PHOTOREACTIVATION OF ULTRAVIOLET-IRtRADIATED ESCHER-ICHIA COLI, WITH SPECIAL REFERENCE TO THE

DOSE-REDUCTION PRINCIPLE AND TOULTRAVIOLET-INDUCED MUTATION'

ALBERT KELNER

7'he Biological Laboratory, Cold Spring Harbor, L. I., ANewv lYork2

Received for p)ublication July 26, 1949

A sufficient dose of ultraviolet light (2,0537 A) inactivates most microorgan-isms. Exposure of inactivated cells to suitable visible light results in the recoveryof a large portion of the cells from their otherwise fatal ultraviolet-inducedinjury. The discovery of light-induced recovery (Kelner, 1949) and its confirm-ation for bacteriophage by Dulbecco (1949) gives us fresh hope for solving thefundamental radiobiological problems of the lethal and mutagenic action ofultraviolet radiation. The effect of reactivating light will be referred to in thispaper as photoreactivation.3 The clear-cut and sweeping nature of photoreactiva-tion is illustrated in figure 1.Numerous workers have reported on the "antagonism" of various radiations

to ultraviolet light (see review by Prat, 1936). However, the effects notedhave usually been so small, and many experiments so undecisive, that theirsignificance has been understandably overlooked. Perhaps the most pertinentwork was that of Whitaker (1942), w0-ho showed that the ultraviolet-inducedlengthening of the lag phase in Fucus eggs was counteracted in part by illumina-tion wvith wvhite light. Unfortunately, the effect was comparatively slight, andthe phenomenon was apparently not investigated further.That some ultraviolet-irradiated cells may recover if stored in suspension after

irradiation has been observed by many (Hollaender and Emmons, 1941; Robertsand Aldous, 1949; see Kelner, 1949, for other references). The degree of recoveryin stored suspensions has been relatively small, and since the possible reactivatingeffect of light from the room has not been controlled in such experiments, thedata must be re-evaluated.

METHODS

Details of the method for the ultraviolet irradiation and the preparation ofcells are found in a previous publication (Kelner, 1949), with the following addi-tions for the bacteria studied:

Escherichia coli B/r (a strain chosen because it wvas used in genetic studiesby Demerec and Latarjet, 1946) was grown in a synthetic liquid medium, M-9

'Preliminary reports of some of these data given at the May, 1949, meetings of the So-cietv of Ameiican Bacteriologists, and at the AEC Information Meeting for Bliology andMledicinie at Oak Ridge National Laboratory, in April, 1949.

2Present address: Biological Lab)oratories, Harvard lniversity, Caambridge 38, MIassa-chusetts.

3The use of this term was suggested b}y Dr. IMax Delblrtick.511

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(the ammonium medium of Anderson, 1946) for 48 hours with aeration at 37 C.The grown cultures had reproducible titers of about 4.5 X 109 cells per ml andwere diluted with saline to about 2 X 109 per ml before ultraviolet irradiation.Cultures were irradiated directly after removal from the incubator, or afterpreliminary chilling at 5 C for an hour. After irradiation the suspensions wereimmediately chilled and kept dark until treated with reactivating light. Theycould be kept chilled and dark for at least 8 hours without significant effecton subsequent reactivation.

Cells were irradiated with ultraviolet light at 20-cm distance from the source,a General Electric 15-watt germicidal lamp (intensity roughly about 50 ergs X

Figure 1. A. Plate spread on the surface with Streptomyces griseus ATC 3326 conidiathen irradiated with ultraviolet light. Following irradiation, the lid of the petri dish, withblack tape in the pattern of an inverted T, was replaced, and the spores were illuminatedwith reactivating light. Dark survivors are seen in the shadow of the tape, and a mass ofphotoreactivated cells wherever visible light struck the cells. B. As in A, except plate wasspread with E. coli B/r. Following irradiation, the petri dish lid, entirely covered withblack tape except for a small square, was replaced, and cells illuminated with reactivatinglight. Dark survivors seen in shadow of tape, and photoreactivated cells in the image oftungsten filament. Visible light was projected on the square pattern through a pro-jection lantern.

