EFFECTS OF URACIL AND THYMIDINE ON THE DEVELOPMENT OFRESISTANCE TO 5-FLUOROURACIL IN PEDIOCOCCUS CEREVISIAE

P. J. WHITE' AND C. A. NICHOLDepartment of Experimental Therapeutics, Roswell Park Memorial Institute, Buffalo, New York

Received for publication 3 August 1962

ABSTRACT

WHITE, P. J. (Roswell Park Memorial In-stitute, Buffalo, N.Y.) AND C. A. NICHOL. Effectsof uracil and thymidine on the development ofresistance to 5-fluorouracil in Pediococcus cer-evisiae. J. Bacteriol. 85:97-105. 1963.-Growthof Pediococcus cerevisiae ATCC 8081 in a basalsemidefined medium was inhibited by 5-fluorouracil (FU) and 5-fluoro-2'-deoxyuridine(FUDR). Addition of uracil slightly decreasedsensitivity to FU, but did not affect inhibition byFUDR. Addition of thymidine alone increasedsensitivity to FUDR 500-fold, but did not appre-ciably affect sensitivity to FU. This organism wasable to grow in the presence of high concentra-tions of either drug when both uracil and thymi-dine were added to the medium. Substrainsresistant to FU were developed in the basalmedium, or in basal medium with added uracil,or with thymidine. The substrains developed inthe presence of uracil or thymidine were notresistant when these compounds were omittedfrom the medium. Cross resistance to FUDR wasshown only when thymidine was present. Theability to use thymidine as a growth factor inthe absence of leucovorin was lost in the sub-strains developed in the presence of uracil, thoughnot in the other substrains. Washed suspensions oforganisms of all strains hydrolyzedFUDR to FU,the parent strain being most active in this respect.The cellular uptake of fluorouracil-2-C14 wasless in all of the resistant substrains than in theparent strain, whether or not uracil or thymidinewas present during the incubation. This uptakeof FU-C'4 was decreased most markedly in thesubstrains which became resistant to FU in theabsence of uracil.

5-Fluorouracil (FU) and 5-fluoro-2'-deoxyuri-dine (FUDR) were introduced as tumor-inhibit-

I Present address: Twyford Laboratories Ltd.,Twyford Abbey Road, London, England.

97

ing compounds by Heidelberger and co-workers(Duschinsky, Pleven, and Heidelberger, 1957;Heidelberger et al., 1957). The drugs are alsoinhibitors of bacterial growth (Scheiner, Kostelak,and Duschinsky, 1957), and the inhibition maybe overcome by the addition of uracil, thymine,or thymidine (Cohen et al., 1958; Edinoff, Knoll,and Klein, 1957). Resistance to FU has beenobserved in tumor cells and in bacteria, and hasbeen associated with loss of enzymes concernedin uracil anabolism. Reichard, Skold, and Klein(1959) found that Ehrlich ascites tumor cellsthat became resistant to FU lost uridine phos-phorylase activity. Brockman, Davis, andStutts (1960) showed that enzyme preparationsfrom FU-resistant Escherichia coli failed tocatalyze the reactions of uracil or FU with 5-phosphoribosyl-1-pyrophosphate to form 5'-ribonucleotides, whereas similar enzyme prepara-tions from FU-sensitive E. coli did catalyze thesereactions. In both of these cases, it is believedthat the administered drug is no longer convertedenzymatically to an inhibitory ribonucleotidederivative by the resistant strains. Heidelbergeret al. (1960) found that resistance to FU in a lineof Ehrlich ascites cells may be due to a decreasedsensitivity of the enzyme that converts deoxyuri-dylic acid to thymidylic acid (thymidylatesynthetase) to inhibition by fluorodeoxyuridylicacid, into which administered FU is still con-verted by the resistant cells.The present study concerns the effects of

uracil and thymidine on the sensitivity of Pedio-coccus cerevisiae 8081 to FU and FUDR, and onthe emergence and properties of resistant sub-strains of this organism. P. cerevisiae was usedbecause of its high initial sensitivity to bothdrugs, and because it can be grown under con-ditions (in the absence of leucovorin) in whichthymidine is an essential growth factor(Sauberlich, 1949). A preliminary report of thiswork has been presented (White, 1960).

