JOURNAL OF BACTERIOLOGY, Sept. 1971, P. 623-632Copyright 0 1971 American Society for Microbiology

Vol. 107, No. 3Printed in U.S.A.

Temperature-Sensitive Recovery of a Mutant ofEscherichia coli K-12 Irradiated with

Ultraviolet LightY. SHIMAZU, M. MORIMYO, AND K. SUZUKI

Biochemistry Laboratory, Department of Chemistry, National Institute ofRadiological Sciences, AnagawaChiba-shi, Japan

Received for publication 12 April 1971

URT-43 is a mutant of Escherichia coli K-12 which gives a much larger numberof survivors when ultraviolet (UV)-irradiated bacteria are incubated on agar me-dium at 30 C than when they are incubated on the medium at 41 C, although inboth cases the number of survivors is fewer than that given by its wild-typeancestor. The UV sensitivity of this mutant was found to be markedly influencedby the presence of a high concentration of NaCl or sucrose in the plating medium.Thus, when irradiated bacteria were plated on agar medium containing 2% NaClor 0.5 M sucrose at 30 C, they exhibited a resistance similar to that of their wild-type ancestor. At 30 C, there was also an extensive recovery in liquid M9 mediumsupplemented with all of the nutrients required for growth and NaCl or sucrose. At41 C, however, the recovery was greatly inhibited. Direct chemical analysis ofthymine dimers has revealed that no significant amount of the dimer was releasedfrom deoxyribonucleic acid during the period of extensive recovery. It was

concluded, therefore, that the temperature-sensitive recovery of URT-43 does notaccompany excision of the bulk of pyrimidine dimers. To learn the gene functioninvolved in the recovery, double mutants carrying an additional mutation either ina uvr or a rec gene have been investigated for their UV sensitivities and recovery inliquid medium. It was found that recA- and recB- derivatives retain the ability ofundergoing an efficient recovery at a low temperature, whereas uvrB- and uvrC-derivatives have completely lost this ability. For these reasons, it was concludedthat the mechanism responsible for the recovery of URT-43 involves the functioncontrolled by the uvr genes. The results of photoreactivation suggested that most ofthe entities dealt with during recovery were pyrimidine dimers.

In previous papers, the authors have reportedisolation and some properties of a mutant ofEscherichia coli K-12 (URT-43), the sensitivityof which to ultraviolet (UV) light is strongly in-fluenced by the temperature of postirradiationincubation (12, 24). In brief, this mutant gave amuch larger number of survivors on agar platesat 30 C than on plates at 41 C, although at bothtemperatures it showed fewer survivors than thewild-type ancestor (KMBL 49). The fact thathigher survival was obtained on plates at a lowtemperature than that obtained on plates at ahigh temperature was interpreted as being due toa recovery at a low temperature, because thenumber of survivors was also markedly increasedwhen irradiated bacteria were incubated in liquidM9 medium at a low temperature, whereas theincrease was very small by incubation at a hightemperature. At either temperature, however,

UV-irradiated bacteriophage was not reactivated(Hcr4). The mutation giving rise to these pheno-types has been mapped at the malB-uvrA region(12).The investigations in this report were per-

formed to learn the nature of recovery of URT-43, which occurs efficiently at low temperatures.The most important result obtained was that therecovery does not necessarily result from releaseof appreciable amount of thymine dimers fromdeoxyribonucleic acid (DNA). Results were alsoobtained suggesting that the mechanism of re-covery involves the function controlled by the uvrgenes, whereas the defect in the rec gene does notinfluence the characteristic recovery of URT-43.

MATERIALS AND METHODSBacterial strains. The bacterial strains used in this

study are listed in Table 1. Isolation and general prop-623

SHIMAZU, MORIMYO, AND SUZUKI

TABLE 1. Bacterial strains useda

Mutation in Genotype concerning auxo- MatingStrain uvr or rec trophic characteristics type Remarks Referencegene

KMBL 49 thr-leu-pyr-thy-thi- F- Ancestor strain of URT-43 25URT-43 thr-leu-pyr-thy- F- 12, 24URT-43 thr-leu-his-try-thy-pyr- Hfr Obtained by cross:

P4X6 Hfr x URT-43URT-433 recB21- thrileu-pyr- F- Obtained by transduction This reportURT-4328 recAl- thr-leu-pyr- F- Obtained by cross: This report

N211 x URT-43URT-4315 uvrBS- thr-leu-arg-his-pro- F- Obtained by cross: This report

URT-43 Hfr x AB1885URT-4335 uvrC34- thr-leu-arg-his-pro- F- Obtained by cross: This report