sec-1 X mm-2). Ninety-five per cent of the ultraviolet radiation of this sourcewas at 2,537 A.For photoreactivation, cell suspensions were illujminated for 45 to 60 minutes

in small glass tubes suspended in a glass-fronted water bath at 37 C. The reac-tivating light source was a 500-watt tungsten projection lamp (GE 500T20-120V)in a projection lantern with the bellows fully contracted. To obtain the highestlight intensity, the cells were placed at the focal point of the beam. The sourceemitted radiations in the long ultraviolet as well as in the visible and infrared.A filter of 0.03 N aqueous CuCl2 in a 3.2-cm deep cell was routinely used to absorba large part of the infrared.4 Suspensions were assayed for viable cells by spread-

4While the reactivating light is referred to in this paper as visible light, it should beremembered that long ultraviolet, as well as visible light, reached the cells.

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ing 0.1 ml of suitable dilutions on the surface of plates containing Difco nutrientagar to which had been added 0.5 per cent NaCl, and 1:2,500,000 gentian violetto inhibit contaminants. Incubation was at 37 C in the dark, for 24 to 48 hours.

Complications due to unwanted photoreactivation of cells during handling,from light necessary for working, were avoided by illuminating the laboratorywith yellow light only. A preliminary experiment showed that wave lengthslonger than about 5,100 A did not cause appreciable photoreactivation. A con-venient source of yellow light was a General Electric "gold" fluorescent bulbwrapped in amber gelatin filter sheets.

RESULTS

Some of the terms used in this paper may need clarification. In an ultraviolet-irradiated suspension kept in the dark, the population consists of (a) viablecells, or the dark survivors, and (b) inactivated or non-colony-forming cells.If the irradiated suspension is treated with reactivating light, the populationwill consist of (a) light survivors, comprising the original dark survivors plusphotoreactivated cells, and (b) inactivated cells which have not recovered despite

TABLE 1Photoreactivation in four microbial species

Streptomyces Escherichia Penicillium Saccharomycesgriseus coli notatum cerevisiac

Dark survival*......... 2.1 X 10-6 4.5 X 10-6 5.5 X 10-4 1.0 X 10-5Light survivalt.......... 6.6 X 10-1 1.2 X 10-1 2.5 X 10-1 1.0 X 10-8

* Fraction of cells surviving in suspensions kept dark after ultraviolet irradiation.t Fraction of cells surviving in suspensions illuminated with reactivating light after

ultraviolet irradiation.

exposure to visible light. Dark survival is the number of dark survivors dividedby the total number of cells before ultraviolet irradiation; light survival is thenumber of light survivors divided by the total nuimber of cells before ultravioletirradiation.

Generality of photoreactivation. Table 1 demonstrates photoreactivation inStreptomyces griseus ATC 3326, Escherichia coli B/r, Penicillium notatum 1951.B25, and Saccharomyces cerevmsiae, a diploid strain received from Dr. C. Linde-gren. All were irradiated in suspension with a dose of ultraviolet light giving thedark survival shown in table 1 and then reactivated with visible light, with a re-sulting increase in survival rate. The maximum degree of photoreactivationpossible for P. notatum and S. cerevisiae may be greater than indicated, for thesespecies were not studied intensively.

Photoreactivation in E. coli. As in S. griseus (Kelner, 1949) the degree ofphotoreactivation in E. coli was proportional to the time times the intensity ofreactivating light, and the effect increased with rise in temperature, withinlimits, the Qio between 20 and 40 C being about 3. Experiments with Wrattenfilters showed that the most effective wave lengths for photoreactivation werebelow 5,100 A. Slight recovery occurred at times in ultraviolet-irradiated sus-

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pensions incubated at 37 C in the dark. The increase in survival rate, or darkrecovery, was always slight, never exceeding in our experiments a value of1.5- to 2-fold. Illumination of cells with visible light before ultraviolet irradiationhad no effect on subsequent survival. Simultaneous irradiation of cells with ul-traviolet and reactivating light, or use of strictly monochromatic ultravioletlight was not studied.

