The range of action of genes controlling radiation sensitivity in Escherichia coli

MUTATION RESEARCH

III

T H E R A N G E OF ACTION OF GENES C O N T R O L L I N G R A D I A T I O N S E N S I T I V I T Y IN E S C H E R I C H I A COLI INA E. MATTERN, MARIA P. VAN WINDEN AND A. R(3RSCH

Medical Biological Laboratory of the National Defence Research Organization TNO, Rijswijk, Z.H. (The Netherlands) (Received December Ioth, 1964)

SUMMARY The range of action of the genes hcr, darl, dar~, dar3, dar4, dars, and dar s, which occur in various radiation-sensitive mutants of Escherichia coli, has been studied. From a comparative examination of these mutants and their corresponding wild types it was deduced that all the genes must be involved in the repair of lethal UV damage in DNA. The extremely sensitive mutants hcr-, dar~, dar'; and dar~ showed, in contrast to the wild type, neither host-cell reactivation nor UV reactivation of phage 4. The moderately sensitive mutants d a ~ and dar¥ showed appreciable host-cell reactivation but no UV reactivation. The moderately sensitive mutant darL on the contrary, showed UV reactivation but no host-cell reactivation. UV irradiation of the wild type and the mutants reduced their capacity to propagate non-irradiated phage ~t to the same extent. The mutants were also indistinguishable from the wild type in their ability to propagate various irradiated RNA phages; UV irradiation reduced the capacity of the strains to propagate irradiated and non-irradiated RNA phages to the same extent. All mutants as well as the wild type were equally susceptible to thymineless death and to induction of prophage ~t by thymine starvation. Ultraviolet induction of prophage 2 however was achieved in sensitive mutants at a lower UV dose than in the wild type ; in addition, the vegetative multiplication of the prophage after induction was more radiation sensitive in the mutants than in the wild type. All the mutants were mutually indistinguishable from each other in their prophage induction pattern. The radiation sensitivity of F' particles in the various strains was studied. The F' particles were far more radiation resistant than the survival of the cells themselves, in the wild type as well as in the sensitive mutants, and appeared to be subject to repair in the wild type. Irradiation of the F' strains reduced their ability to transfer the F' factor to female strains rather strongly; this transfer ability was more radiation sensitive in any of the mutants than in the wild-type strain, and no great differences were observed among the various sensitive mutants. From transduction experiments with an irradiated ~ g lysate it was concluded that the processes leading to the formation of gal+ transductants were only slightly influenced by the presence of the hcr and darl genes. Abbreviations: UV, ultraviolet light of wavelength 254 m/~; p.f.u., plaque forming units.

Mutation Research 2 (1965) I 11-131

112

I, E. MATTERN, M. P. VAN WINDEN AND A. RORSCH

INTRODUCTION

During the past five years several aspects of the mutations which affect the sensitivity to ultraviolet irradiation in Escherichia c0lin,14,15,22,23, 29 have been studied. Attention was focussed especially on mutations which lead to an increased sensitivity, whose appearance has been interpreted to originate from a loss of the capacity to restore lethal UV damage in chromosomal DNA ~2,23, 25. These mutations also result in a decreased capacity to propagate certain UVirradiated bacteriophages% 12, 23, 25. However other functions than survival, scored by the ability to form colonies or plaques, appear to be subject to repair. In order to investigate the range of action of the repair processes concerned, we compared (a) the survival of several DNA and RNA containing phages, (b) the efficiency of induction of prophage 2, (c) the inactivation of transducing particles and episomes and (d) the efficiency of F' transfer, in strains capable or not of restoring UV lesions. For this study we had at our disposal several mutants with different levels of UV sensitivity that have been described in detail in the preceding paper in this journal 3°. As has been shown by the work of HOWARD-FLANDERS et al. 15 and VAN DE PUTTE et al. 22, 30 the UV sensitivity of a bacterium is controlled by a number of genes located at different sites on the bacterial chromosome. It seemed of interest to investigate how these different genes, occurring in the various UV-sensitive mutants, influence the various cell functions mentioned above, in order to obtain more information regarding the precise nature of these mutations. MATERIALS AND METHODS

Bacterial strains and bacteriophages

The origin and the genetic markers of the mutants used can be seen from Table I. The parental strains* KI2S hcr+ (KA I5), KI2S hcr- (KA 16) and KI2S 2+2a~2r (KA 17) were kindly provided by Dr. W. HARM (Dallas, Texas, U.S.A.); the strain E. coli Bs-1 (CBX 13) and E. coli Bs-2 (CBX 23) by Dr. R. F. HILL (New York, N.Y., U.S.A.); the strains CR 34 leu-thr-Bythy-lac-T~ (KA 22) and 200 PS F'lac (KA 36) by Dr. R. DEVORET (Gif-sur-Yvette, France); the strains Hfr AB 312 leu-thr-S r (KA IO), KI2 met-T~ F'lac (KA 5) and KI2 met-2rF'gal by Dr. P. G. DE HAAN (Utrecht, The Netherlands) and the strain HfrH B~ gal~s 1 (KA 52) by Dr. G. BUTTIN (Paris, France). The RNA phages#, MS 2, f 2, and R 17 were kindly provided by Dr. R. DETTORI (Milan, Italy), Dr. R. L. SINSHEIMER (Pasadena, Calif., U.S.A.), Dr. N. D. ZINDER, (New York, N.Y., U.S.A.), and Dr. W. PARANCHYCH (Philadelphia, Pa., U.S.A.) respectively. Phage 2 and its virulent mutant came from the Institut fiir Genetik, Cologne, Germany. Media

The following media were used: M9 medium (ref. 21) : I g NH,C1, 6 g Na2HP04, 3 g KH2PO,, 5 g NaC1, o.i g MgSOa per 1. Tryptone broth: 5 g Difco bactotryptone, * The s y m b o l s in p a r e n t h e s e s refer to t h e n u m b e r i n g of the culture collection of the Medical Biological L a b o r a t o r y (Rijswijk) 24. M u t a t i o n Research 2 (1965) 111-131

RANGE OF ACTION OF GENES AND RADIOSENSITIVITYIN E. coli

II 3

5 g NaC1, 8 g peptone per I. MS 2 broth (ref. 4) : io g bactotryptone, 8 g NaC1, I g yeast extract, I g glucose, 0.2 g CaC12, o.oi g BI per 1. Nutrient broth: 8 g Difco nutrient broth, 5 g NaC1 per 1. EMB agar: 12.5 g Difco EMB broth base, I g yeast extract, 5 g NaC], supplemented with I°/O sugar, sterilized separately. Bottom agars were 1.5% agar, top agars, used for phage assay, contained 0.7% agar.

Inactivation and U V reactivation of phages UV irradiation was performed in M9 medium in I - m m thick layers b y illumination with a low vapour mercury tube (Philips 30 W TUV) with a dose rate of 15 erg/mm2/sec for the wavelength 254 m#. Suitable dilutions of irradiated phage samples were plated with log-phase indicator bacteria. Phage # and f~ were propagated and plated in tryptone broth and tryptone agar, phage MS 2 and R 17 were propagated and plated in MS 2 medium. For the titration of phage 2 tryptone agar supplemented with o.I~/o MgSO4 was used. UV reactivation of phages was performed b y plating UV-irradiated phages, inactivated to o.1-1.o% survival, on irradiated hosts. The host cells were harvested in the logarithmic growth phase, centrifuged, washed twice and resuspended in M 9 medium at a concentration of 5 • Ios cells/ml, and irradiated. At various doses aliquots were taken, preadsorbed with suitable dilutions of irradiated and non-irradiated phages, and plated with indicator bacteria. In the same way the capacity of the bacteria to propagate non-irradiated phage was measured. In some experiments unadsorbed phage was removed b y centrifugation, which led essentially to the same results. Transduction / In the KI2S strains the gal- marker (epimerase negative) was introduced'by manganese treatment; the gal- CR 34 strains were obtained b y crossing the wild-type and the dar~ m u t a n t with KA 52 in order to introduce the same point mutation (kinase 138-1 ) in both strains (see Table I). Transduction of ga/- strains b y 2dg was performed as described b y ARBER 1. The hog lysate was obtained b y UV induction of the double lysogenic strain KA 17. The lysate was irradiated in a dilution of I • lO8-5 • lO 8 plaque forming units (p.f.u.) per ml; receptor strains were infected with a multiplicity of infection of 1-5. The bacteria to be transduced were grown in tryptone broth, harvested in the logarithmic growth phase, washed and resuspended in o.oi M MgSO4 to a concentration of approx. I • lO 7 cells/ml. After incubation for I h at 37°--to afford a maxim u m adsorption of p h a g e - - I - m l aliquots were added to o.I ml transducing phage lysate, irradiated previously with various doses up to 18000 erg/mm 2. After incubation for 3o min at 37 °, o.I-ml samples of suitable dilutions were spread 0~n M9-glucose and M9-galactose agar to measure the number of surviving and transduced bacteria respectively. The transduction efficiency is expressed as the fraction of gal+ cells among the survivors at each dose. Abortive transductants were neglected. Prophage induction by UV irradiation Radiation-sensitive strains and their corresponding wild types were lysogenized with phage 4. Log-phase lysogenic cells, grown in tryptone broth, were collected b y centrifugation, washed twice and resuspended in M 9 to a concentration of approx, lO 6 Mutation Research 2 (1965) i I i-i 31