URT-43 Hfr x AB1884AB2470 recB21 thr-leu-arg-his-pro-thi- F- 8N211 recAl - arg-his-met- Hfr CAB1885 uvrBS- thr-leu-arg-his-pro-thi- F- 9AB1884 uvrC34- thr-leu-arg-his-pro-thi- F- 9W3876C F- malB-lac-Smr 13P4X6 met- Hfr 14X64-4 HfrH

a Some of the strains listed in this table were kindly provided from the following laboratories: KMBL 49, A.R6rsch(Rijswijk,The Netherlands): AB2470, A. J. Clark (California University, Berkeley); AB1885 and AB1884,P. Howard-Flanders (Yale University, New Haven); W3876 and P4X6, H. Ogawa (Osaka University, Osaka);X64-4, N. Otsuji (Kyushu University, Fukuoka); N21 1, T. Kato (Osaka University, Osaka) (originally isolated byH. Ogawa, Osaka University)."The symbol of genotype concerning auxotrophy represents that bacteria require the substance in parenthesis

for growth: thr- (threonine), leu- (leucine), his- (histidine), try- (tryptophane), arg- (arginine), pro- (proline),met- (methionine), thi- (thiamine), thy- (thymine), and pyr- (pyrimidine).

e ma/B- and lac- represent that bacteria cannot use maltose nor lactose for growth, respectively. Smr, resistantto streptomycin.

erties of URT-43 have been described previously (12,24). URT-4328, a recA- derivative of URT-43, wasprepared by mating the latter with N21 1 (HfrC recA -).Among 34 recombinants that were prototrophic bothto thymine and histidine (Thy+ His+), a very UV-sen-sitive recombinant was isolated and designated asURT-4328. It was unable to reactivate UV-irradiated Xbacteriophage and was more sensitive to X rays thanURT-43. The dose of UV light reducing the number ofviable cells to 37% of that of unirradiated cells was es-timated to be approximately 0.24 erg/mm2, indicatingthat an average of 1.7 pyrimidine dimers per chromo-some are enough to kill a single cell. This value is con-sistent with the data obtained by Howard-Flanders etal. (10) for a uvrA-recA- double mutant of E. coli K-12. URT-433, a recB- derivative of URT-43, was iso-lated by using a transducing bacteriophage P1 (11).The phage particles grown on AB2470 (thy+recB-)were used to transduce URT-43 (thy-rec+), and Thy'transductants were selected. These Thy+ transductantswere then scored for X-ray sensitivity. After repeatedpurifications, 2 X-ray-sensitive, UV-supersensitive de-rivatives were isolated among 10 Thy+ isolates tested.URT-433 was one of these two.

URT-4315, a double mutant carrying the mutationof URT-43 and a mutation in the uvrB gene, was pre-pared by mating AB1885 (thy+thi-uvrB5-) with HfrURT-43 (thy-thi+), which had been obtained by thecross: P4x6 Hfr x URT-43. Thy+Thi+ (prototrophic

both to thymine and thiamine) recombinants were iso-lated, and each recombinant was checked for the pres-ence of the URT-43 mutation by transduction with P1bacteriophage (11). The phage grown on eachThy+Thi+ recombinant was used to transduce W3876(malB-), and the infected bacteria were plated on min-imal agar containing maltose as a sole carbon source toscore Mal+ transductants that are capable of usingmaltose for growth. These transductants were thenchecked for their UV sensitivity. Since the mutatedgene of URT-43 had been found to be closely linked(40%) to the malB gene (12), it could be assumed that,if an original Thy+Thi+ recombinant carries the muta-tion of URT-43, the P1 phage particles grown in therecombinant must give temperature-dependent, UV-sensitive Mal+ transductants in high frequency. Threesuch Thy+Thi+ recombinants were isolated, and one(URT-4315) was further checked for the presence ofmutation in the uvrB gene. This check was necessarybecause the UV sensitivity of URT-4315 was notlargely different, either from that of URT-43 or ofAB1885; therefore, measurements of UV sensitivityalone could not serve for demonstrating the existenceof the two mutations. If URT-4315 carries a mutationin the uvrB gene, its replacement by the wild-typeuvrB+ allele should result in the characteristic tempera-ture-dependent UV sensitivity of the URT-43 type.Replacement was accomplished by mating URT-4315(hs-Smr) with x64-4 (Hfr H Sm). To prevent en-

624 J. BACTERIOL.

UV RECOVERY OF E. COLI MUTANT

trance of the wild-type allele of the URT-43 gene by a

prolonged contact of these strains, mating was inter-rupted at 60 min, when His+Smr (prototrophic to histi-dine and resistant to streptomycin) recombinants first

begin to appear (9). Out of 60 His+Smr recombinantstested, 23 (38%) were found to have the UV sensitivityof the URT-43 type, a value consistent with the 41% ofHis+ Smr recombinants found by Howard-Flanders et

al. (9) to inherit the donor's uvrB- allele in a similarcross. It was concluded, therefore, that URT-4315 car-

ries mutations both in the mutated gene of URT-43and in the uvrB gene. URT-4335, a double mutant

carrying an additional mutation in the uvrC gene, was

isolated according to the same procedure. In this case,

93% of 40 His+Smr recombinants tested revealed theUV sensitivity of the URT-43 type. This value was alsoconsistent with the data of Howard-Flanders et al. (9).