The dose-reduction ratio. Suspensions of E. coli irradiated with varying dosesof ultraviolet light were subjected to the same amount of reactivating light (60minutes of intense illumination at 37 C, as described under "Methods"). Foreach ultraviolet dose, an assay was made of the suspension before and afterphotoreactivation, and from these data, the dark-, and light-survival-ultraviolet-dose curves in figure 2 were plotted. It is clear that the two curves have similarshapes.

In table 2 the ultraviolet dose giving a specific dark survival (D) is comparedwith the ultraviolet dose giving numerically the same light survival (L). Thedoses were determined graphically from figure 2. As expected from the similarityin curves, the ratio L/D is remarkably constant throughout the whole rangeof ultraviolet dosages, averaging in this case 2.5. We will call L/D the "dose-reduction ratio."Knowing the dark survival curve and the dose-reduction ratio for a given

amount of reactivating light, one can predict the degree of photoreactivationafter various ultraviolet doses. For example, if a culture of E. coli B/r is irradiatedwith an ultraviolet dose of 35 seconds, giving a dark survival of 1 X 102, the

light survival will be the same as the dark survival for the dose 35 or 14

seconds, equaling a survival of 3.2 X 10-1, or a 32-fold increase in survival dueto photoreactivation.Without implying anything as to the mechanism of photoreactivation, we may

say that in E. coli B/r a given amount of reactivating light removes a constantpercentage (60 per cent in the experiments described here) of the lethal effectsof the ultraviolet dose.The applicability of the dose-reduction principle to organisms other than

E. coli B/r and the variation of the dose-reduction ratio with the amount of re-activating light were not studied.

Loss in ability to be photoreactivated. Whatever the primary effect of ultravioletlight, photoreactivation shows it is not immediately lethal to the cell. In cellsincubated in the dark in a favorable medium after irradiation, when does theultraviolet injury become irreversible?An ultraviolet-irradiated suspension of E. coli (dark survival, 1.6 X 10w)

was diluted 1:10 with sterile broth, then incubated at 37.C, with aeration in thedark. Samples were removed at intervals, diluted 1:10 with chilled saline tostop growth, and treated as follows: one portion of the sample was assayedimmediately for viable cells; a second was assayed after being incubated 45minutes at 37 C in the dark (furnishing data for the "dark" curve in figure 3);and a third was assayed after being illuminated with light 45 minutes at 37 C

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(curve marked "light" in figure 3). These assays disclosed the degree of photo-reactivation after various periods of preincubation in the dark. There was noappreciable increase in cell count during the control incubation for 45 minutes

TRACTION SIJRVIYG

80 40 0 s lo1 120SECONDS OF ULTRAVIOLT IPRADIATION

FigureS. Ultraviolet dose-survival curves of E. coli B/r. Dark (black circles): suspensionleft in dark after irradiation. Light (open circles): suspensions reactivated with constantamount of visible light after ultraviolet-irradiation.

in the dark, except in the last (132-minute) sample, in which the count increasedtwofold, possibly because of division. The relative photoreactivation after vari-ous periods of preincubation is shown by the line of dashes in figure 3 and re-presents the quotient of the "light" divided by the "dark" assays.

Figure 3 shows that preincubation of ultraviolet-irradiated cells in nutrient

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broth at 37 C in the dark results in an exponential loss in the ability to be photo-reactivated. After 2 hours, or at about the time the dark survivors start dividing,reactivating light has scarcely any effect. In other experiments, photoreactiv-ability disappeared after 3 hours of preincubation.

Cells incubated in saline prior to visible light illumination also lost theirability to be photoreactivated, but at a much slower rate. After 2.4 hours ofpreincubation in saline, instead of broth, in one experiment, the suspensionrecovered 113-fold after visible light illumination, as compared to a 4,100-foldrecovery with no preincubation. Nutrient broth is thus not essential for thechanges leading to the loss in ability to be photoreactivated, although it doesaccelerate the loss. A direct relation of metabolism to loss in recoverability is

TABLE 2Dose-reduction ratio of E. coli

ULTRAVIOLET DOSE (SECONDS) DOSE-REDUCTIONSURVIVAL RATE 3Si-rio (LID)

Light (L) Dark (D)

6.3 X 10-1 15 6 2.53.2 X 10-1 36 15 2.41.0 X 10-1 67 24 2.85.0 X 10-2 76 28 2.71.0 X 10-2 91 36 2.55.0 X 10-' 96 38 2.51.0 X 10-3 106 44 2.46.3 X 10-4 108 46 2.43.2 X 10-4 112 48 2.38.9 X 10-i 120 53 2.3

Average ..................................................... 2.5

L-Ultraviolet-irradiated suspension treated with reactivating light.D-Ultraviolet-irradiated suspension kept dark.

not excluded by the saline experiment, since sufficient reserve foods may havebeen present in cells to allow considerable metabolic activity during incubation.

