4-2, ura3-52, leu2-3, leu2-112, adel, MELI[15]), and D13-1A (a his3-532 trpl-289 gal2 [33]).

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Media. YPD medium contained 20 g of glucose, 20 g ofpeptone, and 10 g of yeast extract per liter. Syntheticmedium contained (per liter) 3.4 g of yeast nitrogen base(Difco Laboratories) without amino acids or ammoniumsulfate, 20 g of glucose, auxotrophic supplements at 20jig/ml, and as nitrogen sources, either 2.0 g of glutamine(GGln), 1.25 g of potassium glutamate (GGlt), 2.0 g ofammonium sulfate (GN), 2.0 g of ammonium sulfate and 1.25g of potassium glutamate (GNGlt), or 1.0 g of proline(GProl). YPDgln contained 3.0 g of glutamine per liter.

Preparation of yeast extracts. Log-phase cells were washedand suspended in breaking buffer (5 mM morpholine-ethanesulfonic acid [MES], 5 mM MnCl2, pH 6.3, and 1 mMphenylmethylsulfonyl fluoride or 0.1 M Tris, 0.2 mM ADP,pH 7.0, and 1 mM phenylmethylsulfonyl fluoride) at about100 A6w units per ml. Cells were disrupted by vortexing with1/2 volume of 0.5-mm glass beads for six 30-s periods at 40C.After vortexing, the liquid phase was removed to a 1.5-mlmicrofuge tube and centrifuged for 5 min at 4°C. Thesupematant fluid was diluted and used immediately formeasuring enzyme activity, or it was stored undiluted for upto 2 months at -800C without loss of enzyme activity.Enzyme activities. The transferase activity of glutamine

synthetase was measured as described by Mitchell andMagasanik (25). One unit of transferase activity catalyzesthe formation of 1 ,uM of y-glutamylhydroxamate per min at30°C. NAD-dependent glutamate dehydrogenase activitywas measured as an a-ketoglutarate- and ammonium ion-dependent oxidation of NADH (5). ADP (0.2 mM) wasincluded in the reaction mnixture to stabilize the enzyme. Thedecrease in absorbance at 340 nm was measured simulta-neously in the test sample and in the control which lackeda-ketoglutarate. One unit of enzyme activity represents 1nM of NADH oxidized per min per mg of protein at 250C.Permease activity. Early-log-phase cells, grown in GNGlt

medium at 30°C, were collected by filtration and washedwith and suspended in minimal medium (3.4 g of yeastnitrogen base without amino acids and ammonium sulfateand 20 g of glucose per liter with auxotrophic supplements)prewarmed to 30°C, at a density of 1 to 2 A6w units per ml.The cells were aerated for 2 min at 300C. A sample wasremoved to measure the protein concentration. The radio-active compound was added, and samples of the culturewere rapidly filtered (0.4-,um pore size; Nuclepore Corp.,Pleasanton, Calif.) at 30-s intervals for 2 min. Filters werewashed with ice-cold minimal medium containing a 20-foldexcess of the unlabeled compound and dried, and the radio-activity was counted to determine the initial rate of trans-port. Units of permease activity are expressed as nanomolesof compound accumulated per minute. [35S]methionine (400Ci/mmol; New England Nuclear Corp.), [35S]MSO (2.3 x 106cpm/4mol), and ['4C]citrulline (40 mCi/mmol; AmershamCorp.) were used to measure methionine, MSO, and thegeneral amino acid permease (GAP), respectively.

Yeast transformation. S. cerevisiae was transformed bythe method of Hinnen et al. (13). Strain 745-glnl wastransformed by the procedure of Ito et al. (14).

Plasmid copy number. Total DNA was isolated (33) fromexponential-phase yeast cultures grown in GNGlt mediumsupplemented with leucine for YEp24 transformants oruracil for YEp13 transformants. Further purification wasobtained by extraction with phenol and then chloroform.The ratio of plasmid DNA to genomic DNA was determinedby the DNA dot-blotting procedure of Gillespie and Bresser(10). A dot of 2 ng of plasmid pTCM DNA (9) was made oneach nitrocellulose filter so that filters hybridized to different

probes could be compared. Plasmid pTCM carries pBR322sequences which are shared with pMSO plasmids, and it alsocarries the gene coding for the ribosomal L protein (9). Twoidentical blots were made. One was hybridized to the 32p_oligolabeled BamHI-AvaI fragment from the ribosomal Lprotein coding region. This blot was used to normalize theamount of total yeast DNA per blot, since the L gene ispresent in one copy per cell (9). The other blot was hybrid-ized to 32P-oligolabeled pBR322, which would detect pMSOsequences. Radioactivity in each dot was determined byscintillation counting. All transformants contained 3 to 6copies of the plasmid, with the exception of SJ21(YEp13)and SJ21(pMSO1), which contained 9 and 11 copies, respec-tively. In all transformed strains at least 90% of the cellscontained a plasmid, as measured by plating samples onYPD plates and minimal uracil or leucine selection plates.

Glutamine synthetase mRNA. To measure the relativeamount of glutamine synthetase mRNA, total yeast RNAwas isolated by the method of Carlson and Botstein (2) anddot-blotted to duplicate nitrocellulose filters under condi-tions for preferentially binding polyadenylated [poly(A)+]RNA (10). To normalize the amount of mRNA bound foreach sample, one filter was hybridized with the same 32p_oligolabeled L protein probe that was used for the plasmidcopy number determinations. The other blot was hybridizedto a 32P-oligolabeled 0.8-kilobase (kb) EcoRI DNA fragmentfrom GLNJ. Autoradiograms were quantitated by densitom-etry, and the data were normalized to the glutamine synthe-tase mRNA level of strain SJ21.

