A STRAIN OF ESCHERICHIA COLI WITH AN UNUSUALLYHIGH RATE OF AUXOTROPHIC MUTATION'

AVRAM GOLDSTEIN' AND JANE S. SMOOTDepartment of Pharmacology, Harvard Medical School, Boston, Massachusetts

Received for publication April 25, 1955

The spontaneous rates of various well-definedmutations in bacteria generally range between10-10 and 10-' per cell per generation (Braun,1953). Although some kinds of heritable changeapparently occur much more frequently, thesemay not be truly mutational in origin. Thustransduction or other types of induced transfor-mation, the emergence of recessive traits indiploid organisms, and recombination maysometimes yield high proportions of stable vari-ants. As far as we are aware, the spontaneousoccurrence of auxotrophs, each presumably carry-ing a single mutant gene, at a total frequency ofseveral per cent in a wild-type population, hasnot been described.

In November 1950 we obtained a smoothmotile strain of Escherichia coli from the De-partment of Bacteriology, Harvard MedicalSchool, to use in studies on streptomycin de-pendence (Goldstein, 1954). Subsequently wediscovered that populations of this organism,on nutrient medium, normally contain a varietyof auxotrophic mutants, at a mean frequency ofabout four per cent. The mutability has per-sisted through several single-colony isolationsand through many transfers on miniimal medium,for more than four years. Our purpose here is toreport some of the important properties of thisstrain.

METHODS

The organism used was a motile gram negativerod, which produced smooth round opalescentwhite colonies on nutrient agar. Glucose, lactose,maltose, mannitol, arabinose, and salicin arefermented, with gas production. Litmus milk isacidified and coagulated without peptonization.Gelatin is not liquefied. The strain is indole-

1 This investigation was supported by U. S.Public Health Service Grant E-339 and by theEugene Higgins Trust.

2Present address: Department of Pharma-cology, Stanford University School of Medicine,San Francisco, California.

positive and urease-negative. It is catalase-positive when grown on nutrient broth or a glu-cose-inorganic salt medium. The auxotrophicmutants also contain catalase. The organismcorresponds to the type description (Breed et at.,1948) of E. coli and has been designated as theHarvard strain, ATCC 11887, NCTC 9561.Two kinds of solid and liquid media were used,

nutrient broth and agar (Difco), and a "mini-mal" broth and agar of the following composition:Na2HPO4, 0.04 M; NaH2PO4, 0.022 M; KCI,0.01 M; Na2SO4, 0.001 M; MgSO4, 0.4 mm; NH4Cl,9.2 mm (0.05 per cent); glucose, 11.1 mm (0.2per cent); agar (when desired), 1.5 per cent;pH 7.0. Incubation was at 38 C. Colonies onnutrient agar were about 1.5mm in diameter after24 hours, whereas those on minimal agar ap-peared more slowly and were invisible in 24 hours.By 48 hours, however, the colonies on minimalagar were larger than those on nutrient agar andmuch more mucoid. The heavy final growth andcreamy consistency on minimal agar favor spread-ing so that adjacent colonies readily coalesce.This property makes it easy to miss auxotrophsthat show even a moderate tendency to revert,because as few as 20 or 30 colonies growing in asmall test streak quickly fill the area with heavyconfluent growth.The rapid growth, after a 24-hour lag on mini-

mal agar, does not reflect a true adaptation tominimal medium, since such rapidly growingcells fail to form colonies in 24 hours when trans-ferred to fresh minimal agar. The lag is moststriking when relatively few cells are implantedon a plate, and disappears entirely when largeimplants are used; thus a direct streak with aloop, or a replication by means of velvet growsout heavily as early as 16 hours. All the experi-ments described in this paper were done underconditions permitting wild type growth on mini-mal agar within 24 hours.The high mutant frequency made special

screening methods unnecessary. Instead a dilutionof the population to be tested could be plated on

