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Mutation induction with UV- and X-radiations in spores and vegetative cells of Bacillus subtilis
179
Mutation Research, 49 (1978) 179--186 © Elsevier/North-Holland Biomedical Press
MUTATION INDUCTION WITH UV- AND X-RADIATIONS IN SPORES AND VEGETATIVE CELLS OF BACILLUS SUBTILIS
HIROSHI T A N O O K A , N O B U O M U N A K A T A
and S H I G E Y O K I T A H A R A
Radiobiology Division, National Cancer Center Research Institute,Tsukiji, Chuo-ku, Tokyo 104 (Japan) (Received 10 June 1977) (Revision received 15 July 1977) (Accepted 1 September 1977)
Summary Spores and vegetative cells of Bacillus subtilis strains with various defects in DNA-repair capacities (hcr-, ssp-, hcr-ssp-) were irradiated with UV radiation or X-rays. Induced mutation frequency was determined from the observed frequency of prototrophic reversion of a suppressible auxotrophic mutation. At equal physical dose, after either UV: or X-irradiation, spores were more resistant to mutations as well as to killing than were vegetative cells. However, quantitative comparison revealed that, at equally lethal doses, spores and vegetative cells were almost equally mutable by X-rays whereas spores were considerably less mutable by UV than were vegetative cells. Thus, as judged from their mutagenic efficiency relative to the lethality, X-ray-induced damage in the spore DNA and the vegetative DNA were equally mutagenic, while UVinduced DNA p h o t o p r o d u c t s in the spore were less mutagenic than those in vegetative cells. Post-treatment of UV-irradiated cells with caffeine decreased the survival and the induced mutation frequency for either spores or vegetative cells for all the strains. In X-irradiated spores~ however, a similar suppressing effect of caffeine was observed only for mutability of a strain lacking DNA polymerase I activity.
Introduction Bacterial spores are known to be more resistant to inactivation exerted by radiation and chemicals than are cells in the vegetative form. Spores of Bacillus subtilis are highly resistant to radiation: the transforming activity of the spore DNA is highly resistant to UV as well as to ionizing radiation when it is irraAbbreviation: TDHT, 5-thyminyl-5,6-dihydzothymine.
180 diated in situ [6,8,9], and ionizing radiation induces DNA-strand breaks much less frequently in spores than in vegetative cells [10]. On the other hand, in spore DNA, UV produced no cyclobutane-type pyrimidine dimers but TDHT [12]. These observations indicate that the resistance of spores is partly due to structural alterations of DNA. Such spore-specific DNA structure is expected to be reflected in the mutability of spores. Furthermore, the mutability of spores is expected to depend on t h e genetically controlled functions known to operate in repairing DNA of irradiated B. subtilis spores upon their germination such as excision repair, spore repair, polA and recA functions in the case of UV [2,3], and polA function in the case of ionizing radiation [11]. In the present study, the difference in the frequency of radiation-induced mutation between spores and vegetative cells was demonstrated on strains proficient or deficient in DNA-repair function. It is further asked whether the m u t a n t yield in irradiated spores is affected by post-irradiation treatment in the same manner as in vegetative cells. Caffeine was used in this study, since it is one of a few agents to affect the m u t a n t yield in Escherichia coli when present in the selective medium during post-UV irradiation incubation [13]. Materials and methods
Bacterial strains Strains of B. subtilis used were: HA101 (his met leu) [4], excision repair deficient TKJ5211 (hcr-1 * his met) [7], spore-repair-deficient TKJ6324 (ssp-1 his met), double repair-deficient TKJ6312 (hcr-1 ssp-1 his met leu), and DNA polymerase I deficient TKJ6321 (hcr-1 ssp-1 polA his met). The characteristics of the first two strains were previously described by Tanooka [7]. The latter three strains will be described elsewhere (Munakata et al., in preparation). The test strains all carried suppressible auxotrophic mutations in hisB and met genes. Irradiation with UV and X-rays A thin layer of a suspension of vegetative cells in buffered salt solution or spores in deionized water (5 ml in an 8-cm diameter petri dish) were irradiated with UV or X-rays as described previously [7]. The X-rays were generated by a soft X-ray generator {Softex type CMBW, Softex Co., Tokyo) at 60 kVp and 3 mA. The dose rate of X-rays measured by the dosimetric solution of Fricke in the same geometry as samples was 1.2 krad/min. Measurement of mutant yields The numbers of His* revertants and surviving cells were measured as described previously [7]. The selective medium used for assaying His* mutants and surviving cells was SMM plus L-leucine (glucose, 5 g; K2HPO4, 14 g; KH:PO4, 6 g; a m m o n i u m sulfate, 2 g; sodium citrate, 1 g; MgSO4" 7H20, 0.2 g; casein hydrolyzate, 0.2 g; L-methionine, 0.1 g; L-leucine, 0.1 g; agar, 15 g; all per liter). For m u t a n t detection, about l 0 s cells were plated on the * The mutation,
hcr-1, will b e r e n a m e d
as u v r A l O ( M u n a k a t a ,
Mol. Gen. Genet., in press).
