STUDIES OF MUTABILITY IN NUTRITIONALLY DEFICIENT STRAINSOF ESCHERICHIA COLI

I. GENETIC ANALYSIS OF FIVE AUXOTROPHIC STRAINS'

M. DEMEREC AND ELISE CAHN

Department of Genetics, Carnegie Institution of Washington, Cold Spring Harbor, New York

Received for publication May 15, 1952

Five auxotrophs (mutant strains requiringgrowth factors) were selected for study, and ananalysis was made of their spontaneous and in-duced mutability to the prototrophic state(wild type with regard to nutritional character-istics), particular attention being given to theproblem of delayed appearance of induced mu-tants. It had been found in previous studiesinvolving phage resistance that mutants inducedby radiations continued to appear after thetreated bacteria had divided a number of times,and that the mutation frequency returned to thespontaneous level only after 10 to 12 cell divi-sions (Demerec, 1946). Subsequent work indi-cated that delayed appearance of induced mu-tants is not limited to phage-resistance mutationsbut is a general phenomenon in Escherichia colialthough there are considerable differences in thepatterns of delay. The present study has shownthat the pattern of appearance of these inducednutritional mutants differs in each of the strainsstudied.The mutations observed in each case were from

a particular growth factor requirement to theprototrophic state. As a matter of convenience,they are referred to as reversions although it isrealized that the mutants were probably geneti-cally heterogeneous and not necessarily truereversions to the original parent type.

DESCRIPTION OF STRAINS

Four of the strains used in these experimentswere derived from strain B/r of E. coli by treat-ment with ultraviolet light. The fifth was derivedin a similar manner from the streptomycin-de-pendent mutant of B/r designated Sd4.

Methionineless (strain M12-11). Prototrophscan be isolated readily from this strain, occurring

1 This study was conducted in part under agrant-in-aid from the American Cancer Societyupon recommendation of the Committee onGrowth of the National Research Council.

both spontaneously and as a result of ultravioletirradiation or treatment with manganous chlo-ride. Some of these reversions are feeders; that is,they excrete into the medium a substance thatcan be utilized by the methionineless strain.This results in a circle of heavier backgroundgrowth around the mutant colony. Among thereversions observed, there were all gradationsfrom nonfeeders to heavy feeders.

Methionineless-threonineless (8train MT1J-66).Since complete reversions, both spontaneous andinduced, occur in this strain with easily obser-vable frequency, it is probable that both thesedeficiencies are due to one mutation. Among thereversions, distinct large and small colony typeswere observed. This difference in colony typeswas not analyzed further, but it serves to demon-strate the fact that reversions may be a veryheterogeneous group.

Leucineless and phenylalanineless (8train L,P12-72). Even though this strain, like the others,was obtained after one irradiation of B/r, itprobably carries two mutations since no proto-trophs could be obtained in one step by ultra-violet treatment. Doses up to 1,000 ergs did notyield a single mutant colony on either minimalmedium or minimal medium enriched with 0.01per cent nutrient broth. When the medium wasenriched with either leucine or phenylalanine,however, spontaneous or induced mutations toindependence of the other growth factor wereobtained easily. In these experiments, only theleucineless locus was studied, phenylalanine al-ways being supplied in the medium.

Histidineless (strain H12-23). Although thisstrain gives rise to reversions spontaneously, nodetectable number could be induced, either byultraviolet irradiation or by MnCl2 treatment.

Histidineless and streptomycinependent (8trainH88, Sd-4). This strain is like H12-23 in that itsrate of mutation to prototrophy is not perceptiblyincreased by the radiation or chemical treat-

27

on May 16, 2018 by guest

http://jb.asm.org/

Dow

nloaded from

M. DEMEREC AND ELISE CAHN

ments that increase the mutability of the otherthree strains. A reversion of the Sd character wasobtained in the strain to facilitate tests for feed-ing between the two histidineless strains. Thesetests were carried out by making adjacent streaksof the two strains on minimal medium enrichedwith 0.01 per cent nutrient broth, a medium onwhich both can grow slightly. No evidence offeeding was observed, and therefore the questionof whether these two strains carry the same muta-tion or different mutations remains open.