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nutrient media so as to yield about 100 coloniesper plate. The colonies were then fished in-dividually onto minimal and nutrient plateseither directly or through a dilution step. Inlater experiments, the velvet replica-plate method(Lederberg and Lederberg, 1952) was used totransfer all colonies simultaneously onto the testplates. After incubation for 24 hours at 38 C,inspection revealed which colonies did not growon minimal agar. These could be isolated fromthe original nutrient plate ("master plate")for further study. Whenever a colony on themaster plate gave rise to an unusually smallcolony on the minimal replica-plate, it was testedfurther because many such proved to beauxotrophs. When colonies were replicated byvelvet, the implant was not only large but wasconfined to a relatively small area. Though thecolony being replicated might consist almostwholly of auxotrophs, a few reverted prototrophsmight result in a small replica colony onminimal agar.Our data on auxotrophic mutants refer only

to those strains which persistently fail to growon minimal agar even after subculture on nu-trient agar. We also regularly observed strainsthat grew poorly or not at all in early testing, butafter a cycle of growth on nutrient agar couldno longer be distinguished from wild types. Thisfinding implies frequent back-mutation to proto-trophy, an interpretation consistent with thefollowing additional observations: When colonieson a nutrient master plate were tested by streak-ing, fewer auxotrophs were found when theorganisms were streaked directly on test platesthan when they were diluted prior to plating.The apparent mutant yield also was lower whenvelvet replication was used, presumably becausethe implants were unavoidably large. Therefore,auxotrophic colonies growing on nutrient masterplates are best tested while they are small andbefore many back-mutant clones develop.

Evidently, our method does not detect mu-tants with very high reversion rates, mutantsthat form colonies of normal size when fedsyntrophically by adjacent colonies on theminimal test plate, nor incompletely blockedauxotrophs that grow slowly on minimal agar.Consequently, the mutant frequencies reportedhere must be minimum estimates, the exactdegree of underestimation of the true frequenciesbeing unknown.

We identified the growth requirements of theauxotrophs by streaking or by replication onminimal plates enriched with acid hydrolyzate ofcasein, and mixtures of purines, pyrimidines,nucleosides, and vitamins; and then onto platesenriched with amino acids and other componentsas described by Lederberg (1950), except asfollows: Vitamin K was not included, but p-hy-droxybenzoic acid, pimelic acid, and j3-alaninewere. Nicotinamide was used instead of nicotinicacid. Cytosine was omitted from the purine-pyrimidine group; adenosine and guanosine werethe only nucleosides, and yeast adenylic acid theonly nucleotide used. Spot-testing the supple-ments on a minimal plate heavily seeded with theauxotroph in question was equally effective. Eachmutant was tested on all supplements, even aftershowing growth on one supplement, in order todetect alternative requirements. Mutants failingto grow on individual components were tested onmixtures, in order to detect multiple require-ments. Mutants faiJing to grow on any definedmedium were tested with the addition of yeastextract.

RESULTS

Frequency of auxotrophic mutants in wild typepopulations. When a broth tube is inoculated froma nutrient slant, the resulting population includesmutants from two sources: (a) the progeny fromthe inoculum, and (b) the progeny from new cellsarising during growth in the broth. The highmutant frequency made it almost impossible toavoid the introduction of mutants with theinoculum. The number introduced also varied,reflecting the fluctuation in mutant frequencybetween different stock slants and even betweendifferent areas of the same slant. In most studiesof mutant frequency, we considered isolatedcolonies on nutrient agar as our populations forstudy. Each colony grew independently, wasabout 4 mm in diameter at 48 hours, and con-tained 100 to 200 X 106 viable cells. After ap-propriate dilution, a colony was suspended inwater and plated on nutrient agar. The resultingdaughter colonies were then tested. If the colonyunder test had originated from an auxotrophicmutant, most of its cells would be auxotrophs.This situation could be recognized easily.The results in table 1, section I, show a mutant

frequency fluctuating between 1.3 per cent and15.0 per cent of the total number of viable cells

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TABLE 1Frequency of auxotrophic mutants in

wild-type population8No. of Auxo- Mutant

Source of Colonies Tested Cols. trophs Frequen-Tested Found cy (%)