I. E. MATTERN, M. P. VAN WINDEN AND A. RORSCH

II4 TABLE I STRAINS

Strain

Marker controlling

Origin

Sex

Other markers

FFFFFFFF" lac F" lac F' lac F" lac F" lac F' lac FFFFF" FFFFFFFFFFF" gal F" gal F+ F+ FFFFF" lac F' lac Hfr

thrthrthr thrthrthrthrthrthrthrthrthrthrthrthrthrthrthrthrthr 2+

radiation sensitivity K M B L 49 K M B L 90 K M B L 91 K M B L 92 K M B L 99 K M B L IOO K M B L IOl K M B L lO 5 K M B L lO6 K M B L 193 K M B L 137 K M B L 186 K M B L 138 K M B L 83 K M B L lO 4 K M B L 16o K M B L 161 K M B L 162 K M B L 163 K M B L 164 K M B L 18 K M B L 19 K M B L 14o K M B L 13o K M B L 132 K M B L 134 K M B L 135 K M B L 136 K M B L 139 K M B L I65 K M B L 41 K M B L 42 KMBL 5 KMBL 9 K K 13 K K 14 K K 15 K K 16 AB 312 B,_I*

KA 22 KMBL KMBL KMBL KMBL KMBL KMBL KMBL KMBL KMBL KMBL KMBL KMBL KMBL KMBL KMBL KMBL KMBL KMBL KMBL K A 15 K A 16 KMBL KMBL KMBL KMBL KMBL KMBL KMBL KMBL K A 15 K A 16 K A 16 K A 15 K A 15 K A 16 KA 16 K A 15 K A IO

49 49 49 49 49 49 49\ 9 o] 91j × K A 36 92 99 90 × IKA 5 49 9o 49 91 92 99 IOO

135 × K A 52 83~ 83] lO 4~ × KA 52 Io4j lO 4135 } × K A 6 13o J } × 58-161

X Bs- 1

dar + darldaredar4dar3da*5dar 6dar + daridar3dar 4darsdarl-dar + dar~dar + dar2dar 4darsdarshcr+ hcrdartdar + dar + darldarldaridartdar + hcr+ hcrhcrhcr+ hcr+ hcrhcrhcr+ Bs- t

leu-, l,:u-, leu-, leu-, leu-, leu-, leu-, leu-, leu-, leu-, leu-, leu-, leu-, leu-, leu-, leu , leu-, leu-, leu-, lei¢ ,

thr-, leu-, thr-, leu-, B 1 , thy-, BI-, thy , thr-, leu-, thr-, leu-, thr-, leu-, thr-, leu-, isoleuisoleugalgallac-, Tlr, lac-, S r lac , leu-, lac-, leu-,

Bx-,thy-, Bl-,tky-, Bl-,tky-, B1 , t k y - , Bl-,tky-, Bl-,tky-, Bl-,tky-, Bl-,tky-, Bl-,thy-, Bx-, tky-, Bl-,thy-, Bl-,thy-, B l - , thy-, Bx-,tky-, Bl-,tky-, Bl-,tky-, B~-,tky-, Bl-,thy-, Bt-,tky-, Ba-, thy-,

pyr-, p)r-, pyr-, pyr-, pyr-, pyr-, pyr-, pyr-, pyr-, pyr-, pyr-, pyr-, pyr-, pyr-, pyr-, pyr-, pyr-, pyr-, pyr-, pyr-,

BI-, thy-, BI-, thy-, pyr-,gal , p y r - , gal-, B l - , thy-, Bx- , thy-, BI-, thy-, B~-, lhy-,

lac-, lac-, lac-, lac-, la¢-, lac-, lac-, lac-, lac-, lac-, lac-, lac-, lac-, lac-, lac-, lac-, lac-, lac-, lac-, lac-,

lac-, gal , TlS, lac-, lac-, gal-, lac-, gal-,

T, r T1 r T1 r, prolT1 r T1 r T1 r Tir Tt r T1 r T1 r, prolTj r Tx r T1 r Tar, S r T1 r , S r T1 r,2 + Tx r, 2 +, prolTI r, 2 + Tx r,2 + T1 r, 2 +

T1 r, T1 r, Sr TIS , gal-, Tlr , gal-, T~r

S r, gal-, 2 + Sr Sr T1 r, S r Sr Tar, S r Sr

Sr T1 r, S r thr-, Tlr,

Sr

* Strain lost.

cells/ml. Aliquots were irradiated with various doses of UV and immediately diluted in fresh tryptone broth. After incubation for 30 min at 37°--to afford full expression of induction--o.2-ml samples were plated with E. coli C as indicator strain.

Thymineless death and thymineless prophage induction Thymine-requiring strains, grown in M9 medium supplemented with 1% glucose and all growth requirements, were harvested in the log phase, thoroughly washed and resuspended in the same medium without glucose and thymine. After I-h incubation at 37°--to deplete endogenous sources of thymine--glucose was added to a concentration of 1% and thyminetess death was followed by plating suitable diluM u t a t i o n Research 2 (1965) 1117.131

R A N G E OF ACTION OF G E N E S A N D R A D I O S E N S I T I V I T Y IN E . coli

115

tions on tryptone agar at various time intervals. Prophage induction in lysogenic strains was studied by determination of infective centres in the dilutions on E. coli C as indicator bacteria. The amount of free phage in the thymine-starved cultures was measured after shaking the samples with chloroform. The number of induced cells was calculated after correction for the amount of free phage. The induction efficiency is expressed as the fraction induced cells per cells present originally. Sexduction The cultures of F' strains were inoculated with cells from a single colony on EMB agar. Both F' and F - strains were grown in nutrient broth supplemented with 1°/0 glucose to a concentration of approx. 2 • lO 8 cells/ml. After centrifugation and washing, the F' strains were resuspended in M9 medium, and the F - strains in fresh nutrient broth, both to the same original density. The F' strains were irradiated with various doses of UV; o.I-ml aliquots were taken for measuring episome inactivation and cell survival by plating on EMB agar and 0.5 ml was mixed with 0.5 ml of the female strain. The mixtures were incubated for 30 min at 37 °, and suitable dilutions were plated on appropriate selective media. In crosses with KI2S strains of the type leu-F'lac + × leu+lac-F -, sexduced cells were selected on M9 medium lacking leucine supplemented with lactose as the sole carbohydrate source. In crosses with CR 34 strains of the type SsF'lac + × Srlac-F -, sexduced cells were selected on.EMB or M9 agar supplemented with IOOO/~g/ml streptomycin and lactose as the sole carbohydrate source. Colonies on EMB agar were counted after 24-h incubation at 37 °. RESULTS