181 medium in a gamma-ray-sterilized petri dish (8 cm diameter, Shionogi, Osaka) and incubated for 3 days at 37°C. For the experiments with caffeine, the selective medium contained various concentrations of caffeine (Sigma). Results Mutation induction in spores and vegetative cells with U V Induced mutation frequency in UV-irradiated spores and vegetative cells was plotted as a function of UV fluence for three different strains (Fig. 1), i.e. wildtype HA101, excision-repair-deficient TKJ5211 (hcr-1), and e x c i s i o n - a n d spore-repair-deficient.TKJ6312 (hcr-1, ssp-1). In the double logarithmic plot, the slope of lines gives the number of causative events required to produce the particular effect in question. All the curves in Fig. 1 are two-hit type no matter whether cells are in the spore form or in the vegetative form, or cells are deficient or proficient in the DNA-repair capacity. Spores showed much lower mutation frequency than vegetative cells and this difference was greatest for TKJ5211 and least for TKJ6312. To examine this difference quantitatively, the doses required to give 10 -s His+ mutation frequency were read from Fig. 1 and the ratios of the doses for g
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I L I 1(~7 L I0 100 1 10 X ray dose, krad UV f l u e n c e , J / m2 F i g . 1. C o m p a r i s o n o f i n d u c e d m u t a t i o n f r e q u e n c y w i t h U V in s p o r e s ( o p e n s y m b o l s ) a n d v e g e t a t i v e cells ( c l o s e d s y m b o l s ) o f B. s u b t i l i s s t r a i n s , H A 1 0 1 wild t y p e [•, A], T K J 5 2 1 1 ( h c r ) [o, e l , a n d T K J 6 3 1 2
I
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Fig. 2. C o m p a r i s o n o f i n d u c e d m u t a t i o n f r e q u e n c y w i t h X - r a y s in s p o r e s [A] a n d v e g e t a t i v e cells [ e l o f B. s u b t i l i s w i l d - t y p e s t r a i n H A 1 0 1 .
182 TABLE 1 DOSE-MODIFICATION FACTORS FOR MUTATION PARE SPORES WITH VEGETATIVE CELLS a Radiation
Strain
UV
AND CELL KILLING
TO COM-
S p o r e / v e g e t a t i v e cell r a t i o
HA101 T K J 5 2 1 1 (hcr-1) T K J 6 3 1 2 (her-1 ssp-1 ) HA101
X-rays
INDUCTION
F o r d o s e to give 10 -5 m u t a t i o n frequency
F o r d o s e to give 10% s u r v i v a l
15.3 304 5.4 7.1
5.7 60 1.8 8.5
a T h e s e l e c t i v e m e d i u m ( S M M [ 7 ] s u p p l e m e n t e d w i t h L - l e u c i n e ) w a s u s e d f o r all t h e assays.
spores and vegetative cells (dose-modification factor) are listed in Table 1. The dose-modification factors were determined for 10% cell survival measured under the same assay conditions; they are also listed in Table 1. The differences in the induced mutation frequency between spores and vegetative cells are much greater than those in the survival. The m u t a t i o n frequency of spores was not significantly affected by the lack of excision-repair capacity. However, the frequency was greatly increased in the spores of strain TKJ6312 which, in addition, lacked spore repair. These results are in accord with those obtained on the UV-survival of the spores [3].
HAl01
TKJ5211, hc_.._Er no UV
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10-5
I
1()6 0
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0.1 0.2 0 0.1 Caffeine concentration,
I
J
0.2 °/o
Fig. 3. E f f e c t o f p o s t - i r r a d i a t i o n t r e a t m e n t w i t h c a f f e i n e o n s u r v i v i n g f r a c t i o n ( u p p e r f i g u r e s ) a n d i n d u c e d m u t a t i o n f r e q u e n c y ( l o w e r f i g u r e s ) in U V - i r r a d i a t e d v e g e t a t i v e cells. [ o ] w i t h o u t U V , [ o ] w i t h U V . ( F l u e n c e , 22 J / m 2 f o r H A L O 1 a n d 3 J / m 2 f o r T K J 5 2 1 1 . )
183
Mutation induction in spores and vegetative cells with X-rays X-ray-induced His* mutation frequency of spores and vegetative cells of strain HA101 is shown as a function of dose in Fig. 2. Again, the difference between spores and vegetative cells was evident. However, this difference coincided with that observed in the cell survival {Table 1). The dose--response curve in Fig. 2 is of the one-hit t y p e for both spores and vegetative cells. Effect of caffeine on induced mutation frequency After spores and vegetative cells were irradiated with UV or X-rays so that their survival was reduced to a b o u t 10%, they were immediately plated on the selective medium containing various amounts of caffeine. Fig. 3 shows the results on UV-irradiated vegetative cells. Caffeine had little effect on the colony-forming ability and the spontaneous mutation rate in unirradiated cells. However, it enhanced the killing of UV-irradiated cells, being more effective in the excision-repair-deficient strain TKJ5211 than in the wildtype. The induced mutation frequency was slightly increased at lower concentrations of caffeine and decreased at higher concentrations. This decrease was greater in TKJ5211 than in the wild-type strain. In spores, the effect of postUV-irradiation treatment with caffeine was also greater in repair-deficient strains than in the wild-type strain (Fig. 4). The greatest effect was seen in the spores of the excision- and spore-repair-deficient strain TKJ6312. In X-irradiated spores, the effect of caffeine was n o t observed with the wildtype strain HA101, but it was clearly seen with the poIA strain TKJ6321 (Fig. 5). Induced mutation frequency was suppressed at higher concentrations of caffeine b u t the survival was unaffected.