Media. Throughout the present investigations, thebacteria to be irradiated were grown overnight in M9medium (1) supplemented with appropriate growthrequirements and 0.2% Difco Casamino Acids (com-plete M9). They were harvested by centrifugation,washed with M9 buffer, resuspended in the same bufferto give a concentration of about 5 x 107 bacteria/ml,and subjected to irradiation. The bacteria to be usedfor experiments of recovery in liquid medium were

starved for amino acids for 90 min at 37 C before irra-diation, since unstarved cells had been found to un-

dergo a significant recovery at a nonpermissive temper-

ature for starved cells. The liquid media used for re-

covery were made of M9 with appropriate supple-ments, which will be described in explanations of eachexperiment. The solid medium used for survival meas-

urements was NBY agar containing 0.8% nutrientbroth (Difco), 0.75% yeast extract (Difco), and 1.5%agar (5). NaCl (2%) or 0.5 M sucrose was added to

this medium in some experiments. The temperature ofincubation was at 30 or 41 C.

Determination of thymine dimers. Thymine dimerswere determined by two-dimensional paper chroma-tography. Bacteria were uniformly labeled by growingovernight in complete M9 medium containing 10 ACiof 8H-thymidine per ml (20 Ci/mmole). They were

washed twice by centrifugation and resuspended in M9buffer. They were starved for amino acids for 90 minat 37 C in the presence of thymine and uracil (10sg/ml for each) and glucose (0.4%). The washed bac-

teria were again resuspended in M9 buffer and UV ir-radiated (1,000 ergs/mm2). A portion of bacterial sus-

pension was left unirradiated. The irradiated suspen-

sion was divided into several portions depending on

experimental protocol. All nutrients for growth (thre-onine and leucine, 30 ug of each per ml; thymine anduracil, 10 lAg of each per ml; and glucose, 0.4%) were

added to each portion, and sucrose was added to some

portions to give a final concentration of 0.5 M. Theseportions were then incubated at 30 or 41 C. At timeintervals, samples were withdrawn, and the cells were

harvested and washed several times with ice-cold 5%trichloroacetic acid by centrifugation. The residue was

sealed in a tube with 6 N HCI in the presence of CO2gas and hydrolyzed for 3 hr in a boiling-water bath(27). The hydrolysate was evaporated to dryness in a

rotary evaporator, dissolved in a small quantity of dis-

tilled water, and subjected to two-dimensional paperchromatography on Whatmann 3MM paper. The sol-vent system for the first run was n-butanol-acetic acid-water (80:12:30) (2), and for the second run it wassaturated (NH )2SO,-1 M sodium acetate-isopropylalcohol (40:9:1; reference 22). The thymine and thy-mine dimer regions were cut out, and radioactivity wasdetermined in a liquid scintillation counter by using thesame fluor solution described previously (6). Authenticthymine and its dimer were chromatographed withhydrolysates. The dimer position was located over anUV lamp after heavy UV irradiation of dried papers.

Otber methods. UV irradiation and photoillumi-nation were carried out by the procedures previouslyreported (12). The irradiated bacteria were handledunder a sodium vapor lamp or under a very reducedlight to prevent photoreactivation. The uptake of 3H-thymidine into the acid-insoluble fraction was meas-ured by the procedure described previously (6).

RESULTSEffect of NaCI and sucrose on the UV sensi-

tivity and recovery of URT-43. When 2% NaCIor 0.5 M sucrose was added to NBY agar, via-bility of irradiated URT-43 was greatly enhancedas compared to the viability on the same mediumwithout these substances. Typical results for theNaCl addition are shown in Fig. 1. It can beseen that, on agar with 2% NaCl, URT-43 givesnearly the identical survival curve to that of UV-resistant ancestor strain (KMBL 49) when platesare incubated at 30 C. NaCI also reduces thesensitivity of URT43 determined at 41 C. Thus,it is apparent that URT43 can resist UV irra-diation nearly to the same extent as KMBL 49on agar medium containing a high concentrationof NaCI at 30 C, whereas it is as sensitive as isthe very sensitive uvr- mutants thus far isolatedon agar without NaCl at 41 C. Addition of su-

crose (0.5 M) produced a similar effect. Con-sistent with these results is the finding that thenumber of survivors of UV-irradiated URT43cells was increased more than a thousand-foldwhen they were incubated for 2 hr at 30 C inliquid M9 containing necessary nutrients forgrowth and 0.35 M (2%) NaCI or 0.5 M sucrose.