The radiation sensitivity of the bacterial strains The colony-forming ability after UV irradiation of the strains of E. coli used in this study, has been described previously. For the survival curves of E.coli B8-1 and E.coli B,_z the reader is referred to HILL AND S I M S O N 14, for E.coli B syn- to R6RSCH et al. ~4 and for E.coli KI2S hcr- to HARM18. The survival curves of the darmutants derived from E.coli CR 34 are described by VAN DE PUTTE et al. 3°, and reproduced in Fig. Ia. According to their radiation sensitivity the mutants were divided into two classes: (a) the extremely radiation-sensitive strains B,-1, syn-, darL dar~, dar~ and hcr-, which we shall indicate as belonging to the "hcr class" and (b) the strains with intermediate radiation sensitivity, dar-2, dar-4 and B,-~, which we shall indicate as belonging to the "Bs-~ class". Though the strain dar~ shows an intermediate radiation sensitivity it will be considered to belong to the hcr class because of its reaction on UV-irradiated temperate phage (see Fig. Ib). In order to characterize the reproductive capacity of the radiation-sensitive mutants immediately after irradiation we compared their growth rates with that of the wild type by measuring the absorbancy of growing cultures at 700 m/~ as described previously 2~. At a dose of 300 erg/mm ~ the growth rate of the wild types was found to be reduced to approx. 70% of the non-irradiated control whereas the growth rate of all the sensitive mutants was reduced to 2o-300/0 . No significant differences in their immediate response after irradiation were observed among the various mutants.

Mutation Research 2 (1965) 111-131

116

I. E. MATTERN, M. P. VAN W l N D E N AND A. RORSCH

/

o

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Fig. i . a . U l t r a v i o l e t s u r v i v a l c u r v e s for t h e v a r i o u s dar m u t a n t s . O, K M B L 49 dar+; [2, K M B L 9 9 dar3-; A, K M B L 9 I dar=-;V , K M B L 92 dare-; T[, K M B L i o i dare-; O, K M B L 9 o darl-; ®, K M B L ioo dars-, b. U l t r a v i o l e t s u r v i v a l c u r v e s for p h a g e ~t p l a t e d o n t h e v a r i o u s dar m u t a n t s • T h e s t r a i n s a r e i n d i c a t e d a s i n a.

The radiation sensitivity of D N A phages The plaque-forming ability of DNA-containing phages propagated after UV irradiation on several of the radiation-sensitive mutants has been described beforeg, =3. The survival of irradiated phage Jt on the various dar mutants is represented in Fig. lb. UV-irradiated non-virulent phages such as ]'1, T3 and TT, and a temperate phage such as ~t, form less plaques b y propagation on hcr class mutants than b y plating on the corresponding wild types. We also confirmed that the temperate phages 82c and 434 seem more radiation sensitive on hcr class mutants than on wild-type strains 8. This difference was interpreted to be due to the absence of the ability to repair UV lesions in phage DNA b y the radiation-sensitive mutants. Only phages that contain double-stranded DNA appeared to be reactivated since the single-stranded DNA phages ~ X I 7 4 and S 13 showed the same UV sensitivity on wild-type strains as on hcr- mutants ~. However the double-stranded replicative form of # X I 7 4 again showed a much higher survival on wild-type strains than on a hcr- or a syn- strain 17. The B,-2 class mutants did not show significant differences with the wild-type strains in their capacity to propagate UV-irradiated phages although their own survival was greatly reduced after UV irradiation. The mutant dar~ showed almost the reverse response. Its UV sensitivity was intermediate between the wild-type strain and the hcr class mutants, but it did not reactivate UV-irradiated phages (Fig. I). U V reactivation of phage 2 in dar mutants UV reactivation is the phenomenon that the survival of some UV-irradiated phages, e.g. 4, is higher on slightly UV-irradiated bacteria than on non-irradiated Mutation Research 2 (1965) i I 1--13 I

RANGE OF ACTION OF GENES AND RADIOSENSITIVITY IN

117

E. coli

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5

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10"

10-"

10"

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I 3.0

10" 0

I

I

I

I

I

0.3 0.6 Q9 1.2 1.5 UV dose on bocterio (10 3 e r g / m r n 2 )

Fig. 2. U V reactivation of p h a g e ~. b y dar m u t a n t s , a. P h a g e irradiated to 3% survival, b. Phage i r r a d i a t e d to 50% survival. Abscissa, U V dose on bacterial strains; ordinate, fraction p.f.u, on these s t r a i n s b y p r e - a d s o r p t i o n on 1KMBL 49 dar+ (©), K M B L 9o darx-(A), K M B L 91 dar,-(&), K M B L 92 dar,- (V), K M B L 99 data- (x7), KM]3L IOO da%- (D), and K M B L IOi dar e- (0).

ones1°, al. Since the hcr- and syn- mutants showed a reduced UV reactivation, and the B8-1 mutant none, HARMTM assumed a close correlation between host-cell reactivation and UV reactivation. Therefore we studied the occurrence of UV reactivation in the various dar mutants by plating phage ~, irradiated with doses to o.1-5o% survival, on the various hosts irradiated with doses up to 3000 erg/mmL as described under METHODS. The results of a representative experiment are collected in Fig. 2a. Here the phages were inactivated to a survival of 3%; essentially the same results were obtained at o.1% survival. When a low dose of UV (600 erg/mm ~) was applied to the wild-type strain KMBL 49 or its mutant KMBL 99 dar~, the survivat of the irradiated phage was enhanced. This enhancement was not observed with the mutants KMBL 9 ° dar;, KMBL Ioo dar~ and KMBL IOI dar~. Surprisingly the UV-irradiated Bs-2 class mutants KMBL 91 dar~ and KMBL 92 dar~ showed a steep decrease in their ability to propagate UV-irradiated phage, even at low doses on the hosts. With slightly irradiatedphages--inactivated to a survival of 5o% ---only a small decrease was observed on the irradiated dar-; and dart mutants, similar to that on the dar~ mutant (see Fig. 2b). Under these conditions in the wild type and the dar~ mutant, UV reactivation has not yet been observed. Next the capacity of irradiated strains to reproduce non-irradiated phage was measured. The results are shown in Fig. 3 a. It is clear that the irradiation of the hosts with doses up to 5000 erg/mm ~ had no drastic effect on the capacity to reproduce non-irradiated phage. No significant differences are observed in this respect among any of the radiation-sensitive mutants and the wild type. In summary we see that the sensitive mutants dar-;, dar~ and dar~, show, like the hcr- mutant of HARMTM, no UV reactivation. The wild-type dar+ strain indeed shows the expected UV reactiMutation Research 2 (1965) 111-I31

I18

I. E. MATTERN, M. P. VAN WINDEN AND A. RORSCH

1.0 ~

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Fig. 3. T h e U V s e n s i t i v i t y of t h e c a p a c i t y of E. coli to p r o p a g a t e D N A p h a g e ~ a n d I~NA p h a g e 1~ 17. Abscissa, U V dose on b a c t e r i a ; ordinate, fraction p . f . u . a . T h e c a p a c i t y to p r o p a g a t e n o n i r r a d i a t e d ~ of K M B L 49 da r+ (©), K M B L 90 darl- (&), K M B L 91 dar2- (&), K M B L 92 dar4- (V). K M B L 99 dam- (V), K M B L i o o dar-5 (~), a n d K M B L i o i dar e- ((>). B r o k e n lines, r e c o n s t r u c t e d s u r v i v a l c u r v e s for t h e s e strains, b. T h e c a p a c i t y of E. coli K I 2 wild t y p e to p r o p a g a t e n o n i r r a d i a t e d p h a g e R 17 (O), a n d p h a g e R 17 irradiated w i t h 72o0 e r g / m m 2 (A). [~, ratio p.f.u, bet w e e n i r r a d i a t e d a n d n o n - i r r a d i a t e d R I7 ( = U V reactivation).

vation just like the dar-; mutant though the latter is more like the hcr class mutants in its lack of host-cell reactivation. B y contrast the non-irradiated dar-; and darx mutants are able to repair UV damage in ~ DNA 3° but they readily lose this capacity upon irradiation.