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MIATERIALS AND MIETHODS

Organism. P. cerevisiae (Leuconostoc citrovorum),ATCC 8081, was maintained in Tryptose-glucose-agar stab cultures which had been grown over-night at 37 C. Stock cultures were transferredmonthly and stored at 2 C. Substrains resistant toFU and FUDR were similarly maintained.Medium. A semidefined medium, containing

acid-hydrolyzed casein but without added py-rimidines, was used for growth experiments. Thebasal medium contained (per liter): Vitamin FreeCasamino Acids (Difco), 7.5 g; DL-tryptophan,80 mg; DL-phenylalanine, 50 mg; DL-tyrosine,100 mg; L-cysteine hydrochloride, 50 mg; D-glucose, 25 g; CH3COONa-3H2O, 20 g; NH4Cl,3 g; KH2PO4, 600 mg; K2HPO4, 600 mg; MgSO4.7H20, 200mg;MnSO4 4H20, 20mg; FeSO4 * 7H20,10 mg; NaCi, 10 mg; adenine sulfate, 10 mg;guanine hydrochloride, 10 mg; xanthine, 10 mg;pyridoxine hydrochloride, 2 mg; pyridoxalhydrochloride, 2 mg; pyridoxamine hydro-chloride, 2 mg; riboflavine, 1 mg; thiamine hydro-chloride, 1 mg; calcium pantothenate, 1 mg;nicotinic acid, 1 mg; biotin, 15 ,g. Then, 10 g ofDifco yeast extract or 0.4 Mg of leucovorin(calcium salt of 5-formyltetrahydropteroylgluta-mate) or 100 ,g of thymidine had to be added toeach liter of medium. The medium was preparedat double strength, adjusted to pH 6.5, filteredthrough Whatman no. 50 paper, and brought tothe desired volume with water (or other experi-mental additions), before autoclaving at 120 Cfor 7 min.

Growth conditions. Organisms for inocula weregrown overnight in basal medium containingyeast extract, centrifuged, and washed twice insterile saline of equivalent volume. The washedsuspension in saline was diluted to about 2 X106 organisms per ml, and 0.1 ml of this suspen-sion was used to inoculate 5 ml of medium.Inoculated medium was incubated in air at 37 Cin plugged tubes (150 X 19 mm) set up in dupli-cate. When large quantities of organisms wererequired for experiments with washed suspen-sions, medium (500 ml) containing leucovorin,in a 1-liter conical flask, was inoculated with anovernight culture (1 ml) of P. cerevisiae grown inbasal medium containing yeast extract, andincubated overnight at 37 C.

Assessment of growth. Growth was estimated bymeasurement of turbidity with a Klett-Summer-son photoelectric colorimeter with a red filter,

in matched colorimeter tubes (125 X 16 mm);the uninoculated medium was used to give thezero setting. The relation between the colorimeterreading and the dry weight of organisms waslinear from 0 to 150 on the colorimeter scale, and areading of 100 was equivalent to 0.24 mg (dry wt)of organisms per ml. A 1-ml sample of a suspen-sion of cells, which read 100 on the colorimeterscale, gave 2 X 108 colonies on solid medium.

Sensitivity to FU and FUDR. Tubes of mediumwere prepared, containing the drug at concen-trations increasing about threefold betweeneach pair of tubes in a series. Medium was auto-claved after addition of the drug, inoculated,and incubated for 20 hr (or 40 hr when thymidinereplaced leucovorin). The concentration of druginhibiting growth by 50% after this time wasdetermined by plotting the level of growth againstconcentration of inhibitor in the medium (Fig. 2).

