JouRNAL oF BACFROLOGY, June 1972, p. 823-830Copyright 01972 American Society for Microbiology

Vol. 110, No. 3Printed in U.S.A.

Protection of Normal, Lysogenic, andPyocinogenic Strains Against Ultraviolet

Radiation by Bound AcriflavineTIKVAH ALPER, A. J. FORAGE, AND BRENDA HODGKINS

Medical Research Council, Experimental Radiopathology Unit, Hammersmith Hospital, London W12OHS, England

Received for publication 27 December 1971

The presence of bound acriflavine protects bacteria against the lethal effectsof ultraviolet (UV) light, presumably because pyrimidine dimer formation isinhibited. Although acriflavine present in plating medium usually results inreduced viable counts from irradiated bacteria, no enhancement of lethal ef-fects is observed when acriflavine is added to irradiated bacteria left in sus-pending buffer for 45 min before plating. Acriflavine remaining bound to thedeoxyribonucleic acid of irradiated bacteria at the time they are plated likewisedoes not affect their survival. Protection is precisely dose-modifying unlesssome killing of bacteria by UV results from induction of prophage, againstwhich bound acriflavine is less protective, or from induction of pyocin, againstwhich there is no protection at all. It is inferred that prophage induction pro-ceeds in part, and pyocin induction wholly, by virtue of effects of UV otherthan pyrimidine dimerization. The response of Escherichia coli strain B to ra-diation has been postulated to be attributable in part to induction of a pro-phage or a lethal protein; but exact dose modification was observed for thisstrain, to about the same extent, whether or not the irradiated organisms were

grown in conditions thought to enhance the expected contribution to killing ifsuch a mechanism were involved. Our results support the hypothesis that theinhibition by acriflavine of dimer formation is attributable to energy transfermechanisms. They fail to support the hypothesis that shapes of survival curves(in particular the manifestation of "shoulders") can be attributed to inactiva-tion by radiation of repair enzymes.

Although agreement is lacking on the precisemode of binding of acridine molecules to nu-cleic acid (12, 16), it is well established thatthe presence of these dyes reduces the yield ofthymine dimers in ultraviolet (UV)-irradiateddeoxyribonucleic acid (DNA) (6, 20, 21).Setlow and Carrier (20) presented dose-effectcurves for the inhibition of thymine-thymineand cytosine-thymine dimers in Escherichiacoli DNA by proflavine at 1.5 x 10-5 M. Theirdata suggest that the reduction factor was con-stant for doses of UV at 280 nm up to about104 ergs/mm2, in which range dimer formationproceeds linearly with dose, and preliminarymeasurements of our own for irradiation at 254nm, with acriflavine as protective agent, like-wise demonstrate a factor for reduction whichis constant up to doses much larger than anywe used in the biological experiments to be

reported. If that basic assumption is valid, andif the killing of a given fraction of a populationrequired the induction of a given averagenumber of dimers per bacterium, it could beexpected that the inhibition of dimer forma-tion would result in the requirement for anincrease in dose by the same factor at all levelsof effect, i.e., in precise "dose-modification"(14) of the survival curve: two survival curves,taken with and without the dye present,should be superimposable by the use of dif-ferent dosage scales. If, however, the shape ofthe survival curve depended on the operationof biochemical "repair," the mechanisms forwhich were themselves subject to damage byUV, dose modification could not be expectedunless the reactants responsible for repairwere protected by exactly the same factor aswas dimerization. Also, if killing by UV were

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ALPER, FORAGE, AND HODGKINS

attributable in part to some indirect effect ofthe radiation, due to products other than py-rimidine dimers, dose modification shouldagain not be observed. Thus the mode of pro-tection by acridines can yield information onthe mechanisms of cell killing, and, similarly,can be used to test the involvement of pyrimi-dine dimerization in effects of UV irradiationother than destruction of colony-formingability. In this paper we report observations onthe killing of strains of E. coli, Salmonellatyphimurium, and Pseudomonas aeruginosa;on bacteriophage induction in lysogenicstrains; and on the induction of pyocins in P.aeruginosa. The behavior of the highly re-sistant strain Micrococcus radiodurans in re-sponse to acriflavine treatment differed sub-stantially from that of the other strains, andwill be reported in a separate communication,as will observations on mutation induction.