Cells kept dark and cold after ultraviolet irradiation retain their photoreactiv-ability for many hours. However, under otherwise favorable conditions thereis a short period only, 2 or 3 hours, during which the ultraviolet-induced lethalprocesses can be reversed by light, even in part.

Genetic studies. Ultraviolet radiation of 2,537 A wave length is one of themost powerful mutagenic agents known. An important consequence of the dis-covery of photoreactivation may be the ability ultimately to relate the mutagenicaction to some precisely characterized enzyme system or compound in the cell.The experiments described here were designed to answer the question, Are

induced mutants present among photoreactivated cells? If so, are they presentat a frequency of the same order of magnitude as among the dark survivors?

Techniques for the quantitative study of ultraviolet-induced mutation in

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o 20 40 60 80 100 120 UM TESINCUBATION PERIOD PRIOR TO VISIBLE LIGHT ILLUMINATION

Figure 8. Loss in ability to be photoreactivated, with incubation of ultraviolet-irradiatedE. coli B/r cells in the dark in nutrient broth prior to treatment with visible light. Dark:assay in suspensions incubated in dark after ultraviolet irradiation. Light: assay of suspen-sions treated with constant amount of reactivating light after various periods of preincu-bation. Relative recovery: the quotient of the light divided by the dark assays. Note expon-ential loss in ability to recover.

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E. coli B/r from sensitivity to resistance to coliphage T-1 have been worked outby Demerec and Latarjet (1946). They have distinguished two types of pheno-typic expression of the mutant-(1) "zero-point" mutants, or those whosemutant character is phenotypically expressed within a few minutes after irradi-ation, and (2) "delayed" mutants, which express their mutant character only af-ter incubation on nutrient medium for a variable length of time, and, perhaps,only after undergoing one or two divisions. The delayed mutants are alwayspresent at a greater frequency than the zero-point mutants for any given doseof ultraviolet light. Demerec and Latariet (1946) studied the frequency of in-duced mutants in what we have called the dark survivors, except that theyprobably did not take precautions to exclude visible light in their experiments.

Induced zero-point mutants in photoreactivated ceUs. A culture of E. coli B/rdiluted to a titer of about 2 X 109 per ml, as described previously, was dividedinto several parts. In one, the frequency of spontaneous phage-resistant mutantswas determined by spreading 0.1-ml portions of appropriate dilutions of thesuspension on nutrient agar plates previously coated with 1 to 2 X 109 T-1phage particles each. The colonies appearing after 48 hours of incubation in thedark represented resistant mutants, and from the total number of cells seededto the plate (as determined by suitable assay), the frequency of spontaneousphage-resistant mutants could be calculated. In the suspensions used the fre-quency varied from 5 per 108 to 85 per 108.A second portion of the suspension was irradiat with ultraviolet light as

indicated in table 3, an asay made for dark survival, and 0.1-mnl portions ofthe undiluted suspension spread on phage-coated plates to determine the numberof resistant mutants among the dark survivors contained in this volume ofultraviolet-irradiated suspension. No resistant mutants were found on any ofthe plates, as could be expected from the small nuimber (900 to 76,000) of darksurvivors in 0.1 ml of irradiated suspension.At the same time, a portion of the ultraviolet-irradiated suspension was photo-

reactivated as described previously, an assay made for total viable cells, and0.1-ml portions of undiluted suspension spread on phage-coated plates. Thephage-resistant colonies ou the latter plates could come from three sources,(1) spontaneous mutants which had been inactivated by ultraviolet light andreactivated by visible light, (2) spontaneous and induced mutan in the darksurvivors included in the 0.1 ml of photoreactivated suspension seeded to eachplate, and (3) induced mutants from photoreactivated cells. In our experimentsthe correction for (2) was tero. After correction for (1) the frequency of inducedmutants in photoreactivated cells was calculated and is shown for 6 experimentsin table 3.