HAl01
TKJ5211, hcr
TKJ6324, ssp
TKJ6312, hc_[r ssp
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no UV
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Fig. 4. E f f e c t o f caffeine o n surviving f r a c t i o n ( u p p e r figures) a n d i n d u c e d m u t a t i o n f r e q u e n c y (lower figures) in UV-irradiated spores. [ o ] w i t h o u t UV; [ e ] w i t h UV. (Fluence: 1 6 5 J i m 2 f o r H A 1 0 1 , 1 1 0 J / m 2 f o r T K J 5 2 1 1 , 33 J / m 2 f o r T K J 6 3 2 4 , a n d 6 J / m 2 f o r T K J 6 3 1 2 ) ; [ a ] w i t h UV (3 J / m 2.)
184 HAl01
TKJ6321, hcr ssp poiA
no irrad. E 0
~io-I
X ray
0") E
"~ 10-2 >
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,
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~- 10-5 e--
.9 .~ 156
10-7 0
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0.1 Caffeine
, I , I 01
02
concentration,
°/o
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O2 0
Fig. 5. E f f e c t o f c a f f e i n e o n s u r v i v i n g f r a c t i o n ( u p p e r f i g u r e s ) a n d i n d u c e d m u t a t i o n f r e q u e n c y ( l o w e r f i g u r e s ) in X - i r r a d i a t e d s p o r e s . [ o ] w i t h o u t X - r a y s ; [ o ] w i t h X - r a y s . (Dose: 7 2 k r a d f o r H A 1 0 1 , 36 k r a d for TKJ6321.)
Discussion Radiation resistance of B. subtilis spores was established for mutation induction in the present experiments. The resistance was expressed in terms of the ratios of the doses required to produce the same effects on the survival and the induced m u t a t i o n frequency in spores and vegetative cells (Table 1). In UVirradiated spores, the induced m u t a t i o n frequency was much lower than that of the vegetative cells (Fig. 1). The difference is equally evident for strain TKJ6312, which is defective in both excision repair and spore repair, as well as for strain HA101, which is proficient in both repair functions. To see how the induced m u t a t i o n frequency is related to the cell survival, the data given in Figs. 1 and 2 are summarized in Fig. 6, where the mutation frequency is plotted against relative doses normalized to the 90% lethal dose (doses divided by the 10% survival dose) for each strain. (A similar plot was used previously by Ishii and K o n d o [1].) It is clear from Fig. 6 that, at the same lethal-hit level, spores are less mutable by UV than are vegetative cells for all the strains tested. The enhanced mutability of the spores of TKJ6312, which is incapable of repairing TDHT, indicates that TDHT was responsible for the m u t a t i o n induction in UV-irradiated spores. The yield of TDHT per unit dose in a spore is comparable to or even higher than that of pyrimidine dimers in a vegetative cell [2]. Nevertheless, the killing and the yield of mutants were both lower in the spores than in the vegetative cells. It is concluded that the spore-DNA photoproducts, mainly TDHT, are less mutagenic than pyrimidine dimers produced in vegetative-cell DNA.
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I 0.1 dose
,
,
(dose / DIO for cell survival )
Fig. 6. I n d u c e d m u t a t i o n f r e q u e n c y e x p r e s s e d as a f u n c t i o n o f n o r m a l i z e d dose (dose divided by 10% ceUsurviving d o s e ) in s p o r e s a n d v e g e t a t i v e cens of v a r i o u s B. subfilis strains, i r r a d i a t e d w i t h U V and X-rays.
The induced mutation frequency of the spores irradiated with X-rays was also lower than that of the vegetative cells (Fig. 2). Contrary to the effect of UV irradiation, the difference in the induced mutation frequency between the spores and the vegetative cells corresponded to that in the colony-forming ability. Thus, two curves in Fig. 6 for the induced mutation frequency of X-irradiated spores and vegetative cells plotted against the normalized doses coincide, indicating a parallelism in sensitivity between the mutation induction and the cell killing. These results suggest that the DNA damage produced by ionizing radiation in the spore is similar to or identical with the damage in the vegetative cell, despite the quantitative diffdrence in their production rate. The curves of mutation frequency versus radiation dose are one-hit type for X-rays and two-hit type for UV as recognized in previous works on the vegetative cells of B. subtilis [7] as well as E. coli [1]. These relationships hold whether the cells are in the spore or vegetative form, and whether the cells are proficient or deficient in the DNA-repair capabilities. These results may well be explained by the "overlapping gap" model of radiation-induced mutagenesis as discussed by Sedgwick [5]. The effects of caffeine on mutation induction have been extensively investigated in E. coli and reviewed by Witkin [13]. Our results showed that the suppression of mutation induction and cell survival by post-UV treatment with caffeine was most pronounced in repair-deficient strains either in the spore or vegetative form. This concurs with the results obtained in E. coli by Witkin and
286 Farquharson [14]. Moreover, the effect of caffeine was seen in polA spores irradiated with X-rays. The mutagenic mechanism seems to be c o m m o n for spores and vegetative cells as judged from the response of the induced mutation frequency to post-irradiation treatment with caffeine and also to the cellularrepair deficiencies. Finally, the pre-mutagenic damage in the dormant spore is thought to be unexpressed unless the spore germinate and outgrow. Analysis of mutagenic processes from the developmental aspects of the spores would be an intriguing field of future investigations.