MATERIALS AND METHODSBacterial strains. The following have been used

previously in these laboratories and their originshave been described in relevant reports: E. colistrains B (3, 4), Bs-1 and B-H (5), K-12S and K-12-6(A) (lysogenic for X) (7); S. typhimurium strainsSL427 and SL427 (P22) (lysogenic for phage P22) (7);P. aeruginosa strains HCR-13, HCR-5, and IC, whichwere selected by Holloway (11) and were, presum-ably, derived from a pyocinogenic parent (10). Anadditional strain used in the present investigationwas P. aeruginosa strain 5781, sensitive to pyocin,from the Wellcome Laboratories' culture collection.Media. Cultures were grown before irradiation in

nutrient broth made up either from Oxoid or Difcodry granules, the latter with added NaCl, 5 g/liter.Viable counts were on blood agar base (Oxoid) or onnutrient agar (Difco) with added NaCl, 5 g/liter. Inone set of experiments, E. coli B was plated also onminimal salts-glucose agar, previously described byAlper and Gillies (4).Growth and preparation of bacteria for irra-

diation. Cultures were grown at 37 C with moderateshaking for 15 to 18 hr, by which time they were instationary phase. Cells were harvested, washed threetimes, and suspended in phosphate buffer, pH 7.The buffer was 6.7 x 10-4 M with respect to phos-phate because Webb and Petrusek (23) had reportedthat binding of acridine to bacterial DNA occurredreadily only when the suspending buffer was of lowionic strength. Suspensions were diluted appropri-ately for the radiation experiments, and acriflavine(British Drug Houses, Ltd.) was added to give therequired final concentration, in most experiments 5pg/ml. After preliminary experiments to determinethe time of contact for maximal protection, the usualprocedure was to allow half an hour, keeping thesuspensions in the dark to obviate photodynamicaction.

Irradiation. Irradiation was by a Hanovia "ger-micidal" lamp, delivering about 90% of its energy at

254 nm. Suspensions 2 mm deep were exposed at anincident dose rate of about 900 ergs per mm2 permin when the more resistant strains were used. Forsensitive ones, the UV light was used in a positionwhich gave a steady dose rate of 500 ergs per mm2per min, with a rotating shutter such that the expo-sure lasted for 1/15 of each rotation. In all the exper-iments reported here, the suspensions were in equi-librium with atmospheric air.

Plating. Control or irradiated suspensions wereappropriately diluted and one or more drops, 0.016ml/drop, were dispensed from calibrated droppingpipettes and spread over the surface of agar plates,which were prewarmed to 37 C when strains wereused such that incubation temperature affected sur-vival (4). All operations were performed in subduedlight to obviate photodynamic action. In some in-stances suspensions containing acriflavine were fil-tered through membrane filters (Oxoid, grade 0.45),and the bacteria were resuspended in buffer beforethey were plated; in other experiments, the bacteriawere irradiated in sufficiently high concentration sothat the suspensions which received the highestdoses could be diluted in buffer by a factor of 100and still yield counts of the order of 50 or more colo-nies per plate. It is worth noting that survival of UV-irradiated bacteria was in any case not detectablyaffected when they were plated together with vol-umes of the order of 0.05 ml of acriflavine in a concen-tration of 5 jig/ml.

Bacteriophage induction. Bacteriophage induc-tion was scored by the soft-agar layer method (1). Amodification of the streptomycin technique origi-nally devised by Marcovich (13) was used and hasbeen described before (7).

Determination of pyocin. Two milliliters each ofunirradiated and irradiated suspensions was inocu-lated into test tubes containing 2 ml of double-strength nutrient broth (Oxoid) prewarmed to 37 C.After incubation at 37 C for 1 to 2 hr, 2 to 3 ml ofchloroform was added and the tubes were wellshaken. The two phases were allowed to separate,and the upper layers containing bacterial debris andpyocin were collected and centrifuged. The superna-tant was tested for pyocin by pipetting 0.01- and0.02-ml volumes of serial dilutions onto a lawn of theindicator strain spread on nutrient agar. Tle plateswere incubated for 18 hr at 37 C. The most dilutesample of pyocin that visibly inhibited growth ofthe indicator strain was taken as the amount ofpyocin per milliliter induced by the UV irradiation.