The radiation sensitivity of RNA phages In order to examine the influence of the dar genes on UV damage in RNA, the survival of the four RNA phage s/~, R 17, MS 2 and f 2 after UV irradiation was measured by plating them on wild-type and radiation-sensitive male strains, viz. KA IO Hfr AB 312 hcr+, Hfr AB 312 Bs-1, KMBL 41 F+ her+, KMBL 42 F+ hcr-, KA 36 dar+ F'lac, KMBL 137 F'lac dar;, KMBL 186 F'lac dar~, KMBL 193 F'lac dar~ and KMBL 138 F'lac darT. Some of the results are illustrated in Fig. 4. These survival curves represent the ability of the wild-type strain KMBL 41 and the mutant KMBL 42 hcr- to propagate each of the four irradiated RNA phages. It is clear that no difference in survival is found when the irradiated phages are plated on the wild-type strain or on the sensitive mutant. Exactly the same results were obtained with any of Lhe other Mutation Research 2 (1965) 111-131

RANGE OF ACTION OF GENES AND RADIOSENSITIVITY IN

E. coli

119

1.0

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2

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2

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2

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Fig. 4- UV survival curves for R N A p h a g e s plated on K M B L 41 wild t y p e (®) a n d K M B L 4 2 her- ( × ) . Abscissa, UV dose on p h a g e ; ordinate, fraction p.f.u.

radiation-sensitive mutants, dar; and dar; included. These results are in accord with those of WINKLER aa who found no dark reactivation of the RNA phage fr. We also observed that the survival curves of the four different phages are quite similar and that these phages are rather resistant to UV irradiation (minimal lethal dose approx. 850 erg/mm2). Next we studied the effect of irradiation of the wild-type host on its capacity to propagate irradiated or non-irradiated phage. These results are collected in Fig. 3b. The strain loses its capacity to propagate the phage progressively with increasing dose and at approximately the same rate whether or not the phage was previously irradiated. Therefore the phenomenon of UV reactivation was not observed. Again exactly the same results were obtained with each of the other radiation-sensitive mutants.

UV induction of prophage 2 A number of radiation-sensitive mutants and their corresponding wild types were lysogenized with phage 2, giving the strains KMBL 18, KMBL 19, KMBL 14o, K M B L 16o, KMBL 161, KMBL 162, KMBL 163 and KMBL 164. The prophages in these strains were induced b y UV irradiation and the number of induced cells was measured as described under METHODS. The results are collected in Fig. 5- As usually observed, the number of induced cells increased gradually with increasing dose until a certain maximum, at 40-60% induction, was achieved; at higher doses the number of plaque-forming centres decreased again. In cultures of any of the radiation-sensitive mutants the number of induced cells increased more rapidly with increasing dose than in the corresponding wild type, and the m a x i m u m of induction was achieved at a lower dose. Thereafter the number of plaque-forming centres also decreased more rapidly than in cultures of wild-type strains. No significant differences in the shapes of the induction curves were observed among the various radiationsensitive mutants, despite the differences in their own survival and that of extracellularly irradiated, virulent phage 2 plated on them. Mutation Research 2 (1965) i I I - I 3 I

120

I. E. MATTERN, M. P. VAN WINDEN AND A. RORSCH

1.0

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(10 ~ erg/rnm 3)

Fig. 5. The induction of p r o p h a g e ~ in various m u t a n t s b y U V irradiation. Abscissa, U V dose on bacteria; ordinate, induction, expressed as t h e fraction plaque forming units of t h e n u m b e r of cells originally p r e s e n t with K M B L i6o dar + (©), K M B L I4o dar x- (A), K M B L 161 dar~- (A), K M B L I62 dar 4- (V), K M B L 163 darD- (V), K M B L 18 hcr + (®), and K M B L I9 her- ( × ) .

Induction of prophage by thymine starvation In order to determine whether the difference in the kinetics of prophage induction between the wild-type strain on the one hand and the various mutants on the other was specific for the induction process itself or for UV-mediated induction only, the lysogenic strains described in the former section were induced by thymine starvation as described under METHODS. The results of these experiments are collected in Fig. 6a. The course of the induction process appeared to be similar for all the strains studied. The number of infective centres increased linearly during thymine starvation; for the wild-type strain as well as for the radiation-sensitive mutants a maximum of induction (9O-lOO%) was observed after 12o miD. Having achieved the maximum, the number of plaque-forming centres decreased slowly, for all the strains at the same rate. Under starvation conditions the amount of free phage remained constant. When thymine was added at the time of maximum induction the first burst was observed after a time lag of 30-4 ° miD; a maximum phage yield (1-1o9-5 • lO9 p.f.u./ml starting from approx, lO 7 cells/ml) was obtained after an additional period of 60 rain. During thymine starvation the colony-forming ability of the lysogenic strains was also measured (Fig. 6b). No significant differences in the viable count among Mutation Research 2 (1965) I I ~ - I 3 I

coli

RANGE OF ACTION OF GENES AND RADIOSENSITIVIT¥ IN E .

tO

V

V

/ , 0

•

12I

1.0

o'x,. •

•

0

V

v

0 5

,

/o

•

i 10'

t

10 &

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~o. 10-:

'

1I

I 2 Starvotion

, time

] 3 (h)

,

[ 4

10

I 0

I 1

, I 2 Storvotion

, time

I 3

,

I 4

(h)

Fig. 6. T h e i n d u c t i o n of p r o p h a g e ~, in v a r i o u s m u t a n t s b y t h y m i n e s t a r v a t i o n , a. I n d u c t i o n curv e s ; abscissa, t i m e of t h y m i n e s t a r v a t i o n a f t e r t h e a d d i t i o n of glucose; ordinate, fraction p.f.u, of t h e n u m b e r of cells p r e s e n t originally, b. Cell s u r v i v a l d a r i n g t h y m i n e s t a r v a t i o n ; ordinate, f r a c t i o n colony f o r m i n g cells. T h e s t r a i n s are i n d i c a t e d as in Fig. 5.

the strains were observed. Moreover, in a parallel experiment we studied the effect of thymine starvation on the viability of non-lysogenic strains, and again no significant differences among the wild-type and sensitive strains were found. In accord with the work of SICARD AND DEVORET 27, viability was more rapidly lost in the lysogenic than in the non-lysogenic strains. Thus it is clear that the genes controlling UV sensitivity have no bearing on thymine starvation, whether scored by prophage induction or by survival.

The radiation sensitivity of transducing phage The strains KMBL 9 hcr+gal- and KMBL 5 hcr-gal- were infected with a mixture of ~ and ;tdg, obtained from the double lysogenic strain KA 17 hcr+,~+,~+o~by UV induction. The number of gal+ transductants in infected cultures was measured on M9 agar with galactose as the sole carbohydrate source as described under METHODS. It was found that irradiation of the ,~/,~dg mixture with a comparatively low dose of UV stimulates its transducing capacity; the number of gal+ recipient bacteria recovered increased over ten-fold at a dose of approx, iooo erg/mm 2. Exposure of the transducing phage to a higher dose led to a subsequent decrease in its transducing capacity. These results are in agreement with those described by ARBER2. No dramatic difference in reaction on irradiated ~.dgwas observed between the hcr+ and its Mutation Research 2 (1965) 111-131

I22

L E. MATTERN, M. P. VAN WINDEN AND A. RORSCH

10-'~

\ .A

10-2

+_.

&

/

& 10-3

Af

"~"

.~.-A

•

. . . . .