Acknowledgements We thank Miss M. Nagase for assistance in preparing the manuscript. This work was supported by grants-in-aid from the Ministry of Health and Welfare and from the Ministry of Education, Science and Culture, Japan.
References 1 Ishii, Y., a n d S. K o n d o , C o m p a r a t i v e a n a l y s i s o f d e l e t i o n a n d b a s e - c h a n g e m u t a b i l i t i e s o f Escherichia coli B s t r a i n s d i f f e r i n g in D N A - r e p a i r c a p a c i t y ( w i l d - t y p e , u v r A - , p o l A - , r c c A - ) b y v a r i o u s m u t a g e n s , M u t a t i o n R e s . , 27 ( 1 9 7 5 ) 2 7 - - 4 4 . 2 M u n a k a t a , N., a n d C.S. R u p e r t , D a r k r e p a i r o f D N A c o n t a i n i n g " s p o r e p h o t o p r o d u c t " in Bacillus subtilis, Mol. G e n . G e n e t . , 1 3 0 ( 1 9 7 4 ) 2 3 9 - - 2 5 0 . 3 M u n a k a t a , N., a n d C.S. R u p e r t , E f f e c t o f D N A p o l y m e r a s e - d e f e c t i v e a n d r e c o m b i n a t i o n - d e f i c i e n t m u t a t i o n s o n t h e u l t r a v i o l e t s e n s i t i v i t y o f Bacillus s u b t i l i s s p o r e s , M u t a t i o n R e s . , 27 ( 1 9 7 5 ) 1 5 7 - - 1 6 9 . 40kubo, S., a n d T. Y a n a g i d a , I s o l a t i o n o f a s u p p r e s s o r m u t a n t in Bacillus subtilis, J. B a c t e r i o l . , 9 5 (196S) 1187--1188. 5 S e d g w i c k , S . G . , G e n e t i c a n d k i n e t i c e v i d e n c e f o r d i f f e r e n t t y p e s o f p o s t r e p l i c a t i o n r e p a i r in Escherichia coli B, J. B a c t e r i o l . , 1 2 3 ( 1 9 7 5 ) 1 5 4 - - 1 6 1 . 6 T a n o o k a , H., U l t r a v i o l e t r e s i s t a n c e o f D N A in s p o r e s p h e r o p l a s t s of Bacillus s u b t i l i s as m e a s u r e d b y the transforming activity, Biochim. Biophys. Acta, 166 (1968) 581--583. 7 T a n o o k a 0 H . , D e v e l o p m e n t a n d a p p l i c a t i o n s o f Bacillus subfilis t e s t s y s t e m s f o r m u t a g e n s , i n v o l v i n g DNA-repair deficiency and suppressible auxotrophic mutations, Mutation Res., 42 (1977) 19--32. 8 T a n o o k a , H . , a n d F. H u t c h i n s o n , M o d i f i c a t i o n s o f t h e i n a c t i v a t i o n b y i o n i z i n g r a d i a t i o n s o f t h e t r a n s f o r m i n g a c t i v i t y o f D N A in s p o r e s a n d d r y cells, R a d i a t i o n R e s . , 2 4 ( 1 9 6 5 ) 4 3 - - 5 6 . 9 T a n o o k a , H . , a n d Y. S a k a k i b a x a , R a d i o r e s i s t a n t n a t u r e o f t h e t r a n s f o r m i n g a c t i v i t y o f D N A in b a c terial s p o r e s , B i o c h i m . B i o p h y s . A c t a , 1 5 5 ( 1 9 6 8 ) 1 3 0 - - 1 4 2 . 1 0 T a n o o k a , H . , a n d T. T e r a n o , R e s i s t a n c e o f D N A a g a i n s t r a d i a t i o n - i n d u c e d s t r a n d b r e a k a g e in b a c t e r i a l spores, Radiat. Res., 43 (1970) 613--626. 11 T e r a n o , T., a n d H . K a d o t a , R a d i o s e n s i t i v i t y t o g a m m a r a y s o f s p o r e s o f a D N A p o l y m e r a s e d e f i c i e n t m u t a n t o f Bacillus subtilis, M u t a t i o n R e s . , 2 4 ( 1 9 7 4 ) 3 7 9 - - 3 8 1 . 12 Varghese, A.J., 5-Thyminyl-5,6-dihydrothymine from DNA irradiated with ultraviolet light, Biochern. Biophys. Res. Commun., 38 (1970) 484--490. 13 Witkin, E.M., Ultraviolet-induced mutation and DNA repair, Ann. Rev. Microbiol., 23 (1969) 525-552. 1 4 Witl~n0 E . M . , a n d E . L . F a r q u h a r s o n , E n h a n c e m e n t a n d d i m i n u t i o n o f u l t r a v i o l e t - l i g h t - i n i t i a t e d m u t a genesis b y p o s t - t r e a t m e n t w i t h c a f f e i n e in E s e h e r i e h i a coli, in: W o i s t e n h o l m e a n d O ' C o n n e r ( E d s . ) , M u t a t i o n as C e l l u l a r P r o c e s s , C i b a F o u n d a t i o n S y m p o s i u m , C h u r c h i l l , L o n d o n , 1 9 6 9 , p p . 3 6 - - 4 9 .