At a high temperature or in the absence ofgrowth nutrients, there was only a very smallincrease, if any. The results are shown in Table2. The survival of KMBL 49, AB1886 uvrA- (9),JC1569 recA- (4), and of the radiation-re-sistant parent strains of the latter two was not

significantly influenced either by temperature or

by the addition of either NaCl or sucrose toplating medium.

In the presence of 0.5 M sucrose, the incorpo-ration of 3H-thymidine into the acid-insolublefraction of irradiated and unirradiated cells as

well as into the wild-type bacteria was greatly

625VOL. 107, 1971

SHIMAZU, MORIMYO, AND SUZUKI

UV dose (ergs/mm2)

10-5_9

FIG. 1. Effects of NaCl and temperatures on theUV-survival of URT-43. Bacteria were grown over-night in complete M9, harvested by centrifugation,washed with and resuspended in M9 buffer. Dilutionsof this suspension with M9 buffer were UV irradiatedand plated on NBY agar at 30 C (A, curve 3) or at 41C (A, curve 1), or they were plated on NBY agar con-

taining 2% NaCi and incubated at 30 C (0, curve 5)or at 41 C (0, curve 2). A portion of irradiated bac-terial suspension was brought to 0.35 M NaCl, andthreonine and leucine (30 ug/ml for each), thymineand uracil (10 gg/ml for each), and glucose (0.4%)were added. The mixture was incubated for 4 hr at 30C, and bacteria were plated on NBY agar at 41 C (0,curve 4). Survival ofKMBL 49 was measured on NBYagar containing 2% NaCl at 30 C (x) or at 41 C (@;curve 6). This strain gave a similar survival curve onNBY agar without NaCl.

inhibited (Fig. 2). As a result, there is no signifi-cant incorporation into the cells of URT-43 atleast for 3 hr. It appears, therefore, that the inhi-bition of DNA synthesis does not interfere with,but rather seems to enhance, the recovery. Con-sistent with these results, preliminary experi-ments showed that nalidixic acid (to 100 gg/ml)does not inhibit the recovery (unpublished data).

Effect of a mutation in uvr or rec gene on therecovery of URT43. To learn the genetic back-ground controlling the thermosensitive recoveryof URT-43, properties of double mutantscarrying mutation in a uvr or rec gene in addi-

tion to the URT-43 mutation were investigated.The uvr and rec genes are believed to control themechanism of excision repair (8) and recombina-tional repair (20) of DNA, respectively (7). Forthis purpose, recA- and recB- derivatives ofURT-43 have been prepared by transferring themutated genes by mating and transduction, re-spectively. The double mutants carrying a muta-tion in uvrB or uvrC gene were obtained by thecross using Hfr URT-43. The fact that strainscarry both a uvr- gene and the URT-43 gene hasbeen established by genetic and survival analyses,as was previously described. Extensive tests wererequired, since, in contrast to the double mutantswith a rec- gene, double mutants with a uvr-gene could not be distinguished from URT-43 orfrom the corresponding uvr- single mutant solelyby measurements of UV-irradiation survivals.The survival data from UV irradiation of thesemutants determined before and after incubationfor 2 hr at 30 or 41 C in liquid complete M9containing 0.35 M NaCl are shown in Table 3.

It is seen that the rec- derivatives are muchmore sensitive to UV irradiation than URT-43.They are also more sensitive to UV lightthan the corresponding rec- single mutant. Espe-cially, in agreement with the observations ofHoward-Flanders et al. (10) for a uvrA-recA-double mutant, the recA- derivative is very sen-sitive. Like the original URT-43, both rec- de-

TABLE 2. Effects of temperature, growth nutrients,and ofsucrose on the recovery of UR T-43 from

ultraviolet irradiationa

Temp of Growth SS0incubation nutrients" Sucrosec 2 hr 4 hr

30 C + - 0.98 32_ _ 1.0 1.0+ + 1,940 860_ + 3.6 6.7

41 C + - 0.49 0.42_ _ 1.0 1.1+ + 11 16- + 2.1 3.7

a The bacteria were starved for amino acids beforeirradiation (60 ergs/mm2); survival was determined onNBY agar plates at 41 C.

b Threonine and leucine (30 Ag of each per ml),thymine and uracil (10 ug/ml of each per ml), and glu-cose (0.4%). Plus and minus symbols represent thepresence and absence of these nutrients, respectively.

c Concentration, 0.5 M. Plus and minus symbols rep-resent the presence and absence of sucrose, respec-tively.

d The ratio of the number of survivors determined attimes indicated just below to the number determined attime zero.