1.0

O&'

T

/•

i i

/ / / / /

O

2

4

6

8

10

12

14

10 ~1

UV dose (10 3 e r g / m m 2)

Fig. 7. The U V sensitivity of t h e t r a n s d u c i n g capacity of a m i x t u r e of ). and ;tag e s t i m a t e d either o n K M B L 132 dar + (©) or K M B L 135 dar 1- (&). Abscissa, U V dose on t r a n s d u c i n g phage; right h a n d ordinate, fraction recipient cells (gal + 4- gal-) surviving t h e infection w i t h p h a g e (closed symbols) ; left h a n d ordinate, fraction of t h e surviving cells t h a t is transduced.

her- mutant. The absence of a difference in the inactivation of the transducing capacity of ~d~ on a her + or her- recipient is rather surprising in view of the large difference in survival of normal phage ~, propagated either on a her + or her- host. The UV sensitivity of phage ;tdg and the influence upon it of the recipient strain, was studied in more detail with CR 34 strains. The result of a typical experiment, performed with KMBL 132 dar+gal- and KMBL 135 dar-gal-, is represented in Fig. 7. The transducing phage lysate was irradiated with doses up to 14 ooo erg/mm2; the transducing capacity of the defective phage was tested on non-lysogenic and lysogenie recipients. Moreover the survival of the recipient bacteria, including those that were not transduced, was measured. The m a x i m u m transduction efficiency was achieved at a somewhat lower dose in the strain lacking the repair capacity than in the wild type. Generally the number of transductants formed at zero dose was higher with the dar~ mutants than with the wild-type strains as acceptor strain. However, when the strain KA 52--used to introduce the gal- marker in our strains--was taken as acceptor strain, approximately the same number of transductants as with the dar~ mutants was obtained. After the maximum, the number of transductants recovered decreased in the wild type exponentially with a minimal lethal dose of 2350 erg/mm ~. The dar~ m u t a n t showed an initial decrease two to three times steeper than the wild type (minimal lethal dose 80o erg/mm~), but at higher doses the slope approached that Mutation Research 2 (1965) i i i - i 3 i

RANGE OF ACTION OF GENES AND RADIOSENSITIVITY IN E. coli

123

of t h e wild t y p e . The e x p e r i m e n t was r e p e a t e d w i t h o t h e r dar-; strains a n d several o t h e r w i l d - t y p e strains. T h o u g h some s p r e a d i n g was observed, it follows from t h e results collected in T a b l e I I t h a t t h e i n a c t i v a t i o n curve for t r a n s d u c t i o n of dar-~ m u t a n t s always consists of two p a r t s w i t h different slopes. W i t h s t r a i n K M B L 14o, a lysogenic d e r i v a t i v e of K M B L 135 as recipient, t h e same t r a n s d u c t i o n p a t t e r n w a s o b s e r v e d as w i t h t h e non-lysogenic strain. T h o u g h a small effect of t h e choice of t h e a c c e p t o r s t r a i n on the r a d i a t i o n s e n s i t i v i t y of t h e ~dg function is observed, it is f a r less p r o n o u n c e d t h a n t h e effect on the s u r v i v a l of n o r m a l p h a g e )~. TABLE II SLOPES OF THE SURVIVAL CURVES FOR THE TRANSDUCING

Wild-type strains Strain MLD* ( erg /mm 2)

dar x- mutants Strain

KMBL 13o KMBL 132 KA 52

KMBL KMBL KMBL KMBL

18oo 235 ° 195°

134 135 135 ~.+ 136

CAPACITY OF ~dg

MLD* Ist part ( erg /mm 2)

MLD* 2nd part ( erg /mm ~)

I17O 800 76o 152o

2500 2380 172o 18oo

* MLD = minimal lethal dose. The transfer of F ' episomes In o r d e r to s t u d y the effect of t h e genes controlling UV s e n s i t i v i t y on the t r a n s fer of e p i s o m a l factors, d o n o r strains of the wild t y p e a n d the various sensitive m u t a n t s were i r r a d i a t e d w i t h v a r i o u s U V doses a n d c o n j u g a t e d w i t h n o n - i r r a d i a t e d females, as d e s c r i b e d u n d e r METHODS. The effect of i r r a d i a t i o n on t h e f r e q u e n c y of t r a n s f e r of t h e F'lac factor w a s s t u d i e d p r e v i o u s l y b y DEVORET AND R6RSCH 8 w i t h K I 2 S strains. Their results are r e p r o d u c e d in Fig. 8a. The t r a n s f e r of t h e F ' factor was s t u d i e d from t h e s t r a i n s K K 15 lac-hcr-/F'lac a n d K K 16 lac-hcr+/F'lac to t h e strains K K 14 lac-hcr-F- a n d K K 13 lac-hcr+F -. I t was found t h a t t h e f r e q u e n c y of F ' t r a n s f e r from w i l d - t y p e male to w i l d - t y p e female decreased g r a d u a l l y w i t h increasing U V dose w i t h a slope comp a r a b l e w i t h the s u r v i v a l of t h e m a l e s t r a i n itself. The slope of t h e curve for t h e t r a n s f e r of t h e F ' factor from t h e r a d i a t i o n - s e n s i t i v e male (hcr-) to a w i l d - t y p e female a p p e a r e d to be a p p r o x i m a t e l y twice as steep. This difference in s e n s i t i v i t y of F ' t r a n s f e r is however m u c h less t h a n the difference in s u r v i v a l of t h e hcr+ m a l e strains. F u r t h e r m o r e it was found t h a t t h e genetic c o n s t i t u t i o n of t h e female s t r a i n (hcr + or hcr-) h a d no a p p r e c i a b l e effect on t h e efficiency of sexduction. Similar e x p e r i m e n t s were t h e n p e r f o r m e d w i t h t h e v a r i o u s dar- m u t a n t s . W e s t u d i e d t h e t r a n s f e r of t h e F'lac factor from t h e w i l d - t y p e donor s t r a i n K M B L lO5 dar + a n d t h e donor strains K M B L lO6 darl, K M B L 193 dar'i, K M B L 186 dar~ a n d K M B L 137 dar¥ to t h e w i l d - t y p e female s t r a i n K M B L 83 lac-. The results are repres e n t e d in Fig. 8b. No g r e a t differences were f o u n d a m o n g t h e various sensitive m u t a n t s in t h e i r c a p a c i t y to t r a n s f e r t h e F ' factor a f t e r i r r a d i a t i o n , o n l y t h e dar~ s t r a i n a p p e a r s to be s o m e w h a t m o r e sensitive at lower doses. I n accord w i t h t h e results of DEVORET AND R~RSCH it was f o u n d t h a t t h e difference in efficiency of episome t r a n s fer b e t w e e n wild t y p e a n d dar¥ donor strain was m u c h less t h a n t h e difference in s u r v i v a l b e t w e e n b o t h strains. This could be due to a differential r e p a i r of U V d a m a g e Mutation Research 2 (I965) I I I - I 3 I

124

I. E. MATTERN, M. P. VAN WlNDEN AND A. RORSCIt

1.0

1.0

°~ ,>

"--,...

10-1

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10-1

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10-2[

10-3

10-3

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'

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,

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--

~

I 6

,

l 8

,

10-4 0

, UV

I 2 dose

, (10 2 e r g /

¢ 4

L

I 6

m m 2)

Fig. 8. F r e q u e n c y of F ' lac t r a n s f e r f r o m irradiated male to non-irradiated female strains. Abscissa, U V dose on male s t r a i n s ; ordinate, fraction of female strains h a v i n g received the F ' factor. •a. O, t r a n s f e r of F ' f r o m K K 16hcr + t o K K 13hcr+; e , f r o m K K 1 6 h c r + t o K K 1 4 h c r ; D, from K K 15 hcr- to K K 13 hcr+; , , f r o m K K 15 hcr- to K K 14 hcr-. Broken lines, survival curves for K K 16 hcr+ F ' lac (®) and K K 15 hcr- F" lac ( × ) . b. O, t r a n s f e r of F ' from K M B L lO 5 dar + to K M B L 83 dar+; A, from K M B L lO6 dar t- to K M B L 83 dar+; A, f r o m K M B L 193 dar~- to K M B L 83 dar+; A, f r o m K M B L I37 d a r c to K M B L 83 dar+; V, f r o m K M B L i86 d a r , - t o K M B L 83 dar +.