sodium citrate, 0.05 per cent; and glucose, 0.2 percent (B. D. Davis) and lacking the factors re-quired by the deficient strains; (2) medium Aenriched with 0.0025 per cent dehydrated nu-trient broth (Difco); and (3) medium A enrichedwith 0.01 per cent dehydrated nutrient broth.All three of these contained 1.5 per cent agar.Various dilutions of cells were used in plating.The procedure of the experiments was to cen-trifuge 24 hour cultures grown in nutrient brothunder aeration at 37 C, pour off the supernatant

TABLE 1Number of divisions undergone by three deficient strains of Escherichia coli when various numbers of cells

were plated on minimal (A) and enriched media; and number of bacteria determined after48 hours of growth

MEDIUM A A + 0.01*STRAN CRLLS ATD-

Cells after 48 hours Divisions 48 hours Divisions

M12-11 2.3 X 108 1.7 X 108 0 _2.3 X 107 -3.9 X 107 0.5 1.9 X 109 6.42.3 X 10' 5.1 X 106 1.0 1.5 X 109 9.42.3 X 10' 4.6 X 106 4.4 1.6 X 109 12.82.3 X 10' 1.6 X 109 16.0

MTf 12-66 1.7 X 10' 1.9 X 108 0 2.1 X 109 3.51.7 X 107 5.7 X 107 1.7 1.8 X 109 6.71.7 X 106 3.6 X 107 4.4 2.5 X 109 10.41.7 X 10' 1.9 X 107 6.7 2.6 X 109 13.71.7 X 104 3.4 X 10' 8.2 1.8 X 10' 16.7

L,P12-72t 3.7 X 10' 5.9 X 10' 0.5 4.1 X 10' 3.43.7 X 107 1.8 X 10' 2.3 5.2 X 109 7.13.7 X 10' 7.8 X 107 4.4 3.8 X 109 10.03.7 X 10' 1.2 X 108 8.3 4.9 X 109 13.53.7 X 104 4.8 X 107 10.2 2.3 X 109 15.9

* Indicates the percentage of dehydrated broth added to medium A.t For this strain, 20 pg of phenylalanine per ml was added to medium A.

ESTIMATES OF NUIBER OF DIVISIONS

All five strains undergo slight residual growthon minimal synthetic medium but grow well

when that medium is enriched by adding thefactors for which the strain is primarily deficientor by adding broth. We found enrichment withbroth more satisfactory.

Estimates of the numbers of divisions under-gone by the different strains on minimal and on

enriched medium were obtained. The media em-

ployed were of the following three types: (1)medium A, a synthetic medium contng:

K,HPO4, 0.7 per cent; K-H2PO4, 0.3 per cent;MgSO4, 0.01 per cent; (NH4)2SO4, 0.1 per cent;

broth, resuspend the celLs in saline, and spreadthe desired dilutions of these suspensions on theappropriate media with a glass spreader. Theplates were incubated for 24, 48, or 72 hours,after which they were washed with 10 ml of sa-line. The numbers of living bacteria present inthe resulting cell suspensions were determinedby assaying on nutrient agar. To minimie theexperimental error, colonies derived from spon-taneous reversions were cut out of the agar beforewashing. To check the possibility that a largernumber of divisions might be masked by a corre-sponding death of cells, the colonies on the en-riched media were examined under the micro-

28 [VOL. 65

on May 16, 2018 by guest

http://jb.asm.org/

Dow

nloaded from

STUDIES OF MUTABILITY

scope after 12, 24, and 36 hours of incubation,and camera-lucida drawings were made. Thearea of the colony was measured by means of aplanimeter, and in all cases it was found thatgrowth had stopped after about 12 hours of in-cubation. The colonies on minimal medium weretoo small to be measured by this method. Since

TABLE 2Number of divisions undergone by unirradiated and

irradiated bacteria of strain L,PIS-72

NO. O1 CELS NO. OF

(KEIGS) MEDIUM DIV-Plated After 48 hours SIONS

None A 2.6 X 108 4.5 X 108 0.82.6 X 107 2.2 X 108 3.12.6 X 106 1.5 X 108 5.82.6 X 106 1.0 X 103 8.6