Purification of tabtoxin. Tabtoxin and tabtoxinine wereisolated from culture filtrates of Pseudomonas syringae pv.tabaci CHS-2 by the procedure of Stewart (32), except thatthe first ion-exchange column was equilibrated with distilledwater acidified to pH 2.5 with HCI instead of 1 M NaCl andthe second ion-exchange column was eluted with 0.2 Mammonium formate, pH 3.4, instead of pyridine acetate.Tabtoxinine eluted at 1.5 to 2 column volumes and tabtoxinat 4 to 5 column volumes. Tabtoxin and tabtoxinine werelocated by analysis with ninhydrin, by assay with an aminoacid analyzer, or by a tobacco leaf bioassay (18). In animproved and more rapid assay we used a Bio-Rad AminexA9 cation-exchange high-pressure liquid chromatographycolumn equilibrated and eluted in 0.2 M NaH2PO4, pH 3.3,prepared by adjusting the pH with NaOH. The column wasmonitored at 205 nm with a Schoeffel Spectraflow UV-vismonitor, and tabtoxin was located in the eluate by compar-ison with purified tabtoxin. A typical preparation from 5liters of culture filtrate containing 75 mg of tabtoxin yielded56 mg of tabtoxin from the second ion-exchange column(0.82 by 90 cm) along with 2.5 mg of tabtoxinine.

Miscellaneous procedures. L-[35S]MSO was synthesizedfrom L-[35S]methionine (400 Ci/mmol) by the procedure ofBentley et al. (1). Small- and large-scale preparations ofplasmid DNAs were obtained from E. coli by the alkalinelysis method described by Maniatis et al. (21), and plasmidDNA was purified by CsCl-ethidium bromide density gradi-ent centrifugation. Southern blotting and hybridization pro-cedures have been described (31). Protein concentration wasmeasured by the procedure of Lowry et al. (19). DNAprobes were oligolabeled with [32P]dCTP (3,000 Ci/mmol;New England Nuclear) by the method of Feinberg andVogelstein (7). For selecting MSO-resistant transformants,cells were first plated in 8 ml of GGlt regeneration agar (13)on a 25-ml GGlt base plate. The layer of regeneration agarwas overlaid with another 8 ml of GGlt regeneration agar,and after 6 h of incubation at 30°C, MSO was spread onto the

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FIG. 1. L-Methionine and L-glutamine reverse the growth-inhibiting affect of MSO. A culture of SJ21(YEp13) was grown to thelate exponential phase in GNGlt medium and then diluted to 500cells per ml in the same medium with or without 1 mM MSO. After3 h of growth at 30°C, 14 mM glutamine or 1 mM methionine was

added to the cultures containing MSO at the time indicated by thearrow. At various times samples were removed, diluted, and platedon YPD plates to calculate the number of colony-forming cells.Symbols: *, no MSO; O, 1 mM MSO; A, 1 mM MSO plusglutamine; 0, 1 mM MSO plus methionine.

plate (41 ml total volume) to give a final concentration of 1mM MSO.

RESULTS

Effect of L-MSO and tabtoxin on growth of S. cerevisiae. Apreliminary estimation of the minimum concentration ofMSO that inhibits growth of S. cerevisiae was made byspreading 102 to 103 cells, pregrown to saturation in YPDmedium, onto GN plates containing various concentrationsof MSO. The concentration of MSO that prevented colonyformation was specific for each strain. The most sensitivestrains, DBY745 and D13-1A, failed to grow on 1 mM MSOin the presence of 1% ammonium sulfate. The most resistantstrain, DC5, did not grow on 20 mM MSO when 1%ammonium sulfate was used as a nitrogen source. When theconcentration of ammonium sulfate was lowered to 0.1%,the strain became more sensitive to MSO, and growth was

inhibited by 2 mM MSO. Growth inhibition was also ana-

lyzed by growing cells in liquid GN medium containingdifferent concentrations of MSO. In all strains, the growthrate was proportional to the concentration of MSO, up to a

drug concentration that completely inhibited growth (datanot shown). The inhibitory concentration correlated with thedata obtained on plates.

If cell growth was inhibited due to inactivation ofglutamine synthetase activity by MSO, then glutamineshould relieve growth inhibition. We found that 14 mMglutamine rapidly reversed the growth-inhibiting effect ofMSO (Fig. 1). Other concentrations of glutamine were notexamined.

Yeast might also be spared from killing by MSO if the drugwere excluded from the cell. It has been reported thatChlorella vulgaris transports MSO by an energy-dependent,membrane-bound carrier. Methionine can prevent the toxic

effect of MSO, presumably by competing for this membranecarrier and blocking MSO uptake (23). To determinewhether MSO and methionine use the same membranetransporter(s) in S. cerevisiae, we examined the toxic effectof MSO in the presence and absence of methionine. L-Methionine relieved MSO inhibition when added to themedium at the same concentration as MSO (Fig. ]). As acontrol, we studied growth inhibition by MSO in iediumcontaining a mixture of amino acids (except L-glutamine andL-methionine) that are not transported by the methioninetransporter and that are not structurally similar to MSO. Fullgrowth inhibition was observed in all media, showing thatmethionine specifically overcomes the inhibitory effect ofMSO. More evidence that MSO is transported by a methio-nine transport system is given below.The growth-inhibiting effect of tabtoxin on strain SJ21 was