Mlleasurement of uptake of 5-fluorouracil-2-C14.P. cerevisiae 8081, and its resistant substrains,were grown in basal medium (50 ml), containingleucovorin (0.9 m,ug/ml), in 125-ml Erlenmeyerflasks at 37 C for 14 hr. Organisms were centri-fuged and washed twice with saline (20 ml),then resuspended in double-strength basalmedium to a concentration of about 1 mg (drywt) of organisms per ml. The suspension (1.0 ml),double-strength basal medium (1.5 ml), 5-fluor-ouracil-2-C'4 (0.5 ml; 1 mg/ml; 100 counts permin per Mug), and water or other addition, to afinal volume of 5.0 ml, were incubated in dupli-cate in tubes (130 X 15 mm) at 37 C for 1 hr,along with controls without organisms. Afterincubation, the mixture was filtered through aMillipore filter (Millipore Filter Corp., Bedford,Mass.) into a Millipore disc, type HA (diameter,25 mm; pore size, 0.45 ,). Liquid was removedfrom the disc under slight vacuum for 2 min.Then the disc was transferred to a planchetteand dried in a desiccator over calcium chloride.Radioactivity was measured in a gas flow counter(RCL Mark 12, Model 2; Radiation CounterLab., Inc., Skokie, Ill.) connected to a scalerunit (Model 183A; Nuclear-Chicago Corp.,Des Plaines, Ill.).The difference in counts between discs with

and without cells was attributed to fluorouracil-2-C'4 taken up by the organisms. Washing oncewith water (2 ml) reduced the readioactivity ofdiscs without organisms almost to the limit ofdetection. A second wash did not further de-

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RESISTANCE OF P. CEREVISIAE TO 5-FLUOROURACIL

crease the radioactivity of discs with cells. Thisresidual radioactivity was equivalent to theamount of fluorouracil-2-C'4 calculated to bepresent in the organisms by subtraction of theblank without organisms from the counts ofunwashed discs. There was a linear relation, afterblank correction, between the radioactivity of adisc and the dry weight of organisms collected onthe disc, up to at least 5 mg of organisms (dry wt)per disc. Organisms were similarly incubated inbasal medium containing polyglucose-C'4 (1,350counts per min per ml) to which the organismswere assumed to be impermeable (Werkheiserand Bartley, 1957). The suspension was filteredthrough a Millipore disc. The radioactivity ofthis disc was the same as that of a control discthrough which medium containing polyglucose-C14 without organisms was filtered. The contri-bution of interstitial liquid to the count of adisc was, therefore, negligible when the weights oforganisms collected were small.

Metabolism of fluorodeoxyuridine. Washedsuspensions (1 ml) containing about 50 mg (drywt) of P. cerevisiae 8081, or the resistant sub-strains, in 0.02 M phosphate buffer (pH 6.5) wereincubated with FUDR (1 ml, 1 mg/ml) for 1 hrat 37 C. Organisms were centrifuged and thesul)ernatant fluids were diluted 1:50 in 0.1 NKOH. Controls without organisms and withoutFUDR were included.

Estimation of base mixtures. When presenttogether, FU and FUDR were estimated bymeasurement of optical density (OD), at 290,282, 266, and 255 m,u, of solutions in 0.1 NKOH. The concentrations were calculated fromthe following equations, derived by the methodof Loring (1955), after comparison of the absorp-tion spectra of the two compounds in alkalinesolution (Fig. 1).

FU (moles/liter) = 7.00 OD2 - 2.42 OD2662.28 X 104

5.66 OD266- 7.00 OD2Mor0.492 X 104

FUDR (moles/liter) = 4.65 OD26 -4.05 0D2902.28 X 104

4.05 OD255- 2.58 OD266or

0.492 X 104

Total FU plus FUDR (moles/liter) = 055810.515 X 104

z

w

5

hi

0

z0

X

0

z0I-C.

-J0

-J-i

5

4

3

2

250 260 270 280 290

WAVELENGTH (mp)FIG. 1. Ultraviolet absorption of fluorouracil

(FU) and fluorodeoxyuridine (FUDR) in 0.1 Npotassium hydroxide. 0, FU; 0, FUDR.