0

C.)

toC._cn

626 J. BACTERIOL.

VOL. 107, 1971 UV RECOVERY OF E. COLI MUTANT

(a)

g____4~~-a

D 60 120 180

incubation time (min)

(b)

KMBL-49(41'C)

180Iincubation time (min)

60 120 180

FIG. 2. Effect of sucrose and temperatures on the incorporation of 3H-thymidine into UV-irradiated and un-

irradiated UR T-43 and KMBL 49. The amino acid-starved bacteria were irradiated with UV (50 ergs/mm2) andincubated at 30 C or at 41 C in complete M9 medium containing 0.5 M sucrose (triangles) or in the same mediumwithout sucrose (circles). At time intervals, portions were taken on filter paper discs, and the radioactivity in theacid-insoluble fraction was determined by a liquid scintillation system (13). Unirradiated bacteria (open symbols)were treated in parallel with irradiated bacteria (filled symbols). Panel (a), UR T-43; panel (b), KMBL 49.

rivatives give much higher survivals after incuba-tion in liquid at 30 C than before incubation.Incubation at 41 C does not enhance the survivalof the recB- derivative, although it also stimu-lates the survival of the recA- derivative to a

considerable extent (see also Table 4). Thereason for this difference is not known. Theseresults seem to show that the characteristic fea-ture of URT-43 remains in the strains carrying

an additional mutation in the rec gene. It may beconcluded, therefore, that the recovery of URT-43 is not directly controlled by the rec genes.

In contrast to the case of the rec- derivatives,an additional mutation either in the uvrB or

uvrC gene appears to have deprived URT-43 ofits characteristic property, conditional recoveryin liquid. In addition, contrary to the case of therec- derivatives, double mutants with a mutation

40001URT-43(301C)

627

3000[

2000~

cs

Cs

E

1000

KMBL-49(30tC)

3000k

2000

cR1

a

Ca

1000

60

SHIMAZU, MORIMYO, AND SUZUKI

TABLE 3. Ultraviolet (UV) irradiation survival and recovery of UR T-43 carrying an additional mutation in theuvr or rec gene

Survival determined

S|Additional | UV dose Before incubation on plates After incubation ataStrain mutation in (ergs/mm2) NBY NBY + NaCI.______ 30C 41C

30C 41C 30C 41C

URT-43 60 1.2 x 10-1 4.1 x 10-' 9.1 x 10-' 1.1 x 10-1 5.3 x 10- 2.1 x 10-2URT-433 recB21 14 1.3 x 1o-4 5.0 x 10-' 2.6 x 10-2 3.3 x 1o-4 1.3 x 10-' 3.4 x 10-4URT-4328 recAI 1.5 1.4 x 1o-4 1.5 x 1o-4 6.7 x 1o-3 5.4 x 1o-4 4.0 x 10-' 1.5 x 10-2URT-4315 uvrB5 75 1.3 x 10-4 7.8 x 10-6 4.8 x 1o-4 5.3 x 1o-4 1.1 x 10-' 6.0 x 10-6URT-4335 uvrC34 75 5.1 x 10-4 2.2 x 10-' 1.6 x 10-3 9.7 x 10-4 7.8 x 10-' 4.9 x 10-'

a Incubation for 4 hr at 30 or 41 C in complete M9 medium containing 0.35 M NaCI. Plating medium was NBYat41 C.

in the uvr gene do not show distinguishable su-persensitivity. These results strongly suggest thatthe mechanism of the recovery of URT-43 is re-lated, in some way, to the function controlled bythe uvr genes.

Fate of thymine dimers. It was of interest,then, to know whether pyrimidine dimers are re-leased from DNA during extensive recovery ofURT-43, since there was a possibility that theUV sensitivity of this mutant which is greatlyaffected by environmental conditions might bedue to the conditional ability to excise pyrimi-dine dimers. In contrast to this expectation, thebulk of thymine dimers remained in the acid-in-soluble fraction, keeping high dimer to monomerratios during prolonged incubation. The resultsare shown in Table 4.

It can be seen that the ratio of thymine dimerto thymine (TT/T) of the wild-type ancestorbacteria is markedly decreased during 2 hr, indi-cating approximately 85% dimers are lost fromDNA, whereas the same ratio for URT-43 re-mains high even after 18 hr of incubation at 30 Cin the presence of 0.5 M sucrose. Therefore, itseems very unlikely that dimer excision is neededfor the recovery to take place, although there is apossibility that the release of only a very smallfraction of dimer is enough, so that the releasewhich might have been affected by temperaturesor by sucrose could not be detected by the proce-dure adopted in the present investigation. How-ever, this possibility seems not to be plausible inview of the data of photoreactivation given in thenext section.

