Metabolism of Resistant Mutants of Streptococcusfaecalis

I. Isolation and Characterization of the Mutants*

DORRISJ. HUTCHISON

(Division of Experimental Chemotherapy, Slaan-Kettering Institute for Cancer Research, New York, N.Y.)

The currently unavoidable phenomenon of theappearance of drug-refractory leukemic cells inchildren whose disease was at one time responsiveto that drug has served as a stimulus for the studyof strains of microorganisms resistant to suchagents. A series of mutants of Streptococcusfaecalishas been selected, and these mutants exhibit various degrees of resistance to A-methopterin, 6-mer-captopurine, and 2,6-diaminopurine—all of whichhave been shown to be temporarily effectiveagainst certain mouse leukemias and human leu-kemias (2, 3, 9, 15,16, 21). The general proceduresfor selection of these resistant S. faecalis mutantsas well as maintenance and growth characteristicsof all cultures will be discussed.

MATERIALS AND METHODSThe growth media used throughout these experiments are

various modifications of the folie acid assay medium describedby Flynn et al. (10) ; this folie acid-free medium will be referredto as the F medium. The omission of the purines, adenine,guanine, and xanthine, and of the pyrimidine, uracil, results ina medium which will be designated as F-PP; supplements offolie acid, 1 m^g/ml, or thymine at l /ig/ml will be indicated asF-PP + PGA and F-PP + T, respectively.

S. faecalis ATCC 8043 (SF/O, which indicates a lack of resistance to the antimetabolites studied), which requires folieacid or a purine and thymine for growth, has served as theparent culture for all the mutants.

The method, which has been described in detail (5,18), usedfor the selection of the resistant mutants was that of serialtransfer through an appropriate basal medium that had beensupplemented with increasing concentrations of an anti-metabolite. The solubility of the antimetabolite controlled, tosome extent, the degree of resistance of the mutant. In general,when maximum resistance was attained, the culture was transferred daily or every other day in the medium which containedthe antimetabolite until there was as much growth of the resistant mutant in 18 hours in the antimetabolite medium asthere was of the parent in 18 hours in the absence of the anti-metabolite. At this time the mutant was transferred to agarstabs which contained the same concentration of the antagonistas the selection medium, and it was then maintained and

* These studies were supported by research grants C679 andC2699 from the National Cancer Institute of the National Institutes of Health, Public Health Service, and institutionalgrants from the American Cancer Society.

Received for publication September 19, 1957.

handled for experimental work according to the recommendedprocedures for lactic acid bacteria (20).

Conventional microbiological vitamin assay technics wereemployed, i.e., at the beginning of each week broth cultures(5 ml.) were made from the agar stabs and incubated at 35°C.

for 24 hours. Subsequent cultures for the following day weremade from this broth culture, which in turn served as theinoculum for the following day. The seed culture for each experiment was incubated for 18-20 hours at 35°C., after whichinterval it was spun down and washed with 5 ml. sterile saline.The washed suspension was then diluted so that it containedapproximately 1.6 X IO7 cells per milliliter. This suspensionwas used as the inoculum, 0.1 ml. of which was pipetted tosterile 13 X 100-mm. assay tubes which contained 2 ml. ofmedium. The rack of tubes was shaken vigorously to assure aneven distribution of inoculum and was then placed in a 35°C.incubator for growth. Optical density measurements weremade on a Coleman Junior Spectrophotometer at various tuneswhich will be indicated in each experiment.

The names of all the cultures will be appropriately abbreviated to the initial letters of the generic and specific names, andthen these initials are followed by an abbreviation of the anti-metabolite to which primary resistance was attained.1

RESULTSThe selection of the A-methopterin-resistant

mutant, <S.faecalis/A with a 20,000-fold increasein resistance over the parent SF/O strain has beenreported (4). This mutant was subsequently transferred 18 more times in the P medium supplemented with A-methopterin, and an additional15-fold increase in resistance was attained. ThisSP/A mutant, which has a 300,000 increase inA-methopterin tolerance over the parent strain inthe F medium, has been used in these and subsequent studies. The procedure used in the selectionof S. faecalis/MP has been described (11), and asecond 6-mercaptopurine-resistant mutant S.

1The strains studied have been designated as follows:SF/O = parent (ATCC No. 8043).SF/MP = resistant to 6-mercaptopurine.SF/MP/A = double mutant, resistant to 6-MP and A-

methopterin.SF/MPcc = resistant to 6-MP, isolated independently of

SF/MP.SF/DAP = resistant to 2,6-diaminopurine.SF/A = resistant to A-methopterin.SF/A/O = partial revert of SF/A.SF/A/MP = double mutant, resistant to A-methopterin

and 6-MP.