RESULTSFigures 1 and 2 show how the extent of pro-

tection of E. coli strain Bs-1 varied respec-tively with acriflavine concentration and timeof contact. Similar results were obtained withother strains. Alper and Hodgkins (5) reportedthat acriflavine was dose-modifying for all thevariants of E. coli strain B they tested, toabout the same extent. In those experimentsthey used the same plating medium for all thecolony counts. Since the lethal effect of radia-

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ACRIFLAVINE PROTECTION AGAINST UV

E

C

cs

0u

.0.a

A4

FIG. 1. RelationBs-1 and concentbacteria were in cto UV, 32 ergs/rmdiated bacteria, x,

plated in parallel on rich nutrient agar andsalts-glucose agar. In agreement with previousobservations (e.g., reference 4), the initial slope

x x of the survival curve was about seven times asx steep when nutrient agar was used as when

salts-glucose agar was used. The protectionafforded by the acriflavine, however, was effec-tively independent of the plating medium.Protection ratios were 10 and 11, respectively,for the conditions in which the lesser andgreater sensitivity were evoked.

Alper and Hodgkins (5) noted briefly thatacriflavine present during irradiation failed tobe dose-modifying for three strains of P. aeru-ginosa (11) which they tested (Fig. 3). Of thesestrains the parent, IC, had been shown by Hol-

_____,___,____,___,__ loway (11) to be Hcr+, while the other two had3 6 9 12 is been selected for their Hcr- property and were

,crifhvine concentration,pg/rv more UV-sensitive than the parent. However,as previously observed with variants of E. coli

zship between survival of E. coli strain B, mutation to UV sensitivity, or to thetration of acriflavine with which Hcr- state, or to both, does not result in loss ofontact for 30 min before exposure dose modification by acriflavine nor in lowerm.2 or with no irradiation. Unirra- protection ratios than those for the resistantirradiated bacteria, 0. parents (5). Lack of dose modification and low

protection ratios were observed with all three

9=1

6:..mL-

2LV=3u

wMa5

-X -X --------- X-

C0V0

U1)

5 10 15 20 25 30

Time of contact before irradiation (minutes)

FIG. 2. Relationship between survival of E. coliBs-1 and time of contact with acriflavine, 5 ug/ml,before exposure to UV, 32 ergslmm 2, or no exposure.Unirradiated bacteria, x; irradiated bacteria, 0.

tion on E. coli strain B, in particular, is pro-foundly influenced by postirradiation condi-tions of growth (3, 4, 15), we checked for a pos-sible modification of the protection ratio asso-ciated with the influence of plating medium onthe lethal effect of UV radiation on that strain.Suspensions of E. coli strain B, irradiated inthe presence or absence of acriflavine, were

5.

c

2a

,

m

3

0.a0.

5.060

0w

0

C<

250 500 750 -pyocln induction

UV dose, ergs/mm'

FIG. 3. Survival and pyocin induction for acri-flavine-treated or untreated P. aeruginosa IC ex-posed to UV irradiation. Survival of untreated bac-teria, 0; survival of acriflavine-treated bacteria, 0;pyocin induction in untreated bacteria, &, pyocininduction in acriflavine-treated bacteria, A. Arrowsindicate relevant ordinate scale.

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ALPER, FORAGE, AND HODGKINS

P. aeruginosa strains, IC, HCR-5, and HCR-13, and these features were associated withtheir pyocinogenicity. With a nonpyocinogenicstrain, sensitive to pyocin, protection wasstrictly dose-modifying (Fig. 4). The lack ofdose modification for the killing of pyocino-genic strains is probably accounted for by thefailure of acriflavine to provide any protectionagainst the induction of pyocin, as demon-strated in Fig. 3. If lethal proteins are inducedthey concomitantly kill the host bacterium, soit is plausible to attribute some of the killingof pyocinogenic strains at low doses to photo-chemical lesions that induce the pyocin, ratherthan to those (mainly thymine dimers) thatordinarily kill bacteria. Those bacteria thatsurvived the dose giving maximal pyocin in-duction were increasingly protected by the ac-riflavine with increasing dose.The difference in the protective effect of ac-

riflavine against phage induction and bacterialkilling was less marked but was readily ap-parent. Exact dose modification was not ob-served with lysogenic strains of E. coli K-12and S. typhimurium, but their nonlysogenicderivatives showed dose modification. Theprecision of that quality is particularly strikingwith the latter, for which the survival curve isof unusual shape (Fig. 5). The loss of exactdose modification due to phage induction isillustrated by Fig. 6. In the low-dose region,the survival curves are superimposed if thedoses given to suspensions in buffer are multi-