A + 0.01* 2.6 X 108 4.3 X 109 4.02.6 X 107 4.7 X 109 7.42.6 X 106 4.1 X 109 10.82.6 X 106 4.6 X 109 14.1

300 A 2.1 X 108 3.2 X 108 0.72.1 X 107 1.6 X 108 3.02.1 X 106 1.2 X 10' 5.92.1 X 106 9.0 X 107 8.8

A + 0.01 2.1 X 108 3.8 X 109 4.22.1 X 107 3.4 X 10' 7.32.1 X 106 4.5 X 10' 11.12.1 X 10' 4.2 X 109 14.3

800 A 6.1 X 107 1.4 X 108 1.26.1 X 106 1.4 X 108 4.56.1 X 106 1.6 X 108 8.1

A + 0.01 6.1 X 107 3.8 X 109 5.96.1 X 106 3.9 X 109 8.96.1 X 10' 6.5 X 109 13.3

* Indicates percentageadded to medium A.

of dehydrated broth

the assays made after 24, 48, and 72 hours ofgrowth did not differ significantly, only the 48hour data will be given here.The divisions undergone by various numbers

of M12-11, MT12-66, and L,P12-72 bacteriaplated on minimal and enriched media are shownin table 1. These data indicate that, for eachstrain and each medium, the final number ofbacteria on all plates was about the same, pro-

vided the number originally plated was within arange specific for each medium. This suggeststhat each plate can sustain a certain number ofbacteria and that growth stops when that num-ber has been reached. Thus a smaller number ofcells will pass through more divisions than alarger number plated on the same medium, andthe number of divisions bacteria will undergocan be regulated by varying the medium and thenumber of cells per plate.

Calculations for irradiated and MnCl2-treatedmaterial were based on these experimental esti-mates of number of divisions undergone by un-treated bacteria. It would not be practical touse the plate-washing method of estimation withmutagen-treated material of the M12-11 orMT12-66 strains (which carry only one mutationeach and can undergo reversion in a single step)because of the large number of mutant coloniesthat would have to be cut out of the agar beforewashing. Using the double mutant L,P12-72,however, we were able to test for any significantdifference in number of divisions between treatedand untreated material. Since neither phenylala-nine nor leucine was supplied in the medium, nomutants appeared. The results of this experimentare summarized in table 2, where it is apparentthat irradiated and unirradiated bacteria, platedunder comparable conditions, do not differ sig-nificantly in the number of divisions they passthrough.

MUTAGEN-STABIIITY

In strains M12-11, MT12-66, and L, P12-72the spontaneous rate of reversion was readilyaugmented by exposure to either ultraviolet radi-ation or MnCI2. It was quite unexpected, there-fore, to find that no increase in mutation ratecould be detected when H12-23 and H88, Sd4were treated with these mutagens (table 3). Inthe experiments summarized in the table, variousnumbers of cells were plated on minimal and en-riched media in order to create conditions inwhich bacteria would undergo different numbersof divisions after treatment with a mutagen. It isknown that in some cases ten or more cell divi-sions may be required for the manifestation of allinduced mutants. Since these experiments werecarried through at least 12 divisions, it can beassumed that if mutants had been induced bythe treatments they would have been detected.We use the term "mutagen-stable" to describe

291953]

on May 16, 2018 by guest

http://jb.asm.org/

Dow

nloaded from

M. DEMEREC AND ULISE CAHN

TABLE 3Mutagen-stability of HIS-fl and H88, Sd-4

H12-23 H188, Sd4tTR1ATMENT MEDIUM

Cellplted No. of Mutants Clspae No. of Mutants|CellsPlated |Nofplates per plate Cellsplated plates per plate

None A 2.4 X 108 2 0 3.2 X 108 2 0.52.4 X107 2 0 3.2 X 107 1 02.4 X 106 2 0.5 3.2 X 106 1 02.4 X 105 2 0