I. Diluted water sus- 292 4 1.4pension of colony (275)* (272)* (98.9)*growing on nutrient 175 11 6.3agar 362 6 1.7

318 4 1.3183 27 14.8311 5 1.6237 3 1.3180 27 15.0223 6 2.7

Total.2,281 93 4.1Mean of the individualfrequencies.- - 5.1

II. Diluted 24-hour nu- 576 8 1.4trient broth growth of 1,057 16 1.5inoculum from nutri- 1,666 74 4.4ent slant 1,916 32 1.7

Total for experiments onnutrient-grown popula-tions (I, II).7,496 223 3.0

Mean of the individual fre-quenciesn... 4.2

III. Diluted 48-hour nu- 512 5 1.0trient broth growthfrom single cell grownon minimal agar

IV. Diluted water sus- 198 1 0.5pension of colony 287 0 0.0growing on minimal 257 1 0.4agar 141 1 0.7

354 0 0.0278 1 0.4

* The colony under test here was obviously anauxotroph itself. This experiment is, therefore,excluded from all calculations and from thefluctuation test referred to in the text.

in independently grown wild-type colonies onnutrient agar. The mean frequency in this serieswas about 5 per cent, but the most probable fre-quency is closer to 2 per cent, since the distribu-tion is asymmetric. If the data are treated as afluctuation test, Chi-square analysis indicates

marked inhomogeneity (P <0.001). Thus, theauxotrophs under investigation arise de novo,spontaneously, independently, and in the fluc-tuating manner typical of mutational events,within wild-type colonies growing on nutrientagar. The data of section II, from experimentswith nutrient slants, are wholly consistent withthe above estimates of mutant frequency. Themean of the individual frequencies for all experi-ments on nutrient-grown populations (SectionsI and II combined) is 4.2 per cent and this is ourbest estimate of the degree of mutability in theHarvard strain. Section III shows that mutantscan also originate in a nutrient broth culturetube inoculated with a single wild-type cell andcontaining a final population of 515 x 106 viablecelLs in 5 ml after 48 hours. Section IV demon-strates that colonies growing on mininal agarmay also contain auxotrophs but the frequencyis much lower, presumably because the mutantscannot form clones on the minimal medium.

Origin of the increased mutability. Having es-tablished the unusually high frequency ofauxotrophs originating de novo in wild-typepopulations of this organism, we investigated thehistory of the strain with a view to discovering,if possible, when its peculiar genetic behaviorhad originated. We were fortunate to find severalstock cultures with well-documented historiesin the Department of Bacteriology. The parentstrain had been obtained by the late Dr. J. H.Mueller about 20 years ago. A stock slant wasmaintained on tryptic digest agar, in his ownrefrigerator, for his own use. Shortly after itsacquisition, a subculture was made for teachinguse. This slant (also on tryptic digest agar) wasmaintained separately, with other classroommaterials, and, from time to time, was purifiedthrough single-colony isolations. Our strain wasobtained in November 1950, from this class slant,and maintained by us, through several single-colony purifications on nutrient agar, and onminimal agar. A broth culture of the class strainwas frozen in June 1950 and stored. Finally, asubculture of the class slant had been main-tained by Dr. H. E. Umbarger since 1948. Thus,we had available for study five different stockcultures which had originated at different times,more or less directly, from the original parentstrain.Having established (table 1) that the best esti-

mate of mean mutant frequency in the Harvard

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TABLE 2Origin of the increased mutability of the Harvard strain

The genealogy of each culture is shown in the usual way as a line diagram. In the columns, for thevarious strains, are shown results of testing for mutant frequency. The arrow indicates when, and inwhat culture, the unusual mutability of the Harvard strain originated.