.5

.c.,

id*'

x

-s A, xMi 379 f" AVi r,nAvlw uv .99 OW IUV u

UV dose (ergs/mm2)

FIG. 4. Survival curves for acriflavine-treated anduntreated P. aeruginosa 5781 (nonpyocinogenic) ex-

posed to UV irradiation. Untreated bacteria, 0; acri-flavine-treated bacteria, *; doses to untreated bac-teria multiplied by 8.5, x.

C

10

-4I0

I I2000 4000 6000 8000

uv dose, ergs/mm2FIG. 5. Survival curves for acriflavine-treated and

untreated S. typhimurium SL427 (nonlysogenic)exposed to UV irradiation. Untreated bacteria, 0;acriflavine-treated bacteria, 0; doses to untreatedbacteria multiplied by 6.3, x.

plied by 6.3, the protection ratio for the nonly-sogenic strain; but that factor is too small togive superposition at low survival. For buffersuspensions exposed to 930 ergs/min 2, the sur-viving fraction was 2 x 10-4; but there were 18times as many survivors from acriflavine-treated suspensions exposed to 6.3 times thatdose.

Figure 7 shows how the presence of acri-flavine affected phage induction and killing inE. coli strain K-12 6(X). The ratio 5:1 in UVdose scales suffices to superimpose the as-cending parts of the prophage inductioncurves, but that ratio is too small for superpo-sition of the survival curves except at thelowest doses.The results for all strains dealt with in this

communication are summarized in Table 1.Effect of acriflavine remaining bound

after the washing of acriflavine-treatedsuspensions. It has been observed that acri-dines bind firmly to nucleic acid (12, 16, 22),and acriflavine remained protective, though toa reduced extent, when bacterial suspensionswere washed after acriflavine treatment (Fig.8). The protective action decreased slowly with

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ACRIFLAVINE PROTECTION AGAINST UV

a -2

.20

UV dose ergs/mm2FIG. 6. Survival curves for acriflavine-treated and

untreated S. typhimurium SL427 (P22) (Iysogenic)exposed to WT irradiation. Untreated bacteria, O;acriflavine-treated bacteria, *; doses given to un-treated bacteria multiplied by 6.3, x.

time in buffer. It is well known that acriflavinepresent in plating medium acts synergisticallywith radiation in killing bacteria (2, 24), and itmight have been expected that, when bacteriahad been irradiated while in contact with acri-flavine, and then diluted and plated immedi-ately thereafter, the acriflavine remainingbound to the DNA might have affected sur-vival similarly. However, no effect of acri-flavine bound at the time of plating could bedetected. In some experiments, bacteria wereirradiated and then left in contact with acri-flavine (5 jug/ml) for 45 min before plating.Survival was the same as that of bacteria fromirradiated but untreated suspensions (Fig. 9).Results of other experiments provide sup-porting evidence that acriflavine bound to DNAat the time of plating does not act in the sameway as acriflavine in plating medium. Bacteriawere plated on medium containing acriflavine,and on ordinary nutrient agar, immediatelyafter irradiation in the presence or absence ofthe dye. As shown by Fig. 10, the presence ofacriflavine in the plating medium had pre-cisely the same relative effect on the two irra-diated suspensions.

DISCUSSIONVarious internal checks show that the pro-

tective action of acriflavine could not havebeen due to its acting as a simple physical UV"filter," to which the precision of its dose-modifying properties might have been attrib-utable. One such check is provided by Fig. 1,which shows that for E. coli strain Bs-1 theprotective action reached a maximum at a

concentration of about 9 pg/ml. Further checksare provided by the demonstration that thedye failed to be dose-modifying when killingwas due in part to the induction of pyocin or

phage. Thus the protection seems to be specifi-cally due to inhibition of dimerization. It isunlikely that acriflavine could protect enzymesagainst UV radiation to precisely the same

extent, if at all, and experiments in these labo-ratories on certain enzymes irradiated in thepresence of acriflavine have failed to demon-strate protection. It follows that the shapes ofsurvival curves for "resistant" bacteria shouldnot be attributed to inactivation by radiationof constitutive "repair enzymes," as has beenpostulated (9). This evidence complementsthat of Rude and Alper (17), whose results af-ford independent evidence that that hypoth-esis is untenable.