studied. However, the experiments could not be conductedin the same way as for MSO because tabtoxin is notcommercially available and we had limited amounts. Typi-cally, S. cerevisiae were inoculated at 1,000 cells per ml in 1ml of GNGlt medium, and growth of the culture was moni-tored by counting colonies plated on YPD plates. In this typeof experiment we found that 1 mM tabtoxin inhibited growthby 60 to 80%. As with MSO, growth inhibition did not occurwhen the medium was supplemented with 14 mM L-glutamine, suggesting that tabtoxin was inhibiting glutaminesynthetase. Addition of 1 mM L-methionine to the mediumdid not restore normal growth of the culture, suggesting thattabtoxin does not use the methionine transporter.

In vitro effects of MSO and tabtoxin on glutamine synthe-tase activity in cell extracts. The results of the precedingsection predicted that MSO and tabtoxin should inhibitglutamine synthetase activity in yeast. The inhibitory effectof these two compounds was studied in cell extracts of S.cerevisiae by measuring the transferase activity of glutaminesynthetase. Inhibition of enzyme activity by MSO was veryrapid and was complete after 10 min of preincubation (Fig.2). The inhibitory effect of tabtoxin increased with prein-cubation time (Fig. 2). Maximal inhibition of glutaminesynthetase activity by tabtoxin was observed only when theprotease inhibitor phenylmethylsulfonyl fluoride was omit-ted from the extraction buffer. With this compound in thebuffer, only about half as much inhibition of glutaminesynthetase was obtained (data not shown). These data indi-cate that yeast, like plants (35), must activate tabtoxin byproteolytic cleavage and that the peptidase(s) necessary forthis activation is present in yeast. This result, combined withthe preceding results, indicates that glutamine synthetase isthe primary target of MSO and tabtoxin in yeast.

Multiple copies of a glutamine synthetase gene protect S.cerevisiae from killing by MSO and tabtoxin. The precedingresults suggested that cells overproducing glutamine synthe-tase will show increased resistance to MSO and tabtoxin. Tocreate such an overproducing strain, we transformed S.cerevisiae SJ21 with the recombinant plasmid pJL106 (A.Mitchell, personal communication). The plasmid has a3.5-kb fragment of S. cerevisiae DNA containing GLNI, theglutamine synthetase gene, cloned in the BamHI site ofYEp24. As a control we used SJ21 transformed with YEp24.Transformed cells were selected in two ways. First, since

pJL106 and YEp24 carry the URA3 gene that is able tocomplement the ura3-52 mutation of strain SJ21, we selectedUra+ transformants on uracil-deficient medium. Second, weselected transformants directly for resistance to 1 mM MSO(MSO') by the plate overlay technique described in Materialsand Methods.

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FIG. 2. Inhibition of glutamine synthetase activity by MSO andtabtoxin. S. cerevisiae SJ21 was grown in GGlt medium to an A6N of1. Cell extracts were made as described in Materials and Methods,except that phenylmethylsuflonyl fluoride was not included in theextraction buffer. Extracts were preincubated at 30°C with tabtox,inor MSO for the times shown, and then glutamine synthetasetransferase activity was measured over a 30-min period. Controlextracts were preincubated for various times without MSO ortabtoxin before glutamine synthetase activity was measured. Sym-bols: O, control; 0, 1 mM MSO; *, 1.2 mM tabtoxin.

Plasmid pJL106 gave 4,700 Ura+ transformants and 3,100MSO' transformants per jig of vector DNA. The parentplasmid YEp24 gave 4,000 Ura+ transformants per ,ug ofDNA and no MSO' transformants, as expected. TwentyUra+ and 20 MSOr pJL106 transformants were colonypurified on selective plates and then spotted on a plate withthe opposite selective condition. The 20 MSO' transformantswere all Ura+, and of 20 Ura+ transformants, 18 were MSO'.These data strongly suggest that the two phenotypes are dueto pJL106.To calculate the degree of resistance to MSO and tabtoxin,

we determined the concentration of drug that inhibitedgrowth by 50% (ID50). Cells having multiple copies of theglutamine synthetase gene [SJ21(pJL106) transformants] hada12- to 15-fold increase in resistance to MSO and tabtoxinand overproduced glutamine synthetase activity sixfold (Ta-ble 1). This increased level of enzyme activity was directlyrelated to plasmid copy number, which we determined to be

TABLE 1. Multiple copies of GLNJ confer MSO and tabtoxinresistance on S. cerevisiaea

Strain ID,0 (mM) Glutamine synthetaseMSO Tabtoxin activityb (no. of expt)

SJ21(YEp24) 0.025 0.08 2.8 ± 0.2 (2)SJ21(pJL106) 0.38 1.0 17.3 ± 2.14 (3)

a Cells were inoculated at a density of 103/ml into GNGlt medium containingMSO or tabtoxin at concentrations ranging from 0 to 1 mM. After 19 h ofgrowth, samples were plated on YPD plates to calculate the percentage ofcolony-forming cells per ml.

b Glutamine synthetase transferase activity was measured in cell extractsmade from cultures grown to mid-log phase in GGlt medium (derepressedconditions) and is expressed as units (micromoles of -y-glutamylhydroxamateformed per min at 30°C).

c ~~RNFIG. 3. Restriction map of MSO plasmids. Plasmids were iso-

lated from an S. cerevisiae genomic library made by insertingpartially Sau3A digested DNA from S. cerevisiae D13-lA (33) intothe BamHI site of the yeast shuttle vector YEp13. A, B, and C showthe insert regions of pMSO2, pMSO5, and pMS06, respectively. Inall cases the BamHI site was lost because ofjoining to a Sau3A end.The orientation of the yeast DNA insert relative to the vector is asshown. YEp13 carries the pBR322 genes for ampicillin and tetracy-cline resistance, the LEU2 gene from S. cerevisiae, and the Saccha-romyces 2,um plasmid region allowing autonomous replication inyeast. Abbreviations: R, EcoRI; B, BamHI; C, ClaI; H, HindIll; P,PstI; Pv, PvuII; N, NruI.