A + 0.01* 2.4 X 105 1 8.0 3.2 X 108 2 112.4 X 107 2 2.5 3.2 X 107 1 82.4 X 106 2 4.0 3.2 X 106 1 62.4 X 10" 2 5.5

300 ergs A 1.5 X 105 3 1.7 2.1 X 108 4 11.5 X 107 3 0 2.1 X 107 4 0

A + 0.01 1.5 x 10' 3 5.0 2.1 X 10' 3 16.01.5 X 107 2 2.0 2.1 X 107 3 6.3

600 ergs A 2.3 X 107 3 2.0 1.3 X 108 4 2.22.3 X 10' 3 0.0 1.3 X 107 4 1.0

A + 0.01 2.3 X 107 3 7.0 1.3 X 108 3 17.72.3 X 10' 3 3.67 1.3 X 107 3 7.0

800 ergs A 6.0 X 10' 3 1.3 3.3 X 107 4 2.26.0 X 10 3 0.3 3.3 X 106 4 0.5

A + 0.01 6.0 X 10' 3 6.0 3.3 X 107 3 12.76.0 X 10 3 3.7 3.3 X 10s 3 6.3

1,000 ergs A 7.4 X 105 3 0.7 2.2 X 10' 4 07.4 X 104 3 0.3 2.2 X 105 4 0

A + 0.01 7.4 X 10' 3 3.7 2.2 X 106 3 7.07.4 X 104 3 6.7 2.2 X 105 3 3.0

MnCl2 A + 0.01 7.8 X 107 5 6.0

None A + 0.01 9.0 X 107 5 1.8

MnCl2 A 1.6 X 108 5 1.8

None A 2.0 X 108 5 0.6

* Indicates percentage of dehydrated broth added to medium A.t For this strain, 50 ug of streptomycin per ml was added to medium.

this resistance of the H12-23 and H88 strains to aerated broth cultures grown at 37 C were centri-mutagenic influence. fuged, the supernatant was discarded, and the

cells were resuspended in saline. From this cellSPONTANEOUJS-MUTATION FREQUJENCIES suspension, samples of about 4 X 106 cells were

In experiments to determine frequencies of spread on a series of plates containing the mini-spontaneously occurring reverse mutants, 24 hour mal and enriched media. These plates were incu-

30 [VOL. 65

on May 16, 2018 by guest

http://jb.asm.org/

Dow

nloaded from

STUDIES OF MUTABILITY

bated for 6 days, and the mutant colonies werecounted. The spontaneous-mutation frequenciesshown in table 4 were computed on the basis ofestimates, made by plate washings, of the finalnumbers of bacteria present on the plates and areexpressed in terms of mutants per 10' cells.

It was necessary to ascertain that all the col-onies counted represented mutants that had actu-ally arisen on the plates and not been carried overfrom the original culture (background mutants).Accordingly, the cultures were tested for back-

66, which gave an average of 12.5 colonies percontrol plate. This means that the probabilityof plating even a single mutant cell on a testplate, in a sample containing only 1/100 thenumber of cells in the control platings, was verylow.The data of this table also suggest a higher

frequency of mutants appearing during the earlydivisions following the resting stage than duringlater divisions. Since such results could be theconsequence of underestimating the number of

TABLE 4Spontaneous-mutation frequencies

NO. OF CzLLS PER PIATE NO. OPF MTANTS ESTIMTEDSTRAWNM NO.xOP_T_ NO. OPr

Plated Final Per plate Per los cells DIVISIONS

M12-11 A 2 4.0 X 108 2

A 20 4.0 X 106 5.8 X 106 0.1 1.7 0.4A + 0.01* 20 2.2 X 109 5.3 0.24 9.1

MT12-66 A 2 2.6 X 10' 12.5

A 19 2.6 X 106 2.5 X 107 0.47 1.9 3.3A + 0.01 20 2.0 X 10' 5.0 0.25 9.6

L,P12-72t A 2 3.9 X 10'| 10

A 19 3.9 X 10" 7.8 X 107 1.0 1.29 4.3A + 0.01 17 4.0 X 109 7.5 0.19 10.0

H12-23 A 2 3.8 Xl0OO

A 20 3.8 X 10' 1.0 X 108 0.15 0.15 4.8A + 0.01 14 2.0 X 1010 4.6 0.023 12.4

* Indicates percentage of dehydrated broth added to medium A.t For this strain, 20 jg of phenylalanine per ml was added to the medium.