I 1934

Nov. 1948

June 1950

Nov. 1950

Mueller Umbarger Class Harvard (Fss) K-12

No. mutants/total cells in different experi- 0/249 0/200 17/350 38/500 0/200 1/523ments 0/505 3/183 0/569 0/755

0/607 6/5151/638

Max. per cent mutants in stable strains 0.34 2.30 0.60 0.52(upper 99% confidence limit)

Per cent mutants in unstable strains 4.9 3.9*

* An estimate based on frequencies in a larger number of experiments (table 1) is 4.2 per cent.

strain was 4.2 per cent, we sought to determinewhich of the other strains displayed a comparabledegree of mutability. A fresh nutrient-grownpopulation was tested in each experiment. Theresults are summarized in table 2. Only the classstrain currently in use yielded a mutant fre-quency of the same order as the Harvard strain.Populations of the other strains contained onlyan occasional auxotroph. Upper. 99 per cent con-fidence limits, calculated readily from the Poissondistribution, show that the mutant frequency isvery significantly lower in these strains than inthe class and Harvard strains.The relationship of the class and Harvard

strains to the others (table 2) indicates clearlythat the mutability of the Harvard strainoriginated between June and November of 1950in the Department of Bacteriology. The culturefrozen in June 1950 (designated MSFB) is thenearest genetically stable ancestor of the Harvardstrain. It is available on request from the authors.As there is no record or recollection of any extra-ordinary environmental circumstance at thistime, it must be assumed that the mutability

itself, having arisen as a mutational event, wasselected accidentally during a single-colony purifi-cation and thus perpetuated. The negative resultswith other cultures (including the K-12 strain)exclude artifacts, misinterpretations, and unusualconditions in our laboratory as explanations ofthe apparent mutability of the Harvard strain.

Types of auxotroph obtained. Table 3 presentsthe results of two typical experiments, performedat different times, with different stock cultures.The auxotroph frequencies were 4.4 and 1.7 percent, the significant difference (P < 0.001) re-flecting the expected fluctuation between inde-pendent cultures. Characterization of the auxo-trophs obtained in these experiments establishestwo important facts: (1) The nutritional require-ments are diverse. The pathways for synthesis ofamino acids, vitamins, purines, and pyrimidinesare involved. The finding that requirements foramino acids (especially histidine) are most fre-quent accords with general experience. (2) Thenutritional requirements are single in most cases.Only 5 to 6 per cent have a multiple requirement.Such a pattern is characteristic of auxotrophic

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mutation involving single genes, in contrast tolinkage-group effects such as result from recom-bination. The diversity of the single requirementssuggests that the genetic instability is not con-fined to the vicinity of any single gene locus.

TABLE 3Analysis of growth requirements of auxotrophs

obtained from two wild-type populationsExperiments A and B were done at different

times, using different nutrient slants of wild-typeHarvard strain. Inocula (about 10' viable cells)were grown in nutrient broth to 84.5 and 97.2 X106 viable cells per ml, respectively. Dilute im-plants were made on nutrient plates and the re-sulting colonies were replicated by velvet ontominimal and nutrient agar. Subsequent testingwas done by enriching minimal agar as describedin the text. Data in the table are the numbers ofcolonies of each mutant type obtained, each suchcolony representing a single cell of that type inthe wild-type population. One cannot distinguishhere between a single large mutant clone andseveral smaller clones of the same mutant type.

Requirement Experiment A Experiment B

None* ............ 14 9Histidinet .......... 53 14Proline.................. 3 0Methionine ......... 2 0Cysteinet........ . .. .......3 1Isoleucine ............... 1 0Arginine .......... 3 0Lysine.................. 0 5Tryptophan ......... 0 3Tyrosine ................ 1 0Tyrosine or phenylalanine 0 2B12 or methionine .. 1 0Nicotinamide .. 1 0Purine§.................. 1 1Uracil.................. 1 2Arginine plus" Pyridox-amine.................. 1 0