EL-o

0

0.L-o

0

I._0U0

La.

UVdose, ergs/mm ii

2500 5000

FIG. 7. Survival and prophage induction curvesfor acriflavine-treated and untreated E. coli K-12strain 6(X) exposed to UV irradiation. UV dose scalesin the ratio 5:1 were selected so as to superimposethe ascending parts of the prophage inductioncurves. Survival of untreated bacteria, 0; survival ofacriflavine-treated bacteria, 0; prophage inductionin untreated bacteria, A; prophage induction in acri-flavine-treated bacteria, A.

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ALPER, FORAGE, AND HODGKINS

TABLE 1. Ratios of doses for the same effects onbacteria irradiated with and without acriflavinebound to DNA (acriflavine present in suspendingmedium during irradiation except where stated)

Strains with which killing was precisely dose-modified

Dose-Strain Plating medium modi-fying

factor

Escherichia coli Bs-1 Nutrient agar 10

E. coli B Nutrient agar 11Salts-glucose agar 10

E. coli B-HAcriflavine present Nutrient agar 9

during irradiation Nutrient agar + 9acriflavine,5 pg/ml

Acriflavine washed Nutrient agar 4out before irradia-tion

E. coli K-12 Nutrient agar 8

Pseudomoras aeru- Nutrient agar 9ginosa 5781

Salmonella typhimu- Nutrient agar 6rium SL427

Strains with which precise dose modificationwas not observed

Ratio of doses to give sameeffects

Initial (low dose)region High-Strain eat

Pyocin Pro- doseinduc- phage Kill- used:tion induc- ing killingin tion

Pseudomonas aeruginosa 1 1 3IC

P. aeruginosa IC HCR-5 1 1 2

P. aeruginosa IC HCR-13 1 1 2

Escherichia coli K-12 5 5 86(X)

Salmonella typhimu- 5 6 8rium SL427 (P22)

As shown in Table 1, protection ratios forthe strains we tested, other than lysogenic or

pyocinogenic ones, ranged from about 6 to 10under the aerobic conditions employed. The

0

x

.- | | I

2000 4000 6000 8000 10000

UV dose, ergs/mm2FMG. 8. Survival curves for acriflavine-treated or

untreated E. coli B-H exposed to LV irradiation.Untreated bacteria, 0; acriflavine-treated bacteria,dye present during irradiation, 0; acriflavine-treatedbacteria, suspensions washed before irradiation, x.

results of Webb and Petrusek (23) showed thatprotection of UV-irradiated bacteria by acri-dine orange was greater for irradiation in an-

oxic than aerobic conditions, to an extent thatvaried somewhat with conditions and the spe-

cific test of damage. Alper and Hodgkins (5)confirmed that protection by acriflavine was

greater when suspensions were anoxic duringirradiation, the dose-modifying factor beingincreased by about 1.5. With some strains,therefore, protection factors as high as 15 havebeen observed. If dimer formation were inhib-ited by specific intercalation between pairs ofthymine residues, a protection ratio as high as15 would require that a dye molecule be in-serted between 14 out of every 15 such pairs, aresult in conflict with the observations ofLerman (12) and Sutherland and Sutherland(21) that the maximum ratio of tightly boundacridine molecules to bases in DNA was some-what less than one dye molecule to every twobase pairs. On the other hand, the protectionratios we have observed are in reasonably goodagreement with the deduction of Sutherlandand Sutherland (21) that an acridine moleculecan accept energy from 10 to 14 base pairs.The induction of pyocins by UV can evi-

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ACRIFLAVINE PROTECTION AGAINST UV

1id

V

0

._

3

x300 600 9501200 01so 1800

UV dose ergs/mm2

FIG. 9. Survival curves for E. coli WP2 Hcr+ ex-posed to UV irradiation. Bacteria washed and platedimmediately after irradiation, 0; bacteria after irra-diation left in suspension in contact with acriflavinefor 45 min, then washed and plated, 0; bacteria heldin suspension in the dark for 45 min, then washedand plated, x.

dently not be accounted for by pyrimidine di-merization, whereas phage induction is to someextent dependent thereon. However, sincethat form of damage is less reduced by thepresence of acriflavine than killing, which maybe almost wholly attributable to pyrimidinedimerization, there must be some pathway ofUV-induced damage leading to phage induc-tion which would not otherwise be lethal.