Photoreactivation. Experiments of photoreacti-vation were performed to estimate the validity ofthe results of chemical analyses of thymine di-mers and to infer the identity of the event re-sponsible for the thermosensitive recovery ofURT-43, which appears not to involve the re-lease of pyrimidine dimers from DNA. The bac-

teria irradiated with various doses of UV werephotoilluminated for 2 hr at 30 C under a whitefluorescence lamp to give the maximum photo-reactivation. Survival was determined on NBYagar at 30 or 41 C. Control experiments usingunirradiated bacteria showed that there was nokilling nor growth during the illumination period.The results are shown in Fig. 3. The survivalcurves without photoillumination are also shownin this figure. The efficiency of photoreactivation(21) obtained from two curves, the one for pho-toilluminated and the other for nonphotoil-luminated cell survivals, is approximately 75%on agar at 41 C over a wide range of UV doses.When photoilluminated bacteria are plated onagar at 30 C, a very high survival level is at-tained.To determine whether the recovery in liquid is

competitively inhibited by preceeding photoillu-mination, experiments were performed in whichUV-irradiated bacteria were first illuminated andthen incubated in liquid at 30 C, or vice versa.The results are shown in Table 5. In these experi-ments, URT-43 was irradiated with a large doseof UV light (900 ergs/mm'). To detect the phe-nomenon, which occurs at low doses of UV light,the results of URT-4328 (recA -) are also shownin this Table 5. In the case of URT-43, there is avery large difference in the survivals determinedat high as compared to low temperatures afterphotoillumination. This difference is greatly de-creased after incubation in liquid after photoillu-mination. The situation is the same when thesetreatments are given to bacteria in the reversesequence. The recovery by photoillumination andthe recovery induced by incubation in liquid maybe related to the same photoproduct, namely py-rimidine dimers. The situation is much moresimple in the case of URT-4328 recA -. A largefraction of the sensitive bacteria that have beenkilled by a very low dose of UV light (1.5

628 J. BACTERIOL.

VOL. 107, 1971 UV RECOVERY OF E. COLI MUTANT

TABLE 4. Fate of thymine dimers in the acid-insoluble fraction of UR T-43 and KMBL 49after ultraviolet (UV) irradiation

Temp of Time of Counts/mm Counto/minUV incubation Sucrosea incubaton .in Cu/inTt/T(ergs/MM2) (C) (hr) infT in f-I' (%

URT-430 - - - 93,397 155 0.166

1,000 - + 0 59,261 308 0.5201,000 30 + 2 46,865 305 0.6511,000 30 + 4 40,703 243 0.5971,000 30 + 18 40,623 221 0.5431,000 41 - 2 55,819 335 0.6001,000 41 - 4 52,618 276 0.5251,000 41 - 18 39,273 237 0.604

UR T-430 - - 96,613 115 0.119

1,000 - + 0 79,811 330 0.4131,000 30 + 2 90,796 347 0.3821,000 41 - 2 97,170 374 0.406

KMBL 49

0 - - 93,930 38 0.0401,000 - + 0 90,370 447 0.4951,000 30 + 2 68,743 95 0.1381,000 41 - 2 19,090 27 0.141

a 0.5 M. Plus and minus symbols represent the presence and absence, respectively.h T and fT represent thymine and thymine dimer, respectively. The volume of samples applied to filter papers

was not always constant. Therefore, only the ratio of radioactivities has meaning.

ergs/mm2) can recover most of their viabilityeither by photoillumination or by incubation inliquid nearly to the same extent.

In conclusion, the results of photoreactivationstrongly suggest that the thermosensitive re-

covery of URT-43 in liquid is brought about bythe tolerance to or repair of photorepairable UVphotoproducts, namely pyrimidine dimers.

DISCUSSION

URT-43, a mutant of E. coli K-12, revealsvarious sensitivities to UV irradiation, dependingon the conditions of postirradiation incubationeither on agar or in liquid medium. Therefore, itseems to provide a good system for studying themechanism controlling the UV sensitivity of E.coli cells. An important factor modifying sur-

vival is the temperature of incubation: high sur-

vival at a low temperature and low survival at a

high temperature (12, 24). Such an effect oftemperatures also holds in the case of recovery inliquid (Table 2). It has also been found thatNaCl (0.35 M or 2%) or sucrose (0.5 M) added tothe plating agar medium markedly enhances sur-

vival (Fig. 1) and that they can stimulate re-

covery in liquid as well. Therefore, when appro-

priate combinations of these factors are applied,we can give a very large contrast to survival or

recovery for a given dose of UV irradiation. Infact, when irradiated URT-43 cells are plated on

agar containing 2% NaCl and are incubated at30 C, they give nearly the same survival level as

does its UV-resistant ancestor, KMBL 49,whereas, when they are plated on agar withoutNaCl and are incubated at 41 C, they respond toUV irradiation very sensitively. As far as hasbeen tested, only dar3- of E. coli K-12 exhibiteda similar property (24, 25).The mechanism of action of NaCl or sucrose

is not known. According to Ricard and Hirota(18), a temperature-sensitive mutant of E. coliwith respect to DNA synthesis can resume DNAsynthesis at a nonpermissive temperature when a

high concentration of NaCl or sucrose is addedto incubation medium. They also found that cer-

tain uvrC mutants of E. coli give higher survivalvalues in the presence of high concentrations ofNaCl or sucrose. They thought that the mutantsof this type might possess altered membranestructure. The effect of NaCl or sucrose couldalso be accounted for by their inhibitory effecton DNA synthesis. Recent investigations made