ground mutants by plating samples of about4 x 108 cells on medium A. Because of the largenumber of cells plated, the bacteria underwentless than one division, and only an occasionalmutant could be expected to arise on the plates.The colonies appearing on these control plates,therefore, represented almost exclusively back-ground mutants and provided an estimate of thefrequencies of background mutants to be ex-pected on test plates that was perhaps slightlytoo high but certainly not too low. Table 4, whichsummarizes these data, shows that the highestnumber of background mutants estimated bythis method was in the culture of strain MT12-

early divisions, interpretation of this observationwill be postponed until after the completion ofexperiments especially designed to test this point.

INDUCED MUTATIONS

Reversions were induced either with ultravioletlight or with MnCl2. As bacteria of the strainsused must pass through a number of divisionsbefore all induced mutants show up, it is evidentthat data concerning rates of induced mutationcan be obtained only under conditions allowingfor an adequate number of divisions. To establishthe rate of appearance of induced mutants, and

311953]

on May 16, 2018 by guest

http://jb.asm.org/

Dow

nloaded from

M.7DEMEREC AND ELISE CAHN

the number of divisions required for them all toappear, the following,procedure was employed.A 24 hour aerated broth culture of bacteria

was centrifuged, the supernatant was poured off,

100

90r80s

Cn(0)wna.X

70

60

50

40

30

20

10

prevent photoreactivation, all work following theirradiation was done in a darkened room with aninsect-repellent yellow bulb as the only lightsource. The irradiated material was assayed on

Figure 1. Rate of appearance of induced reverse mutants of strain M12-11 during approximately 14bacterial divisions following treatment.

100

90

80I

70O

60° 60Co

w 500.

1&J 40be

301

20

10

0 ~ ~ 0

_ ~ ~ ~ ~~° 0

O /

a

a O

0

° 300 ERGS* 300 ERGS' 600 ERGS0 MC16O2

I I ~I~__ I I I13 14 15

Figure 2. Rate of appearance of induced reverse mutants of strain MT12-66 during approximately 14bacterial divisions following treatment.

and the cells were resuspended in saline andassayed on Difco nutrient agar. Samples (7 ml)of the cell suspension were placed in the bottomof a petri dish and irradiated with the desired doseof ultraviolet light with constant shaking. To

Difco nutrient agar and then permitted to un-

dergo various numbers of divisions by beingplated in different dilutions on the minimal andenriched media. All dilutions were made in saline.The plates were incubated at 37 C for 6 days,

32 [VOL. 65

00

0 600 ERGS* 600 ERGSo 300 ERGSaMnC12/

0

o 0

2 0

0 0 0~~~Ds o

I 2 3 4 5 6 7 8 9 10 II 12 13 14

NUMBER OF BACTERIAL DIVISIONS

I I I I I I II 2 3 4 5 6 7 8 9 tO II

NUMBER OF BACTERIAL DIVISIONS

I

on May 16, 2018 by guest

http://jb.asm.org/

Dow

nloaded from

STUDIES OF MUTABILITY

100

90?-

701Fw

C)V)w

XIL

at

50o-

10

v 9I 2 3 4 5 6 7 8 9 10NUMBER OF BACTERIAL DIVISIONS

It1 2e 3

Figure S. Rate of appearance of induced reverse mutants of strain L,P12-72 during approximately12 bacterial divisions following treatment.

and then the mutant colonies were counted.Mutation frequencies were calculated per 108survivors plated, and the numbers of divisionsundergone by the cells under these conditionswere estimated on the basis of the numbers ofdivisions undergone by unirradiated materialplated in comparable samples on the same media.For MnCl2 treatment, the 24 hour culture was

centrifuged, the supernatant broth poured off,and the cells resuspended in 0.3 M NaCl. Thissuspension was centrifuged again, and the pelletwas resuspended in 0.04 per cent MnCl2 and in-cubated at 37 C for 1 hour. Then the celLs were

plated, and the results calculated as in the irra-diation experiments except that liquid minimalmedium was used for dilutions.