Casein hydrolysate(acid)¶ .................. 3 0

Casein hydrolysate plusvitaminsl . .. 0 2

Yeast extractil........... 0 2

(a) Total colonies origi-nally tested ......... 1,666 1,916

(b) Total apparent auxo-trophsonfirst test.... 88 41

(c) Total stable auxo-trophs............... 74 32

as per cent of (a) 4.4% 1.7%

TABLE S-Continued

Requirement Experiment A ExperimentB

Type of growth require-ment

Single requirement oralternative singlerequirements ........ 70 28

as per cent of (c) . 94.6% 87.4%Not completely deter-mined ............... 2as percent of (c) . 0%o So6.3%

Definite or probablemultiple require-ment.. .. 4 2as percent of (c) . 5.4% 6.3%

* These failed to grow on minimal agar in 24hours in the initial test but subsequently grew ina manner indistinguishable from wild type. Theymay have been mutants that reverted during thesingle passage on nutrient agar.

t There were at least two types of histidinerequirement. Both could be met by acid-hydro-lyzed casein but only one by enzyme-hydrolyzedcasein.

t For one mutant in each experiment -methi-onine plus a vitamin mixture replaced cysteine.

§ Adenine or guanine or hypoxanthine.¶ As these would not grow on any single amino-

acid or any one screening group of amino-acidsthey presumably required two or more from dif-ferent groups. Some also had a vitamin require-ment. They were not further studied.

11 No other supplement or mixture of supple-ments sufficed.

In studies on streptomycin resistance and de-pendence, we found that the unusual mutabilitydoes not involve all genetic systems to the samedegree. The frequency of mutants resistant to 10,ug streptomycin per ml, estimated from data ofa great many experiments, was about one in60,000 cells; and of mutants dependent uponstreptomycin, about one in 10,000 cells (Gold-stein, 1954). Since the resistant mutants areknown to grow at about the same rate as thewild type in ordinary nutrient medium, one cancalculate the mutation rate to be approximately7 X 10-7 per cell per division cycle. This is withinthe range found by other invesitgators (Braun,1953). On the other hand, the mutation rate fromwild type to dependence must be phenomenallyhigh. Dependent mutants form clones of verylimited size in the absence of streptomycin (Gold-

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stein, 1954), and it follows that the mutation rateis not very much lower than the observed mutantfrequency, or about 1 X 10-4 per cell per divisioncycle. In contrast to the situation found (New-combe and Hawirko, 1949) in other strains ofE. coli mutation to dependence, in the Harvardstrain, is much more frequent than mutation toa moderate degree of resistance.Of 25 streptomycin-dependent strains, each

isolated in a different experiment, four were

found to be auxotrophs, each with a differentsingle requirement. Likewise, a streptomycin-de-pendent but otherwise prototrophic populationwas found to contain 4.9 per cent of cells thatwere both streptomycin-dependent and auxo-

trophs. These results seem to show that the muta-tions to streptomycin dependence and to auxo-

trophy, in this strain, are quite independentprocesses. We do not consider four auxotrophsamong 25 streptomycin-dependent strains to besignificantly higher than expected, especiallysince the data in table 1 show the particularwild-type slant used as starting material for theisolation of dependent mutants may have con-

tained as many as 16 per cent auxotrophs.The independence of successive mutations is

also shown in the experiment summarized intable 4. A uracil-less auxotroph was used as

starting material. Progeny of this mutant were

screened for the presence of cells with additionalgrowth requirements, and several types of doubleauxotroph were found. The unusual mutabilityof the strain persists after a first mutation hasoccurred, so that double auxotrophs arise as

frequently in a population of single auxotrophs,as did the latter in a wild-type population.

Effects of ultraviolet irradiation. In two experi-ments summarized in table 5 we compared thefrequency of auxotrophic mutants in a wild-typepopulation before and after ultraviolet irradi-ation. A turbid supension was exposed to a

General Electric germicidal lamp for one minute,killing 97 to 99 per cent of the cells, as measuredby viable count on nutrient agar. In experimentA the irradiated organisms grew on minimal agarand only a few auxotrophs were initially present.In experiment B a nutrient slant contaning a

high proportion of auxotrophs was used. In bothcases there was an apparent increase in auxotrophfrequency. At least in experiment B some new

auxotrophs must have been produced, unlessthose initially present were killed more slowly

TABLE 4Frequency and types of double auxotrophs

The populations studied were three colonies ofa uracil-less auxotroph of the Harvard strain,growing on nutrient agar. The minimal test mediacontained uracil and suspected mutants weretested by adding further supplements. All themutants isolated still required uracil and had asingle additional requirement, as shown below.