It has been suggested that the great suscep-tibility of the radiation response of E. colistrain B to modification by growth conditions,as well as its greater radiosensitivity than thatof E. coli strain B/r, might be accounted for ifstrain B carried an "inducible (supposedlydefective) prophage" (18) or some analogouspotentially lethal agent, perhaps of the natureof a colicin, the activity of which is normallyrepressed, but which may be derepressed byradiation and other agents (25). According tothe hypothesis of Witkin (25), the effects ofderepression would be the less, the more pro-tein synthesis was slowed after the dere-pressing stimulus: that subsidiary hypothesiswould accommodate the observation that theradiation response of E. coli strain B is greatlyreduced in conditions of reduced postirradia-

tion rates of protein synthesis (4, 8). Byanalogy with our observations on the inductionby UV of prophage in E. coli and S. typhimu-rium, and of pyocin in P. aeruginosa, it couldbe expected that acriflavine would be lesseffective as a protective agent if part of thelethal effect of radiation were contributed bythe induction of such agents, so Witkin's hy-pothesis would yield the prediction that leastprotection would be observed when postirra-diation conditions were such as to evoke thegreatest radiation response. Our experimentswith E. coli strain B showed, however, that theprotection ratio was, if anything, slightlygreater when the postirradiation growth me-dium was highly nutritive than when a "shift-down" medium was used. If a part of the ra-diation response of E. coli strain B is indeed tobe attributed to the induction of a lethalagent, it seems unlikely that it is of the natureof a prophage or a lethal protein. It may betentatively concluded that, if such an agentexists, its induction by UV is more dependenton pyrimidine dimerization than is the induc-tion of prophage in lysogenic strains of E. coli

I0

c0

40'

C._

.5

61

i0

0

200 4006 030 1000

UVdose,ergs/mm2: Buffer t, +Acriflaorve, sjg /ml4

1200

2000 4000 6000 8000 10.000

FIG. 10. Survival curves for E. coli B-H plated onmedium with or without acriflavine after UV irradia-tion in the presence or absence of the dye. Dosescales in the ratio 9:1 were used so as to superim-pose the survival curves for bacteria irradiated in thepresence and in the absence of the dye. Untreatedbacteria plated on medium without acriflavine, 0;acriflavine-treated bacteria plated on mediumwithout acriflavine, 0; untreated bacteria plated onmedium containing acriflavine (5 pg/ml), A, acri-flavine-treated bacteria plated on medium con-taining acriflavine (5 pg/ml), A.

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and S. typhimurium and much more de-pendent on dimerization than the induction ofpyocin, which indeed seems from our results toproceed independently thereof.

It was seen (Fig. 8) that bacteria were pro-tected against UV after acriflavine had beenrigorously washed out of the suspending me-dium, an observation attributable to the tightbinding of the dye to the DNA. It has, indeed,been emphasized that, once acridine moleculesare bound, "drastic measures" are required fortheir removal (12). It is reasonable to assume,therefore, that bacteria irradiated in the pres-ence of acriflavine, and then diluted andplated immediately, must still contain dyebound to the DNA during much of the periodbefore first division. Yet, as shown by Fig. 10,the presence of the bound acriflavine at thetime of plating did not reduce survival: if ithad, the effect of additional dye in the platingmedium would have been relatively less thanon bacteria irradiated in buffer. Furthermore,when irradiated bacteria were held for 45 minin buffer containing acriflavine before theywere plated survival, was not affected (Fig. 9).No specific hypothesis has been advanced

for the action of acriflavine in plating mediumin bringing additional lethal events to lightafter ionizing radiation (2); but it is generallybelieved that its effects after UV irradiationare attributable to its acting as an inhibitor ofdimer excision. As evidence in support of thisbelief, Setlow (19) quoted experiments showingthat the presence of acridines affected the rateof dimer excision in strains of E. coli. By im-plication, therefore, lower survival must beattributable to a competitive process thatwould kill the cells if dimers were not removedfast enough. On the other hand, our resultsshow that the presence of acriflavine tightlybound to the DNA of irradiated bacteria at thetime of plating does not interfere with repairmechanisms operating in resistant strains; interms of the hypothesis quoted above, thiswould suggest that irradiated bacteria can sur-vive delay in the operation of excision-repair.If this paradox were resolved, additional in-sight might be gained into the mechanism bywhich acriflavine in growth medium enhancesthe effects of radiation on microorganisms.