It was necessary to estimate the number ofbackground and spontaneous mutants per platein each experiment so that this number could besubtracted from the total number of mutantscounted on the test plates. For this purpose,suitable dilutions, generally from 10-1 to 10-', ofthe untreated cell suspension were plated on eachmedium used in the experiment.

Figures 1, 2, and 3 show the rate at which in-duced reversions of strains M12-11, MT12-66,and L,P12-72 appeared. Evidently, the rate ofappearance is an inherent characteristic of themutation itself and not a result of the intensity

o E*4 ~ ~ ~

Co103 K

0

o102CoLI--

z

I- 10DM12-1I MOREDISON

T20L,P12-72' 6

_i50 300 600 800 1000 12001

ERGS PER MM2Figure 4. Relation between ultraviolet radia-

tion dose and frequency of induced mutants forthree strains. Only values obtained after a num-

ber of divisions adequate for complete or nearlycomplete manifestation of mutants are plotted.

or type of treatment. Mutations of strains M12-11 (figure 1) and MT12-66 (figure 2) appearcomparatively slowly, in terms not only of thetotal number of divisions needed for the mani-

festation of all mutants, but also of the percen-

_~~~~~~~&

000

@/0~~~~

o 300 ERGS* 300 ERGSa MndI2o Mn Cie

I I 9 1 I I-- I I I I I

19531 33

0O

60F

40 .

30 .

20 "

on May 16, 2018 by guest

http://jb.asm.org/

Dow

nloaded from

M. DEMEREC AND ELISE CAHN

tage appearing during the early divisions. Thepattern for strain L,P12-72 (figure 3) is quitedifferent. Fewer divisions are necessary for com-plete manifestation of mutants, and also the per-centage appearing during the early divisions isconsiderably higher than in the other two strains.

Figure 4 shows curves representing the fre-quencies of reversions induced by various dosesof ultraviolet radiation in strains M12-11, MT12-66, and L,P12-72. Only values obtained after anumber of divisions sufficient to allow for theappearance of all, or nearly all, induced mutantsare indicated on these curves.

DISCUSSION

In quantitative studies of spontaneous and in-duced mutability, involving either single loci orspecific types of mutants, it is essential that thematerial fulfill the following requirements. (1)The phenotypic change must be well defined sothat mutants can be classified with ease and cer-tainty. (2) It should be possible to handle theorgism efficiently in large numbers because mu-tation rates are low and many individuals arenecessary to provide reliable data. (3) Treatedcells should undergo a well controlled number ofdivisions before selection for the mutant pheno-type, to allow for delayed appearance of inducedmutants.

In the B strain of E. coli, two systems havebeen employed for the quantitative study ofmutability: the phage system, in which the mu-tations observed are from sensitivity to resistanceto phage T1; and the streptomycin system, inwhich the mutations scored are from streptomy-cin dependence (strain Sd4) to nondependence.The work reported here shows that now we alsomay make use of the reverse mutation systems ofa large number of auxotrophic strains.

Since the number of mutants appearing on aplate is a function of the total number of bac-teria present at the time of selection for the mu-tant phenotype, and since-where induced mu-tations are concerned-it is also a function of thenumber of treated cells plated and the numberof divisions undergone by these cells, it is impor-tant in studies of induced mutability to havegood control over all these factors. The data pre-sented here, supported by later studies of a con-siderable number of other auxotrophs, indicatethat each plate containing a given medium (mini-mal or enriched) can sustain a certain number of

bacteria of any particular auxotrophic strain.Consequently, the number of divisions such bac-teria will undergo on a certain medium is deter-mined by the number of cells put on a plate. It ispossible therefore to vary, within a wide rangeand with a great deal of precision, the averagenumber of divisions of treated bacteria simply byvarying the number of cells plated and the me-dium on which they are plated, as was done inour experiments. With this technique it is pos-sible to study both spontaneous and inducedmutation from auxotrophy to prototrophy inmany nutritionally deficient strains.Of the five strains studied by us, two (H12-23

and H88 ,Sd4) showed an unexpected resistanceto mutagenic influence (mutagen stability). Thatis, we were unable to induce in them any detec-table number of reversions by treatment withultraviolet radiation or manganous chloride al-though reversions occurred spontaneously with afrequency similar to that found in a number ofother strains investigated in this laboratory.Strains H12-23 and H88,Sd-4 are both histidine-requiring; but they are independent in origin,one having been isolated from a B/r strain andthe other from its streptomycin-dependent de-rivative, Sd4. Since recombinants are not to befound in our strains of E. coli, we could not usethe recombination technique for a test of allelism.We did determine, however, that bacteria of thetwo histidineless strains do not feed each otherwhen plated close together on minimal medium,a finding to be expected in the case of identicalalleles but also possible with nonalleles.