Po]Ua Total Numbers and Types of MutantColonies Auxotroph Found Frequen-Tested cy

I 388 2 histidine1 nicotinamide2 casein hydrolysate

(acid)*1 yeast extractt

Total .. 6 1.5

II 218 2 histidine1 lysine1 thiamin

Total .. 4 1.8

III 199 9 purine$ 4.5

* Would not grow on uracil plus any singleamino acid.

t Would not grow on any defined medium.$The purine requirement was met alterna-

tively by adenine, guanine, or hypoxanthine.

than the wild type. Irradiation also produced, inboth experiments, a great many colonies unableto grow on minimal agar when first tested. Later,after further growth on nutrient agar, the coloniesregained this capacity. Such apparently unstableauxotrophs are characteristic of the Harvardstrain under normal conditions. Their usual fre-quency is much increased by irradiation, herefrom 16.4 to 42.0 per cent and from 13.4 to 39.6per cent in experiments A and B, respectively.

DISCUSSION

The findings indicate that in a certain strain ofE. coli an abrupt change occurred, leading topersistent genetic instability. The unstable line,designated the Harvard strain, does not differobviously from its parent, except in the frequencywith which various mutants arise. Auxotrophicmutants have been studied in some detail andhave been shown to arise spontaneously in an

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TABLE 5Effects of ultraviolet irradiation upon auxotroph frequency

Experiment A Experiment B

Source of organisms ................. minimal slant nutrient slant

Per cent killed by U.V............... 99 97

Conditions of first test............... Direct streak from colonies Replication of colonies byonto minimal and nutrient velvet onto minimal agaragar at 21 hours. at 24 hours. Master plates

incubated a further 24hours.

Conditions of retest.............. Dilute streak from growth on Direct streak from colonies onnutrient plate of first test, master plate, onto minimalonto minimal and nutrient and nutrient agar.agar.

Before U. V. After U. V. Before U. V. After U. V.

Total colonies tested............... 195 200 463 187

Number failing to grow, or growingvery poorly on minimal agar in 24hours in first test.................. 32 (16.4%) 84 (42.0%) 62 (13.4%) 74 (39.6%)

Number of stable auxotrophs after re-test ............................ 1 (0.5%) 2 (1.0%) 46 (9.9%) 29 (15.5%)

ordinary wild-type population growing on nu-trient media. Their frequency fluctuates signifi-cantly from population to population about amean of approximately 4 per cent of the totalnumber of viable cells present. Assuming equalgrowth rate of auxotrophs and wild type innutrient medium, this would represent an over-allmutation rate of 1.5 X 10-3 per cell per divisioncycle. A wide variety of nutritional deficiencies isrepresented, but the requirements for growth onminimal medium can usually be met by single (oralternative single) substances. The frequency ofauxotrophic double mutants in a single-mutantpopulation is just as phenomenally high as that ofinitial mutants in a wild-type population, anddiverse types of second mutation occur. There issome evidence of a very high frequency of strep-tomycin-dependent mutants, but other mutationshave not yet been studied.The evidence seems to indicate that the changes

which occur in the Harvard strain are in factsingle-gene mutations. The resulting auxotrophsare apparently like those occurring in normalstrains at much lower spontaneous frequencies.Because the genetic instability of the Harvardstrain is itself a stable hereditary characteristic it

may be assumed to have a genetic basis. Com-parable instances of the actions of mutator geneshave been described in Drosophila (Demerec,1937; Ives, 1950), maize (McClintock, 1951),yeast (Ephrussi and Hottinguer, 1951) and Strep-tomyces (Newcombe, 1953). Treffers et al. (1954)reported a similar case in the K-12 strain of E.coli, characterized by high mutation rates tostreptomycin resistance as well as resistance tocertain other agents.What heritable biochemical anomaly might