4 to 6 copies per cell (data not shown). These data supportedthe hypothesis that S. cerevisiae with multiple copies ofGLNI would overproduce glutamine synthetase and becomeincreasingly resistant to MSO and tabtoxin.

Selection of yeast genes that confer MSO resistance. To seewhether any yeast genes besides GLNJ would confer MSOand tabtoxin resistance on their host when carried on amulticopy vector, we transformed strain DC5 with an S.cerevisiae clone bank made in YEp13 (Fig. 3). The leu2defect in strain DC5 can be complemented by the LEU2 genecarried on YEp13. Seven thousand Leu+ transformants wereselected and pooled, and then MSOr cells were selected intwo ways. First, 104 Leu+ transformants were grown for 4days at 30°C in 1 ml ofGN medium containing 50 mM MSO.

TABLE 2. Isolation and classification of plasmids carrying yeastgenes that give an MSO-resistant phenotype

Selection conditionsa Plasmid(s) isolated'

Glutamate (0.1%) + 2.5 mM MSO.......pMSO2 (8)Glutamate (0.1%) + 25 mM MSO .......pMSO1 (12), pMSO2 (8),

pMSO5 (1)MSO (2.5 mM) ...................pMSO2 (4), pM$05 (1)MSO (25 mM) ...................pMSO1 (2),. pMSO2 (9),

pMSO6 (1)a All selection plates contained 2x yeast nitrogen base, 1% glucose, 0.1%

ammonium sulfate, and the nutrients required by strain DC5.b The number of isolates obtained (of 46 analyzed) is shown in parentheses.

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1 2 :3 4 5 6 7FIG. 4. Southern blot hybridization ofMSO genes. DNA from S.

cerevisiae DC5 was digested with EcoRI or BamHl, electropho-resed on a 0.9% agarose gel, and blotted to nitrocellulose. Blotswere probed with the 32P-labeled EcoRt fragments carrying thepMS02, pMS05, or pMS06 insert-specific probes described in thetext. Lanes: 1, HindIII fragments of lambda end-labeled with 32p; 2,EcoRI-cleaved yeast DNA probed with the pMS02 probe; 3,BamHI-cleaved yeast DNA probed with the pMS02 probe; 4,EcoRI-cleaved yeast DNA probed with the pMS05 probe; 5,BamHI-cleaved yeast DNA probed with the pMS05 probe; 6,EcoRI-cleaved yeast DNA probed with the pMS06 probe; 7,BamHl-cleaved yeast DNA probed with the pMS06 probe. Frag-ment sizes are indicated (in kilobases).

Samples were plated, and four individual colonies thatshowed cosegregation of the Leu+ and MSOr markers werestudied. They all contained the plasmid designated pMSO1(Table 2). In the second procedure, MSO-resistant cells wereselected by plating 106 Leu+ transformants on plates con-taining either 2.5 or 25 mM MSO (Table 2). One set ofselection plates contained glutamate to facilitate selection oftransformants with elevated glutamine synthetase activity. Ithas been noted that increased glutamine synthetase activitycan cause intracellular depletion of glutamate, a substrate forthis enzyme, and subsequent growth inhibition. Inhibitioncan be overcome by adding glutamate to the medium (A.Mitchell, personal communication).

Twenty-five of the largest colonies from each of the fourtypes of plates were colony purified. Total DNA preparedfrom each isolate was used to transform E. coli DG75, withselection for ampicillin resistance. Forty-six samples gave E.coli transformants. Plasmid DNA was isolated from eachtransformant and digested with EcoRI. From these data theplasmids could be categorized into four classes (Table 2).Surprisingly, none of the isolates appeared to contain GLNJ,since none of them contained the 0.8-kb EcoRI fragmentpresent in GLNJ (A. Mitchell, personal communication).Also, we found that while pJL106 complemented the glnldefect in strain 745-glnl, as expected for a plasmid carryingthe wild-type GLNJ gene, none of the MSO plasmids com-

plemented glnl. These results also ruled out the possibilitythat one or more of our plasmids coded for a gene thatallowed glutamine synthesis by a novel pathway. The GLNJgene was probably not selected by these procedures becausetoo few Leu+ cells were examined. Also, only the selectiondone with 2.5 mM MSO in the presence of 0.1% glutamate(Table 2) would have favored selection of GLNJ. Since wepicked only the largest colonies from these selection plates,we may have overlooked transformants carrying GLNJ. Theother three selection conditions were Unfavorable for select-ing GLNJ either because the MSO concentration was toohigh or because no glutamate was added to the medium andtransformants carrying multiple copies of GLNJ would bestarved for glutamate (A. Mitchell, personal communica-tion).