214

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HUTCHISON—Metabolismof S. faecalis Mutants. I 215

/aecaZts/MPcc was selected according to the sameregimen. Both organisms show a 50-fold increasein resistance to 6-mercaptopurine in comparisonwith SF/O in the F-PP + PGA medium.

S. faecalis/A/O is a subculture of SF/A whichwas obtained after 350 transfers in F + PGAmedium. The mutant has a 50-fold increase inA-methopterin resistance over SF/O and is thus

MP/A, had the same quantitative requirement asSF/O; and two mutants, SF/A and SF/A/MP, required more folie acid and an additional 28 hoursof incubation before maximum growth was obtained; the other three mutants had a lower quantitative requirement than SF/O, which observation suggests that they are more efficient in the denovo pathway of total nucleic acid biosynthesis.

TABLE1MAINTENANCEMEDIAFORStreptococcusfaecalis

SUPPLEMENTS(PG/ML) TOF-PP MEDIUM*

PGA(0.001)

Adenine (5)Guanine (S)

Xanthine (10)Uracil (Õ)

A-meth.

(100)

6-MP

(300)DAP

(1000)ORGANISMS. faecalis ATCC 8043 (SF/O) + + - - -S./aeca/¿»/A(SF/A) - + + - -S./aeca/w/MP (SF/MP) + - +5. faecalis/MPcc (SF/MPcc) + - - -|- -5. faecalis/DAP (SF/DAP) + - - - +S./oecaKs/A/O(SF/A/O) + + _5. faecalis/A./MP (SF/A/MP) + - - + -S./a«caZw/MP/A(SF/MP/A) + +

The following abbreviations have been used: PGA, pteroylglutamic acid; A-meth., A-methopterin;6-MP, 6-mercaptopurine; DAP, 2,6-diaminopurine.

* The amount of each supplement (in Mg/ml) is given in parentheses. F-PP medium = folie acid-free medium without purines (adenine, guanine, and xanthine) and without the pyrimidine, uracil.

1/6000 as resistant as its immediate parent, SF/A.Mutant S. faecalis /DAP, which has more than a300-fold increase in resistance to 2,6-diaminopurine in the F-PP + PGA medium, was isolatedafter 84 transfers in F-PP + PGA medium supplemented with 2,6-diaminopurine.

S. faecalis/A/MP is an isolate from SF/A,which was selected after 49 transfers on theF-PP + PGA medium containing 6-MP. S.faecalis/MP/A was selected from SF/MP after70 transfers in an A-methopterin-supplementedF-PP + PGA medium.

The maintenance media for SF/O and the sevenmutants are summarized in Table 1. The basalmedium in all cases was the F-PP medium; allmedia were completely formulated and then sterilized at 121°C. for 10 minutes. Daily transfers of

each organism were made on the above liquidmedia, and stock cultures were maintained instabs on the appropriate medium to which 1.5 percent agar had been added.

The quantitative requirements of the eight organisms for folie acid are compared in Table 2.Three media (F-PP, F-PP + T, and F) were compared. These amounts of folie acid in m//g/ml forhalf-maximum growth were determined after anincubation period of 18-20 hours, except in thecase of SF/A and SF/A/MP, in the F-PP andF-PP + T media, both of which were incubatedfor 48 hours. Two mutants, SF/MP and SF/

The addition of thymine to the F-PP mediumspares the folie acid requirement of SF/O (22) buthas no sparing effect for any of the mutants exceptSF/A/MP. The results obtained in F-PP + Tmedium suggest that SF/A/MP is most similar to

TABLE 2

QUANTITATIVEFOLICACIDREQUIREMENTS(m^g/ml)FORHALF-MAXIMUMGROWTHIncubation period: 18-20 hours

F-PP*1.05.o§1.00.40.50.255.0§1.0PGAMediumF-PP+Tt0.55.0§1.00.40.50.252.0§1.0Ft0.80.10.250.40.50.250.250.3

THTHINIMedium

F430200150300200380320300

above,except

ORGANISMSF/OSF/ASF/MPSF/MPccSF/DAPSF/A/OSF/A/MPSF/MP/A

* F-PP medium = see footnote *, Table 1.t F-PP + T medium = same medium as

supplemented with thymine, 1 jug/ml,t F medium = folie acid-free medium.§Incubation period, 48 hours.

the parent strain in that it used preformed thymine in preference to de novosynthesis of thymine ;the other mutants apparently utilized the de novoroute preferentially. Quantitative thymine requirements in the F medium are also given inTable 2 and all mutants required less thyminethan SF/O.