Mutation Research, 49 (1978) 179--186 © Elsevier/North-Holland Biomedical Press
MUTATION INDUCTION WITH UV- AND X-RADIATIONS IN SPORES AND VEGETATIVE CELLS OF BACILLUS SUBTILIS
HIROSHI T A N O O K A , N O B U O M U N A K A T A
and S H I G E Y O K I T A H A R A
Radiobiology Division, National Cancer Center Research Institute,Tsukiji, Chuo-ku, Tokyo 104 (Japan) (Received 10 June 1977) (Revision received 15 July 1977) (Accepted 1 September 1977)
Summary Spores and vegetative cells of Bacillus subtilis strains with various defects in DNA-repair capacities (hcr-, ssp-, hcr-ssp-) were irradiated with UV radiation or X-rays. Induced mutation frequency was determined from the observed frequency of prototrophic reversion of a suppressible auxotrophic mutation. At equal physical dose, after either UV: or X-irradiation, spores were more resistant to mutations as well as to killing than were vegetative cells. However, quantitative comparison revealed that, at equally lethal doses, spores and vegetative cells were almost equally mutable by X-rays whereas spores were considerably less mutable by UV than were vegetative cells. Thus, as judged from their mutagenic efficiency relative to the lethality, X-ray-induced damage in the spore DNA and the vegetative DNA were equally mutagenic, while UVinduced DNA p h o t o p r o d u c t s in the spore were less mutagenic than those in vegetative cells. Post-treatment of UV-irradiated cells with caffeine decreased the survival and the induced mutation frequency for either spores or vegetative cells for all the strains. In X-irradiated spores~ however, a similar suppressing effect of caffeine was observed only for mutability of a strain lacking DNA polymerase I activity.
Introduction Bacterial spores are known to be more resistant to inactivation exerted by radiation and chemicals than are cells in the vegetative form. Spores of Bacillus subtilis are highly resistant to radiation: the transforming activity of the spore DNA is highly resistant to UV as well as to ionizing radiation when it is irraAbbreviation: TDHT, 5-thyminyl-5,6-dihydzothymine.
180 diated in situ [6,8,9], and ionizing radiation induces DNA-strand breaks much less frequently in spores than in vegetative cells [10]. On the other hand, in spore DNA, UV produced no cyclobutane-type pyrimidine dimers but TDHT [12]. These observations indicate that the resistance of spores is partly due to structural alterations of DNA. Such spore-specific DNA structure is expected to be reflected in the mutability of spores. Furthermore, the mutability of spores is expected to depend on t h e genetically controlled functions known to operate in repairing DNA of irradiated B. subtilis spores upon their germination such as excision repair, spore repair, polA and recA functions in the case of UV [2,3], and polA function in the case of ionizing radiation [11]. In the present study, the difference in the frequency of radiation-induced mutation between spores and vegetative cells was demonstrated on strains proficient or deficient in DNA-repair function. It is further asked whether the m u t a n t yield in irradiated spores is affected by post-irradiation treatment in the same manner as in vegetative cells. Caffeine was used in this study, since it is one of a few agents to affect the m u t a n t yield in Escherichia coli when present in the selective medium during post-UV irradiation incubation [13]. Materials and methods
Bacterial strains Strains of B. subtilis used were: HA101 (his met leu) [4], excision repair deficient TKJ5211 (hcr-1 * his met) [7], spore-repair-deficient TKJ6324 (ssp-1 his met), double repair-deficient TKJ6312 (hcr-1 ssp-1 his met leu), and DNA polymerase I deficient TKJ6321 (hcr-1 ssp-1 polA his met). The characteristics of the first two strains were previously described by Tanooka [7]. The latter three strains will be described elsewhere (Munakata et al., in preparation). The test strains all carried suppressible auxotrophic mutations in hisB and met genes. Irradiation with UV and X-rays A thin layer of a suspension of vegetative cells in buffered salt solution or spores in deionized water (5 ml in an 8-cm diameter petri dish) were irradiated with UV or X-rays as described previously [7]. The X-rays were generated by a soft X-ray generator {Softex type CMBW, Softex Co., Tokyo) at 60 kVp and 3 mA. The dose rate of X-rays measured by the dosimetric solution of Fricke in the same geometry as samples was 1.2 krad/min. Measurement of mutant yields The numbers of His* revertants and surviving cells were measured as described previously [7]. The selective medium used for assaying His* mutants and surviving cells was SMM plus L-leucine (glucose, 5 g; K2HPO4, 14 g; KH:PO4, 6 g; a m m o n i u m sulfate, 2 g; sodium citrate, 1 g; MgSO4" 7H20, 0.2 g; casein hydrolyzate, 0.2 g; L-methionine, 0.1 g; L-leucine, 0.1 g; agar, 15 g; all per liter). For m u t a n t detection, about l 0 s cells were plated on the * The mutation,
hcr-1, will b e r e n a m e d
as u v r A l O ( M u n a k a t a ,
Mol. Gen. Genet., in press).