cause the instability? It is hard to imagine anintrinsic structural instability of chromosomaldesoxyribonucleic acid or protein that wouldinvolve a great many different loci and at thesame time spare others, e. g., those controllingstreptomycin resistance. More plausible mecha-nisms currently under investigation include pro-duction of endogenous mutagen, abnormalamount or localization of a protective enzyme,and excessive desoxyribonuclease activity. Thesurprising inability of ultraviolet irradiation tocause a substantial increase in the frequency ofstable auxotrophs perhaps suggests that whatevermechanism is activated by irradiation of normalbacteria is already maximally active in the Har-

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vard strain. The significance of the normally oc-curring unstable auxotrophs whose frequency isincreased by irradiation remains obscure al-though very similar observations have been re-ported by Peacocke and Hinshelwood (1948),Miller et al. (1949), and Lederberg (1950).

ACKNOWLEDGMENTS

The authors are indebted to Alexandra Shed-lovsky and Barbara Wolff for assistance withsome of the experiments, to Marjorie Jewell forassistance in establishing the genealogy of thestrain, and to Dr. H. E. Umbarger for helpfuldiscussions at various stages of the investigation.

SUMMARY

A strain (ATCC 11887) of Escherichia coli hasbeen described in which various auxotrophicmutants arise at phenomenally high rates, leadingto mutant frequencies of several per cent in ordi-nary wild-type populations. This strain may serveas a useful tool in studying the causes of "spon-taneous" mutation.

REFERENCES

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BREED, R. S., MURRAY, E. G. D., AND HITCHENS,A. P. 1948 Bergey's Manual of DeterminativeBacteriology. 6th ed. The Williams andWilkins Co., Baltimore, Md.

DEMEREC, M. 1937 Frequency of spontaneousmutations in certain stocks of Drosophilamelanogaster. Genetics, 22, 469-478.

EPHRUSSI, B., AND HOTTINGUER, H. 1951 Cyto-plasmic constituents of heredity. On anunstable cell state in yeast. Cold SpringHarbor Symposia Quant. Biol., 16, 75-85.

GOLDSTEIN, A. 1954 The origin of streptomycin-dependent variants of Escherichia coli. J.Pharmacol. Exptl. Therap., 112, 326-340.

IVES, P. T. 1950 The importance of mutationrate genes in evolution. Evolution, 4, 236-252.

LEDERBERG, J. 1950 Isolation and characteriza-tion of biochemical mutants of bacteria.Methods in Medical Research, 3, 5-22.

LEDERBERG, J., AND LEDERBERG, E. M. 1952Replica plating and indirect selection of bac-terial mutants. J. Bacteriol., 63, 399-406.

MCCLINTOCK, B. 1951 Chromosome organiza-tion and genic expression. Cold SpringHarbor Symposia Quant. Biol., 16, 13-48.

MILLER, H., FARGHALY, A. H., AND MCELROY,W. D. 1949 Factors influencing the recoveryof biochemical mutants in luminous bacteria.J. Bacteriol., 57, 595-602.

NEWCOMBE, H. B., AND HAWIRKO, R. 1949Spontaneous mutations to streptomycin-resistance and dependence in Escherichia coli.J. Bacteriol., 57, 565-571.

NEWCOMBE, H. B. 1953 Radiation-induced in-stabilities in Streptomyces. J. Gen. Micro-biol., 9, 30-36.

PEACOCKE, A. R., AND HINSHELWOOD, C. N. 1948The changes induced in Bacterium lactis aero-genes by irradiation with ultraviolet light.Proc. Roy. Soc. (London), B135, 454-461.

TREFFERS, H. P., SPINELLI, V., AND BELSER, N. O.1954 A factor (or mutator gene) influencingmutation rates in Escherichia coli. Proc.Nat. Acad. Sci. U. S., 40, 1064-1071.

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