Chromatographic procedure. FU and FUDR(about 10 ,ug per spot) were separated by de-scending chromatography on Whatman no. 1paper for 24 hr at room temperature, using assolvent three parts (by volume) of n-butanol toone part of 28% (w/v) ammonia (Reichard,1955). The compounds were detected by exami-nation of the paper in the dark under an ultra-violet lamp. The RF values for FU and FUDRwere 0.20 and 0.15, respectively. After elutionfor 18 hr with 0.1 N HCI at 37 C, both compoundswere estimated by measurement of absorbancies(Beckman DU spectrophotometer) of the solu-tions in 0.1 N hydrochloric acid at the maxima,266 and 269 m,u (millimolar extinction co-efficients 7.6 and 9.0) for FU and FUDR, re-spectively. The ratio OD230:OD260 was used as

7

VOL. 85, 1963 99

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WHITE AND NICHOL

a criterion of purity (0.71 and 0.89 for FU andFUDR, respectively). Determinations afterseparation agreed with estimations for the mix-tures described previously.

Source of chemicals. FU and FUDR were sup-plied by H. Bond of the Cancer ChemotherapyNational Service Center, Bethesda, Md. 5-Fluor-ouracil-2-C'4 was purchased from the CaliforniaCorporation for Biochemical Research, LosAngeles. Orotidylic acid was a gift from R. E.Handschumacher, Department of Pharmacology,Yale University, New Haven, Conn. Deoxyuri-dylic acid was prepared from deoxycytidylic acidby the method of Flaks and Cohen (1959). Asolution of polyglucose-CI4 was a gift from W. C.Werkheiser of this department. Other materialswere commercial samples.

RESULTSSensitivity to FU and FUDR. In the basal

medium containing leucovorin, growth of P.cerevisiae 8081 was inhibited 50% by FU (5 X10-8 M) and by FUDR (109 M), after incubationfor about 20 hr. The concentration of eitherdrug needed for inhibiton increased about three-fold, when incubation was prolonged for 3 days.When the inoculum was increased 1,000-fold,sensitivity to FUDR decreased 30-fold but thesensitivity to FU decreased only 3-fold.

Addition of thymidine to the basal mediumcontaining leucovorin increased by 400-fold the

0

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FIG. 2. Effects of uracil and thymidine on sensi-tivity of Pediococcus cerevisiae 8081 fluorouracil(FU) and fluorodeoxyuridine (FUDR) after 20 hrof incubation. 0, no addition to basal medium; C,uracil added to basal medium; Q, thymidine added tobasal medium; *, uracil and thymidine added tobasal medium; solid line, FU; dashed line, FUDR.

TABLE 1. Effects of pyrimidines and derivatives onsensitivity of Pediococcus cerevisiae 8081 tofluorouracil (FU) and fluorodeoxyuridine

(FUDR)

Concn (ms) of drugpermitting 50% of maximal

Addition (20 pg/ml) to basal growth (20 hr ofmedium containing leucovorin incubation)*

FU FUDR

No addition..............Orotic acid...............Orotidylic acid...........Uracil ....................Uridine ..................Deoxyuridine .............Uridylic acid.............Deoxyuridylic acid.......Thymine.................Thymidine ...............Cytosine .................Cytidine..................

Uracil + thymidine.Uracil + uridine....Uracil + deoxyuridine....Uracil + thymine........Uridine + deoxyuridine...

Thymidine + uridine.....Thymidine + deoxyuri-

dine....................Thymidine + uridylic

acid....................Thymidine + deoxyuri-

dylic acid..............Thymidine + orotic acid..Thymidine + orotidylic

acid ....................

Uracil + uridine + deoxy-uridine .................

Uracil + uridine +thymidine..............

Uracil + deoxyuridine +thymidine..............