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216 Cancer Research Vol. 18, February, 1958

The addition of adenine, guanine, xanthine, anduracil to the F-PP medium, which results in theF medium, spared the folie acid requirement ofall organisms except SF/MPcc, SF/DAP, andSF/A/O.

The purine-sparing effect on the folie acid requirement of SF/O was marked, and about thesame effect was observed with SF/MP and SF/MP/A. A more pronounced effect was noted withSF/A, which required one-third the amount offolie acid that the parent strain needed for half-maximum growth (14).

purines and purine ribosides. Growth of SF/A/MP was similar to SF/A; the ribosides, with theexception of adenosine, were equal to the purinein stimulating growth. However, when 42-hourincubation periods of similar cultures were carriedout, the growth on all purine derivatives was essentially the same.

The growth of SF/A was slightly less on eachliboside than on the corresponding free base, andat the concentration used in these experimentsxanthine was the least effective base. When higherconcentrations of xanthine were added to the

TABLE3GROWTHRESPONSETOPURINESANDFURINERIBOSIDES

Optical density at 600 m^ORGAN»»

SF/OSF/ASF/MPSF/MPccSF/DAPSF/A/OSF/A/MPSF/MP/A

SF/OSF/ASF/MPSF/MPccSF/DAPSF/A/OSF/A/MPSF/MP/A

NONE

.380

.30

.34

.33

.400

.30

.47

.40

.41

.43

.40

.41

.41

.37

.91

.¿00

.Si

.14

.23.31.12

ADS

.43

.34

.42

.30

.37

.30

.34

.40

.22

.190

.24

.18

.20

.30

.14

G GUS HF-PP+PGA medium*

.44.33.41.31.30.40.36.34.18.190.25.15.20.26.10.45.24.39.32.37.39.36.37F-PP+T

mediumf.23.190.22.18.22.27.21.4233.36,3737.4043.40231002510182321

INS

.44

.20

.36

.32

.38

.41

.43

.39

.23

.230

.25

.21

.23

.30

.21

.47

.24

.40

.30

.30

.42

.41

.41

.27

.25

.34

.23

.10

.20

.21

.21

XNS

.46

.22

.46

.41

.40

.40

.41

.41

.22

.21

.21

.24

.17

.21

.82

.26

The following abbreviations have been used: A, adenine; ADS, adenosine; G, guanine; GUS, guanosine;H, hypoxanthine; INS, inosine; X, xanthine; XNS, xanthosine. These purine derivatives were added to themedium at a concentration of 3 X 10~6moles/liter.

* F-PP + PGA medium = folie acid-free medium without purines (adenine, guanine, and xanthine)and without the pyrimidine, uracil; supplemented with folie acid, 1 m^g/ml. Incubation period, 18-20hours.

t F-PP + T medium = see footnote f»Table 2. Incubation period, 42 hours.

The quantitative folie acid requirement of certain cultures as shown in Table 2 was affected bythe purine supplementation; therefore, experiments were carried out to determine the stimulatory effect of purines and purine ribosides in theF-PP + PGA medium and the growth-supporting effect they might have in the F-PP + Tmedium. The results (Table 3) show that duringan incubation period of 18-20 hours in the folieacid medium (F-PP + PGA), the growth of onlytwo organisms (SF/A and SF/A/MP) was stimulated by the addition of the purine derivatives tothis medium. The results with SF/A indicate thatthere was little difference among the purine derivatives but that the growth on adenine and adenosine was somewhat better than on the other

medium, greater growth was obtained; but thegrowth was always less than that with an equivalent concentration of any of the other purines.

The results obtained in the F-PP + T mediumwere essentially the same for all organisms, withthe notable exception of SF/MP. As we had previously reported (11, 12), this mutant grows wellonly on xanthine and xanthosine, and a trace ofgrowth was noted on isoguanine. The lack of sucha marked change in SF/MPcc, which was selectedunder conditions identical to those used for theisolation of SF/MP, was surprising; however, thealterations in the double mutants which allowthem to grow on all purines suggest that theremay be several pathways which are responsiblefor resistance to 6-MP. In the F-PP + T medium

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HUTCHISON—Metabolismof S. faecalis Mutants. I 217

the concentration of purine derivative (3 X IO"6

moles/liter) was the growth-limiting factor; in allcases when the concentration of purine was increased threefold there was an increase in growthof about 50 per cent.