The size of the yeast DNA insert was found from restric-tion digestions to be 2.3 kb for pMS02, 2.8 kb for pMS05,2.1 kb for pMS06 (Fig. 3), and 9.3 kb for pMS01. BecausepMS01 carried a large insert and conferred a low level ofdrug resistance, we did not characterize it further. Southernblot analysis was used to determine whether the yeast DNAinserts in pMS02, pMS05, and pMS06 were unique. Forthese experiments, total genomic DNA from S. cerevisiaeDC5 was cleaved with the restriction endonuclease EcoRI orBamHI. When the EcoRI-cleaved DNA was hybridized to a32P-labeled probe specific for the yeast DNA insert inpMS02 (the fragment extends from the EcoRI site in thepMS02 insert to the EcoRI site at the pBR322-2,um junc-tion), a major band of hybridization was observed at about 6kb and two less intense bands were observed at 3.8 and 2.1kb (Fig. 4, lane 2). With BamHI-cleaved yeast DNA, thisprobe detected a major band of 2.3 kb and a minor band of6.8 kb (Fig. 4, lane 3). With a probe for the pMS05 yeastinsert (an EcoRI fragment spanning the pBR322-yeast por-tion of pMS05), there was an EcoRI band of 4.1 kb (Fig. 4,lane 4) and a BamHI band of about 3.2 kb (Fig. 4, lane 5). Aprobe representing one-half of the pMS06 yeast insert (anEcoRI fragmnent going from the EcoRI site in the middle ofthe insert to the EcoRI site at the pBR322-2,um junction)hybridized to EcoRI bands of 1.3, 3.8, and 2.1 kb (Fig. 4,lane 6), while it hybridized to a BamHI band of 2.1 kb (Fig.4, lane 7). Since each probe hybridized primarily to a uniqueEcoRI or BamHI chromosomal DNA fragment and becausetheir restriction maps were different (Fig. 3), pMS02,pMS05, and pMS06 probably carry different genes for MSOresistance.We compared the level ofMSO resistance that each MSO

plasmid conferred on its host by measuring growth rates inthe presence of increasing MSO concentrations (Fig. 5). Forthese experiments all plasmids were transferred to strainSJ21, which is tnore sensitive to MSO than strain DC5.Growth of the control strain, SJ21(YEp13), was inhibited50% by 0.04 mM MSO. Plasmids pMS01, pMS02, and

I-I

1.0

mmoles MSOFIG. 5. MSO plasmids confer MSO resistance on yeast. Strain

SJ21 was transformed with YEp13 or MSO plasmids. Cultures werestarted at 103 cells per ml in GNGlt medium and grown at 30°C in thepresence of increasing concentrations of MSO. After 19 h of growth,samples were plated on YPD plates to calculate the number ofcolony-forming cells. Growth without inhibitor was considered100%. Symbols: L, SJ21(YEp13); 0, SJ21(pMSO1); A,SJ21(pMSO2); 0, SJ21(pMSO5); *, SJ21(pMSO6).

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40-

20-

O- I I I I I>0 0.2 0.4 0.6 0.8 1.0

mM tabtoxin

FIG. 6. MSO plasmids confer tabtoxin resistance on yeast. Thesame procedures were used as described in the legend to Fig. 5

except that a filter-sterilized solution of purified tabtoxin dissolvedin water was used in place of MSO. Growth without inhibitor wasconsidered 100%. Symbols: [, SJ21(YEp13); *, SJ21(pMSO1); A,

SJ21(pMS02); 0, SJ21(pMSO5); *, SJ21(pMS06).

pMSO5 made their host much more resistant to MSO thanthe control strain was, but the ID50 concentration of drugcould not be determined because the growth of these cellswas not inhibited as much as the control strain even at thehighest drug concentrations used, greater than 5 mM (datanot shown). Plasmid pMSO6 made its host at least fivefoldmore resistant to MSO, the same level of resistance that wasconferred by pJL106, which carries GLNI. Similar growthexperiments were repeated with tabtoxin in place of MSO.pMSO6 made its host at least fivefold more resistant totabtoxin than the control plasmid YEp13, while pMSO1 andpMSO5 increased the resistance of their hosts even more,and pMSO2 made its host resistant to all concentrations oftabtoxin tested (Fig. 6).The higher level of host resistance conferred by some

MSO plasmids could be due to a larger number of plasmidsper cell or to integration of the plasmid into a host chromo-some in a manner that would cause increased expression ofthe plasmid-borne resistance gene(s). Southern blot analysisshowed that all strains contained autonomous, not inte-grated, copies of the plasmid (data not shown) and that therewere 3 to 5 plasmid copies per cell, except for YEp13 andpMSOl, which were present at 9 and 11 copies per cell,respectively (data not shown).Glutamine synthetase activity in MSO-resistant transform-

ants. We measured glutamine synthetase transferase activityin transformants under growth conditions that repressed,derepressed, or gave intermediate levels of enzyme activity(Table 3). This was done to determine whether MSO plas-mids conferred MSO resistance by stimulating the level ofglutamine synthetase activity, which might occur if an MSOplasmid carried a positive regulatory gene such as GLN3(26). Under growth conditions that repressed glutaminesynthetase activity (GGln medium), all strains showed lowglutamine synthetase activity except SJ21(pJL106) (GLNJ),SJ21(pMSO2), and SJ21(pMSO5). Under growth conditionsthat derepressed (GGlt medium) or gave intermediate en-

zyme levels (GNGlt medium), only strain SJ21(pJL106)showed elevated enzyme activity. These data confirmed thatMSO plasmids did not carry GLNJ, because they did not