It is seen that, at the ultraviolet dosages used, induced zero-point mutantsare present among photoreactivated cells, but at a very low frequency.Demerec and Latarjet (1946) had found that at ultraviolet dose comparable

to those used by us, the frequency of zero-point mutants (in dark survivors)was of the order of 10,000 per 108, or over 500 times higher than we foundin photoreactivated cells. As a further check on our results, an ultraviolet-irradi-

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ated suspension was concentrated by centrifugation, and then the inducedzero-point mutants were determined, after the suspension was incubated 45minutes in the dark at 37 C as the control, and for comparison after photoreacti-vating as usual. The dark survival in this experiment was 1.2 X 10<, the recoveryafter photoreactivation 1,200-fold. The frequency of induced zero-point mutantsamong the dark survivors was 2,900 per 108, a figure in fair agreement with thedata of Demerec and Latarjet (1946); the frequency in the photoreactivatedcells was as expected only 11 per 108.We may infer conservatively that for the ultraviolet dose used, and the

techniques employed, induced zero-point mutants are present at a much smallerfrequency among photoreactivated cells than in dark survivors.

Delayed mutants. The methods for determining delayed mutants in E. coliB/r were adapted from those of Demerec (1946), Demerec and Latarjet (1946),and Beale, (1948). One-tenth ml of suitable dilutions of nonirradiated, ultra-violet-irradiated, and photoreactivated E. coli B/r suspensions were spread on

TABLE 3Induced zero-point mutants in photoreactivated E. coli

ULTRAVIOLET DOSE DARKX LIGHT INDUCED MUTANTS PER 108SURVIVAL SURVIVALt PHOTOREACTIVATED CELLS

SeC

65 1.2 X 10-5 1.3 X 10-1 2165 3.1 X 10-6 2.0 X 101 065 4.5 X 10 1.2 X 10-1 655 3.8 X 104 3.3 X 101 1355 6.7 X 105 2.5 X 101 1655 4.3 X 10-c 2.8 X 10-1 12

* Fraction of cells surviving in suspensions kept dark after ultraviolet irradiation.t Fraction of cells surviving in suspensions illuminated with reactivating light after

ultraviolet irradiation.

the surface of nutrient agar plates. After various periods, up to 5 to 6 hours, ofincubation at 37 C in the dark, plates were sprayed with 1 to 3 X 10" T-1 phageparticles in the form of a fine mist of the phage lysate. The plates were then in-cubated further for 48 hours, and the phage-resistant colonies were counted.The average total number of viable cells on the plate when sprayed with phagewas determined by assaying the bacteria washed off of comparable plates withsaline (Beale, 1948). From these data, the mutation rate from sensitivity toresistance during various increments of the first 5 to 6 hours of incubation wascalculated.As Demerec (1946) has shown, the mutation rate is highest during the first

few hours of incubation, presumably during the first few divisions of irradiatedcells, then drops off to the spontaneous rate. We therefore felt that measurementof the mutation rate during the first 5 hours of incubation would give us a sensi-tive measure of delayed mutation and would avoid complications due to spray-ing phage on older plates that had excessive numbers of bacteria and the attend-ant problems of incomplete adsorption of phage and the like.

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In table 4 are shown delayed mutation rates in photoreactivated cells; andas controls, in dark survivors and nonirradiated cells. Complications due tothe presence of dark survivors among photoreactivated cells were avoided byseeding plates with high enough dilutions of photoreactivated suspensions todilute the dark survivors out. The frequency of delayed mutation, calculated

TABLE 4Effect of photoreactivation on delayed mutation in E. coli Blr

PREINCUBATION Bfr/1o INCREMENT IN DELAYEDBEFORE TOTAL CELLS (AVG. NO. INCEMENT ____________ _MUTATION

WIHPHAYIGE O LTPER PLATE) TotRlcell RATEt

STHPEAYIGE ONoPLTEceERIO B/r/1*

(Expt. 59-A4t Control-nonirradiatedhr ~~~~~~~~~~~~hr

o 8.8 X104 0 --- -3 2.1 X 106 0.75 0-3 2.0 X lO1 0.75 374 2.2 X107 0.5 3-4 2.O0X 107 0 05.8 7.0 X 10O 3 4-5.8 6.8 X l01 2.5 0.4