181 medium in a gamma-ray-sterilized petri dish (8 cm diameter, Shionogi, Osaka) and incubated for 3 days at 37°C. For the experiments with caffeine, the selective medium contained various concentrations of caffeine (Sigma). Results Mutation induction in spores and vegetative cells with U V Induced mutation frequency in UV-irradiated spores and vegetative cells was plotted as a function of UV fluence for three different strains (Fig. 1), i.e. wildtype HA101, excision-repair-deficient TKJ5211 (hcr-1), and e x c i s i o n - a n d spore-repair-deficient.TKJ6312 (hcr-1, ssp-1). In the double logarithmic plot, the slope of lines gives the number of causative events required to produce the particular effect in question. All the curves in Fig. 1 are two-hit type no matter whether cells are in the spore form or in the vegetative form, or cells are deficient or proficient in the DNA-repair capacity. Spores showed much lower mutation frequency than vegetative cells and this difference was greatest for TKJ5211 and least for TKJ6312. To examine this difference quantitatively, the doses required to give 10 -s His+ mutation frequency were read from Fig. 1 and the ratios of the doses for g
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I L I 1(~7 L I0 100 1 10 X ray dose, krad UV f l u e n c e , J / m2 F i g . 1. C o m p a r i s o n o f i n d u c e d m u t a t i o n f r e q u e n c y w i t h U V in s p o r e s ( o p e n s y m b o l s ) a n d v e g e t a t i v e cells ( c l o s e d s y m b o l s ) o f B. s u b t i l i s s t r a i n s , H A 1 0 1 wild t y p e [•, A], T K J 5 2 1 1 ( h c r ) [o, e l , a n d T K J 6 3 1 2
I
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Fig. 2. C o m p a r i s o n o f i n d u c e d m u t a t i o n f r e q u e n c y w i t h X - r a y s in s p o r e s [A] a n d v e g e t a t i v e cells [ e l o f B. s u b t i l i s w i l d - t y p e s t r a i n H A 1 0 1 .
182 TABLE 1 DOSE-MODIFICATION FACTORS FOR MUTATION PARE SPORES WITH VEGETATIVE CELLS a Radiation
Strain
UV
AND CELL KILLING
TO COM-
S p o r e / v e g e t a t i v e cell r a t i o
HA101 T K J 5 2 1 1 (hcr-1) T K J 6 3 1 2 (her-1 ssp-1 ) HA101
X-rays
INDUCTION
F o r d o s e to give 10 -5 m u t a t i o n frequency
F o r d o s e to give 10% s u r v i v a l
15.3 304 5.4 7.1
5.7 60 1.8 8.5
a T h e s e l e c t i v e m e d i u m ( S M M [ 7 ] s u p p l e m e n t e d w i t h L - l e u c i n e ) w a s u s e d f o r all t h e assays.
spores and vegetative cells (dose-modification factor) are listed in Table 1. The dose-modification factors were determined for 10% cell survival measured under the same assay conditions; they are also listed in Table 1. The differences in the induced mutation frequency between spores and vegetative cells are much greater than those in the survival. The m u t a t i o n frequency of spores was not significantly affected by the lack of excision-repair capacity. However, the frequency was greatly increased in the spores of strain TKJ6312 which, in addition, lacked spore repair. These results are in accord with those obtained on the UV-survival of the spores [3].
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Fig. 3. E f f e c t o f p o s t - i r r a d i a t i o n t r e a t m e n t w i t h c a f f e i n e o n s u r v i v i n g f r a c t i o n ( u p p e r f i g u r e s ) a n d i n d u c e d m u t a t i o n f r e q u e n c y ( l o w e r f i g u r e s ) in U V - i r r a d i a t e d v e g e t a t i v e cells. [ o ] w i t h o u t U V , [ o ] w i t h U V . ( F l u e n c e , 22 J / m 2 f o r H A L O 1 a n d 3 J / m 2 f o r T K J 5 2 1 1 . )
183
Mutation induction in spores and vegetative cells with X-rays X-ray-induced His* mutation frequency of spores and vegetative cells of strain HA101 is shown as a function of dose in Fig. 2. Again, the difference between spores and vegetative cells was evident. However, this difference coincided with that observed in the cell survival {Table 1). The dose--response curve in Fig. 2 is of the one-hit t y p e for both spores and vegetative cells. Effect of caffeine on induced mutation frequency After spores and vegetative cells were irradiated with UV or X-rays so that their survival was reduced to a b o u t 10%, they were immediately plated on the selective medium containing various amounts of caffeine. Fig. 3 shows the results on UV-irradiated vegetative cells. Caffeine had little effect on the colony-forming ability and the spontaneous mutation rate in unirradiated cells. However, it enhanced the killing of UV-irradiated cells, being more effective in the excision-repair-deficient strain TKJ5211 than in the wildtype. The induced mutation frequency was slightly increased at lower concentrations of caffeine and decreased at higher concentrations. This decrease was greater in TKJ5211 than in the wild-type strain. In spores, the effect of postUV-irradiation treatment with caffeine was also greater in repair-deficient strains than in the wild-type strain (Fig. 4). The greatest effect was seen in the spores of the excision- and spore-repair-deficient strain TKJ6312. In X-irradiated spores, the effect of caffeine was n o t observed with the wildtype strain HA101, but it was clearly seen with the poIA strain TKJ6321 (Fig. 5). Induced mutation frequency was suppressed at higher concentrations of caffeine b u t the survival was unaffected.