with respect to the F' factor and the chromosome or to a differential effect on the conjugation process itself. To decide between these two possibilities the inactivation of the episomes F'gal and F'lac after UV irradiation in the strains themselves was studied. After irradiation of the male strains with various UV doses, they were plated on EMB agar, and the fraction of the surviving cells that lost or retained the episome was .determined. At zero dose the fraction of white colonies in an F' culture was approx. IO 2, due to a spontaneous loss of the episome. With the F'gal-containing strains KMBL 165 and KMBL 139 a gradual increase in the number of white colonies with increasing dose was observed, until a plateau was reached at approx, lO% gal- cells (see Fig. 9a). This increase was approximately three times more rapid in the dar~ mutant than in the wild type, and the plateau was reached at a lower dose, indicating that the F'gal particle is more radiation-sensitive in the dar{ mutant than in the wild-type strain. Moreover, comparing the decrease in the fraction of cells that retained the F'gal episome (i.e. the inactivation of the episome) with the survival of the cells themselves, scored by the ability to form colonies, we found that the radiation resistance of the episome is much greater in both strains than the resistance of the cells themselves. Similar results were obtained with F'lac strains (Fig. 9b). The episome in the dar7, dar~ and dar; mutants was somewhat more sensitive than in the dar~ and wild-type strain, but in all strains at least 90% of the cells retained the F' factor at a dose .of 500 erg/mm 2. Mutation Research 2 (1965) 111-131

E. coli

RANGE OF ACTION OF GENES AND RADIOSENSITIVITY IN 1.0

"A~A~.L..~--__~ •

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125

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10-3

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Fig. 9- a. The UV sensitivity of the F ' gal factor in K M B L I65 dar+ F' gal (©, $) and K M B L 139 dar 1- F'gal (&, &). Abscissa, UV dose on F ' strain. R i g h t h a n d ordinate (broken lines), increase fraction gal- clones a m o n g survivors ; left h a n d ordinate, closed symbols, fraction of cells a m o n g the s u r v i v o r s retaining the F ' factor, open symbols, survival of the irradiated male strains, b. The UV sensitivity of the F ' lac episome in K M B L lO 5 dar + (©), K M B L lO6 dar 1- (A), K M B L 193 dar=(A), K M B L 137 dar4-(V), and K M B L 186 dar3- (V). Abscissa, UV dose on F ' strains; ordinate, either fraction of F ' s t r a i n s retaining F ' lac episome ( u n b r o k e n lines) or the survival of the F ' s t r a i n s (broken lines).

Thus it is clear that the F' particles are much more UV resistant than the cells themselves, a result to be expected on account of the great difference in the sizes of the targets concerned. Therefore the observed UV sensitivity of the transfer frequency, as described above, cannot be due to the UV sensitivity of the F' particle itself but must be mainly determined by the UV sensitivity of the processes that lead to conjugation and transfer. Lastly it was found that the rate of transfer of the F' factor was not responsible for the reduced transfer observed after UV irradiation, since this rate was similar in irradiated and non-irradiated wild-type strains and mutants as can be seen in Fig. IO. The maximum of transfer was always reached within 30 rain of contact. Since the processes that lead to conjugation and transfer are mainly determined by the donor strain, and practically only undamaged episomes are transferred, we can understand why the genetic constitution of the female (hcr + or h c r - ) has hardly any influence on the observed sensitivity of sexduction for UV irradiation. Mutation Research 2 (I965) i i I - i 31

I26

I. E. MATTERN, M. P. VAN WINDEN AND A. RORSCH

DISCUSSION

In this paper we have collected a number of experiments with hcr class and Be-2 class mutants which were performed in order to gain more information on the range of action of the genes that seem to be involved in the recovery of the strains 1.0

°

10_1

~ 1(j2 ~5

._g

10-0

10

20

30 40 Incubotion time (rain)

Fig. IO. T h e effect of U V i r r a d i a t i o n on t h e r a t e of t r a n s f e r of t h e F ' lae factor. Abscissa, t i m e

a f t e r m i x i n g e q u a l a m o u n t s of t h e F ' s t r a i n s w i t h t h e F - strains. Ordinate, fraction of F - cells h a v i n g received t h e F ' factor; @, n o n - i r r a d i a t e d K K 16 hcr+ × K K 13 hcr+; I+l, n o n - i r r a d i a t e d K K 15 her- × K K 13 her+; O, K K 16 hcr +, i r r a d i a t e d w i t h 225 e r g / m m ~, × K K 13 hcr+; I , K K 15 hcr-, i r r a d i a t e d w i t h 225 e r g / m m 2, × K K 13 hcr +.

from lethal UV damage in their DNA 3°. The results are briefly summarized in Table III. Though the mutants hcr-, darL dar~ and dar~ differ in their genotype, they were indistinguishable from each other phenotypicaJly. For example their growth rate was found to be inhibited to the same extent after UV irradiation, and lysogenic derivatives of the mutants showed the same prophage induction pattern. In these respects the mutants were more UV sensitive than the corresponding wild-type strains; some other important cell functions were found not to be influenced by any of these mutations. Irradiated mutant strains were indistinguishable from the irradiated wild type in their ability to propagate non-irradiated phage ~ (see the capacity curve, Fig. 3a), to propagate irradiated or non-irradiated RNA phages (Fig. 4) and all strains studied were equally susceptible to thymine starvation (Fig. 6). M u t a t i o n Research 2 (1965) 111-131

RANGE OF ACTION OF GENES AND RADIOSENSITIVITY IN E .

coli

127

TABLE III PROPERTIES OF RADIATION-SENSITIVE MUTANTS

Wild type Strain sensitivity scored b y cell survival I n h i b i t i o n of g r o w t h after UV irradiation Sensitivity of p h a g e ~ on non-irradiated h o s t Capacity to p r o p a g a t e non-irradiated phage ~. Capacity to p r o p a g a t e irradiated phage ). (UV reactivation) Sensitivity of R N A p h a g e s I n d u c t i o n of p r o p h a g e ~. b y UV Sensitivity for t h y m i n e s t a r v a t i o n Sensitivity of ~dg f u n c t i o n E p i s o m e sensitivity Sensitivity of episome t r a n s f e r

hcr class dar t- etc.

dara-

Bs-, class

+ + + +

+ + + +

---+ +

+ --+ +

± -+ + +

+ + + + + + +

+ + + + + + +

~ + + -+ + + ~: +

+ + + + -+ +

-+ + -+ +

+ +

± +

An important difference between the dar~ mutant on the one hand and the other hcr class mutants on the other hand was observed. Though the capacity to form colonies is less radiation sensitive in this strain than in the others, the dar-, mutant was classed among the hcr class strains because of its inability to propagate UV-irradiated phage ;t (no host-cell reactivation) ~. Now we found that the dar~ mutant also shows a deviated UV-reactivation pattern (Fig. 2a). HARM13 argued that UV reactivation and host-cell reactivation must be strongly correlated since the B,-1 mutant showed, in contrast to its wild type, neither of these phenomena for phage T v We confirm such a correlation: the mutants darG dar~ and da~ showed no UV reactivation nor host-cell reactivation for phage ~. Recently however KNESER et al. is found that under certain conditions UV reactivation and host-cell reactivation could be separated from each other. It seems that host-cell reactivation is suppressed by caffeine whereas UV reactivation is not. The existence of the dar~ mutant proves that UV reactivation and host-cell reactivation are not identical and can be separated from each other on the genetic level also. The same applies to the mutants darT~and dar;, that show no UV reactivation but do show host-cell reactivation for phage ;t. Intentionally we incorporated in our study research on the B,_, class mutants dar~ and dar; although the evidence collected until now was rather poor that these mutations also concern repair processes. According to BOYCE et al? thymine dimer excision--the process that underlies the recovery from lethal UV damage in D N A - occurs in B,_,, and this class of mutants is quite able to reactivate UV-irradiated DNA phages. We now consider the possibility that the high UV sensitivity of these strains is due to some kind of irreversible damage in their RNA. The survival of four different RNA phages on any of the mutants available was measured, but no effect of the type of the host on the radiation sensitivity of these phages was observed. This negative result does not completely exclude an influence of the dar genes on a particular bacterial RNA fraction. The UV "reactivation" pattern for phage ;~ of these mutants however again pointed to a specific interaction of the dar, and dar4 gene with irradiated DNA. We found that the dar-; and dar; m u t a n t s - - t h o u g h quite able to propagate UV-irradiated phage ~t--rapidly lose that capacity after irradiation of the strains. Therefore we consider it likely that the dar2 and dar4 genes are involved also Mutation Research 2 (1965) i 11-131