(Expt._59-A4$ Photoreactivated cells

0 8.8 X104 0 --- -3 4.1 X 106 4.5 0-3 3.2 X 101 4.5 1,4004 4.1X 106 14 3-4 3.7 X106 9.5 2605.1 3.3 X 107 21 4-5.1 2.9 X 107 7 24

(Expt. 61-A)§ Dark survivors

0 6.4 X104 0 --- -3.1 5.3 X104 0.25 0 -3.1-- -4.1 4.8 X 101 1.5 3.1-4.1 4.3 X 101 1.25 2905.0 3.8 X 106 8 4.1-5 3.3 X 106 6.5 200

(Expt._61-A)I Photoreactivated cells

0 1.4 X105 0 --- -3.1 6.0 X 105 1.5 0 -3.1 4.6 X 106 1.5 3304.1 6.9 X 106s 22 3.1-4.1 6.3 X 106 20.5 3265.0 5.5 x 107 31 4.1-5 4.8 X 107 9 19

*B/r/1-E. coli B/r mutant resistant to phage T-1.t Number of B/r/i mutants per 101 new cells that appeared during the increment period

indicated.$ In expt. 59-A the dark survival equaled 6.7 X 10-'; light survival, 2.5 X 10-1.§ In expt. 61-A the dark survival equaled 3.8 X 10-4; light survival, 3.3 X 10-1.

on the basis of the number of cells seeded to the plate (see Demerec and Latarjet,1946) was determined for photoreactivated suspensions in experiments not shownin table 4 and showed the same order of magnitude for both dark survivors andphotoreactivated cells, 1 to 2 X 104 per 10g. This figure agrees fairly well withthe values (about 7 X 104 per 108) given by Demerec and Latarjet (1946) forcomparable doses of ultraviolet light.The data in table 4 show that, at the ultraviolet dosages used by us, the

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mutation rates for delayed mutants are of the same high order of magnitudein both dark survivors and photoreactivated cells.The experiments here described show conclusively that induced mutants

appear in photoreactivated cells, but do not decisively answer the questionof whether reactivating light has an effect on mutation comparable to thatwhich it has on the lethal effects of ultraviolet light.

DISCUSSION

The demonstration of photoreactivation in several microbial species increasesthe probability that light reactivation will occur in most microorganisms and,perhaps, in higher forms as well. If this is so, it follows that the cellular com-pounds concerned in the recovery process are common to most cells. The chal-lenge that confronts us is to determine the nature of these compounds and themechanisms involved in photoreactivation.The dose-reduction principle should furnish a basis upon which to compare

different effects of ultraviolet light. For example, on first glance, our resultwith mutations appears inconsistent in that it implies (a) that light reactivationcauses a pronounced reduction in mutation frequency (data on zero-pointmutations) or (b) that it has no effect (data on delayed mutation). However,the results are consistent if we assume the dose-reduction principle to hold formutagenesis as well as for lethal effects. This would mean that the mutationfrequency in light survivors is equal to that found in the dark survivors at anultraviolet dose equal to the actual dose used, divided by the dose-reductionratio. In the dose-mutation frequency curves shown by Demerec and Latarjet(1946, figure 1), at the points corresponding to the ultraviolet doses used by us(giving a dark survival of about 1 X 10- to 1 X 10-5), the curve for delayedmutants rises slowly, whereas the curve for zero-point mutants rises rapidly.If the reactivating light reduces the effective ultraviolet light by a constantratio, as assuimed by the dose-reduction principle, or, in other words, pushesthe mutation frequency back on the curve, we should expect a far greater re-duction in the mutation of zero-points than for delayed mutants. Taking intoconsideration the variation in our technique from that of Demerec and Latarjet(1946), our finding that zero-point mutation is more strongly reduced by reacti-vating light than is delayed mutation is consistent with the hypothesis that thedose-reduction principle holds for mutagenesis. According to the shape of thedose-mutation frequency curve at the ultraviolet dose used, reactivating lightshould have either no effect on mutation frequency (horizontal curve), or causea reduction (rising curve) or increase (falling curve) in frequency.