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Fig. 4. E f f e c t o f caffeine o n surviving f r a c t i o n ( u p p e r figures) a n d i n d u c e d m u t a t i o n f r e q u e n c y (lower figures) in UV-irradiated spores. [ o ] w i t h o u t UV; [ e ] w i t h UV. (Fluence: 1 6 5 J i m 2 f o r H A 1 0 1 , 1 1 0 J / m 2 f o r T K J 5 2 1 1 , 33 J / m 2 f o r T K J 6 3 2 4 , a n d 6 J / m 2 f o r T K J 6 3 1 2 ) ; [ a ] w i t h UV (3 J / m 2.)
184 HAl01
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.9 .~ 156
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, I , I 01
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Fig. 5. E f f e c t o f c a f f e i n e o n s u r v i v i n g f r a c t i o n ( u p p e r f i g u r e s ) a n d i n d u c e d m u t a t i o n f r e q u e n c y ( l o w e r f i g u r e s ) in X - i r r a d i a t e d s p o r e s . [ o ] w i t h o u t X - r a y s ; [ o ] w i t h X - r a y s . (Dose: 7 2 k r a d f o r H A 1 0 1 , 36 k r a d for TKJ6321.)
Discussion Radiation resistance of B. subtilis spores was established for mutation induction in the present experiments. The resistance was expressed in terms of the ratios of the doses required to produce the same effects on the survival and the induced m u t a t i o n frequency in spores and vegetative cells (Table 1). In UVirradiated spores, the induced m u t a t i o n frequency was much lower than that of the vegetative cells (Fig. 1). The difference is equally evident for strain TKJ6312, which is defective in both excision repair and spore repair, as well as for strain HA101, which is proficient in both repair functions. To see how the induced m u t a t i o n frequency is related to the cell survival, the data given in Figs. 1 and 2 are summarized in Fig. 6, where the mutation frequency is plotted against relative doses normalized to the 90% lethal dose (doses divided by the 10% survival dose) for each strain. (A similar plot was used previously by Ishii and K o n d o [1].) It is clear from Fig. 6 that, at the same lethal-hit level, spores are less mutable by UV than are vegetative cells for all the strains tested. The enhanced mutability of the spores of TKJ6312, which is incapable of repairing TDHT, indicates that TDHT was responsible for the m u t a t i o n induction in UV-irradiated spores. The yield of TDHT per unit dose in a spore is comparable to or even higher than that of pyrimidine dimers in a vegetative cell [2]. Nevertheless, the killing and the yield of mutants were both lower in the spores than in the vegetative cells. It is concluded that the spore-DNA photoproducts, mainly TDHT, are less mutagenic than pyrimidine dimers produced in vegetative-cell DNA.
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I 0.1 dose
,
,
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Fig. 6. I n d u c e d m u t a t i o n f r e q u e n c y e x p r e s s e d as a f u n c t i o n o f n o r m a l i z e d dose (dose divided by 10% ceUsurviving d o s e ) in s p o r e s a n d v e g e t a t i v e cens of v a r i o u s B. subfilis strains, i r r a d i a t e d w i t h U V and X-rays.
The induced mutation frequency of the spores irradiated with X-rays was also lower than that of the vegetative cells (Fig. 2). Contrary to the effect of UV irradiation, the difference in the induced mutation frequency between the spores and the vegetative cells corresponded to that in the colony-forming ability. Thus, two curves in Fig. 6 for the induced mutation frequency of X-irradiated spores and vegetative cells plotted against the normalized doses coincide, indicating a parallelism in sensitivity between the mutation induction and the cell killing. These results suggest that the DNA damage produced by ionizing radiation in the spore is similar to or identical with the damage in the vegetative cell, despite the quantitative diffdrence in their production rate. The curves of mutation frequency versus radiation dose are one-hit type for X-rays and two-hit type for UV as recognized in previous works on the vegetative cells of B. subtilis [7] as well as E. coli [1]. These relationships hold whether the cells are in the spore or vegetative form, and whether the cells are proficient or deficient in the DNA-repair capabilities. These results may well be explained by the "overlapping gap" model of radiation-induced mutagenesis as discussed by Sedgwick [5]. The effects of caffeine on mutation induction have been extensively investigated in E. coli and reviewed by Witkin [13]. Our results showed that the suppression of mutation induction and cell survival by post-UV treatment with caffeine was most pronounced in repair-deficient strains either in the spore or vegetative form. This concurs with the results obtained in E. coli by Witkin and
286 Farquharson [14]. Moreover, the effect of caffeine was seen in polA spores irradiated with X-rays. The mutagenic mechanism seems to be c o m m o n for spores and vegetative cells as judged from the response of the induced mutation frequency to post-irradiation treatment with caffeine and also to the cellularrepair deficiencies. Finally, the pre-mutagenic damage in the dormant spore is thought to be unexpressed unless the spore germinate and outgrow. Analysis of mutagenic processes from the developmental aspects of the spores would be an intriguing field of future investigations.