It was probably only by coincidence that boththe histidineless strains we worked with weremutagen-stable, for a comprehensive survey nowunder way, involving about 30 strains, showsthat mutagen stability is not limited to histidine-less strains and that not all histidineless strainsare mutagen-stable.

It is evident that mutagen-stable strains havethe capacity to mutate because spontaneous mu-tants were observed, but the very potent muta-gens used in our experiments were not able toincrease that capacity. If we assume that muta-tions are initiated by some metabolic disturbanceor the accumulation of a certain chemical in thecell, then this failure of ultraviolet radiation andMnCl2 to raise mutation rate above the spon-taneous level might mean one of two things.Either (a) the mutagens do not, in these particu-

34 [VOL. 65

on May 16, 2018 by guest

http://jb.asm.org/

Dow

nloaded from

STUDIES OF MUTABILITY

lar strains, increase the proportion of mutation-producing agents above that normally presentin the cells; or (b) the reactions initiated by thesemutagens are specific, affecting only certain ge-netic loci or certain alleles.

Using strain H88,Sd4, we were able to testthese two possibilities: that is, to determinewhether mutagen stability is due to the proper-ties of the bacterial strain or to the propertiesof the genetic locus. Since strain H88,Sd4 isboth histidineless and streptomycin-dependent,we could study the effect of a mutagen on thefrequencies of two kinds of reversion occurringin one strain. A sample of cells of strain H88,Sd-4, treated with MnCl2, was divided in two andused to determine, in one part, the frequency ofreversions from histidine deficiency and, in theother, the frequency of reversions from strepto-mycin dependence. In every experiment, thetreatment increased the rate of Sd4 mutationsbut left the rate of H88 mutations unaffected.Mutagen stability cannot, then, be a property ofthe bacterial strain as postulated in (a), since inthis case histidine deficiency was resistant to themutagen whereas streptomycin dependence wasnot. With the evidence now available we are notin a position to analyze further the mechanismresponsible for mutagen stability, but work nowunder way may bring us nearer to a solution ofthis problem.

In the three strains in which reversions couldbe induced it was possible to make a detailedstudy of the delayed appearance of the mutants.It was found that each kind of mutant has itsown characteristic pattern of delayed appearanceas shown in figures 1, 2, and 3. It is interestingto note that this pattern is not affected by theuse of different mutagens or different amountsof radiation. Apparently it is determined by theproperties of the genetic locus rather than byconditions external to it.

Several possible explanations of the mechanismresponsible for delayed appearance of inducedmutants have been considered by various inves-tigators. Demerec (1946) and Demerec and La-tarjet (1946) judged it improbable, in view oftheir results, that the delay is brought abouteither by diploidy of the bacterial strain or by amixture of haploid, diploid, and polyploid cellsin the population. They proposed two alternativeexplanations: that mutations occur during treat-ment, but their manifestation is delayed until

the supply of substrate originally manufacturedby the gene is exhausted; or that the mutabilityof the gene system is increased by the treatment.Newcombe and Scott (1949) suggested that "theincreased time need not necessarily be due to agreater number of cell divisions before inducedmutations are expressed, but may be the resultof variability in the time of onset of division oftreated bacteria such that many individuals failto divide until early growth has produced an ap-preciable population rise". Davis' interpretation(1950) is similar to one of the two proposed byDemerec (1946), namely, that the cell in whicha mutation has been induced "must undergosome growth, possibly several generations, beforethe premutational products of the mutated gene(enzymes; possible intermediates between genesand enzymes) are exhausted". Witkin (1951)assumes that the delayed appearance of inducedmutants is a complex phenomenon, involving atleast two, and possibly more, factors. The evi-dence she has accumulated makes the interpreta-tions proposed by Newcombe and Scott andDavis untenable and supports the hypothesisthat delay is due to an induced state of geneticinstability that may persist for several cell gen-erations.Our results demonstrating that each locus has