Consistency of data with the dose-reduction principle is of course insufficientproof that the latter is correct for mutagenesis. Moreover, since the significanceof zero-point and delayed mutation is currently quite uncertain (see Demerecand Latarjet, 1946; Newcombe, 1948) other explanations for our data may provemore accurate.There can be no doubt, however, that the solution of not only the problem

of ultraviolet mutagenesis, but mutagenesis in general, will be considerablyadvanced by the elucidation of the mechanism of photoreactivation.

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SUMMARY

Visible light of wave lengths under 5,100 A will cause the recovery of microbialcells from ultraviolet-induced injury which would otherwise be fatal. Light-induced recovery, or photoreactivation, occurs in at least four diverse species,Escherichia coli B/r, Streptomyces griseus ATC 3326, Penicillium notatum, andSaccharomyces cerevisiae.

In E. coli B/r the ultraviolet dose-survival curves for suspensions kept darkafter irradiation, and for suspensions photoreactivated, have in general similarshapes. From the similarity in shape of the curves there was evolved the dose-reduction principle-the eiYect of a constant amount of reactivating light onsurvival in a suspension irradiated with varying amounts of ultraviolet light isthe same as if it decreased the effective ultraviolet dose by a constant factor.For E. coli B/r this means that the amount of reactivating light used in oneexperiment reduced the dose of ultraviolet light effective in killing cells by60 per cent.

E. coli B/r cells incubated in broth at 37 C, in the dark, after ultravioletirradiation, lost their ability to be photoreactivated. The ability to recoverdecreased exponentially with incubation time, becoming zero after 2 to 3 hoursof incubation.Induced mutants occur in photoreactivated E. coli B/r cells. At the ultraviolet

doses used, reactivating light apparently reduced the frequency of mutantscharacterized by phenotypic expression within a few minutes after irradiation(zero-point mutants) but had little or no effect on mutants characterized byphenotypic expression only after a prolonged period of incubation. The possiblesignificance of these data in the light of the dose-reduction principle is discussed.

REFERENCESANDERSON, E. H. 1946 Growth requirements of virus-resistant mutants of Escherichia coli

strain "B." Proc. Natl. Acad. Sci. U. B., 32, 120-128.BEALE, G. H. 1948 A method of measurement of mutation rate from phage sensitivity to

phage resistance in Escherichia coli. J. Gen. Microbiol., 2, 131-142.DEMEREC, M. 1946 Induced mutations and possible mechanisms of the transmission of her-

edity in Escherichia coli. Proc. Natl. Acad. Sci. U. S., 32, 36-46.DEMEREC, M., AND LATARJET, R. 1946 Mutations in bacteria induced by radiations. Cold

Spring Harbor Symposia Quant. Biol., 11, 38-50.DULBECCO, R. 1949 Reactivation of ultra-violet-inactivated bacteriophage by visible light.

Nature, 163, 949-950.HOLLAENDER, A., AND EMMONS, C. W. 1941 Wave length dependence of mutation production

in the ultraviolet, with special emphasis on fungi. Cold Spring Harbor Symposia Quant.Biol., 9, 179-186.

KELNER, A. 1949 Effect of visible light on the recovery of Streptomyces gri8eU conidia fromultraviolet irradiation injury. Proc. Natl. Acad. Sci. U S.,_35, 73-79.

NEWCOMBE, H. B. 1948 Delayed phenotypic expression of spontaneous mutations in Escher-ichia coli. Genetics, 33, 447-476.

PRiT, S. 1936 Strahlung und antagonische Wirkungen. Protoplasma, 26, 113-149.ROBERTS, R. B., AND ALDous, E. 1949 Recovery from ultraviolet irradiation in Escherichia

coli. J. Bact., 57, 363-375.WHITAKER, D. M. 1942 Counteracting the retarding and inhibitory effects of strong ultra-

violet on Fucus eggs by white light. J. Gen. Physiol., 25, 391-397.

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