give elevated glutamine synthetase activity under derepres-sing conditions, as did pJL106, which carries GLNJ. Underrepressing conditions, strain SJ21(pMS05) had a slightlyelevated level of enzyme activity and strain SJ21(pMS02)had about a fivefold-higher level of enzyme activity than thecontrol strain. These two plasmids could carry genes thatregulate GLNI expression, but this seems unlikely sincethey only elevated enzyme activity under repressing condi-tions. We also measured the activity of NAD-glutamatedehydrogenase, an enzyme that is coregulated withglutamine synthetase by GLN3 (26). The glutamate dehydro-genase activity remained normal in all MSO-resistant trans-formants, indicating that none of the MSO plasmids regu-lated the level of this enzyme (Table 3).To explore further the possibility that MSO plasmids carry

a gene that regulates GLNJ in a positive manner, wemeasured glutamine synthetase activity and mRNA levels intransformed strains after treatment with MSO. We reasonedthat if one of the MSO plasmids carried a positive regulatorygene, then after addition of MSO to the culture there shouldbe an increase in glutamine synthetase mRNA because theMSO would inactivate glutamine synthetase, producing adecrease in the intracellular glutamine concentration. Theintracellular level of glutamine is sensed in an unknownmanner by the positive regulatory gene GLN3 (26), and theGLN3 product would stimulate transcription of GLNI. Theincrease in glutamine synthetase mRNA would not be par-alleled by an increase in glutamine synthetase activity be-cause of inhibition by MSO.For this experiment cells were grown in the presence or

absence of MSO under conditions that gave intermediateglutamine synthetase levels (GNGlt medium). This growthmedium was used because there would be no enzymeinhibition under repressed growth conditions due to thepresence of glutamine in the medium, and under derepressedconditions there would be a high basal level of glutaminesynthetase mRNA, which might obscure an increasedmRNA level. Part of each culture was used for enzymeassays, and part was used for preparation of RNA.None of the MSO plasmids caused a significant increase in

the level of glutamine synthetase mRNA in comparison with

TABLE 3. Glutamine synthetase and NAD-dependent glutamatedehydrogenase activities in yeast transformed

with MSO plasmidsa

Glutamine synthetase NAD-dependentactivity'(U) glutamate dehydrogenase

Strain activityb (U) activityc (U)

Gln GNGlt GGlt GGln GNGIt GGIt

SJ21 0.05 0.50 2.28 3 5 38SJ21(YEp13) 0.04 0.41 2.62 2 4 39SJ21(pMSO1) 0.07 0.45 2.62 6 2 48SJ21(pMSO2) 0.28 0.41 2.74 3 1 21SJ21(pMSO5) 0.12 0.52 3.16 7 4 37SJ21(pMSO6) 0.07 0.48 3.41 6 6 30SJ21(YEp24) 0.08 0.51 3.27 9 2 48SJ21(pJL106) 0.23 1.3 18.1 4 3 47

a Saturated cultures were grown in GGIn medium with auxotrophic supple-ments. Cells were then diluted to an A600 of 0.02 into GGln (repressed), GNGlt(intermediate), or GGlt (derepressed) medium, grown to an A6 of 1.2, andprepared for enzyme assays. The values are the averages from at least threeexperiments for glutamine synthetase activity and two experiments forNAD-glutamate dehydrogenase activities, except for the cultures grown onGNGlt medium, which represent one experiment.

b One unit equals 1 ,uM of y-glutamylhydroxamate formed per min at 30°C.c One unit equals 1 nM of NADH oxidized per min per mg of protein at

25°C.

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TABLE 4. Glutamine synthetase mRNA in transformantsof SJ21a

Relative level of glutamine synthetase mRNAStrain

GNGlt GNGlt + 1 mM MSO

SJ21 1.0 3.0SJ21(YEp13) 0.6 1.2SJ21(pMSO1) 0.6 1.1SJ21(pMSO2) 2.0 2.1SJ21(pMSO5) 0.9 2.6SJ21(pMSO6) 1.7 2.8SJ21(YEp24) 1.7 3.1SJ21(pJL106) 6.3 17.1

a Saturated cultures were grown in GNGlt medium, diluted to an A6Eo of0.15, and grown in the presence or the absence of 1 mM MSO to an A6M of 2.Strains SJ21, SJ21(YEp13), and SJ21(YEp24) are not resistant to MSO andwere inoculated at an A6w of 0.5 in the medium containing MSO. They wereharvested at the same time as the other cultures. The amount of glutaminesynthetase mRNA was measured by hybridization to the 0.8-kb internalEcoRP fragment of the GLNI gene. A fragment of yeast DNA derived from thegene coding for the ribosomal L protein (9) served as an internal control forthe amount of mRNA retained on the filter in each sample. All values werenormalized to that for strain SJ21 grown in GNGlt medium.

the control strains SJ21, SJ21(YEp24), and SJ21(YEp13)(Table 4). Increased mRNA was only seen in cells trans-formed with pJL106, which carries GLNJ. The glutaminesynthetase activity values for these strains are presented inTable 5. As expected, all strains had low enzyme activitywhen MSO was present in the culture medium. However,the MSO-resistant strains all had a slightly higher enzymelevel than the nonresistant strains, which must have enabledthem to grow in the presence of MSO. We also measuredNAD-glutamate dehydrogenase activity and found that itwas increased 20- to 80-fold when MSO was present in theculture medium. This result shows that GLN3 is functioning,since it controls the NAD-glutamate dehydrogenase struc-tural gene, and in the presence of MSO this gene would beturned on due to the decreased intracellular level ofglutamine. We conclude from these data that none of theMSO plasmids carries a gene that regulates transcription ofGLNJ in a positive manner, as does GLN3.MSO plasmids block MSO uptake. Cells transformed with