4 X 10-84 X 10-84 X 10-88 X 10-78 X 10-72 X 10-64 X 10-74 X 10-74 X 10-82 X 10-78 X 10-84 X 10-7

8 X 10-52 X 10-63 X 10-68 X 10-72 X 10-6

3 X 10-4

5 X 10-6

4 X 10-6

4 X 10-72 X 10-7

2 X 10-7

2 X 10-6

8 X 10-10

3 X 10-92 X 10-98 X 10-8

8 X 10-9

3 X 10-7

3 X 10-9

4 X 1O-'

8 X 10-810-9

4 X 10-8

3 X 10-4

8 X 10-6

4 X 10-8

8 X1O-4 1>4 X1o-4

2 X 10-4 I>4 X 10-4

* Sensitivity to FU or FUDR was determined(see Materials and Methods) in medium to whichthe various pyrimidine derivatives were addedbefore autoclaving. Although the growth responsgwas measured after incubation for 20 hr, thesensitivity to FU and FUDR did not decreasemarkedly upon longer incubation.

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VOL. 85, 1963 RESISTANCE OF P. CEREVISIAE TO 5-FLUOROURACIL

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WHITE AND NICHOL

concentration of FUDR needed for inhibition,but thymidine had very little effect in overcomingthe inhibition due to FU (Fig. 2). When uracilwas substituted for thymidine, the concentrationof FU needed to cause inhibition was increased20-fold, but there was very slight effect oninhibition caused by FUDR. When thymidineand uracil were both present, growth occurredin the presence of high levels of either drug (10-4M). Tests were made of the ability of variouspyrimidine derivatives to reproduce or to aug-ment these effects of uracil and thymidine (Table1). Only deoxyuridine was more effective thanuracil in overcoming inhibition due to FU, andonly deoxyuridine approached the effectivenessof thymidine in overcoming inhibition by FUDR.Uridine plus thymidine was the only binarycombination more effective than uracil plusthymidine. In the presence of uracil, uridine, andthymidine, the growth of P. cerevisiae 8081 be-came almost insensitive to FU or FUDR.

Development of resistance to FU and FUDR.Organisms were grown in a series of tubes con-taining medium and inhibitor. The concentrationof inhibitor was trebled at each step in the series.Organisms from the highest level of drug atwhich growth occurred after 2 to 3 days wereused to inoculate a fresh series of tubes. Repeti-tion of this procedure led to a gradual increase ofresistance. Resistance to FU was developed byserial passage in four different media (Table 2).Substrain 1 (1,000-fold resistant) was developedin the basal medium containing leucovorin withuracil added; substrain 2 (4,000-fold resistant)was developed in the presence of leucovorin andthymidine; substrain 3 (400-fold resistant) wasdeveloped in basal medium containing thymidine,but without added leucovorin; and substrain 4(20,000-fold resistant) was developed in basalmedium containing leucovorin with no otheraddition. One substrain (5) resistant to FUDR(100,000-fold) was developed by serial passage ofthe parent organism, in the presence of increasingconcentrations of this drug, in basal mediumcontaining leucovorin and uracil. About tenpassages at intervals of 2 to 3 days were requiredin each medium to develop substrains able togrow in the presence of about 5 X 104 M drug.In the case of substrain 4, it was necessary toincrease the size of the inoculum from 2 X 105to 2 X 106 organisms per 5 ml of medium in theearlier passages to obtain increased resistance.

Properties of resistant strains. All of the sub-

strains were able to grow in the basal medium inthe absence of drug with only leucovorin added;the growth response to leucovorin of the resistantsubstrains and of the parent strain was over thesame range of concentration (0.01 to 0.4 m,ug/ml).Substrains 1 and 5, however, were unable togrow in the basal medium when thymidine re-placed leucovorin. The parent strain and all ofthe other substrains could grow, after a lag ofabout 40 hr, when leucovorin was replaced in thebasal medium by thymidine, although the finallevel of growth (colorimeter reading 80) in thepresence of thymidine was lower than in thepresence of leucovorin (colorimeter reading 250).This relatively long lag and low level of growthwere still shown when organisms were transferredseveral times into fresh medium containingthymidine without leucovorin. Hence, the growthon thymidine is probably not due to the selectionof mutants, but rather to a slow adaptation touse of thymidine as a growth factor.The resistant strains were able to grow in