Acknowledgements We thank Miss M. Nagase for assistance in preparing the manuscript. This work was supported by grants-in-aid from the Ministry of Health and Welfare and from the Ministry of Education, Science and Culture, Japan.
References 1 Ishii, Y., a n d S. K o n d o , C o m p a r a t i v e a n a l y s i s o f d e l e t i o n a n d b a s e - c h a n g e m u t a b i l i t i e s o f Escherichia coli B s t r a i n s d i f f e r i n g in D N A - r e p a i r c a p a c i t y ( w i l d - t y p e , u v r A - , p o l A - , r c c A - ) b y v a r i o u s m u t a g e n s , M u t a t i o n R e s . , 27 ( 1 9 7 5 ) 2 7 - - 4 4 . 2 M u n a k a t a , N., a n d C.S. R u p e r t , D a r k r e p a i r o f D N A c o n t a i n i n g " s p o r e p h o t o p r o d u c t " in Bacillus subtilis, Mol. G e n . G e n e t . , 1 3 0 ( 1 9 7 4 ) 2 3 9 - - 2 5 0 . 3 M u n a k a t a , N., a n d C.S. R u p e r t , E f f e c t o f D N A p o l y m e r a s e - d e f e c t i v e a n d r e c o m b i n a t i o n - d e f i c i e n t m u t a t i o n s o n t h e u l t r a v i o l e t s e n s i t i v i t y o f Bacillus s u b t i l i s s p o r e s , M u t a t i o n R e s . , 27 ( 1 9 7 5 ) 1 5 7 - - 1 6 9 . 40kubo, S., a n d T. Y a n a g i d a , I s o l a t i o n o f a s u p p r e s s o r m u t a n t in Bacillus subtilis, J. B a c t e r i o l . , 9 5 (196S) 1187--1188. 5 S e d g w i c k , S . G . , G e n e t i c a n d k i n e t i c e v i d e n c e f o r d i f f e r e n t t y p e s o f p o s t r e p l i c a t i o n r e p a i r in Escherichia coli B, J. B a c t e r i o l . , 1 2 3 ( 1 9 7 5 ) 1 5 4 - - 1 6 1 . 6 T a n o o k a , H., U l t r a v i o l e t r e s i s t a n c e o f D N A in s p o r e s p h e r o p l a s t s of Bacillus s u b t i l i s as m e a s u r e d b y the transforming activity, Biochim. Biophys. Acta, 166 (1968) 581--583. 7 T a n o o k a 0 H . , D e v e l o p m e n t a n d a p p l i c a t i o n s o f Bacillus subfilis t e s t s y s t e m s f o r m u t a g e n s , i n v o l v i n g DNA-repair deficiency and suppressible auxotrophic mutations, Mutation Res., 42 (1977) 19--32. 8 T a n o o k a , H . , a n d F. H u t c h i n s o n , M o d i f i c a t i o n s o f t h e i n a c t i v a t i o n b y i o n i z i n g r a d i a t i o n s o f t h e t r a n s f o r m i n g a c t i v i t y o f D N A in s p o r e s a n d d r y cells, R a d i a t i o n R e s . , 2 4 ( 1 9 6 5 ) 4 3 - - 5 6 . 9 T a n o o k a , H . , a n d Y. S a k a k i b a x a , R a d i o r e s i s t a n t n a t u r e o f t h e t r a n s f o r m i n g a c t i v i t y o f D N A in b a c terial s p o r e s , B i o c h i m . B i o p h y s . A c t a , 1 5 5 ( 1 9 6 8 ) 1 3 0 - - 1 4 2 . 1 0 T a n o o k a , H . , a n d T. T e r a n o , R e s i s t a n c e o f D N A a g a i n s t r a d i a t i o n - i n d u c e d s t r a n d b r e a k a g e in b a c t e r i a l spores, Radiat. Res., 43 (1970) 613--626. 11 T e r a n o , T., a n d H . K a d o t a , R a d i o s e n s i t i v i t y t o g a m m a r a y s o f s p o r e s o f a D N A p o l y m e r a s e d e f i c i e n t m u t a n t o f Bacillus subtilis, M u t a t i o n R e s . , 2 4 ( 1 9 7 4 ) 3 7 9 - - 3 8 1 . 12 Varghese, A.J., 5-Thyminyl-5,6-dihydrothymine from DNA irradiated with ultraviolet light, Biochern. Biophys. Res. Commun., 38 (1970) 484--490. 13 Witkin, E.M., Ultraviolet-induced mutation and DNA repair, Ann. Rev. Microbiol., 23 (1969) 525-552. 1 4 Witl~n0 E . M . , a n d E . L . F a r q u h a r s o n , E n h a n c e m e n t a n d d i m i n u t i o n o f u l t r a v i o l e t - l i g h t - i n i t i a t e d m u t a genesis b y p o s t - t r e a t m e n t w i t h c a f f e i n e in E s e h e r i e h i a coli, in: W o i s t e n h o l m e a n d O ' C o n n e r ( E d s . ) , M u t a t i o n as C e l l u l a r P r o c e s s , C i b a F o u n d a t i o n S y m p o s i u m , C h u r c h i l l , L o n d o n , 1 9 6 9 , p p . 3 6 - - 4 9 .