MSO plasmids could be resistant to MSO because the drug isprevented from entering the cell. To examine this possibility,we measured the transport of 35S-labeled MSO. Strains were

TABLE 5. Glutamine synthetase and NAD-dependent glutamatedehydrogenase activity in SJ21 transformants grown

in the presence or absence of MSOa

Glutamate synthetase NAD-dependentactivity (U) glutamate dehydrogenase

Strain activity (U)

GNGIt GNGlt + 1 GNGIt GNGlt + 1

SJ21 0.49 0.02 5 420SJ21(YEp13) 0.41 0.07 5 410SJ21(pMSO1) 0.25 0.18 3 73SJ21(pMSO2) 0.43 0.15 4 165SJ21(pMSO5) 0.36 0.19 5 351SJ21(pMSO6) 0.34 0.10 4 193SJ21(YEp24) 0.38 0.07 4 390SJ21(pJL106) 3.3 0.18 5 133

a See Table 4, footnote a, and Table 3, footnotes b and c. Strains SJ21,SJ21(YEp13), and SJ21(YEp24) did not go through a division cycle in thepresence of MSO.

20-

CL

10-0

E5- -n

0 20 40 60 80 100 120time (sec)

FIG. 7. MSO transport in yeast carrying MSO plasmids. Trans-port measurements were made as described in Materials and Meth-ods. Symbols: A, SJ21(YEp13); U, SJ21(pMS01); O,SJ21(pMS02); 0, SJ21(pMS05); *, SJ21(pMS06).

cultivated in GNGlt medium to match the conditions usedfor the growth inhibition studies. In strains SJ21(pMSOl),SJ21(pMSO2), and SJ21(pMSO5), the rate of MSO uptakewas reduced about 80% compared with that in the controlstrain, SJ21(YEp13) (Fig. 7). In strain SJ21(pMSO6), the rateof MSO uptake was reduced about 20%. These resultsindicate that MSO resistance is due to impairment of MSOtransport.The results presented in Fig. 1 suggested that MSO was

being transported into the cell by a methionine transporter.Further evidence favoring this interpretation was obtained.First, we found that 0.1 mM methionine inhibited the rate ofMSO transport by 50% in strain SJ21, whereas 5 mMglutamine or 5 mM alanine reduced the rate of MSO -trans-port by only 20% (data not shown). Second, we measuredthe rate of [35S]methionine transport in SJ21 carrying MSOplasmids (Fig. 8) and found that transport was significantly

00S

It

Q

OD AoC U

E9.E

time (sec)FIG. 8. Methionine transport in yeast carrying MSO plasmids.

Transport measurements were made as described in Materials andMethods. Symbols: U, SJ21(YEp13); A, SJ21(pMSO1); O,SJ21(pMS02); 0, SJ21(pMSO5); *, SJ21(pMS06).

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reduced by pMSO1, pMSO2, and pMSO5. Only a slightreduction in the rate of methionine transport was producedby pMSO6. These are the same results obtained for MSOtransport (Fig. 7), implying that MSO plasmids are reducingthe activity of a transporter that transports both methionineand MSO. Third, we measured the Km for MSO transportand compared it with the Km for methionine transport.Methionine transport in strain SJ21 occurred by at least twosystems, a low-affinity transporter with a Km of 600 ,uM anda high-affinity transporter with a Km of 20 ,uM (data notshown). These results are similar to published values formethionine transport (11). Similar transport studies withMSO gave a single Km value of 800 pLM so MSO could beusing either the high- or the low-affinity methionine trans-porter or both.MSO could also be transported by the GAP (12). Trans-

port by GAP can be measured by using citrulline (28). IfMSO was transported by GAP, we would expect citrullinetransport to be reduced by MSO plasmids, whereas citrullinetransport should be normal if MSO is transported by themethionine-specific transporters. We found that the rate oftransport of ['4C]citrulline was not affected by MSO plas-mids (data not shown). We conclude that MSO plasmids arenot affecting the activity of GAP. MSO is not transported bythis transporter to any large extent under the experimentalconditions used here.

DISCUSSION

The data presented here demonstrate that S. cerevisiaecan be made resistant to MSO and tabtoxin by overproduc-ing glutamine synthetase, the enzyme target of these twodrugs. Overproduction of enzyme (Table 1) was achieved byplacing the glutamine synthetase gene, GLNI, on a yeastshuttle vector, YEp24, that was present at an average of 3 to5 copies per cell. Other cell types, including mouse 3T3 cells(36) and Chinese hamster ovary cells (29), have been shownto be resistant to MSO because of amplification of theirglutamine synthetase gene and consequent overproductionof enzyme activity. In these cases resistant cells wereselected after repeated subculturing in the presence of in-creasing concentrations of MSO. We have not tried to selectyeast cells for increased drug resistance, but it should bepossible to do so.By using MSO resistance as a selection procedure, we