high concentrations of drug only in the presenceof the same additional nutrients that supple-mented the basal medium during the develop-ment of resistance (Table 2). Thus, substrains 1and 5 were resistant to FU only if uracil waspresent; substrains 2 and 3 were resistant only inthe presence of thymidine; whereas substrain 4was resistant in the absence of uracil and thymi-dine. When incubation in the basal medium plusleucovorin was continued beyond the usualperiod of 24 hr to 40 to 45 hr, substrains 1 and 5showed intermediate levels of resistance to FU.and substrains 2 and 3 were able to grow in thehighest levels of FU.When tested in the same medium used for the

development of resistant substrains, 2 and 3were highly cross-resistant to FUDR, whereassubstrains 1 and 4 had only slight cross resistance(Table 2). With longer periods of incubation,substrains 1 and 4 were able to grow at increasedconcentrations of FUDR, but resistance did notreach a very high level. Substrain 4 became highlyresistant to FUDR if thymidine was added to thebasal medium. Substrain 5 was cross-resistant toFU when uracil was present in the medium.

Uptake of fluorouracil-2-C'4 from the basalmedium by washed suspensions of organisms. Theamounts of FU taken up by the various strainsof P. cerevisiae were compared in the absence andin the presence of uracil and thymidine, singly ortogether (Table 3). The parent strain took up

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more FU from the basal medium than did any ofthe resistant strains; in substrain 2, no uptake ofFU was detected. With the parent strain andwith substrain 1, the addition of uracil diminishedthe uptake of drug; in other strains it had a lessmarked effect. The addition of thymidine gener-

ally had only a slight effect. The uptake of FUper mg (dry wt) of organisms did not changeappreciably over a range of FU concentrations inthe incubation mixture from 25 to 200 ,ug perml. The uptake of FU increased with the time ofincubation.

Mlletabolism of FUDR. Washed suspensions ofP. cerevisiae 8081 were incubated in the basalmedium with FUDR (100 ,ug/ml) to measure theuptake of the drug. When organisms were centri-fuged and the supernatant fluid was examined bychromatography, ultraviolet-absorbing spots cor-

responding to FU, as well as to FUDR, were

found on the paper. The presumed FU had thesame OD280:OD260 ratio (0.68) as authentic FU.It was inhibitory to P. cerevisiae 8081 over thesame range of concentrations as was FU, andthis inhibition was partly overcome by theaddition of uracil to the medium. Washed or-

ganisms, incubated in the buffer solution for 1hr, also hydrolyzed FUDR to FU (Table 4).Since no other ultraviolet-absorbing materialwas present in the buffer solution, or formed dur-ing the incubation, both drugs could be estimatedby direct measurement of absorbancy after re-

moval of the cells.Although the parent strain was the most sen-

sitive to FUDR in growth tests, washed cellsuspensions of this strain hydrolyzed FUDR toFU more rapidly than did any of the resistant

TABLE 3. Uptake of fluorouracil (FU) -2-C14 fromthe basal medium by Pediococcus cerevisiae

8081 and resistant substrains

Uptake of FU-2-C'4 (jug/mg dry wt)

Organism* +hyNo + Uracil

Thy-+ Uraciladdition (20 jg/ (20idine and thy-

m) Ml) mi'dine

P. cerevisiae 8081 6.9 2.3 7.8 3.7Substrain 1 4.1 1.1 2.6 1.7Substrain 2 <0. 1 0.6 0.5 0.1Substrain 3 0.5 0.6 0.4 0.1Substrain 4 0.8 0.1 <0. 1 0.9Substrain 5 1.1 1.0 1.1 1.1

* Organisms were incubated 1 hr at 37 C in thebasal medium containing FU-2-C14 (100 ,g/ml).

TABLE 4. Conversion of fluorodeoxyuridine(FUDR) to fluorouracil (FU) by Pedio-

coccus cerevisiae 8081 and substrains

Drug remaining afterStrain Cells (dry wt) incubation*

FUDR FU

mng iAmoles p.rnoles

8081 ........... 111 0 3.893 0 3.856 0.7 3.2116t Ot 4.2t

Substrain 1 56 2.9 0.9Substrain 2....... 92 0.3 3.4Substrain 3....... 105 0.4 3.2Substrain 4 92 0.2 3.8Substrain 5 80 2.6 1.3

* Cells were incubated in 0.01 M phosphatebuffer (pH 6.5) for 1 hr at 37 C with 4.0 ,Amolesof FUDR.

t Cells incubated in basal medium.

substrains (Table 4). The two substrains devel-oped in the presence of uracil were considerablyless active with respect to such degradation thanwere any of the other strains.