629

SHIMAZU, MORIMYO, AND SUZUKI

dose of UV (ergs/mm2)

0

bC2 1(

En

FIG. 3. UV survival curves of URT-43 determinedbefore and after photoreactivation. The method ofpre-paring bacteria to be irradiated has been describedunder Fig. 1. Survival was determined on NBY agar at30 C (open symbols) or at 41 C (closed symbols). Thefigures attached to curves represent the temperatures ofincubation ofplates. Photoreactivated (0, 0); nonpho-toreactivated (A, A).

by Radman and Errera (17) showed that the effi-ciency of excision repair is markedly enhancedby temporary blocking of DNA synthesis in amutant of E. coli at a nonpermissive tempera-ture.The mutation giving rise to the characteristic

property of the present mutant has been mappedat a locus very closely linked (about 40% co-transduction) to the malB gene (12). The uvrAgene, which is believed to control the UV sensi-tivity of E. coli cells through the excision repairof DNA (2, 9), is also closely linked to the malBgene to nearly the same extent (9). Therefore, itwas impossible to distinguish these mutationsonly by transduction experiments. The conclu-sions derived from the present results on doublemutants carrying an additional mutation eitherin the uvr or rec gene as well as from the resultsof previous investigations are as follows. (i) Thefunction controlled by the rec genes seems not tobe involved in the recovery of URT-43, since anadditional mutation in the recA or recB genedoes not abolish the characteristic recovery of

URT43. These results are consistent with thefindings that URT43 is as resistant to X rays asthe radiation-resistant ancestor, KMBL 49, andthat the efficiency of recombination of URT-43with a male bacteria is normal irrespective of thetemperature (12, 24). The fact that the rec- de-rivatives examined are much more sensitive toUV irradiation than either URT-43 or every rec-single mutant may also support this view. Sim-ilar results have been obtained with uvr-rec-double mutants of E. coil (8, 10). (ii) In contrastto the rec genes, the uvr genes appear to have arelationship with the relevant recovery of URT-43. This view is based on the following reasons.First, double mutants carrying an additionalmutation in either the uvrB or uvrC gene do notreveal supersensitivity to UV (Table 3). Similarphenomena have been observed by Howard-Flanders et al. (9) for several double mutants ofthe uvr genes and by Willetts and Clark (26) fordouble mutants with respect to the rec genes. Inevery case, the UV sensitivity of a double mutantdoes not exceed the sensitivity of the most sensi-tive single mutant of the same category. Sec-ondly, the double mutants of URT-43 with anuvr- gene tested have lost the capability to un-dergo recovery (Table 3), suggesting that thecharacteristic recovery of URT-43 is related tothe function controlled by the uvrB and uvrCgenes. In this respect, it is of interest and impor-tant to know whether the uvrA gene is also in-volved. If the mutation of URT-43 is located inthe uvrA gene, the mutant may genetically beexpressed as uvrAt8uvrB+uvrC+rec+ and if themutated gene of URT-43 (urt) is different fromthe uvrA gene, the relevant genetic compositionof URT-43 may be written as urttauvr+rec+, oras urttsuvrA - uvrB+ uvrC+ rec+. In the latter case,urt gene functions as a suppressor specific for theuvrA gene, and it is very closely linked to theuvrA gene so that they are transferred together.The most important and unexpected result was

the absence of the release of thymine dimersfrom DNA under the most favorable conditionsfor recovery (Table 4). At first sight, therefore,dimer excision appears not to be the importantprocess of potentiating cell recovery, whereas, ashas been discussed just above, the investigationson the uvr- or rec- derivatives of URT-43 havegiven a tentative conclusion that the uvr genesshould be functional at least under the permissiveconditions for recovery.There may arise, however, an argument that

release of only a very small fraction of pyrimi-dine dimers, too small to be detected in thepresent method, is enough for the recovery totake place. This view, however, seems to be un-likely. The dose-modifying ratio obtained from

630 J. BACTERIOL.

UV RECOVERY OF E. COLI MUTANT

TABLE 5. Combining effect ofphotoreactivation and reactivation in liquid on survival of ultraviolet (U V)-irradiated UR T-43 and UR T-4328a

UV dose No. of Reactivation Survival fraction atStrain UVedosem' No.oferiment____Strain(ergs/mm2) experiment Primary Secondary 30 C 41 C

URT-4328 1.5 1 _ _ 1.7 x 10-4 1.1 x 10-4PR _ 9.1 x 10-2 4.7 x 10-OPR LR 3.6 x 10-' 2.0 x 10-'

2 1_ 1.2x10-4 9.0x10-5LR _ 8.6 x 102 3.4 x 10-'LR PR 4.1 x 10-' 4.7 x 10-'