128

I. E. MATTERN, M. P. VAN WINDEN AND A. RORSCH

in the recovery of DNA from lethal UV damage. In this connection we recall that the dar~ and dar-; mutants are indistinguishable from the hcr class mutants in their

prophage induction pattern (Fig. 5). I t is clear that none of the mutations that affect repair of UV-damaged DNA has a bearing on the sensitivity of RNA phages, but we m a y still wonder whether UV lesions in RNA are subject to some kind of repair process. These phages are far more UV resistant than the DNA phage (DXI74 which has a comparable molecular weight, In UV-irradiated RNA uracil dimers can be formed 2s like thymine dimers in DNA. According to BOYCE AND HOWARD-FLANDERS3 and SETLOW AND CARRIER2e repair from lethal UV damage in DNA is essentially associated with the excision of thymine dimer-containing oligonucleotides from irradiated DNA followed b y repair replication. A similar process could be assumed to exist for UV-damaged RNA. However only double-stranded DNA can be repaired 17, which is easily explained b y the lack of the complementary strand required as primer for the repair replication. Since the RNA phages contain single-stranded RNA we cannot expect a repair process analogous to the excision/repair-replication mechanism. The replicative form of the RNA phage might be subject to repair but no infectious, biologically active R F - R N A is available to test this possibility. The method of measuring the UV sensitivity of intracellular phage RNA is difficult to apply since only part of the infecting RNA has been demonstrated to be converted into an R F (ref. 32) at a definite time after infection and additional copies of the phage RNA can be formed very soon after infection. The observed absence of UV reactivation for RNA phages m a y suggest that these phages are not subject to host-cell reactivation, but we cannot consider this conclusive as UV reactivation and host-cell reactivation appeared not to be obligatorily concomitant. At the moment we can most easily explain the higher UV resistance of RNA phages in comparison with single-stranded DNA phages by the difference in quantum efficiency for the photochemical reactions involved 20. It is remarkable that the hcr class mutations (dar~ included) as well as the Bs-z class mutations influence the induction of prophage ~ in a similar way. Experiments making use of the B o r e k - R y a n phenomenon demonstrated that the UV-induced vegetative multiplication of lysogenic phage m a y be preceded b y the formation of a factor, the B R factor, that inactivates the repressor for the vegetative multiplication of the prophageS, e. We can imagine that in the wild type the formation of this factor is retarded b y repair processes in this strain 19, so a higher dose of UV is necessary than in the hcr class mutants to obtain the same level of induction. With our current knowledge it is difficult to interpret in more detail the observed effect of the various dar genes on the shift of the induction m a x i m u m to lower UV doses. Future research on the nature of the B R factor m a y contribute to a better understanding of the initiation of prophage induction and the influence upon it of the various dar genes 7. Having achieved the m a x i m u m induction, .the yield of plaque forming units declines in all the radiation sensitive mutants with a steeper slope than in the wild type. We m a y wonder which target determines the radiation sensitivity of the ability to produce plaque-forming centres. When a lysogenic bacterium is irradiated with UV we expect to damage both the prophage and the, bacterial apparatus that multiplies the phage. The capacity of the bacteria to propagate non-irradiated free phage is very resistant to UV and equally resistant in all the mutants and the wild-type Mutation Research ~ (x965) i i i-i 31

RANGE OF ACTION OF GENES AND RADIOSENSITIVITY

IN E . coli

12 9

strain (Fig. 3a). However in the case of prophage induction we are dealing with the multiplication of an irradiated prophage in an irradiated bacterium; therefore we have to compare it with the capacity to propagate phage, irradiated extracellularly with a dose comparable to that applied to the lysogenic strains. Thus, comparing the results illustrated in Figs. 2b and 5a, we see that for all the strains the capacity of lightly irradiated bacteria to propagate lightly irradiated extracellular phage is less affected b y UV than the ability to produce plaque-forming centres after the m a x i m u m of lysogenic induction. On the other hand, the induction curves parallel rather well the survival curves of irradiated ,~ for the wild-type and hcr class mutants (Fig. ib), and, since the sensitivity of the prophage seems to be equal to that of free phage is, we can explain the decrease in the induction curves for the hcr class m u t a n t s (dar~ included) and the wild type as mostly due to damage to the prophage. However in the Bs-~ class mutants dar~ and dar-;, the decrease in the induction curves is much steeper than the survival curves for irradiated free phage. Since their capacity is also more UV resistant, we are led to the conclusion that no reactivation of the irradiated prophage occurs in these strains. Fig. 2 shows that the dar~ and dar~ mutants upon irradiation also lose their ability to reactivate extracellularly irradiated phage ~t and thus behave more like the dart mutant. The induction of the prophage b y thymine starvation was not influenced b y any of the dar genes, so we can conclude that these genes act specifically on UVmediated induction and not on the induction process per se. If we assume that during thymine starvation the repressor for prophage multiplication is no longer synthesized, we can understand why the dar genes do not influence thymineless induction since there is no question of repressor inactivation which can be subject to repair processes. Lastly we have to consider the UV sensitivity of the transfer of F' episomes and of the ~tdgparticle. The ability to transfer the F' episome to a female strain was found to be much more sensitive to UV than the F' particle itself in all the strains tested. For conjugation adaptive enzyme synthesis is required--for example to build the conjugation b r i d g e m a n d we can imagine that the size of the structures that control this proce s is much larger than the target size of the episome. The UV sensitivity of the ability to form colonies approaches the UV sensitivity of conjugation in wild-type and B8-2 mutants; survival and ability to conjugate are not necessarily correlated, since in the dary m u t a n t the ability to form colonies is much more sensitive t o irradiation than the ability to transfer the F' factor, while in the da~ m u t a n t survival is less sensitive than conjugation (see Figs. 8b and 9b). The transduction experiments show that the )~g particle is very resistant to UV irradiation, and that its sensitivityis not strongly influenced b y the dart mutation. At moderate UV doses--after the m a x i m u m has been achieved--the particle appears to be approximately twice as sensitive in a dar; recipient as in a wild-type strain, whereas at high doses of UV no difference was observed. According to ARBER 2, transductants obtained from an infection with heavily irradiated 20, result mainly from recombination between the gal + marker and the bacterial genome, whereas infection without irradiation and with lightly irradiated ~tdg leads mainly t o the formation of defective lysogenic heterogenotes. Furthermore the formation of stable transductants was found to be less UV-sensitive than the ability to form heterogenotes. The shape of the transduction curve for the dary m u t a n t m a y reflect this differential sensitivity, but, as the processes t h a t lead to the formation o I gal+ transM u t a t i o n Research 2 (1965) I 1 1 - 1 3 1

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ductants are still not quite understood and appear to be very complicated, we cannot yet satisfactorily explain the observed effects. Considering all the information obtained on the range of action of the dar genes, we see that they strongly influence the radiation sensitivity of cell survival and DNA phage multiplication (dose reduction 6 to 7), a little less prophage multiplication and episome inactivation (dose reduction 3 to 4), whereas episome transfer, protein synthesis 22 and transducing capacity of 2d~ are much less influenced (dose reduction 2). This suggests that the genes controlling the UV sensitivity in E. coli act more strongly on the replication of a bacterial or phage genome than on its transcription. ACKNOWLEDGEMENT