DIscussIONThe nutrients that are related to metabolic

pathways inhibited by FU or its deoxyribosidecan influence the characteristics of resistantstrains of P. cerevisiae 8081 selected in the pres-ence of these metabolites. Both uracil andthymidine must be added to the basal mediumto permit growth of the sensitive organismin the presence of high concentrations of eitherFU or FUDR. Thus, the action of each drugappears to involve at least two separate sites.At one locus, inhibition is overcome by the addi-tio-n of uracil; at the other, by the addition ofthymidine. When organisms become resistant toFU in the absence of uracil and thymidine (sub-strain 4), mechanisms must be developed tocircumvent two blocked reactions or two differentfunctions. In the presence of uracil (substrain 1),only the block in the synthesis of thymidylicacid has to be overcome; in the presence ofthymidine (substrains 2 and 3), only the impair-ment of uracil metabolism needs to be circum-vented. In fact, substrain 1 is sensitive to FU ifuracil is omitted, and substrains 2 and 3 aresensitive to FU if thymidine is omitted.Not only do uracil and thymidine render

organisms less sensitive to FU, but their presence

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WHITE AND NICHOL

may also lead to the selection of substrains ofhigher levels of resistance. This is in accord withthe postulate of Sevag (1946) that organisms maymore readily become tolerant of inhibitors if theenvironment contains substances that can beused for alternative metabolic pathways to by-pass the blocked reaction. Several observationsare relevant. Kohn and Harris (1942) describeda strain of E. coli that developed a requirementfor methionine during the development of sul-fonamide resistance in medium that containedmethionine. Harrison and Clapper (1950) pro-duced a strain of Streptococcus mitis which usedpteroylglutamic acid as a growth factor, bydeveloping sulfonamide resistance in mediumcontaining pteroylglutamic acid. In two in-stances, a requirement for thymine occurred instrains of Lactobacillus casei selected for resistanceto two different folic acid antagonists,pyrimethamine (Singer, Elion, and Hitchings,1958) or amethopterin (Anton and Nichol, 1959).In each of these cases, the new requirement wasshown in the absence of drug. With the resistantstrains of P. cerevisiae, however, uracil or thymi-dine was required only to permit growth in thepresence of high concentrations of inhibitor. Thus,the presence in the medium of metabolites, whichcan decrease the sensitivity to a growth inhibitor,can condition the selection of resistant substrainswith partial or complete requirements for suchcompounds.The enzymatic conversion of deoxyuridylic acid

to thymidylic acid (thymidylate synthetase) isinhibited by FU, and more powerfully by FUDR,after the administered drug has been converted tofluorodeoxyuridylic acid (Danneberg, MIontag,and Heidelberger, 1958; Bosch, Harbers, andHeidelberger, 1958). Thymidine most probablyacts by overcoming the inhibition of thymidylatesynthetase caused by FU or FUDR. The locusat which uracil acts in preventing inhibition isless definite. FU can be incorporated into theribonucleic acids (Gordon and Stachelin, 1958;Chaudhuri, Montag, and Heidelberger, 1958;Horowitz and Chargaff, 1959) or into uridinecoenzymes (Rogers and Perkins, 1960), anduracil may prevent these incorporations byeffective substrate competition. The requirementfor uracil to overcome inhibition by FUDR isprobably due to conversion of FUJDR to FU, areaction that is carried out rapidly by washedsuspensions of these organisms. Furthermore, in