URT-43 900 3 _ - 5.1 x 10-4 Immeasurablysmall

PR _ 1.6 x 10-2 Immeasurablysmall

PR LR 9.8 x 10-2 6.4 x 10-2

4 _ - 3.2 x 10-4 Immeasurablysmall

LR _ 7.3 x 10-4 1.6 x 10-5LR PR 1.5 x 10'2 1.5 x 10-2

a Bacteria had been starved for amino acids before irradiation. The washed bacterial suspension in M9 bufferwas UV irradiated. Survivals were determined before and after irradiation. The irradiated suspension was thenphotoilluminated for 2 hr at 30 C, and survivals were determined. After photoillumination, the requirements forgrowth described in Table I and glucose (0.4%) were added, and the bacteria were incubated for 4 hr at 30 C.Finally survivals were again determined (experiment I and 3). In experiments 2 and 4, the irradiated bacteria weretreated in the reverse sequence of reactivation. PR represents photoreactivation and LR represents reactivation byincubating in complete M9 medium. Survivals were determined on NBY agar plates both at 30 and 41 C.

the two survival curves in Fig. 3, the one for pho-toreactivated bacteria and the other for nonpho-toreactivated bacteria, indicates that roughly90% of pyrimidine dimers produced in URT-43cells irradiated with 850 ergs/mm2 should bephotoreactivated to give a survival level of ap-proximately 10- 6 on plates at 41 C. In otherwords, more than 90% of pyrimidine dimersmust be repaired to attain the survival level of10-' before plating at 41 C. Since the recovery ofURT-43 cells irradiated with 900 ergs/mm2could attain nearly the same level of survival at41 C (Fig. I and Table 4), we could assume that,also in this case, more than 90% of dimers mustbe dark repaired, if pyrimidine dimers are thesingle entity producing killing of bacteria. Theexcision, or repair, of such a high percentage ofthymine dimers could have been detected in thepresent experiments. Another argument maypoint out that actual excision of dimers, which isnecessary for the recovery, takes place on NBYagar plates containing various nutrients, but itdoes not in liquid M9 medium. This view is alsounlikely, because an extensive thermal effect wasobserved when irradiated URT-43 cells wereplated on minimal agar medium, instead of

NBY. In this respect, the extent of minimal me-dium recovery (MMR) defined by Ganesan andSmith (5) was found to be very small (less than10-fold), if any. The absence of dimer excisionmight also be interpreted by a view that dimersliberated from DNA are present in relativelylarge, acid-insoluble oligonucleotides. On thebasis of this view, the gene urt may control thecutting out of such oligonucleotide pieces andURT-43 must have another mutation in a gene,which exerts its function on these pieces to makethem acid-soluble form. This view seems to beunlikely, but not detectable, since sedimentationanalyses in alkaline sucrose density gradientshave given no evidence to indicate the productionof gaps in parent DNA strands in irradiatedURT-43. For these reasons, the mode of exten-sive recovery of URT-43 appears not to fit themodel of DNA excision repair that is believed tobe controlled by the uvr genes (7, 8).

Unlike the liquid-holding recovery (19), therecovery of URT-43 does not occur in buffer(Table 2), but rather it may require new proteinsynthesis, since chloramphenicol inhibits the re-covery (12). In any case postirradiation replica-tion of the DNA of this mutant must take place

631VOL. 107, 1971

SHIMAZU, MORIMYO, AND SUZUKI

by copying templates carrying a number of pho-toproducts. A similar phenomenon has been ob-served by Prakash and Strauss (15) on Bacillussubtilis cells treated with methyl methanesul-fonate. The fact that URT-43 cells can toleratesuch nonphotoreactivable products has also beenshown for cells UV irradiated under a frozenstate (3, 23) and with cells treated with 4-nitro-quinoline-l-oxide. In every case, there were ex-

tensive recoveries at a low temperature (12). Inview of the survival curves of URT-43 deter-mined at 30 C (Fig. I and 3), cells are killedmore efficiently by low doses of UV irradiation(to 100 ergs/mm2) than by high doses (100 to800 ergs/mm2). This tendency also holds forbacteria that have been photoilluminated beforeplating (Fig. 3). Since the bacteria that origi-nated from a carefully purified single colonygave a similar type of survival curve, the phe-nomenon is not due to a contaminant. A similartype of biphasic curve for UV survival of A bac-teriophage has been analyzed by Radman et al.(16) as being due to a complementary action ofexcision repair and recombinational repair ofDNA. In any case, more investigation is neededto decide whether the recovery of URT-43 is dueto the known repair mechanisms of DNA or toan unknown tolerance mechanism.

ACKNOWLEDGMENTS

We express our sincere thanks to S. Kondo, Osaka Univer-sity School of Medicine, and his co-workers for their helpfuldiscussions and encouragements. This study was supported bya Grant-in-Aid for Scientific Research of the Ministry of Educa-tion of Japan.

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