We gratefully acknowledge the helpful discussions with Dr. R. DEVORET (Gifsur-Yvette, France) and his constructive suggestions and criticisms. REFERENCES I ARBER, W . , T r a n s d u c t i o n des caractGres gal p a r le bactGriophage l a m b d a . Arch. Sci. Geneva, II (1958 ) 259-338. 2 ARBER, W., H o s t specificity of D N A p r o d u c e d b y E. coli. III. Effects on t r a n s d u c t i o n m e d i a t e d b y Adg. Virology, 23 (1964) 173-182. 3 BOYCE, R. P. AND P. HOWARD-FLANDERS, Release of U V - i n d u c e d t h y m i n e - d i m e r s f r o m D N A in E. coli K I 2 . Proc. Natl. Acad. Sci. U.S., 51 (1964) 293-300. 4 DAVIS, J. E. AND R. L. SINSHEIMER, T h e replication of b a c t e r i o p h a g e MS2. J. Mol. Biol., 6 (1963) 203-207. 5 DEVORET, R. AND J. GEORGE, Sur F a c t i o n i n d u c t r i c e d u r a y o n n e m e n t u l t r a v i o l e t apr~s conj u g a i s o n chez Escherichia coli K I 2 . Compt. Rend., 258 (1964) 2227-2230. 6 DEVORET, R. AND J. GEORGE, Sur u n f a c t e u r e x t r a - c h r o m o s o m i q u e r e s p o u s a b l e de l ' i n d u c t i o n u l t r a v i o l e t t e p a r c o n j u g a i s o n chez Escherichia coli K I 2 . Compt. Rend., 258 (1964) 5287-5290. 7 DEVORET, R., M. MONK AND J. GEORGE, I n d i r e c t u l t r a v i o l e t i n d u c t i o n of p r o p h a g e ~t a n d colicin I factor. Zentr. Bakteriol. Parasitenl., in t h e press. 8 DEVORET, R. AND A. RORSCH, u n p u b l i s h e d results. 9 ELLISON, S. A., R; R. FEINER AND R. V. HILL, A h o s t effect o n b a c t e r i o p h a g e s u r v i v a l after U V - i r r a d i a t i o n . Virology, i i (196o) 294-296. io GAREN, A. AND N. D. ZINDER, Radiological evidence for p a r t i a l genetic h o m o l o g y b e t w e e n b a c t e r i o p h a g e a n d h o s t bacteria. Virology, I (1955) 347-376. I I GREENBERG, J. AND P. WOODY-KARRER, R a d i o s e n s i t i v i t y in E. coll. J. Gen. Microbiol., 33 (1963) 283-292. 12 HARM, W., R e p a i r of lethal u l t r a v i o l e t d a m a g e in p h a g e D N A . I n F. H. SOBELS, Repair from Genetic Radiation Damage, P e r g a m o n Press, L o n d o n , 1963, p. lO7-118. 13 HARM, W., O n t h e r e l a t i o n s h i p b e t w e e n h o s t cell r e a c t i v a t i o n a n d U V r e a c t i v a t i o n in U V i n a c t i v a t e d p h a g e s . Z. Vererbungslehre, 94 (1963) 67-79. 14 HILL, R. F. AND E. SIMSON, A s t u d y of radiosensitive a n d r a d i o r e s i s t a n t m u t a n t s of E. coli B. J. Gen. Microbiol., 24 (1961) 1-14: 15 HOWARD-FLANDERS, P., R. P. BOYCE, E. SIMSON AND L. THERIOT, A genetic locus in E. coli K I 2 t h a t controls t h e r e a c t i v a t i o n of U V - p h o t o p r o d u c t s a s s o c i a t e d w i t h t h y m i n e in D N A . Proc. Natl. Acad. Sci., 48 (1962) 21o9-2115. 16 JACOB, F. AND E. L. WOLLMAN, Sexuality and the Genetics of Bacteria, A c a d e m i c Press, L o n d o n , p. 322. 17 JANSZ, H. S., P. H. POUWELS AND C. VAN ROTTERDAM, S e n s i t i v i t y to u l t r a v i o l e t light of single- a n d d o u b l e - s t r a n d e d D N A . Biochim. Biophys. Aeta, 76 (1964) 655-657. I~8 KNESER, H., K. METZGER AND W. SAUERBIER, E v i d e n c e for t h e n o n - i d e n t i t y of U V r e a c t i v a tion a n d h o s t cell r e a c t i v a t i o n . I n t h e press. 19 LIEB, M., D a r k repair of U V i n d u c t i o n in K I 2 (~t). Virology, 23 (1964) 381-388. 20 MAHLER, H. R. "AND D. FRASER, T h e replication of T2 b a c t e r i o p h a g e . Advan. Virus Res., 8 (1961) 81-82. 21 ROBERTS, R. B., P. H. ABELSON, D. W. COWlE, E. T. BOLTON AND R. J. BRITTEN, Studies of b i o s y n t h e s i s in E. coll. Carnegie Inst. Wash. Publ., 607 (1957) I2 a.

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22 R6RSCH, A., A. EDELMAN, C. VAN DER KAMP AND J. A. COHEN, P h e n o t y p i c a n d g e n o t y p i c c h a r a c t e r i z a t i o n of r a d i a t i o n s e n s i t i v i t y in E. coli B. Biochim. Biophys. Acta, 61 (1962) 278-289. 23 R6RSCI~, A., A. EDELMAN AND J. A. COHEN, T h e gene-controlled r a d i a t i o n s e n s i t i v i t y in E. coli. Biochim. Biophys. Acta, 68 (x963) 263-27 o. 24 RORSCH, A., ~D. VAN DE PUTTE, C. A. VAN SLUIS, C. VAN DER KAMP AND J. VAN DILLEWIJN, T h e m u t a n t collection of t h e r e s e a r c h section for microbial genetics. Report of the Medical Biological Laboratory, R V O - T N O , Rijswijk, The Netherlands, M B L / I 9 6 4 ] 7. 25 SAUERBIER, W., T h e bacterial m e c h a n i s m r e a c t i v a t i n g U V - i r r a d i a t e d p h a g e in t h e d a r k (host cell reactivation). Z. Vererbungslehre, 93 (1962) 220-228. 26 SETLOW, R. B. AND W. L. CARRIER, T h e d i s a p p e a r a n c e of t h y m i n e - d i m e r s f r o m D N A : A n error-correcting m e c h a n i s m . Proc. Natl. Acad. Sci. U.S., 51 (1964) 226-231. 27 SICARD, N. AND R. DEVORET, Effects de la carence en t h y m i n e s u r des s o u c h e s d'Escherichia coli lysog~nes K I 2 T - et colicinog~nes 15 T - . Compt. Rend., 225 (1962) 1417-1419. 28 TRAGER, L., G. TURCK, ~¢[. ISHIMOTO AND A. WACKER, S t r a h l e n c h e m i s c h e R e a k t i o n e n z u r A u f k l g r u n g m o l e c u l a r - g e n e t i s c h e r VorgAnge. Biophysik, I (1964) 4o3-406. 29 VAN DE PUTTE, P., C. WESTENBROEK AND A. RORSCH, T h e r e l a t i o n s h i p b e t w e e n gene-controlled r a d i a t i o n r e s i s t a n c e a n d f i l a m e n t f o r m a t i o n in E. coli. Biochim. Biophys. Acta, 76 (1963) 247-256. 30 VAN DE PUTTE, P. C. A. VAN SLUIS, J. VAN DILLEWIJN AND A. R6RSCH, T h e location of g e n e s controlling r a d i o s e n s i t i v i t y in Escherichia coll. Mutation Research, 2 (1965) 97-11o. 31 WEIGLE, J. J., I n d u c t i o n o5 m u t a t i o n s in a bacterial virus. Proc. Natl. Acad. Sci. U.S., 39 (1953) 628-636. 32 WEISSMANN, C., P. BORST, R. H. BURDON, M. A. BILLETER AND S. OCHOA, R e p l i c a t i o n of viral R N A . I I I . D o u b l e - s t r a n d e d replicative f o r m of MS2 p h a g e R N A . Proc. Natl. Acad. Sci. U.S., 51 (1964) 6 8 2 ~ 9 o . 33 WINKLER, U., t3ber die fehlende p h o t o - u n d wirtszell R e a k t i v i e r b a r k e i t des U V - i n a k t i v i e r t e n R N S - P h a g e n fr. Photochem. Photobiol., 3 (1963) 37-43-

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