the presence of thymidine, approximatelyequimolar concentrations of FU and FUDR areneeded to inhibit growth, suggesting that, wheninhibition of thymidylate synthetase is circum-vented, organisms can grow in the presence ofFUDR until its degradation produces an inhibi-tory concentration of FU. The decreasing sen-sitivity to FUDR as the size of the inoculum isincreased may be related to the conversion of thedrug to a less potent inhibitor. The substrainsthat are resistant to FU in the presence of thymi-dine are cross-resistant to FUDR, and substrain 5,developed in the presence of FUDR and uracil,is cross-resistant to FU. Both of these observa-tions are consistent with the degradation ofFUDR to FU by the organisms. Substrains 1 and4, although resistant to FPU in the absence ofthymidine, are not cross-resistant to FUDR.Possibly these two substrains do not convert FUto deoxyribose derivatives.Even in the absence of uracil or thymidine,

all of the resistant substrains took ul) lessfluorouracil-2-C'4 than did the parent strain.This decreased uptake may be the result of adecreased permeability of the organisms to thedrug, or it may be due to a diminished incorpora-tion of FU into ribonucleic acid (RNA) or uridinecoenzymes. Resistance of S. faecalis to anotheruracil analogue, 6-azauracil, was associated withreduced uptake of both uracil and the drug.Also, uridine, but not uracil, was readily incor-porated into RNA of the resistant cells(Handschumacher, 1957). The inability of sub-strains 1 and 5 to use thymidine in place ofleucovorin suggests that the loss of enzymesconcerned in pyrimidine metabolism may have apart in the resistance of these strains of P.cerevisiae. A recent report by Bloch and Hutchi-son (1962) indicates that thymine-2-C'4 wasincorporated into the deoxyribonucleic acid(DNA) of a FU-sensitive strain of S. faecalis, butnot into the DNA of a FU-resistant substrain.Any decrease in the ability of the resistant or-ganisms to use exogenous pyrimidines must becompensated for by accelerated de novo synthesis.Better understanding of the altered metabolismassociated with resistance may indicate means ofreducing the alternative ways by which the lethaleffect of the drug can be evaded.

ACKNOWLEDGMENTS

This work was supported in part by a researchgrant (CY-2906) from the National Cancer

104 J. BACTERIOL.

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Institute, U.S. Public Health Service. The awardof a postdoctoral fellowship to one of us (P. J.W.) from U.S. Public Health Service TrainingGrant CRTY-5016 is gratefully acknowledged.

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105VOL. 85, 1963

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ERRATA

ATTEMPT IN CLASSIFICATION OF THE CATALASE-POSITIVESTAPHYLOCOCCI AND MICROCOCCI

D. A. A. MOSSELLaboratory of Bacteriology, Central Institute for Nutrition and Food Research T.N.O.,

Utrecht, The Netherlands

Volume 84, no. 6, page 1140, title should read: "Attempt in classification of the catalase-positivecocci: staphylococci and micrococci." Col. 1, lines 3 and 4 should read "Netherlands). Attempt inclassification of the catalase-positive cocci: staphylococci and micrococci." Col. 1, lines 22 and 23should read "3%. Of 105 obligately aerobic coagulase-negative cocci (micrococci), none fermentedman-." Page 1143, col. 2, line 31 should read "this may be, our results clearly demonstrate that-."

EFFECTS OF URACIL AND THYMIDINE ON THE DEVELOPMENT OFRESISTANCE TO 5-FLUOROURACIL IN PEDIOCOCCUS CEREVISIAE

P. J. WHITE AND C. A. NICHOLDepartment of Experimental Therapeutics, Roswell Park Memorial Institute, Buffalo, New York

Volume 85, no. 1, page 97, Abstract, lines 11-12 should read "Addition of thymidine alone de-creased sensitivity to FUDR 500-fold."

PROTOPLAST MEMBRANE OF STREPTOCOCCUS FAECALISGERALD D. SHOCKMAN, JOSEPH J. KOLB, BOHDAN BAKAY,

MARGARET J. CONOVER, AND GERRIT TOENNIES

Department of Microbiology, Temple University School of Medicine, and The Institute for Cancer Research,Philadelphia, Pennsylvania

Volume 85, no. 1, page 175, Acknowledgments, lines 6 and 7 should read "The electron micro-graphs were prepared and taken by T. F. Anderson."

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