were able to isolate S. cerevisiae genes that make yeastresistant to MSO. The genes, represented by plasmidspMS01, pMS02, pMS05, and pMS06, are not structuralgenes coding for glutamine synthetase, because they did notcomplement a glnl-defective strain. Plasmids pMS02,pMS05, and pMS06 probably carry unique resistance genes.This conclusion is based on (i) restriction site mapping (Fig.3), (ii) Southern blotting (Fig. 4), and (iii) the drug resistancelevel conferred on yeast cells by each plasmid (Fig. 5 and 6).However, our data do not exclude the possibility that theseplasmids carry the same resistance gene. Further work,including subcloning, transcription mapping of, and se-quencing of the DNA that confers MSO resistance, shouldresolve the issue of whether the MSO plasmids carry iden-tical or unique resistance genes.Our results demonstrate that all four MSO plasmids made

their host resistant to MSO by reducing the rate of MSOtransport across the plasma membrane. To our knowledgethis is the first report of MSO resistance resulting from areduced rate of intracellular drug transport. Several resultssuggest that MSO is using one or both of the two methionine

permeases, rather than GAP, some other amino acid-specificpermease, or another type of permease. First, methioninewas the only amino acid that reduced the rate of MSOtransport. Second, methionine and glutamine were the onlyamino acids that prevented growth inhibition by MSO.Third, MSO plasmids reduced the rate of transport of bothMSO and methionine. Finally, MSO plasmids did not affectthe rate of citrulline transport, indicating that they were notreducing GAP activity, which they would be doing if MSOwas transported by GAP. It is not clear from our resultswhether MSO is transported by the low- or the high-affinitymethionine transporter (11) or by both. Use of a METP-defective strain (11), which lacks the high-affinity trans-porter, might help to resolve this issue.There are three obvious ways in which MSO plasmids

could be reducing MSO transport. They could be (i) prevent-ing transcription of the permease structural gene, (ii) pre-venting translation of the permease mRNA, or (iii) inactivat-ing the permease. These three mechanisms are not mutuallyexclusive, and each MSO plasmid could be using a differentone. It is not possible to propose a more detailed model forthe function of MSO plasmids because the regulation ofmethionine permease activity (reviewed in Cooper [4]) andother methionine-related enzymes (reviewed in reference 16)are not understood. Also, one or more MSO plasmids couldbe influencing methionine permease(s) activity by an indirectaffect such as by changing the size of intracellular methio-nine pools (reviewed in reference 16, p. 249). Inhibition ofMSO and tabtoxin transport probably requires multiplecopies of the resistance genes, because the parental strain,D13-1A, from which the resistance genes were isolated isvery sensitive to each drug and probably has only one copyof each of the genes. Also, YEp13, the vector used in theseexperiments, has a 2,im origin of replication and is present inmultiple copies per cell. Our measurements ranged from 3 to11 copies per cell. Further experiments with the MSO geneson single-copy vectors will be needed to verify that drugresistance is due to increased gene dosage.

Since methionine is part of the aspartate family of aminoacids and since some genes in this family are coregulated(16), it is possible that MSO plasmids regulate otherpermeases besides the methionine permease. They couldregulate nonpermease genes and enzyme activities also.Such coregulation could explain our results for tabtoxin.Since the toxic effects of tabtoxin were not prevented bymethionine, it is likely that tabtoxin is not transported by thesame methionine permease that transports MSO. If thehypothetical tabtoxin permease was coregulated with themethionine permease by MSO plasmids, then our data wouldbe readily explained. We did not analyze tabtoxin transportdirectly because of the difficulty and expense of makingradioactive tabtoxin. Because of this, we cannot eliminatethe possibility that the tabtoxin resistance phenotype con-ferred by MSO plasmids is due to some mechanism otherthan exclusion from the cell.One of our initial reasons for undertaking this research

was to gain insight into the mechanism of wildfire diseaseresistance in N. longiflora and to develope procedures forconstructing tobacco strains that are resistant to wildfire.Based on our results with S. cerevisiae, we would predictthat overproduction of glutamine synthetase activity in to-bacco would lead to MSO, tabtoxin, and wildfire diseaseresistance. However, recent studies by Donn et al. (6)suggest that overproduction of glutamine synthetase activityin alfalfa cells does not yield MSO resistance. They selectedalfalfa cells in culture that were 20- to 100-fold more resistant

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than wild-type cells to the nonselective herbicide L-phosphinothricin, a competitive inhibitor of glutamine syn-thetase. The 3- to 7-fold overproduction of glutamine syn-thetase activity in their resistant cell lines was shown tocorrespond to a 4- to 11-fold amplification of one glutaminesynthetase gene. It is not clear why their cell lines failed toshow increased resistance to MSO. It may be that MSO hasanother target in alfalfa besides glutamine synthetase, thathigher levels of enzyme are necessary for resistance, or thatone of the noncytoplasmic glutamine synthetases in alfalfa isparticularly sensitive to MSO and not to phosphinothricin.Without a better understanding of how MSO resistancegenes block MSO and tabtoxin transport into yeast, wecannot predict whether they would confer MSO, tabtoxin, orwildfire resistance on tobacco; in some cases they might.

ACKNOWLEDGMENTS

We thank Richard E. Galardy, Todd Monroe, and Camilla Blockfor purifying tabtoxin, David Watt for assistance in synthesizingradioactive methionine sulfoximine, Aaron Mitchell for supplyingpJL106 and glutamine synthetase-deficient yeast strains, and R.Durbin for a sample of purified tabtoxin.

This work was supported by grant 7H180-5E532 to R.C.D. andGlenn B. Collins from the Tobacco and Health Research Institute atthe University of Kentucky.

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