Some additional observations (Table 4) onquantitative alterations in mutants were noted ina study of the effects of the antimetabolites,A-methopterin, 6-mercaptopurine, and 2,6-di-aminopurine, on the inhibition of growth. Thesevalues (Table 4) represent the amounts of antagonist in fig/nil that permitted half-maximumgrowth. The results on A-methopterin inhibitionin the P + PGA medium show that SF/A, SF/A/MP, and SF/MP/A had a 300,000-fold increaseover the parent strain in A-methopterin resistance. SF/A/O was next in increased resistanceand had a 50-fold increase. SF/DAP had the sametolerance as SF/O, while the two 6-MP-resistantmutants had a fourfold increase in resistance. Inthe F-PP + PGA medium somewhat different results were obtained. The inhibitory effect ofA-methopterin was essentially the same as it wasin the previous medium, with the exception ofSF/A and SF/A/MP, both of which tolerated lessA-methopterin in this medium than they did inthe F medium; presumably this also may be a reflection of the decreased de novo biosyntheticcapacities of the mutants.

The effects of 6-mercaptopurine and 2,6-di-aminopurine are also enumerated in Table 4;SF/A was more sensitive to 6-MP than was theparent SF/O, while all others showed a markedincrease in 6-MP resistance. There was an increasein resistance to 2,6-diaminopurine in all organismsexcept SF/A/MP.

DISCUSSION

These studies on the selection, cultivation, andmaintenance of resistant mutants of S. faecalisindicate the facility with which such microorganisms can be obtained. If the ease of the selectionof such assorted mutants in a biological systemsuch as S. faecalis is an indication of the distribution of resistant mutants in a population of cellsof mouse leukemia or cells of human leukemia, theproblems of chemotherapy are extremely acute.

Among the seven mutants, SF/A and SF/A/MP are unique in that they are inefficient in thede novo synthesis of purines but have an increasedefficiency in thymine synthesis. The decreasedability to make purines is evidenced in the increased folie acid requirements in the F-PP andF-PP + T media; the preferential utilization ofpreformed purines as shown by the marked stimu

lation of growth when purines such as adenine,guanine, hypoxanthine and xanthine are added tothe F-PP + PGA medium; and the rathermarked alteration in A-methopterin sensitivity inF-PP + PGA medium in contrast to the F +PGA medium. The increased efficiency in thyminesynthesis may be observed in the data which showa decrease in the amount of thymine required forthe growth of SF/A and SF/A/MP in the Fmedium. A further example is the decrease in thefolie acid requirement of SF/A in the F mediumin contrast to its folie acid requirement in theF-PP + T medium (0.1 m^g/ml to 5.0 m/tg/ml).

Another profound difference was observed withSF/MP. It has been reported that the mutantcannot grow on purine derivatives other than xan-

TABLE4GROWTHINHIBITIONOFStreptococcusfaecalis

(¿ig/mlfor 50 per cent inhibition)

ORGANISM

SF/OSF/ASF/MPSF/MPccSF/DAPSF/A/OSF/A/MPSF/MP/A

*F + PGA medium = folie acid-free medium supplemented with folie acid, l m jug/ml.

t F-PP + PGA medium = see footnote *, Table 3.The following abbreviations have been used: A-meth., A-

methopterin; 6-MP, 6-mercaptopurine; DAP, 2,6-diamino-purine.

All incubations were for 24 hours except the 6-MP experiments, which were incubated for 18 hours.

thine and isoguanine (11,12); thus an attempt wasmade to determine whether the alteration would bemanifested in all mutants resistant to 6-mercaptopurine. Indeed this was not the case, as can beseen when three additional mutants of equal resistance, SF/MPcc, SF/MP/A, and SF/A/MP,are compared with SF/MP. Even though thethree mutants are equally resistant to 6-mercaptopurine, they grow on adenine, guanine, hypoxanthine, and their respective ribosides as well ason xanthine; therefore their ability to use preformed purines for growth is identical with thenonresistant parent strain.

These 6-mercaptopurine-resistant organisms,except for SF/A/MP, whose immediate parentwas SF/A, show no difference in quantitative folieacid requirements in the presence or absence ofthymine; this suggests that they are slightly moreefficient in de novo purine biosynthesis than is

F+PGAMEDIUM*A-meth

.0.0013000.0040.0040.001O.OS300300F-PP+PGAMEDIUMtA-meth.0.0005600.0030.0050.0010.050.52006-MP101>500>50050>500>500>500DAP32530>60>1000151>60

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218 Cancer Research Vol. 18, February, 1958

SF/0. SF/MP and SF/MP/A are similar to thewild strain in folie acid requirement in the F medium. Since SF/MPcc needs the same amount regardless of thymine or purine supplementation, itwould appear that SF/MPcc has an equal capacity to carry out de novo synthesis or to utilize preformed nucleic acid precursors.