LITERATURE CITED1. Adams, M. H. 1959. Bacteriophages, p. 40. Interscience

Publishers, Inc., New York.2. Alper, T. 1963. Effects on irradiated micro-organisms of

growth in the presence of acriflavine. Nature (London)200:534-536.

3. Alper, T., and N. E. Gillies. 1958. 'Restoration' of Esch-erichia coli strain B after irradiation: its dependenceon suboptimal growth conditions. J. Gen. Microbiol.18:461-472.

4. Alper, T., and N. E. Gillies. 1960. The relationship be-tween growth and survival after irradiation of Esche-richia coli strain B and two resistant mutants. J. Gen.Microbiol. 22:113-128.

5. Alper, T., and B. Hodgkins. 1969. 'Excision repair' anddose-modification: questions raised by radiobiologicalexperiments with acriflavine. Mutat. Res. 8:15-23.

6. Beukers, R. 1965. The effect of proflavine on UV-in-duced dimerization of thymine in DNA. Photochem.Photobiol. 4:935-937.

7. Forage, A. J. 1971. The dependence of the oxygen en-hancement ratio on the test of damage in irradiatedbacteria. Int. J. Radiat. Biol. 20:427-436.

8. Gillies, N. E, and T. Alper. 1959. Reduction in the lethaleffects of radiations on Escherichia coli B by treat-ment with chloramphenicol. Nature (London) 183:237-238.

9. Haynes, R. H. 1966. The interpretation of microbial in-activation and recovery phenomenon. Radiat. Res.Suppl. 6:1-24.

10. Holloway, B. W. 1960. Grouping Pseudomonas aerugi-nosa by lysogenicity and pyocinogenicity. J. Pathol.Bacteriol. 80:448-450.

11. Holloway, B. W. 1966. Radiation-sensitive mutants ofPseudomonas aeruginosa with reduced host-cell reac-tivation of bacteriophage. Mutat. Res. 3:167-171.

12. Lerman, L. S. 1964. Acridine mutagens and DNA struc-ture. J. Cell. Comp. Physiol. Suppl. 1 64:1-18.

13. Marcovich, H. 1956. Etude radiobiologique du systemelysogene d'E. coli K12. II. Induction par les rayons X:etude des faibles doses. Ann. Inst. Pasteur (Paris) 90:458-481.

14. Read, J. 1952. Effect of ionizing radiations on the broadbean root. X. Dependence of X-ray sensitivity on dis-solved oxygen. Brit. J. Radiol. 25:154-160.

15. Roberts, R. B., and E. Aldous. 1949. Recovery from UVirradiation in Escherichia coli. J. Bacteriol. 57:363-375.

16. Roth, D., and M. L. Manjon. 1969. Studies of a specificassociation between acriflavine and DNA in intactcells. Biopolymers 7:695-705.

17. Rude, J. and T. Alper. 1972. The nature of the changes inthe UV survival curve of Escherichia coli B/r concom-itant with changes in growth conditions. Photochem.Photobiol. 15:51-460.

18. Rupert, C. S., and W. Harm. 1966. Reactivation afterphotobiological damage, p. 1-81. In L. G. Augenstein,R. Mason, and M. R. Zelle (ed.), Advances in radia-tion biology, vol. II. Academic Press Inc., New York.

19. Setlow, R. B. 1964. Physical changes and mutagenesis.J. Cell. Comp. Physiol. Suppl. 1 64:51-58.

20. Setlow, R. B., and W. L. Carrier. 1967. Formation anddestruction of pyrimidine dimers in polynucleotidesby ultra-violet irradiation in the presence of pro-flavine. Nature (London) 213:906-907.

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