its own characteristic pattern with regard to de-layed appearance of induced mutants, togetherwith the earlier finding (Demerec et at., 1951)that the same treated sample of bacteria requiresabout 12 divisions to achieve complete mani-festation of induced phage-resistance mutants,but only 3 or 4 for appearance of all streptomycin-resistance mutants, show conclusively that de-layed appearance cannot be explained on a basisof either multinucleate condition of the bacteria;diploidy, polyploidy, or multistranded structureof bacterial chromosomes; or variability in thetime of onset of division. If a mechanism in-volving any one of these factors were responsiblefor the delay, one would expect to observe anidentical pattern of delayed appearance for allloci.Of the suggestions proposed earlier (Demerec,

1946; Demerec and Latarjet, 1946), the mostplausible explanation of the delayed effect, onthe basis of what is known at present, seems to bethat mutagenic agents induce an instability inthe genomes of treated cells, which persiststhrough several divisions, and that each locus of

19531 35

on May 16, 2018 by guest

http://jb.asm.org/

Dow

nloaded from

M. DEMEREC AND ELISE CAHN

the genome has its own characteristic pattern ofbehavior, which is independent of the mutageninitiating the reaction.We made a study in three strains of the rela-

tion between ultraviolet dose and induction ofmutations; and the data, plotted in figure 4, in-dicate consistent differences among the strains.Care was taken to use only values obtained afterthe treated bacteria had undergone a sufficientnumber of divisions to ensure complete mani-festation of all induced mutants. Otherwise aquite different and erroneous relationship mighthave been indicated.

ACKNOWIEDGMENT

We wish to acknowledge the efficient assistanceof Miss Barbara Powell in carrying out the ex-perimental work.

SUMMARY

A detailed genetical study was made of re-verse mutations in five nutritionally deficientstrains of Escherichda coli.Two strains (H12-23 and H88,8d4, both defi-

cient for histidine) were found to be mutagen-stable; that is, it was not possible to increase therate of their reversion to prototrophy above thespontaneous level by treatment with either ultra-violet light or manganous chloride. Studies nowin progress indicate that not all histidinelesstypes are mutagen-stable, nor is this propertylimited to histidineless types.Delayed appearance of induced mutants was

studied in three strains. The results indicate thateach strain has its own characteristic pattern ofdelay and that this pattern is not a function ofthe mutagen used in treatment. A hypothesisconcerning the mechanism of delayed appearnceis discussed.The rate of induction of mutations by ultra-

violet irradiation differed in each of the threestrains studied.

REFERENCESDAVIS, B. D. 1950 Studies on nutritionally defi-

cient bacterial mutants isolated by means ofpenicillin. Experientia, 6, 41-50.

DEmEREC, M. 1946 Induced mutations and pos-sible mechanisms of the transmission of he-redity in Escherichia coli. Proc. Natl. Acad.Sci., 32, 36-46.

DEmEREc, M., AND LATABJET, R. 1946 Muta-tions in bacteria induced by radiations. ColdSpring Harbor Symposia Quant. Biol., 11,38-50.

DEM1EREC, M., WiXIN, E. M., BECKHORN, E. J.,VISCONTI, N., FLNT, J., CAHN, E., COON,R. C., DOLLNG1ER, E. J., POWELL, B., ANDScHwARTz, M. 1951 Bacterial genetics.Carnegie Institution of Washington Year-book, 50, 181-195.

NzWCOMBz, H. B., AND ScOTT, G. W. 1949 Fac-tors responsible for the delayed appearanceof radiation-induced mutants in Escherichiacoli. Genetics, 34, 475-492.

WITIN, E. M. 1951 Nuclear segregation andthe delayed appearance of induced mutantsin Escherichia coli. Cold Spring Harbor Sym-posia Quant. Biol., 16, 357-372.

36 [VOL. 65

on May 16, 2018 by guest

http://jb.asm.org/

Dow

nloaded from