SF/MP and SF/MPcc show an increase in resistance to A-methopterin, which, in the case ofthe first mutant, may be attributed to its slightdecrease in folie acid requirement; however, thisexplanation certainly cannot be ascribed to thesecond mutant, since no such decrease was observed in the F medium even though it was notedin the F-PP medium.

SF/DAP is of interest in that it requires thesame amount of folie acid in all the media studied;and this observation is further corroborated, sinceA-methopterin was equally effective in the F-PPand F media.

The mutant SF/A/O, which is a partial revertfrom SF/A, exhibits some characteristics that areidentical with those of SF/A/MP and others likethose of SF/MPcc. Therefore, one is left with theconclusion that resistance to one drug does notresult in a set of unique modifications in themutants.

Many biochemical mechanisms have been postulated as explanations of antibiotic and drug resistance in bacteria (6), and examples of each havebeen demonstrated by several investigators (7, 8,17-19, 23). It is necessary to reiterate at this pointthat these mechanisms have been postulated onthe basis of information gained, in most cases,from one resistant mutant. It is important thatone should not conclude that all organisms resistant to a particular drug will have identical modifications—indeed, the results obtained with theseantimetabolite-resistant mutants of S. faecalis indicate myriad alterations. In the present studies,in which emphasis has been placed on the growthprocesses relative to nucleic acid biosynthesis,with particular observations on both the route ofde novo purine formation and the route concernedwith the utilization of preformed purines andpurine derivatives, it is obvious that the mutationswhich were responsible for these resistant strainswere not the same for any two resistant organismsstudied.

The multiplicity of alterations in these S. faecalis mutants suggests that in nature there aremany mechanisms whereby an originally sensitivebacterial population can become refractory to aninhibitory agent. The interrelationships amongthe observations made here make it impossible todevise an unequivocal explanation for the resist

ance to A-methopterin, 6-mercaptopurine, or2,6-diaminopurine in S. faecalis.

These growth data will be further elucidatedby additional experiments dealing with the utilization of a variety of C14-labeled nucleic acidprecursors (1).

SUMMARYThe selection and maintenance of seven anti-

metabolite-resistant strains of Streptococcus faecalis have been described. These mutants werecompared with the parent strains in regard toquantitative folie acid utilization, and two A-methopterin-resistant mutants were significantlydifferent from the other cultures. In media thatdid not contain purines, they required high concentrations of folie acid and an excessively long incubation time for growth. The other mutants inwhich a difference was noted (SF/MPcc, SF/DAPand SF/A/O) showed a shift toward a more efficient utilization of folie acid for de novo purine, orpurine and thymine, synthesis.

SF/A and SF/A/MP were the only mutantswhose growth on folie acid was stimulated by theaddition of purines to the media; and SF/MP wasunique in its lack of ability to grow on adenine,guanine, and hypoxanthine and their respectiveribosides in a thymine medium.

Some degree of resistance to A-methopterin wasobserved with all mutants. Similar increases in resistance were observed toward 6-MP, with theexception of SF/A which, in short-term (18-hour)experiments, appeared to be more sensitive to theantimetabolite. Increased resistance to 2,6-diaminopurine was seen in all cases except SF/A/MP.

The multiplicity of changes observed in thesemutants has been compared with the parent strainin an attempt to elucidate some of the pathwaysinvolved in nucleic acid biosynthesis.

ACKNOWLEDGMENTSThe author is indebted to Mrs. Georgia B. Waring, Mrs.

Marcia Polak Leonard, Mrs. Cecile Chagnon Meloni, and MissM. Katharine Coultas for technical assistance, and to Dr.M. Earl Balis for discussion during the course of this work.

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HUTCHISON—Metabolismof S. faecalis Mutants. I 219

3. BUBCHENAL,J. H.; ROBINSON,E.; JOHNSTON,S. F.; andKUSHIDA,M. N. The Induction of Resistance to 4-Amino-NIO-Methyl-Pteroylglutarnic Acid in a Strain of Transmitted Mouse Leukemia. Science, 111:116-17, 1950.

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1958;18:214-219. Cancer Res   Dorris J. Hutchison  Isolation and Characterization of the Mutants

: I.Streptococcus faecalisMetabolism of Resistant Mutants of

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